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Topic Editors

Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso D’Augusto 237, 47921 Rimini, Italy
Department for Life Quality Studies, University of Bologna, 40126 Bologna, Italy

Neuroprotection by Drugs, Nutraceuticals and Physical Activity

Abstract submission deadline
closed (30 July 2022)
Manuscript submission deadline
closed (30 September 2022)
Viewed by
100136

Topic Information

Dear Colleagues,

Acute and chronic neurodegenerative diseases, such as stroke, brain trauma, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease are associated with high morbidity and mortality rates. A characteristic of these neurodegenerative diseases is selective neuronal dysfunction and death. The symptoms and the exacerbations of these diseases are, however, very different according to their specific pathways of neuronal impairment. Several mechanisms can lead to neuronal dysfunction and death, including calcium overload, excitatory amino acid release, oxidative stress, inflammation and microglial activation, protein misfolding, proteostasis and mitochondrial disfunction. The clinical management of these diseases is currently very critical as therapeutic strategies are often limited to relieving symptoms rather than treating the disease. Hence, the development of neuroprotective strategies to prevent or delay the neuronal impairment is at the epicenter of the current 21st-century research agenda in biomedicine. The scientific community, in addition to focusing on the development of effective new neuroprotective drugs, is also exploring non-pharmacological approaches by food components, such as nutraceuticals, and physical activity. In this regard, several studies show that nutraceuticals and physical activity have similar or complementary neuroprotection mechanisms, suggesting new integrated approaches with the pharmacological interventions to enhance neuroprotective effects. We invite you to submit your research findings to this Special Issue, which has the aim to present the updated state-of-the-art research on the potential mechanisms of neuroprotection at pre-clinical and clinical level mediated by drugs, nutraceuticals and physical activity. Original research articles, review articles, clinical trials, and meta-analyses are welcome.

Dr. Cristina Angeloni
Prof. Dr. Andrea Tarozzi
Topic Editors

Keywords

  • old and new drugs
  • food supplements, nutraceuticals, and functional foods
  • physical activity and exercise
  • integrated neuroprotective interventions
  • neuroprotective mechanisms
  • neuroprotective strategies
  • new targets for neuroprotection
  • prevention of eurodegeneration
  • neurodegenerative diseases

Participating Journals

Journal Name Impact Factor CiteScore Launched Year First Decision (median) APC
Biomedicines
biomedicines
3.9 5.2 2013 15.3 Days CHF 2600
Current Issues in Molecular Biology
cimb
2.8 2.9 1999 16.8 Days CHF 2200
International Journal of Molecular Sciences
ijms
4.9 8.1 2000 18.1 Days CHF 2900
Neurology International
neurolint
3.2 3.7 2009 22.1 Days CHF 1600
Pharmaceuticals
pharmaceuticals
4.3 6.1 2004 12.8 Days CHF 2900

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Published Papers (25 papers)

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7 pages, 220 KiB  
Editorial
Neuroprotection by Drugs, Nutraceuticals and Physical Activity
by Andrea Tarozzi and Cristina Angeloni
Int. J. Mol. Sci. 2023, 24(4), 3176; https://doi.org/10.3390/ijms24043176 - 6 Feb 2023
Cited by 2 | Viewed by 2325
Abstract
Acute and chronic neural injuries, including stroke, brain trauma and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), Parkinson’s disease (PD), and Alzheimer’s disease (AD) are associated with high morbidity and mortality rates [...] Full article
26 pages, 4093 KiB  
Article
Neuroprotective Effects of Some Nutraceuticals against Manganese-Induced Parkinson’s Disease in Rats: Possible Modulatory Effects on TLR4/NLRP3/NF-κB, GSK-3β, Nrf2/HO-1, and Apoptotic Pathways
by Karema Abu-Elfotuh, Ahmed Mohsen Elsaid Hamdan, Asmaa A. Mohammed, Ahmed M. Atwa, Magy R. Kozman, Amany M. Ibrahim, Shaimaa M. Motawea, Heba Mohammed Refat M. Selim, Sally Tohamy Kamal Tohamy, Mahmoud Nour El-Din, Sameh S. Zaghlool, Ayah M. H. Gowifel and Magdy M. Awny
Pharmaceuticals 2022, 15(12), 1554; https://doi.org/10.3390/ph15121554 - 14 Dec 2022
Cited by 19 | Viewed by 3648
Abstract
Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting the substantia nigra where functions controlling body movement take place. Manganese (Mn) overexposure is linked to a neurologic syndrome resembling PD. Sesamol, thymol, wheat grass (WG), and coenzyme Q10 (CoQ10) are potent antioxidants, anti-inflammatory, [...] Read more.
Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting the substantia nigra where functions controlling body movement take place. Manganese (Mn) overexposure is linked to a neurologic syndrome resembling PD. Sesamol, thymol, wheat grass (WG), and coenzyme Q10 (CoQ10) are potent antioxidants, anti-inflammatory, and anti-apoptotic nutraceuticals. We investigated the potential protective effects of these nutraceuticals alone or in combinations against MnCl2-induced PD in rats. Seven groups of adult male Sprague Dawley rats were categorized as follows: group (I) was the control, while groups 2–7 received MnCl2 either alone (Group II) or in conjunction with oral doses of sesamol (Group III), thymol (Group IV), CoQ10 (Group V), WG (Group VI), or their combination (Group VII). All rats were subjected to four behavioral tests (open-field, swimming, Y-maze, and catalepsy tests). Biochemical changes in brain levels of monoamines, ACHE, BDNF, GSK-3β, GABA/glutamate, as well as oxidative stress, and apoptotic and neuroinflammatory biomarkers were evaluated, together with histopathological examinations of different brain regions. Mn increased catalepsy scores, while decreasing neuromuscular co-ordination, and locomotor and exploratory activity. It also impaired vigilance, spatial memory, and decision making. Most behavioral impairments induced by Mn were improved by sesamol, thymol, WG, or CoQ10, with prominent effect by sesamol and thymol. Notably, the combination group showed more pronounced improvements, which were confirmed by biochemical, molecular, as well as histopathological findings. Sesamol or thymol showed better protection against neuronal degeneration and some behavioral impairments induced by Mn than WG or CoQ10, partly via interplay between Nrf2/HO-1, TLR4/NLRP3/NF-κB, GSK-3β and Bax/Bcl2 pathways. Full article
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on motor functions in open-field test of rats treated with MnCl<sub>2</sub>. (<b>A</b>) Latency time, (<b>B</b>) Ambulation frequency, (<b>C</b>) Rearing frequency, and (<b>D</b>) Grooming frequency. Values are means of 12 rats ± S.E.M, as compared with control (a), Mn (b), COMB (c), sesamol (d), and thymol (e) groups. One-way ANOVA with post-test Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 2
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on swimming test of rats treated with MnCl<sub>2</sub>. (<b>A</b>) latency time, (<b>B</b>) swimming time, and (<b>C</b>) swimming direction score. Values are means of 12 rats ± S.E.M, as compared with control (a), Mn (b), COMB (c), and sesamol (d) groups. One-way ANOVA with posttest Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 3
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on spontaneous alternation percent in Y-maze test of rats treated with MnCl<sub>2</sub>. Values are means of 12 rats ± S.E.M, as compared with control (a), Mn (b), and COMB (c) groups. One-way ANOVA with posttest Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 4
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on catalepsy score in (<b>A</b>) grid and (<b>B</b>) bar tests of rats treated with MnCl<sub>2</sub>. Values are means of 12 rats ± S.E.M, as compared with control (a), Mn (b), COMB (c), sesamol (d), thymol (e), and CoQ10 (f) groups. One-way ANOVA with posttest Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 5
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on MnCl<sub>2</sub>-induced changes in (<b>A</b>) Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) and (<b>B</b>) Hemoxygenase-1 (HO-1). Values are means of 6 rats ± S.E.M, as compared with control (a), Mn (b), COMB (c), sesamol (d), thymol (e), and CoQ10 (f) groups. One-way ANOVA with posttest Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 6
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on brain inflammatory markers in rats treated with MnCl<sub>2</sub>. (<b>A</b>) tumor necrosis factor alpha, (<b>B</b>) TLR4, (<b>C</b>) NLRP3, (<b>D</b>) NF-κB, (<b>E</b>) Caspase-1, and (<b>F</b>) IL-1β. Values are means of 8 rats ± S.E.M, as compared with control (a), Mn (b), COMB (c), sesamol (d), thymol (e), and CoQ10 (f) groups. One-way ANOVA with posttest Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 7
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on MnCl<sub>2</sub>-induced changes in (<b>A</b>) <span class="html-italic">Bax</span> mRNA expression level, (<b>B</b>) <span class="html-italic">Bcl2</span> mRNA expression level, and (<b>C</b>) caspase-3 protein content. Values are means of 8 rats ± S.E.M, as compared with control (a), Mn (b), COMB (c), sesamol (d), thymol (e), and CoQ10 (f) groups. One-way ANOVA with posttest Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 8
<p>Effects of sesamol, thymol, CoQ10, wheat grass, or their combination on MnCl<sub>2</sub>-induced changes in mRNA expression levels of (<b>A</b>) Glial fibrillary acidic protein (GFAP), (<b>B</b>) apoptosis inducing factor (AIF), and (<b>C</b>) glycogen synthase kinase 3-beta (GSK-3β). Values are means of 6 rats ± S.E.M, as compared with control (a), Mn (b), COMB (c), sesamol (d), thymol (e), and CoQ10 (f) groups. One-way ANOVA with posttest Tukey’s multiple comparison assessed the statistical differences between the various groups, <span class="html-italic">p</span>-value &lt; 0.05.</p>
Full article ">Figure 9
<p>Photomicrographs of brain sections (cerebral cortex, subiculum and fascia dentata in hippocampus, and striatum areas) stained by Hematoxylin and Eosin (magnification power of 40×). Where: (<b>a1</b>–<b>a4</b>): control group, (<b>b1</b>–<b>b4</b>): Mn group, (<b>c1</b>–<b>c4</b>): Mn + Sesamol group, (<b>d1</b>–<b>d4</b>): Mn + Thymol group, (<b>e1</b>–<b>e4</b>): Mn + CoQ10 group, (<b>f1</b>–<b>f4</b>): Mn + WG group, and (<b>g1</b>–<b>g4</b>): Mn + Combination of Sesamol + Thymol + CoQ10 + WG. Where: (<b>blue arrow</b>) indicates no histopathological alteration, (<b>orange arrow</b>) indicates nuclear pyknosis and degeneration, and (<b>black arrow</b>) indicates focal eosinophilic plagues.</p>
Full article ">Figure 10
<p>Summary of the experimental design and all behavioural and biochemical tests.</p>
Full article ">
14 pages, 1625 KiB  
Article
Milmed Yeast Alters the LPS-Induced M1 Microglia Cells to Form M2 Anti-Inflammatory Phenotype
by Federica Armeli, Beatrice Mengoni, Elisa Maggi, Cristina Mazzoni, Adele Preziosi, Patrizia Mancini, Rita Businaro, Thomas Lenz and Trevor Archer
Biomedicines 2022, 10(12), 3116; https://doi.org/10.3390/biomedicines10123116 - 2 Dec 2022
Cited by 7 | Viewed by 2345
Abstract
Microglial cells polarized towards a proinflammatory phenotype are considered the main cellular players of neuroinflammation, underlying several neurodegenerative diseases. Many studies have suggested that imbalance of the gut microbial composition is associated with an increase in the pro-inflammatory cytokines and oxidative stress that [...] Read more.
Microglial cells polarized towards a proinflammatory phenotype are considered the main cellular players of neuroinflammation, underlying several neurodegenerative diseases. Many studies have suggested that imbalance of the gut microbial composition is associated with an increase in the pro-inflammatory cytokines and oxidative stress that underlie chronic neuroinflammatory diseases, and perturbations to the gut microbiota were detected in neurodegenerative conditions such as Parkinson’s disease and Alzheimer’s disease. The importance of gut-brain axis has been uncovered and the relevance of an appropriate microbiota balance has been highlighted. Probiotic treatment, rebalancing the gut microbioma, may reduce inflammation. We show that Milmed yeast, obtained from S. cerevisiae after exposure to electromagnetic millimeter wavelengths, induces a reversal of LPS-M1 polarized microglia towards an anti-inflammatory phenotype, as demonstrated morphologically by the recovery of resting phenotype by microglia, by the decrease in the mRNAs of IL-1β, IL-6, TNF-α and in the expression of iNOS. Moreover, Milmed stimulated the secretion of IL-10 and the expression of Arginase-1, cell markers of M2 anti-inflammatory polarized cells. The present findings data suggest that Milmed may be considered to be a probiotic with diversified anti-inflammatory activity, capable of directing the polarization of microglial cells. Full article
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Figure 1

Figure 1
<p>(<b>A</b>) BV2 cells area following Lipopolysaccharide (LPS) treatment (1 ng/mL) for 24 h in the presence or absence of untreated yeast grown in different cultural conditions. LPS induced a cell body enlargement which was even more evident in the presence of untreated yeast grown in G2%. (<b>B</b>) BV2 cells area following LPS treatment (1 ng/mL) for 24 h in the presence or absence of Milmed: after Milmed addition cells showed a measure of area similar to that of control in Glucose 2%, Glycerol 3% and SD with LPS. Data are expressed as mean ± SD for each group (<span class="html-italic">n</span> = 3). Statistical analysis was performed by the one-way analysis of variance (ANOVA) method coupled with the Bonferroni post-test. *** <span class="html-italic">p</span> &lt; 0.001; ## <span class="html-italic">p</span> &lt; 0.01; ### <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 2
<p>(<b>A</b>) Immunofluorescence analysis of Inducible Nitric Oxide Synthase (iNOS) expression in BV-2 cells cultured in the presence of untreated yeast grown for 24 h in different metabolic medium without LPS (on the left, first column) or after addition of Lipopolysaccharide (LPS) 1 ng/mL (second column) and (<b>B</b>) Arginase-1 (ARG-1)expression in BV-2 cells cultured in the presence of Milmed grown in different metabolic medium without LPS (on the right, first column) or after addition of LPS 1 ng/mL (second column). Quantification of the median fluorescence intensity was performed by ImageJ software and data were expressed as histograms, normalized to the number of cells for field. 4′,6-diamidino-2-phenylindole (DAPI)was used to counterstain the nuclei. Data are expressed as mean ± SD for each group (<span class="html-italic">n</span> = 3). Statistical analysis was performed by the one-way analysis of variance (ANOVA) coupled with the Bonferroni post-test. *** <span class="html-italic">p</span> &lt; 0.001; ### <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 3
<p>BV2 cells were pretreated with 5 × 10<sup>2</sup> Milmed/well grown in Yeast Extract–Peptone–Dextrose (YPD) medium or regrown in YPD medium from a dried preparation for 45 min and afterward incubated with Lipopolysaccharide (LPS) 1 ng/mL. Inducible Nitric Oxide Synthase (iNOS) and Arginase-1 (ARG-1) mRNAs expressions were evaluated by qRT-PCR at 4 h and normalized to β-actin. Data are shown as mean ± SD from three independent experiments performed in triplicate. Statistical analysis was evaluated by unpaired Student’s <span class="html-italic">t</span> test. Expression profiles were determined using the 2<sup>−ΔΔCT</sup> method. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; ## <span class="html-italic">p</span> &lt; 0.01; ### <span class="html-italic">p</span> &lt; 0.001; #### <span class="html-italic">p</span> &lt; 0.0001 * VS CTRL, # VS LPS.</p>
Full article ">Figure 4
<p>mRNA expression of (<b>A</b>) interleukin-1β (IL-1 β); (<b>B</b>) interleukin-6 (IL-6); (<b>C</b>) Tumor Necrosis Factor-α (TNF-a); and (<b>D</b>) interleukin-10 (IL-10); monitored by qRT-PCR and normalized to β-actin. The decrease of mRNA expression of pro-inflammatory cytokines is associated with increased mRNA expression of IL-10 in BV2 cells in both Milmed and Dried treatment. Data are shown as mean ± SD from three independent experiments performed in triplicate. Expression profiles were determined using the 2<sup>−ΔΔCT</sup> method. Statistical analysis was evaluated by unpaired Student’s <span class="html-italic">t</span> test. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; ## <span class="html-italic">p</span> &lt; 0.01; ### <span class="html-italic">p</span> &lt; 0.001; #### <span class="html-italic">p</span> &lt; 0.0001 * VS CTRL, # VS LPS.</p>
Full article ">
13 pages, 8506 KiB  
Article
The Neuroprotective Effects of Arecae Pericarpium against Glutamate-Induced HT22 Cell Cytotoxicity
by Yun Hee Jeong, You-Chang Oh, Tae In Kim, Jong-Sup Bae and Jin Yeul Ma
Curr. Issues Mol. Biol. 2022, 44(12), 5902-5914; https://doi.org/10.3390/cimb44120402 - 27 Nov 2022
Cited by 6 | Viewed by 2000
Abstract
Arecae Pericarpium has been found to exert anti-migraine, antidepressant, and antioxidative effects. However, the mechanisms involved are unclear. This study explored the possibility that Arecae Pericarpium ethanol extract (APE) exerts neuroprotective effects against oxidative stress-induced neuronal cell death. Since glutamate excitotoxicity has been [...] Read more.
Arecae Pericarpium has been found to exert anti-migraine, antidepressant, and antioxidative effects. However, the mechanisms involved are unclear. This study explored the possibility that Arecae Pericarpium ethanol extract (APE) exerts neuroprotective effects against oxidative stress-induced neuronal cell death. Since glutamate excitotoxicity has been implicated in the pathogenesis and development of several neurodegenerative disorders, we explored the mechanisms of action of APE on oxidative stress-induced by glutamate. Our results revealed that pretreatment with APE prevents glutamate-induced HT22 cell death. APE also reduced both the levels of intracellular reactive oxygen species and the apoptosis of cells, while maintaining glutamate-induced mitochondrial membrane potentials. Western blotting showed that pretreatment with APE facilitates the upregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) phosphorylation; the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2); and the production of antioxidant enzymes, including catalase, glutamate-cysteine ligase catalytic subunits, NAD(P)H quinone oxidoreductase 1, and heme oxygenase (HO)-1. The administration of LY294002, a PI3K/Akt inhibitor, attenuated the neuroprotective effects of APE on oxidative stress-induced neuronal cell damage. This allowed us to infer that the protective effects of APE on oxidative damage to cells can be attributed to the PI3K/Akt-mediated Nrf-2/HO-1 signaling pathway. Full article
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Figure 1

Figure 1
<p>Effects of Arecae Pericarpium ethanol extract on glutamate-induced cytotoxicity in HT22 cells. (<b>A</b>) HT22 cells were incubated with APE at concentrations of 10, 50, 100, or 200 μg/mL. (<b>B</b>–<b>D</b>) After APE pretreatment, the HT22 cells were stimulated with glutamate (5 mM). (<b>D</b>) The images represent the three independent experiments, shown at 1000× magnification. Scale bar = 20 μm. Control was non-treated cells. Data were presented as mean ± standard error of the mean. Statistical significance was set at # <span class="html-italic">p</span> &lt; 0.05 (vs. control), ** <span class="html-italic">p</span> &lt; 0.01, and † <span class="html-italic">p</span> &lt; 0.001 (vs. glutamate). APE, Arecae Pericarpium ethanol extract; Con, control; Glu, glutamate; LDH, lactate dehydrogenase.</p>
Full article ">Figure 2
<p>Effects of Arecae Pericarpium ethanol extract on glutamate-induced intracellular ROS production in HT22 cells. Cells were pretreated with APE at concentrations of 10, 50, 100, or 200 μg/mL and then with 5 mM glutamate. H<sub>2</sub>DCFDA (20 μM), an oxidation-sensitive fluorescent dye, was used to assess ROS levels. The expression of ROS was determined using a fluorescence microscope and fluorescence microplate reader. Scale bar = 100 μm. Control was non-treated cells. All experiments were repeated at least three times, and similar results were obtained. Data were presented as mean ± standard error of the mean. Statistical significance was set at # <span class="html-italic">p</span> &lt; 0.05 (vs. control) and † <span class="html-italic">p</span> &lt; 0.001 (vs. glutamate). APE, Arecae Pericarpium ethanol extract; Con, control; Glu, glutamate; ROS, reactive oxygen species.</p>
Full article ">Figure 3
<p>Effects of Arecae Pericarpium ethanol extract on glutamate-induced mitochondrial dysfunction in HT22 cells. MMP was assessed via microscopy using JC-1 staining. The images represent the three independent experiments, shown at 200× magnification. Scale bar = 100 μm. Red fluorescence indicated normal MMP and green fluorescence, damaged mitochondria with MMP loss. The histogram shows the red/green fluorescence intensity ratio. Control was non-treated cells. Data were presented as mean ± standard error of the mean. Statistical significance was set at # <span class="html-italic">p</span> &lt; 0.05 (vs. control), * <span class="html-italic">p</span> &lt; 0.05, and † <span class="html-italic">p</span> &lt; 0.001 (vs. glutamate). APE, Arecae Pericarpium ethanol extract; Con, control; Glu, glutamate; MMP, mitochondrial membrane potential.</p>
Full article ">Figure 4
<p>Effects of Arecae Pericarpium ethanol extract on glutamate-induced apoptosis in HT22 cells. Cells were pretreated with APE at concentrations of 50, 100, or 200 μg/mL and then treated with glutamate (5 mM). (<b>A</b>) Apoptosis of HT22 cells was evaluated using flow cytometry. The image on the top right shows the percentage of healthy, early apoptotic, late apoptotic, and necrotic cells for each treatment group. (<b>B</b>) The expression levels of BAX, Bcl-2, PARP, and cleaved-PARP were determined by Western blot analysis. Control was non-treated cells. Blot images represent the three independent experiments. Data were presented as mean ± standard error of the mean. Statistical significance was set at # <span class="html-italic">p</span> &lt; 0.05 (vs. control), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and † <span class="html-italic">p</span> &lt; 0.001 (vs. glutamate). APE, Arecae Pericarpium ethanol extract; BAX, Bcl-2-associated X; Bcl-2, B-cell lymphoma 2; Con, control; Glu, glutamate; PI, propidium iodide; PARP, Poly(ADP-ribose) polymerase.</p>
Full article ">Figure 5
<p>Effects of Arecae Pericarpium ethanol extract on the activation of antioxidant enzymes in glutamate-exposed HT22 cells. The cells were incubated with 5 mM glutamate with or without <span class="html-italic">APE</span>. The expression levels of catalase, GCLC subunits, and NQO1 were determined via Western blot analysis. Control was non-treated cells. Blot images represent the three independent experiments. Data were presented as mean ± standard error of the mean. Statistical significance was set at # <span class="html-italic">p</span> &lt; 0.05 (vs. control), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and † <span class="html-italic">p</span> &lt; 0.001 (vs. glutamate). APE, Arecae Pericarpium ethanol extract; Con, control; GCLC, glutamate-cysteine ligase catalytic; Glu, glutamate; NQO1, NAD(P)H quinone oxidoreductase 1.</p>
Full article ">Figure 6
<p>Effects of Arecae Pericarpium ethanol extract on the phosphorylation of PI3K and protein kinase B, the nuclear translocation of Nrf-2, and the expression of HO1 in glutamate-exposed HT22 cells. Cells were pretreated with APE at concentrations of 50, 100, or 200 μg/mL and then treated with glutamate (5 mM). Control was non-treated cells. Blot images represent the three independent experiments. Data were presented as mean ± standard error of the mean. Statistical significance was set at # <span class="html-italic">p</span> &lt; 0.05 (vs. control), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and † <span class="html-italic">p</span> &lt; 0.001 (vs. glutamate). Akt, protein kinase B; APE, Arecae Pericarpium ethanol extract; Con, control; Glu, glutamate; HO1, heme oxygenase-1; Nrf-2, nuclear factor erythroid 2-related factor 2; PI3K, phosphoinositide 3-kinase.</p>
Full article ">Figure 7
<p>The suppressive effects of LY294002 on the neuroprotective action of Arecae Pericarpium ethanol extract. HT22 cells were incubated with or without APE combined with LY294002 before glutamate treatment. (<b>A</b>) Cell viability and, (<b>B</b>) Activation of protein kinase B, Nrf-2, and HO1 were assessed using a cell-counting kit assay and Western blot analysis, respectively. Control was non-treated cells. Blot images represent the three independent experiments. Data were presented as mean ± standard error of the mean. Statistical significance was set at # <span class="html-italic">p</span> &lt; 0.05 (vs. control), † <span class="html-italic">p</span> &lt; 0.001 (vs. glutamate), and *** <span class="html-italic">p</span> &lt; 0.001 (vs. APE). Akt, protein kinase B; APE, Arecae Pericarpium ethanol extract; Con, control; Glu, glutamate; HO, heme oxygenase; LY, LY294002; Nrf-2, nuclear factor erythroid 2-related factor 2.</p>
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27 pages, 4893 KiB  
Article
Identification of a Cardiac Glycoside Exhibiting Favorable Brain Bioavailability and Potency for Reducing Levels of the Cellular Prion Protein
by Shehab Eid, Thomas Zerbes, Declan Williams, Xinzhu Wang, Chris Sackmann, Sammy Meier, Nickolai O. Dulin, Pavel Nagorny and Gerold Schmitt-Ulms
Int. J. Mol. Sci. 2022, 23(23), 14823; https://doi.org/10.3390/ijms232314823 - 26 Nov 2022
Cited by 6 | Viewed by 2521
Abstract
Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets [...] Read more.
Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets Na,K-ATPases (NKAs) with cardiac glycosides (CGs), causing cells to respond with the degradation of these pumps and nearby molecules, including PrPC. Because the therapeutic window of widely used CGs is narrow and their brain bioavailability is low, we set out to identify a CG with improved pharmacological properties for this indication. Starting with the CG known as oleandrin, we combined in silico modeling of CG binding poses within human NKA folds, CG structure-activity relationship (SAR) data, and predicted blood–brain barrier (BBB) penetrance scores to identify CG derivatives with improved characteristics. Focusing on C4′-dehydro-oleandrin as a chemically accessible shortlisted CG derivative, we show that it reaches four times higher levels in the brain than in the heart one day after subcutaneous administration, exhibits promising pharmacological properties, and suppresses steady-state PrPC levels by 84% in immortalized human cells that have been differentiated to acquire neural or astrocytic characteristics. Finally, we validate that the mechanism of action of this approach for reducing cell surface PrPC levels requires C4′-dehydro-oleandrin to engage with its cognate binding pocket within the NKA α subunit. The improved brain bioavailability of C4′-dehydro-oleandrin, combined with its relatively low toxicity, make this compound an attractive lead for brain CG indications and recommends its further exploration for the treatment of prion diseases. Full article
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<p>Study design. (<b>A</b>) In silico identification of a CG with favorable predicted docking characteristics and BBB barrier penetrance. (<b>B</b>) Synthesis and characterization of short-listed CG. (<b>C</b>) Assessment of BBB penetrance and distribution in relevant tissue. (<b>D</b>) In vitro characterization of potency and mechanism of action for PrP<sup>C</sup> reduction of lead CG in cell-based assay.</p>
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<p>In silico prediction of CG binding pose within relevant human NKA α subunits and summary of key CG design considerations. (<b>A</b>) Electron densities of CG binding pose in PDB entry 4HYT [<a href="#B41-ijms-23-14823" class="html-bibr">41</a>]. (<b>B</b>) Structural alignment of ouabain (brown) and bufalin (green) within the CG binding pocket of two well-resolved X-ray crystallography models (PDB entries 4HYT and 4RES [<a href="#B47-ijms-23-14823" class="html-bibr">47</a>]) of NKAs. (<b>C</b>) Sequence identities between porcine and human NKA α subunits known to be expressed in the brain. (<b>D</b>) Comparison of observed porcine and predicted human amino acids lining the CG binding pocket. (<b>E</b>) Generalized Born binding pose and predicted free binding energies for oleandrin within human ATP1A3 model. (<b>F</b>) Surface area-optimized binding pose and predicted free binding energies for oleandrin within human ATP1A3 model. (<b>G</b>) Spatial alignment of observed and predicted binding poses of ouabain and oleandrin in experimentally deduced ATP1A1 and predicted surface-area optimized ATP1A3 models, respectively. (<b>H</b>) Key considerations for the design of potent CGs with optimized brain bioavailability drawn from structure-activity relationship data and medicinal chemistry considerations. Close-ups were produced with UCSF Chimera (v 1.1.2), San Francisco, CA, USA.</p>
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<p>In silico filtering of CG derivatives accessible through chemical derivatization based on their predicted docking scores and Rankovic multi-parameter optimized (MPO, version 2) BBB penetrance scores. (<b>A</b>) Design of CG scaffolds and chemically accessible modifications evaluated, giving rise to a combinatorial total of 270 CGs considered. (<b>B</b>) Workflow for the in silico assessment of candidates. The assignment of protonation states and partial charges increased total CG ligands evaluated to 330, a number that further increased to 850 once alternative binding poses, predominantly caused by freely rotating bonds were considered. The elimination of binding poses with unfavorable internal energy or deviation from the hypothetical oleandrin binding pose led to a 146 CGs that passed these filters. (<b>C</b>) Exemplary chart depicting results from the evaluation of Scaffold 1 CG derivatives. The position of Scaffold 1 combinatorial candidate CGs within the chart can be deduced from the R1 and R chemical modifications defined on the two axes. The color scheme reflects Rankovic MPO.v2 scores [<a href="#B48-ijms-23-14823" class="html-bibr">48</a>] (a high score, represented by a green square, indicates high predicted BBB penetrance) and the size of squares reflects docking strength (a low docking score, represented by a large square, indicates strong binding). The absence of a square indicates that no binding pose that passed filter criteria (see above) was found. Asterisks indicate sites used for the attachment of the respective combinatorial moieties. (<b>D</b>) Summary chart depicting results from the evaluation of Rankovic MPO.v2 scores and docking scores for all five scaffolds. Note that CGs derived from Scaffold 4 had similar docking scores as derivatives from other scaffolds but excelled based on their high predicted BBB penetrance. As a reference, we show the position of oleandrin in this chart at the intersection of the two blue lines.</p>
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<p>Features of chemically accessible Scaffold 4 derivatives-of-interest. (<b>A</b>) Chemical structure and reference Rankovic MPO.v2 and docking scores of oleandrin. (<b>B</b>) oleandrin derivatives of interest that should be chemically accessible with good yields in few steps. Starting with oleandrin, several compounds with high predicted Rankovic MPO.v2 scores can be accessed through C4′-dehydro-oleandrin (here termed KDC203) or C4′-amine-oleandrin [<a href="#B49-ijms-23-14823" class="html-bibr">49</a>]. (<b>C</b>) Chemical structures of compounds listed in Panel B with their respective Rankovic MPO.v2 and Glide scores. Note that structures of KDC204, KDC206 and KDC208 are not depicted. (<b>D</b>–<b>F</b>) Close-up models providing plausible explanations for predicted increases of Glide docking scores (residue numbers as in PDB 4HYT). (<b>D</b>) Salt bridge between the C4′ amine and the sidechain of glutamic acid residue 312. (<b>E</b>) Hydrogen bonding opportunity for the C3′ isopropyl group with the side chain of aspartic acid residue 116. (<b>F</b>) Good fit of C16 trifluoro-acetoxy group within hydrophobic binding pocket shaped from side-chain atoms of phenylalanine 783 and isoleucine 800 residues lining the CG binding pocket. Close-ups were produced with UCSF Chimera (v 1.1.2), San Francisco, CA, USA.</p>
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<p>Improved brain bioavailability of KDC203 relative to oleandrin. (<b>A</b>) Oleandrin enriches in kidney and liver tissue and its brain and heart levels are similar 24 h following its acute subcutaneous injection as a tritium-labeled compound into cohorts of five mice. In contrast, KDC203 reaches the highest levels in the brain and its brain levels exceeded its heart levels 3.5-fold 24 h after subcutaneous injection. ‘ns’ denotes non-significance. Asterisks indicate the level of statistical significance, i.e., one asterisk denotes <span class="html-italic">p</span> &lt; 0.05, with each additional asterisk denoting tenfold lower <span class="html-italic">p</span>-values. (<b>B</b>) KDC203 is a lesser hMDR1 substrate than oleandrin. Recorded values of apparent permeability (Papp) from apical to basolateral (<b>A</b>–<b>B</b>) and vice versa (<b>B</b>–<b>A</b>). The efflux ratio is calculated as Papp<sub>B-A</sub>/Papp<sub>A-B</sub>. Zosuquidar was deployed as a selective inhibitor of MDR1. Measurements of the TEER on the day of experiment (day 5) resulted in a mean value of 107 ± 8 Ω·cm² (±SD, n = 22). Lucifer Yellow Permeability data (n = 22) were generated prior to the bidirectional assay with oleandrin and KDC203 (n = 3). (<b>C</b>) Rapid equilibrium dialyses of brain and plasma samples establish that KDC203 has a lower propensity than oleandrin to associate nonspecifically with components in the respective extracts, predictive of a higher concentration of free KDC203 that is available for specific engagement with its NKA target. (<b>D</b>) Pertinent pharmacological characteristics of oleandrin and KDC203 computed based on their in vivo tissue and plasma concentrations and the in vitro measurement of their unbound fractions in rapid equilibrium dialyses.</p>
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<p>KDC203 reduces steady-state PrP<sup>C</sup> levels in human neural cell lines. (<b>A</b>) Oleandrin and KDC203 reduce steady-state ATP1A1 protein levels to a similar degree. Side-by-side Western blot-based comparison of ATP1A1 signal intensities in cellular extracts derived from ReN VM cells following 7-day treatment with the respective CGs (equal amount of total protein loaded). Because the Western blot was not stripped of the ATP1A1-directed primary and secondary antibodies, before it was stained with Coomassie, a stronger signal can be seen at the 85–90 kDa level, where the ATP1A1 detection antibodies bound. (<b>B</b>) 7-day exposure of ReN VM cells does not only affect ATP1A1 protein levels by also causes a concentration-dependent reduction in the steady-state protein levels of the NKA β subunit ATP1B1 and PrP<sup>C</sup>. Note that the total amount of protein loaded in the two biological replicate series was not identical for the PrP-directed 3F4 blot; to capture an informative linear range, half the amount of total protein was loaded for samples shown on the right hand side. (<b>C</b>) Quantitation of Western blot signal intensities of ATP1A1 and PrP<sup>C</sup> following 7-day KDC203 treatment of ReN VM cells at concentrations indicated, with each value being computed from the analysis of three biological replicates. (<b>D</b>) The KDC203-dependent reduction in steady-state PrP<sup>C</sup> protein levels is not an idiosyncrasy of ReN VM cells but can also be observed in other neural cell models, including differentiated human glioblastoma cells (T98G). Asterisks indicate the level of statistical significance, i.e., one asterisk denotes <span class="html-italic">p</span> &lt; 0.05, with each additional asterisk denoting tenfold lower <span class="html-italic">p</span>-values.</p>
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<p>KDC203 induces a compensatory upregulation of ATP1A3, is less toxic than oleandrin, and requires binding to CG binding pocket within ATP1A1 for reducing PrP<sup>C</sup> levels. (<b>A</b>) The KDC203 concentration-dependent reduction in the steady-state protein levels of ATP1A1, ATP1A2, and PrP<sup>C</sup> is paralleled by an increase in ATP1A3 levels. Total concentrations of cellular extracts were adjusted, and equal volumes of these adjusted extracts were loaded onto the gel as evidenced by the Coomassie stain. Please note the slight rebound in ATP1A1 signals and the splitting of the 3F4-reactive signals into two bands that migrated slower and faster than the corresponding band in vehicle-treated differentiated ReN VM cells exposed to 20–32 nM KDC2039 concentrations. (<b>B</b>) KDC203 exhibits tenfold lower toxicity than oleandrin in three assays that were used to monitor the metabolic health of 7-day differentiated ReN VM cells. Each data point represents the mean of six biological replicates. Concentrations are depicted on a logarithmic axis. (<b>C</b>) Amino acid sequence alignment of an NKA α subunit segment contributing to CG binding in wild-type human ATP1A1 protein and its mutated derivative rendered refractory to CG binding by replacing human residues 118 and 129 (numbering based on human <span class="html-italic">ATP1A1</span> transcript NM_00701.7) with the corresponding mouse Atp1a1 residues. (<b>D</b>) KDC203 causes the expected reduction in the steady-state levels of ATP1A1 and ATP1A2 in differentiated wild-type ReN VM cells but not in the gene-edited ReN VM <span class="html-italic">ATP1A<sup>R/R</sup></span> cells whose ATP1A1 protein does not bind CGs. Despite the decrease in ATP1A2 levels, the NKA α subunit that is predominantly expressed in astrocytes, no reduction in the steady-state levels of astrocytic GFAP is observed.</p>
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11 pages, 2096 KiB  
Article
Effect of Cannabidiolic Acid, N-Trans-Caffeoyltyramine and Cannabisin B from Hemp Seeds on microRNA Expression in Human Neural Cells
by Armando Di Palo, Chiara Siniscalchi, Giuseppina Crescente, Ilenia De Leo, Antonio Fiorentino, Severina Pacifico, Aniello Russo and Nicoletta Potenza
Curr. Issues Mol. Biol. 2022, 44(10), 5106-5116; https://doi.org/10.3390/cimb44100347 - 21 Oct 2022
Cited by 11 | Viewed by 2424
Abstract
Given the increasing interest in bioactive dietary components that can modulate gene expression enhancing human health, three metabolites isolated from hemp seeds—cannabidiolic acid, N-trans-caffeoyltyramine, and cannabisin B—were examined for their ability to change the expression levels of microRNAs in human [...] Read more.
Given the increasing interest in bioactive dietary components that can modulate gene expression enhancing human health, three metabolites isolated from hemp seeds—cannabidiolic acid, N-trans-caffeoyltyramine, and cannabisin B—were examined for their ability to change the expression levels of microRNAs in human neural cells. To this end, cultured SH-SY5Y cells were treated with the three compounds and their microRNA content was characterized by next-generation small RNA sequencing. As a result, 31 microRNAs underwent major expression changes, being at least doubled or halved by the treatments. A computational analysis of the biological pathways affected by these microRNAs then showed that some are implicated in neural functions, such as axon guidance, hippocampal signaling, and neurotrophin signaling. Of these, miR-708-5p, miR-181a-5p, miR-190a-5p, miR-199a-5p, and miR-143-3p are known to be involved in Alzheimer’s disease and their expression changes are expected to ameliorate neural function. Overall, these results provide new insights into the mechanism of action of hemp seed metabolites and encourage further studies to gain a better understanding of their biological effects on the central nervous system. Full article
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<p>Chemical structures of the three metabolites isolated from hemp seeds. Cannabisin B (<b>a</b>), <span class="html-italic">N</span>-<span class="html-italic">trans</span>-caffeoyltyramine (<b>b</b>), and cannabidiolic acid (<b>c</b>) were isolated from <span class="html-italic">Cannabis sativa</span> seeds.</p>
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<p>Structural characterization of hemp seed metabolites by mass spectrometry. (<b>A</b>) <span class="html-italic">N-trans</span>-caffeoyltyramine (i) Total Ion Chromatogram (TIC); (ii) TOF-MS spectrum showing the [M-H]<sup>-</sup> and [2M-H]<sup>-</sup> ions at <span class="html-italic">m/z</span> 298.1084 and 597.2254, respectively; (iii) TOF-MS/MS of the [M-H]<sup>-</sup> ion; (iv) proposed fragmentation pattern; theoretical <span class="html-italic">m/z</span> value is below each chemical structure. (<b>B</b>) Cannabisin B (i) Total Ion Chromatogram (TIC); (ii) TOF-MS spectrum showing the [M-H]<sup>-</sup> ion at <span class="html-italic">m/z</span> 595.2107; (iii) TOF-MS/MS of the [M-H]<sup>-</sup> ion; (iv) proposed fragmentation pattern; theoretical <span class="html-italic">m/z</span> value is below each chemical structure.</p>
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<p>Mitochondrial Redox Activity Inhibition (RAI). SH cells were treated with cannabidiolic acid (CBDA), <span class="html-italic">N</span>-<span class="html-italic">trans</span>-caffeoyltyramine, or cannabisin B for 48 h, and RAI was assessed by MTT assay. Values are the mean ± SD of three independent experiments performed in triplicate.</p>
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<p>Volcano plots of microRNA expression changes in cultured neural cells. SH cells were treated with cannabisin B (<b>A</b>), <span class="html-italic">N</span>-<span class="html-italic">trans</span>-caffeoyltyramine (<b>B</b>), or cannabidiolic acid (<b>C</b>) for 48 h, then microRNA expression changes were detected by next-generation small RNA sequencing. Plots were prepared by the program VolcaNoseR [<a href="#B27-cimb-44-00347" class="html-bibr">27</a>]. Fields outside the vertical broken lines include miRNAs showing a fold change &gt; 1.5; fields above the horizontal broken line contain miRNAs with a highly significant variation (<span class="html-italic">p</span> &lt; 0.001); hsa, homo sapiens.</p>
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<p>Biological pathways potentially affected by a miRNA-based mechanism. The whole targetomes (predicted and validated) of microRNAs deregulated by cannabisin B (<b>A</b>), <span class="html-italic">N</span>-<span class="html-italic">trans</span>-caffeoyltyramine (<b>B</b>), and cannabidiolic acid (<b>C</b>) were analyzed by the program DAVID and the top-20 significantly enriched KEGG pathways were displayed. Biological pathways were considered statistically significant if <span class="html-italic">p</span>-value was less than 0.05 (Benjamini-Hochberg procedure for multiple correction).</p>
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<p>Venn diagram of microRNAs from neural SH cells showing expression levels markedly affected by cannabis compounds. Selection was limited to those microRNAs with an absolute value of log2 fold change ≥ 1. MicroRNAs involved in Alzheimer’s disease are marked in bold; those varying such as to expect an amelioration of neural function in AD are underlined.</p>
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22 pages, 2446 KiB  
Article
Neuroprotective Effect of Artichoke-Based Nanoformulation in Sporadic Alzheimer’s Disease Mouse Model: Focus on Antioxidant, Anti-Inflammatory, and Amyloidogenic Pathways
by Heba A. S. El-Nashar, Haidy Abbas, Mariam Zewail, Mohamed H. Noureldin, Mai M. Ali, Marium M. Shamaa, Mohamed A. Khattab and Nehal Ibrahim
Pharmaceuticals 2022, 15(10), 1202; https://doi.org/10.3390/ph15101202 - 28 Sep 2022
Cited by 43 | Viewed by 3697
Abstract
The vast socio-economic impact of Alzheimer’s disease (AD) has prompted the search for new neuroprotective agents with good tolerability and safety profile. With its outstanding role as antioxidant and anti-inflammatory, alongside its anti-acetylcholinesterase activity, the artichoke can be implemented in a multi-targeted approach [...] Read more.
The vast socio-economic impact of Alzheimer’s disease (AD) has prompted the search for new neuroprotective agents with good tolerability and safety profile. With its outstanding role as antioxidant and anti-inflammatory, alongside its anti-acetylcholinesterase activity, the artichoke can be implemented in a multi-targeted approach in AD therapy. Moreover, artichoke agricultural wastes can represent according to the current United Nations Sustainable Development goals an opportunity to produce medicinally valuable phenolic-rich extracts. In this context, the UPLC-ESI-MS/MS phytochemical characterization of artichoke bracts extract revealed the presence of mono- and di-caffeoylquinic acids and apigenin, luteolin, and kaempferol O-glycosides with remarkable total phenolics and flavonoids contents. A broad antioxidant spectrum was established in vitro. Artichoke-loaded, chitosan-coated, solid lipid nanoparticles (SLNs) were prepared and characterized for their size, zeta potential, morphology, entrapment efficiency, release, and ex vivo permeation and showed suitable colloidal characteristics, a controlled release profile, and promising ex vivo permeation, indicating possibly better physicochemical and biopharmaceutical parameters than free artichoke extract. The anti-Alzheimer potential of the extract and prepared SLNs was assessed in vivo in streptozotocin-induced sporadic Alzheimer mice. A great improvement in cognitive functions and spatial memory recovery, in addition to a marked reduction of the inflammatory biomarker TNF-α, β-amyloid, and tau protein levels, were observed. Significant neuroprotective efficacy in dentate Gyrus sub-regions was achieved in mice treated with free artichoke extract and to a significantly higher extent with artichoke-loaded SLNs. The results clarify the strong potential of artichoke bracts extract as a botanical anti-AD drug and will contribute to altering the future medicinal outlook of artichoke bracts previously regarded as agro-industrial waste. Full article
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<p>TEM micrographs of Poloxamer 407 based (<b>A</b>) uncoated SLNs and (<b>B</b>) CS-coated SLNs.</p>
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<p>(<b>A</b>) Cumulative percentage of ART released in acidic medium and (<b>B</b>) Cumulative percentage of ART permeated through the intestinal mucosa for the prepared SLNs (F1: Poloxamer 407/uncoated—F2: Tween 80/uncoated—F3: Poloxamer 407/chitosan coated—F4: Tween 80/chitosan coated).</p>
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<p>Schematic representation of experimental design illustrating drug dose, duration of the experiment and on which days behavioral tests were carried out.</p>
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<p>Behavioral assessment of the effect of ART on (<b>A</b>) the percentage of spontaneous alternation, (<b>B</b>) mean escape latency in MWM and (<b>C</b>) time spent in target quadrant in MWM. Statistical analyses are performed using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. +: Statistically significant different from the normal control group (Saline) at <span class="html-italic">p</span> &lt; 0.05, * and **: Statistically significant different from the positive control group (STZ, 3 mg/kg) at <span class="html-italic">p</span> &lt; 0.05, #: Statistically significant different from the ART group (50 mg/kg) at <span class="html-italic">p</span> &lt; 0.05. ART-Nanoparticles (equivalent to 50 mg/kg).</p>
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<p>Biochemical analysis of (<b>A</b>) TNF-α level, (<b>B</b>) Amyloid β1-42 level and (<b>C</b>) Tau level. Statistical analyses are performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer post-hoc test, whereby each value was presented as mean ± standard deviation (SD). * Statistically significantly different from the normal control group (<span class="html-italic">p</span> &lt; 0.05); ** statistically significantly different from the STZ group, 3 mg/kg (<span class="html-italic">p</span> &lt; 0.05); # statistically significantly different from the ART group 50 mg/kg (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Anatomical overview of total hippocampal region highlighted examined fixed subregions in different groups (H&amp;E stain, 40X; black boxed area for CA1 subregion and yellow boxed area for Dentate Gyrus subregion). Neuroprotective histological effect of different treatments on CA1 hippocampal subregions (left column micrographs) and Dentate Gyrus subregions (Right column micrographs) with correlated groups. H&amp;E stain, 400X. Black arrows = intact neurons, red arrows = degenerated and necrotic neurons. (<b>A</b>,<b>B</b>) represent group1 (negative control). (<b>C</b>,<b>D</b>) represent group 2 (Positive control). (<b>E</b>,<b>F</b>) represent group 3 (ART). (<b>G</b>,<b>H</b>) represent group 4 (F3).</p>
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<p>Light-microscopic examination of toluidine blue-stained pyramidal neurons in CA1 &amp; Dentate Gyrus hippocampal sub regions in different groups with mean intact neuronal counts, 400X. Data were represented as mean ± SD (<span class="html-italic">n</span> = 6), black arrows = intact neurons, red arrows = damaged neurons. (<b>A</b>) and (<b>B</b>) represent group1 (negative control). (<b>C</b>) and (<b>D</b>) represent group 2 (Positive control). (<b>E</b>) and (<b>F</b>) represent group 3 (ART). (<b>G</b>) and (<b>H</b>) represent group 4 (F3). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. @ Significant as compared to negative control (group 1). % Significant as compared to positive control group (group 2). # Significant as compared to ART (group 3). &amp; Significant as compared to ART formulation (group 4). Significant difference was conducted by one-way ANOVA at <span class="html-italic">p</span> &lt; 0.0001.</p>
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17 pages, 4966 KiB  
Article
Trio-Drug Combination of Sodium Valproate, Baclofen and Thymoquinone Exhibits Synergistic Anticonvulsant Effects in Rats and Neuro-Protective Effects in HEK-293 Cells
by Faheem Hyder Pottoo, Mohammed Salahuddin, Firdos Alam Khan, Batool Taleb Albaqshi, Mohamed S. Gomaa, Fatima S. Abdulla, Noora AlHajri and Mohammad N. Alomary
Curr. Issues Mol. Biol. 2022, 44(10), 4350-4366; https://doi.org/10.3390/cimb44100299 - 20 Sep 2022
Cited by 6 | Viewed by 3042
Abstract
Epilepsy is a chronic brain disorder, with anti-epileptic drugs (AEDs) providing relief from hyper-excitability of neurons, but largely failing to restrain neurodegeneration. We investigated a progressive preclinical trial in rats, whereby the test drugs; sodium valproate (SVP; 150 and 300 mg/kg), baclofen (BFN; [...] Read more.
Epilepsy is a chronic brain disorder, with anti-epileptic drugs (AEDs) providing relief from hyper-excitability of neurons, but largely failing to restrain neurodegeneration. We investigated a progressive preclinical trial in rats, whereby the test drugs; sodium valproate (SVP; 150 and 300 mg/kg), baclofen (BFN; 5 and 10 mg/kg), and thymoquinone (THQ; 40 and 80 mg/kg) were administered (i.p, once/day for 15 days) alone, and as low dose combinations, and subsequently tested for antiseizure and neuroprotective potential using electrical stimulation of neurons by Maximal electroshock (MES). The seizure stages were monitored, and hippocampal levels of m-TOR, IL-1β, IL-6 were measured. Hippocampal histopathology was also performed. Invitro and Insilco studies were run to counter-confirm the results from rodent studies. We report the synergistic effect of trio-drug combination; SVP (150 mg/kg), BFN (5 mg/kg) and THQ (40 mg/kg) against generalized seizures. The Insilco results revealed that trio-drug combination binds the Akt active site as a supramolecular complex, which could have served as a delivery system that affects the penetration and the binding to the new target. The potential energy of the ternary complex in the Akt active site after dynamics simulation was found to be −370.426 Kcal/mol, while the supramolecular ternary complex alone was −38.732 Kcal/mol, with a potential energy difference of −331.694 Kcal/mol, which favors the supramolecular ternary complex at Akt active site binding. In addition, the said combination increased cell viability by 267% and reduced morphological changes induced by Pentylenetetrazol (PTZ) in HEK-293 cells, which indicates the neuroprotective property of said combination. To conclude, we are the first to report the anti-convulsant and neuroprotective potential of the trio-drug combination. Full article
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<p>Effect of SVP (150 and 300 mg/kg), BFN (5 and 10 mg/kg), THQ (40 and 80 mg/kg) alone and in low dose combination on MES induced THLE. The values are expressed as ratio of THLE:NO-THLE. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 versus toxic control rats. Fisher’s exact test (2 × 2 contingency table; One tailed) was used for statistical analysis.</p>
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<p>Effect of SVP (150 mg/kg) + BFN (5 mg/kg) + THQ (40 mg/kg) treatment on hippocampal mTOR levels. The values are expressed as mean ± SEM. ** <span class="html-italic">p</span> &lt; 0.01 versus TC. NC; Normal Control, TC; Toxic Control.</p>
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<p>Effect of SVP (150 mg/kg) + BFN (5 mg/kg) + THQ (5 mg/kg) treatment on hippocampal levels of IL-1β. The values are expressed as mean ± SEM. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 versus TC. NC; Normal Control, TC; Toxic Control.</p>
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<p>Effect of SVP (150 mg/kg) + BFN (5 mg/kg) + THQ (5 mg/kg) treatment on hippocampal levels of IL-6. The values are expressed as mean ± SEM. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 versus TC. NC; Normal Control, TC; Toxic Control.</p>
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<p>The H/E-stained hippocampal section visualized under 40× shows normal arrangements and distributions in the normal control group (Group-I). Plenty of neuronal loss, degeneration and death from electroshock observed in Group-II. Slight cellular changes in CA1, CA2, CA3, DG of the SVP (150 mg/kg) + BFN (5 mg/kg) + THQ (40 mg/kg) injected rats are indicative of minimal neuronal degeneration and nuclear pyknosis in Group-XII.</p>
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<p>Surviving neurons from hippocampal regions, CA1, CA2, CA3 and DG. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 versus TC. NC; Normal Control, TC; Toxic Control.</p>
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<p>% cell viability of HEK-293 cells by MTT assay. The cells were first exposed to PTZ (0.6 µg/mL), and later with different concentrations of SVP (120 µg/mL), BFN (1.50 µg/mL), THQ (12.0 µg/mL) and SVP + BFN + THQ (120 + 1.5 + 12.0 µg/mL) for 24 hr. The % of cell viability is given in the graph is taken from the dose which gives the highest percentage of cell viability. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>The morphological representation of the HEK-293 cells taken by light microscope. (<b>A</b>) Non treated HEK-293 cells; (<b>B</b>–<b>E</b>) PTZ (0.6 µg/mL), SVP (120 µg/mL), BFN (1.50 µg/mL), THQ (12.0 µg/mL); (<b>F</b>) SVP + BFN + THQ (120 + 1.5 + 12.0 µg/mL). The cells were first treated with PTZ for 24 h, then were treated with SVP, BFN and THQ and SVP + BFN + THQ for 24 h. Magnifications 200×.</p>
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<p>Comparative binding positions of ternary supramolecular complex of the SVP (magenta), BFN (blue) and THQ (green) with the reference crystallized ligand (red) bound to Akt active site.</p>
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<p>Binding interactions of co-bound ternary supramolecular complex of SVP, BFN and THQ with Akt allosteric site and active site. The distances are represented by red dotted lines, and are measured in Angstrom.</p>
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<p>Binding interactions of co-bound ternary supramolecular complex of SVP, BFN and THQ with Akt allosteric site and active site and showing key solvating water molecules. (<b>A</b>) first frame in dynamics, (<b>B</b>) last frame in dynamics. Distances are represented by red dotted lines and are measured in Angstrom.</p>
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<p>Poses of the three drugs in the supramolecular ternary complex, (<b>A</b>) alone and (<b>B</b>) after docking in dynamics in Akt enzyme (<b>B</b>).</p>
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17 pages, 4115 KiB  
Article
Kynurenine 3-Monooxygenase Interacts with Huntingtin at the Outer Mitochondrial Membrane
by Aisha M. Swaih, Carlo Breda, Korrapati V. Sathyasaikumar, Natalie Allcock, Mary E. W. Collier, Robert P. Mason, Adam Feasby, Federico Herrera, Tiago F. Outeiro, Robert Schwarcz, Mariaelena Repici and Flaviano Giorgini
Biomedicines 2022, 10(9), 2294; https://doi.org/10.3390/biomedicines10092294 - 15 Sep 2022
Cited by 6 | Viewed by 3146
Abstract
The flavoprotein kynurenine 3-monooxygenase (KMO) is localised to the outer mitochondrial membrane and catalyses the synthesis of 3-hydroxykynurenine from L-kynurenine, a key step in the kynurenine pathway (KP) of tryptophan degradation. Perturbation of KP metabolism due to inflammation has long been associated with [...] Read more.
The flavoprotein kynurenine 3-monooxygenase (KMO) is localised to the outer mitochondrial membrane and catalyses the synthesis of 3-hydroxykynurenine from L-kynurenine, a key step in the kynurenine pathway (KP) of tryptophan degradation. Perturbation of KP metabolism due to inflammation has long been associated with the pathogenesis of several neurodegenerative disorders, including Huntington’s disease (HD)—which is caused by the expansion of a polyglutamine stretch in the huntingtin (HTT) protein. While HTT is primarily localised to the cytoplasm, it also associates with mitochondria, where it may physically interact with KMO. In order to test this hypothesis, we employed bimolecular fluorescence complementation (BiFC) and found that KMO physically interacts with soluble HTT exon 1 protein fragment in living cells. Notably, expansion of the disease-causing polyglutamine tract in HTT leads to the formation of proteinaceous intracellular inclusions that disrupt this interaction with KMO, markedly decreasing BiFC efficiency. Using confocal microscopy and ultrastructural analysis, we determined KMO and HTT localisation within the cell and found that the KMO-HTT interaction is localized to the outer mitochondrial membrane. These data suggest that KMO may interact with a pool of HTT at the mitochondrial membrane, highlighting a possible physiological role for mitochondrial HTT. The KMO-HTT interaction is abrogated upon polyglutamine expansion, which may indicate a heretofore unrecognized relevance in the pathogenesis of this disorder. Full article
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Figure 1

Figure 1
<p><b>Exogenous KMO expression and localisation in HEK293T cells.</b> (<b>A</b>) Schematic representation of KMO ICC constructs: flKMO-RFP and tKMO-RFP. Grey boxes show putative transmembrane domains (TM). tKMO has a deletion of amino acids from position 368 to 380 that resembles the deletion in KMO isoform 2, but is also truncated at its C-terminus, with 67 aa missing from the protein. (<b>B</b>) Immunoblotting of KMO constructs using anti-KMO antibody (10698-1-AP). (<b>C</b>,<b>D</b>) HEK293T cells were transfected with the flKMO-RFP (<b>C</b>), Left panel)) or tKMO-RFP ((<b>D</b>), Left panel) constructs and fixed 24 h after transfection; ((<b>C</b>,<b>D</b>) middle panels): immunolabelling for the mitochondrial protein HtrA2 using anti-HtrA2 antibody (AF1458) (Alexa Fluor 488); ((<b>C</b>,<b>D</b>) right panels): merge of the RFP and anti HtrA2 signal. Nuclei were stained with Hoechst 33342. Scale bar = 8 µm. flKMO-RFP depicts mitochondrial localisation (punctate structures), whereas the tKMO-RFP signal is diffuse throughout the cell. The squares on the images indicate the selected areas for co-localisation analysis (an enlarged view of the selected area is shown on the side of each merge panel). (<b>E</b>,<b>F</b>) Co-localisation analysis of optical z-sections from eight deconvolved confocal images, using JACoP in ImageJ. (<b>E</b>) Pearson’s coefficient shows a significant difference in the mitochondrial co-localisation of flKMO-RFP and tKMO-RFP. (<b>F</b>) Mander’s coefficient correlation. M1 represents the red signal overlapping the green signal, while M2 indicates the green signal overlapping the red signal. **** (<span class="html-italic">p</span> &lt; 0.0001), for unpaired <span class="html-italic">t</span>-test. ns = not significant. Data are expressed as mean ± SEM (<span class="html-italic">n</span> = 8).</p>
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<p><b>Schematic representation of BiFC models and their complementation pairs.</b> (<b>A</b>) Schematic outline of the KMO and HTT BiFC constructs. flKMO/tKMO were fused C-terminally to N-terminus of CFP (CC) via (GGGGS)<sub>2</sub> linker. The HTT BiFC constructs were composed of the N17, polyglutamine (polyQ) and polyproline (polyP) domains fused to either C-terminus or N-terminus halves of Venus (VC or VN). For each of the -VN or -VC HTT BiFC constructs in the illustration there are versions with different polyQ lengths: 25Q-VC, 19Q-VN, 46Q-VN, 97Q-VC and 97Q-VN. (<b>B</b>,<b>C</b>) Illustrations of BiFC combination pairs: (<b>B</b>) KMO and HTT pair that when interacting bring CC and VN together to form a fluorescence protein with 510 nm emission as in enhanced-GFP (E-GFP); (<b>C</b>) HTT pairs upon interactions, the two halves of Venus (VN and VC) re-constitute with emission 527 nm.</p>
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<p><b>Interaction of flKMO-CC and HTT-VN in HEK293T cells.</b> (<b>A</b>) Cells were transfected for 48 h with 0.16 µg of each plasmid and 0.08 µg of RFP. Fluorescence intensities were analysed using ScanR analysis software. Mean green intensity of the fluorescence complementation signal of three independent experiments shows a clear interaction between flKMO-CC and HTT-VN. The histogram shows a significant reduction in the fluorescence complementation as the polyQ length increases, which is significantly different from the background (background = flKMO-CC + VN-backbone); positive control = DJ-1-GN + DJ-1-CC from [<a href="#B34-biomedicines-10-02294" class="html-bibr">34</a>]. **** <span class="html-italic">p</span> &lt; 0.0001, for one-way ANOVA, followed by Tukey’s multiple comparison tests. Data are expressed as mean ± SEM. The number of analysed cells ranged from 15,000 to 18,500 cells per condition. (<b>B</b>) A representative immunoblot of the lysates from one BiFC experiment shows the expression levels of flKMO-CC and the soluble fraction of HTT-VN proteins, using anti-GFP antibody (ab6556). Positive control = DJ-1-GN + DJ-1-CC. (<b>C</b>) Filter trap of cells lysates expressing only HTT-VN to reveal polyQ dependent protein aggregation, using anti-GFP antibody (ab6556; 1:10,000); each lysate was blotted in duplicate. (<b>D</b>) Activity of BiFC KMO constructs after expression in HEK293T cells: truncation leads to complete loss of activity, but flKMO-CC activity is maintained when co-expressed with BiFC-VN constructs. **** <span class="html-italic">p</span> &lt; 0.0001 and ns = not significant for one-way ANOVA, followed by Tukey’s multiple comparison tests. Data are expressed as mean ± SEM. (<b>E</b>) HEK293T cells were transfected with untagged flKMO and either MYC alone, 1–90 amino acid HTT 23Q-MYC or 145Q-MYC constructs. Upon crosslinking, HTT constructs were pulled down by using MYC-Trap and revealed with anti-KMO (Proteintech, 1:1000). A strong interaction is observed between flKMO and 1–90 HTT-Q23-MYC whereas flKMO and 1–90 HTT-Q145-MYC displays a weaker interaction. (<b>F</b>) HEK293T cells were transfected with a construct expressing flKMO-RFP, which was pulled down with the RFP-Trap system and revealed with anti-HTT (4C8) antibody (MAB2166; 1:1000). An interaction between flKMO-RFP and endogenous HTT was detected. TCL = total cell lysate, FT = flow through, W = wash and IP = immunoprecipitation.</p>
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<p><b>Cellular localisation of BiFC complexes in live HEK293T cells, using confocal microscopy.</b> Cells were seeded on ibiTreat dishes and co-transfected with BiFC constructs for 48 h. (<b>A</b>–<b>C</b>) illustrate the HTT signal using the BiFC system (BiFC signal = left image, and internal control RFP signal = right image) in each panel. (<b>A</b>) Cells were transfected with 19Q-VN, 25Q-VC and RFP, the BiFC signal is cytosolic and slightly punctate. (<b>B</b>) Cells were transfected with 97Q pair and RFP; the BiFC signal is generally cytosolic, with HTT inclusions present. (<b>C</b>) Cells were transfected with flKMO-CC, 97Q-VN and RFP; the BiFC signal is mainly mitochondrial as suggested by the dotted appearance of the signal. Scale bar = 8 µm. (<b>D</b>–<b>F</b>) Localisation of KMO-BiFC complexes. Left panels show BiFC signal of the following pairs: (<b>D</b>) flKMO-CC and HTT19Q-VN, (<b>E</b>) flKMO-CC and HTT46Q-VN, and (<b>F</b>) flKMO-CC and HTT97Q-VN. Second column of panels (<b>D</b>–<b>F</b>): mitochondria stained with MitoTracker Red CMXRox (M-7512). Third column of panels (<b>D</b>–<b>F</b>): merge of the BiFC signal and the MitoTracker signal. Scale bar = 8 µm. The BiFC signal in panels (<b>D</b>–<b>F</b>) exhibits dotted structures that co-localise with the MitoTracker signal, as seen in the merge images in the right panels of (<b>D</b>–<b>F</b>). This confirms the mitochondrial localisation of all the BiFC complexes of flKMO-CC with different polyQ lengths of HTT-VN.</p>
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<p><b>Subcellular localisation of HTT-VN constructs.</b> HEK293T cells were transfected for 48 h with either 19Q-VN, 46Q-VN or 97Q-VN, then fixed and immunolabelled. (<b>A</b>) Co-localisation analysis of 19Q-VN and mitochondrial fluorescent signals on deconvolved confocal optical z-sections, using JACoP plugin in ImageJ. Left panel, 19Q-VN immunolabelling of anti-HTT (mEM48) antibody (MAB5374), (Alexa Fluor 647). Middle panel, mitochondrial immunolabelling of anti-HtrA2/Omi antibody (AF1458) (Alexa Fluor 555). Right panels: merge image of the HTT and mitochondrial signals. Nuclei were stained with Hoechst 33342. Scale bar = 8 µm. Co-localisation analyses were carried out on the regions of interest indicated on the images, and an enlarged image is presented on the right of panel (A). The 19Q-VN signal appears punctate and co-localises majorly with mitochondrial, 82.7% (Pearson’s correlation). Analysis was performed on the presented images. (<b>B</b>,<b>C</b>) Left panel: anti-HTT (mEM48) antibody (MAB5374) (Alexa Fluor 647), (B: 46Q-VN, C: 97Q-VN). Middle panel: anti-HtrA2/Omi antibody (AF1458) (Alexa Fluor 555). Right panel: merge of HTT and mitochondrial signals. Nuclei were stained with Hoechst 33342. Scale bar = 8 µm. Anti-HTT signal co-localises with the mitochondrial signal, but the presence of aggregates (bright inclusions) makes images unquantifiable. HTT-VN and mitochondrial signals are presented with red and green signals for Alexa Fluor 647 and 555, respectively.</p>
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<p><b>Fluorescence tag effect on wild type HTT subcellular localisation in fixed HEK293T cells transfected with various constructs for 48 h.</b> (<b>A</b>) N-terminal half of Venus (VN) localisation in dual immunolabelled HEK293T cells. Left panel: anti-GFP (ab6556) (Alexa Fluor 555). Middle panel: anti-mitochondria antibody (MAB1273) (Alexa Flour 647). Right panel: merge of VN and mitochondrial signals, nuclei stained with Hoechst 33342. VN localises mostly in the nucleus, with minor punctate staining in the cytosol which co-localises with mitochondria. (<b>B</b>) Cells expressing 25Q-VC were double immunolabelled as follows: Left panel: anti-HTT (mEM48) antibody (MAB5374) (Alexa Fluor 647). Middle panel: anti-HtrA2/Omi antibody (AF1458) (Alexa Fluor 555). Right panel: merge of HTT and HtrA2 signals; nuclei were stained with Hoechst 33342. 25Q-VC co-localises with mitochondria. (<b>C</b>,<b>D</b>) Cells were seeded in ibiTreat dishes and co-transfected for 48 h with 19Q-VN and 25Q-VC (<b>C</b>) or 25Q-GFP (<b>D</b>). Live cells were stained with MitoTracker Red CMXRox (M-7512) prior to confocal examination. Acquired images were deconvolved. (<b>C</b>) Left panel: BiFC signal 19Q-VN and 25Q-VC. Middle panel: MitoTracker signal. Right panel: merge of BiFC and MitoTracker signals. BiFC signal of WT HTT is mitochondrial (indicated by the co-localisation with MitoTracker) as well as cytosolic. (<b>D</b>) Left panel: GFP signal showing the fused 25Q localisation. Middle panel: MitoTracker signal. Right panel: merge of the 25Q-GFP and MitoTracker signals. Scale bar = 8 µm. 25Q-GFP is expressed in the cytosol, with complete exclusion of mitochondrial localisation.</p>
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<p><b>Electron micrographs of dual immunogold labelling in transfected HEK293T cells for 48 h.</b> Cells were co-transfected and co-probed with anti-KMO antibody (10698-1-AP) and anti-HTT (mEM48) antibody (MAB5374), followed by 30 and 15 nm gold conjugate secondary antibodies, respectively. (<b>A</b>,<b>C</b>) Overview of dual labelling of flKMO-CC and 19Q-VN, respectively. Scale bar = 1 µm. (<b>B</b>,<b>D</b>) Zoomed view of the regions indicated by the black box in (<b>A</b>) and (<b>C</b>), respectively. Mitochondria are all intensely labelled with HTT (15 nm particles), and some particles are seen in the cytoplasm. flKMO-CC labelling is seen on the outer membrane of some HTT-labelled mitochondria (30 nm particles). Scale bar = 1 µm.</p>
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23 pages, 3578 KiB  
Article
Neuroprotective Effects of Phytochemicals against Aluminum Chloride-Induced Alzheimer’s Disease through ApoE4/LRP1, Wnt3/β-Catenin/GSK3β, and TLR4/NLRP3 Pathways with Physical and Mental Activities in a Rat Model
by Ahmed Mohsen Elsaid Hamdan, Fatimah Hussain J. Alharthi, Ahmed Hadi Alanazi, Soad Z. El-Emam, Sameh S. Zaghlool, Kamel Metwally, Sana Abdulaziz Albalawi, Yahia S. Abdu, Reda El-Sayed Mansour, Hoda A. Salem, Zakaria Y. Abd Elmageed and Karema Abu-Elfotuh
Pharmaceuticals 2022, 15(8), 1008; https://doi.org/10.3390/ph15081008 - 17 Aug 2022
Cited by 29 | Viewed by 5913
Abstract
Background: Alzheimer’s disease (AD) is a neurodegenerative disorder that is associated with abnormal cognition. AD is aided in its initiation and progression by hereditary and environmental factors. Aluminum (Al) is a neurotoxic agent that causes oxidative stress, which is linked to AD progression. [...] Read more.
Background: Alzheimer’s disease (AD) is a neurodegenerative disorder that is associated with abnormal cognition. AD is aided in its initiation and progression by hereditary and environmental factors. Aluminum (Al) is a neurotoxic agent that causes oxidative stress, which is linked to AD progression. Additionally, Nrf2/HO-1, APOE4/LRP1, Wnt3/β-catenin, and TLR4/NLRP3 are the main signaling pathways involved in AD pathogenesis. Several phytochemicals are promising options in delaying AD evolution. Objectives: This study aimed at studying the neuroprotective effects of some phytochemicals as morin (MOR), thymol (TML), and thymoquinone (TMQ) on physical and mental activities (PhM) in Al chloride (AlCl3)-induced AD rat model. Another objective was to determine the specificity of phytochemicals to AD signaling pathways using molecular docking. Methods: Eighty male Dawley rats were divided into eight groups. Each group received: saline (control group), AlCl3, (ALAD), PhM, either alone or with a combination of MOR, TML, and/or TMQ for five weeks. Animals were then subjected to behavioral evaluation. Brain tissues were used for histopathological and biochemical analyses to determine the extent of neurodegeneration. The effect of phytochemicals on AlCl3-induced oxidative stress and the main signaling pathways involved in AD progression were also investigated. Results: AlCl3 caused a decline in spatial learning and memory, as well as histopathological changes in the brains of rats. Phytochemicals combined with PhM restored antioxidant activities, increased HO-1 and Nrf2 levels, blocked inflammasome activation, apoptosis, TLR4 expression, amyloide-β generation, and tau hyperphophorylation. They also brought ApoE4 and LRP1 levels back to normal and regulated Wnt3/β-catenin/GSK3β signaling pathway. Conclusions: The use of phytochemicals with PhM is a promising strategy for reducing AD by modulating Nrf2/HO-1, TLR4/NLRP3, APOE4/LRP1, and Wnt3/β-catenin/GSK-3β signaling pathways. Full article
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Graphical abstract

Graphical abstract
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<p>The effect of phytochemical combinations, MOR, TML, and TMQ, with PhM on the behavioral changes induced by AlCl<sub>3</sub> administration for five weeks (70 mg/kg/day, i.p.). (<b>A</b>) The number of trials to avoid the electric shock in CAR test. (<b>B</b>) SAP (%) in Y-Maze test. (<b>C</b>) The escape latency in four days. (<b>D</b>) The time spent in target the quadrant in the MWM test. The data are presented as means ± SD (<span class="html-italic">n</span> = 10). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group. Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they are not significant.</p>
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<p>Photomicrographs of brain sections stained by Hematoxylin and Eosin (magnification 40X). In control group, there was no histopathological alteration in the cerebral cortex, hippocampus, striatum, and substantia nigra (<b>Inserts 1–5</b>). In ALAD group, there were nuclear pyknosis and degeneration in the neuronal cells of the cerebral cortex (<b>Insert 6</b>), subiculum and fascia dentate of the hippocampus (<b>Inserts 7,8</b>). Focal eosinophilic plagues were detected in the striatum (<b>Insert 9</b>). The substantia nigra showed atrophy in the neuronal cells (<b>Insert 10</b>). In ALAD + PhM group, the cerebral cortex and hippocampus showed no histopathological alteration (<b>Inserts 11–13</b>). Nuclear pyknosis and degeneration were recorded in the neurons of the striatum with congestion in the blood vessel (<b>Insert 14</b>). The substantia nigra showed atrophy in some of the neuronal cells (<b>Insert 15</b>). In ALAD + MOR group, there was no histopathological alteration in the cerebral cortex (<b>Insert 16</b>). Nuclear pyknosis and degeneration were observed in some neuronal cells of the subiculum as well as the fascia dentate in the hippocampus (<b>Inserts 17,18</b>). The striatum showed intracellular oedema in the neuronal cells (<b>Insert 19</b>). Mild atrophy was detected in the cells of substantia nigra (<b>Insert 20</b>). In ALAD+ TML group, nuclear pyknosis was observed in the neurons of the cerebral cortex and striatum while the hippocampus was intact (<b>Inserts 21–24</b>). Diffuse gliosis was detected in substantia nigra <b>(Insert 25).</b> In ALAD + TMQ group, the cerebral cortex showed focal nuclear pyknosis and degeneration in the neuronal cells (<b>Insert 26</b>). There was no histopathological alteration in the hippocampus as well as in the striatum (<b>Inserts 27–29</b>). Atrophy was detected in some neurons of the substantia nigra (<b>Insert 30</b>). In ALAD + COM group, the cerebral cortex and hippocampus (subiculum, fascia dentate and hilus) showed normal histological structure (<b>Inserts 31–33</b>). Focal fine plagues were detected in striatum (<b>Insert 33</b>). There was atrophy in some neuronal cells in the substantia nigra (<b>Insert 35</b>). In ALAD + COM + PhM group, there was no histopathological alteration in the cerebral cortex, hippocampus (subiculum, fascia dentate and hilus), striatum and substantia nigra (<b>Insert 36–40</b>). [The data of the effect of MOR, TML and TMQ on the control is not shown as it is not significant].</p>
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<p>The effect of phytochemicals combination, MOR, TML, and TMQ, with PhM on the gene expression of <span class="html-italic">HO-1</span> and <span class="html-italic">Nrf2</span> and their protein levels in AD. (<b>A</b>) Relative gene expression of <span class="html-italic">HO-1</span>, (<b>B</b>) Protein expression of HO-1, (<b>C</b>) Relative gene expression of <span class="html-italic">Nrf2</span>, (<b>D</b>) Protein expression of Nrf2. The data are presented as means ± SD (<span class="html-italic">n</span> = 7). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they were not significant.</p>
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<p>The effect of phytochemicals combination, MOR, TML, and TMQ, with PhM on TLR4 signaling and inflammatory cascade in AD. (<b>A</b>) Protein expression of TLR4, (<b>B</b>) Relative gene expression of TLR4, (<b>C</b>) Protein expression of NF-κb, (<b>D</b>) Relative gene expression of NF-κb, (<b>E</b>) Protein levels of IL-1β, (<b>F</b>) Protein levels of TNF-α. The data are presented as means ± SD (<span class="html-italic">n</span> = 7). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they were not significant.</p>
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<p>The effect of phytochemicals combination, MOR, TML, and TMQ, with PhM on CHI3L1, BDNF, and apoptosis in AD. (<b>A</b>) CHI3L1 levels, (<b>B</b>) <span class="html-italic">Bax/Bcl-2</span> ratio, and (<b>C</b>) BDNF levels. The data are presented as means ± SD (<span class="html-italic">n</span> = 7). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they were not significant.</p>
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<p>The effect of phytochemicals combination, MOR, TML, and TMQ, with PhM on Aβ aggregation and Tau hyperphosphorylation in AD. (<b>A</b>) BACE1 levels, (<b>B</b>) APP levels, (<b>C</b>) Aβ levels, (<b>D</b>) Folds of p-Tau protein expression, (<b>E</b>) p-Tau levels. The data are presented as means ± SD (<span class="html-italic">n</span> = 7). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they were not significant.</p>
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<p>The effect of phytochemicals combination, MOR, TML, and TMQ, with PhM on ApoE4 and LRP1 levels in AD. (<b>A</b>) ApoE4 levels and (<b>B</b>) LRP1 levels. The data are presented as means ± SD (<span class="html-italic">n</span> = 7). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they were not significant.</p>
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<p>The effect of phytochemicals combination, MOR, TML, and TMQ, with PhM on Wnt3/β-catenin/GSK-3β signaling in AD. (<b>A</b>) Wnt3a levels, (<b>B</b>) β-catenin level, (<b>C</b>) Folds of GSK-3β protein expression, and (<b>D</b>) GSK-3β levels. The data are presented as means ± SD (<span class="html-italic">n</span> = 7). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they were not significant.</p>
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<p>The effect of phytochemicals combination, MOR, TML, and TMQ, with PhM on inflammasome signaling in AD. (<b>A</b>) Relative gene expression of NLRP3, (<b>B</b>) Folds of NLRP3 protein expression, (<b>C</b>) Relative gene expression of caspase-1, and (<b>D</b>) Folds of caspase-1 protein expression. The data are presented as means ± SD (<span class="html-italic">n</span> = 7). Significance (a): relative to the control group. Significance (b): relative to the ALAD group. Significance (c): relative to ALAD + PhM group. Significance (d): relative to either ALAD + MOR, ALAD + TML, or ALAD + TMQ group. Significance (e): relative to ALAD + COM group Significance: <span class="html-italic">p</span> &lt; 0.05. The data of the effect of MOR, TML and TMQ on the control are not shown as they were not significant.</p>
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20 pages, 3217 KiB  
Review
ROCK and PDE-5 Inhibitors for the Treatment of Dementia: Literature Review and Meta-Analysis
by Dong-Hun Lee, Ji Young Lee, Dong-Yong Hong, Eun Chae Lee, Sang-Won Park, Yu Na Jo, Yu Jin Park, Jae Young Cho, Yoo Jin Cho, Su Hyun Chae, Man Ryul Lee and Jae Sang Oh
Biomedicines 2022, 10(6), 1348; https://doi.org/10.3390/biomedicines10061348 - 8 Jun 2022
Cited by 6 | Viewed by 3621
Abstract
Dementia is a disease in which memory, thought, and behavior-related disorders progress gradually due to brain damage caused by injury or disease. It is mainly caused by Alzheimer’s disease or vascular dementia and several other risk factors, including genetic factors. It is difficult [...] Read more.
Dementia is a disease in which memory, thought, and behavior-related disorders progress gradually due to brain damage caused by injury or disease. It is mainly caused by Alzheimer’s disease or vascular dementia and several other risk factors, including genetic factors. It is difficult to treat as its incidence continues to increase worldwide. Many studies have been performed concerning the treatment of this condition. Rho-associated kinase (ROCK) and phosphodiesterase-5 (PDE-5) are attracting attention as pharmacological treatments to improve the symptoms. This review discusses how ROCK and PDE-5 affect Alzheimer’s disease, vascular restructuring, and exacerbation of neuroinflammation, and how their inhibition helps improve cognitive function. In addition, the results of the animal behavior analysis experiments utilizing the Morris water maze were compared through meta-analysis to analyze the effects of ROCK inhibitors and PDE-5 inhibitors on cognitive function. According to the selection criteria, 997 publications on ROCK and 1772 publications on PDE-5 were screened, and conclusions were drawn through meta-analysis. Both inhibitors showed good improvement in cognitive function tests, and what is expected of the synergy effect of the two drugs was confirmed in this review. Full article
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Figure 1
<p>The RhoA/ROCK pathway and PDE-5 pathway contribute to smooth muscle contraction. ROCK prevents dephosphorylation of phosphorylated MLC and contributes to smooth muscle contraction by activating the LIMK2/cofilin pathway and the ERM pathway. PDE-5 decomposes increased cGMP from eNOS/NO, contributing to the contraction of VSMC. cGMP: cyclic Guanosine monophosphate; eNOS: endothelial nitric oxide synthase; ERM: ezrin/radixin/moesin; GTP: guanosine triphosphate; LIMK: Lin11-Isl1-Mec3 kinase; MLC: myosin light chain; NO: nitric oxide; PDE-5: phosphodiesterase-5; ROCK: rho-associated protein kinase.</p>
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<p>Improvement of the inflammatory response and recruitment of inflammatory cells due to various factors in the ROCK and PDE family. CyPA: cyclophilin A; ICAM-1: intercellular adhesion molecule 1; MCP-1: monocyte chemoattractant protein 1; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; NO: nitric oxide; PDE-5: phosphodiesterase-5; PKA: protein kinase A; ROCK: rho-associated protein kinase; ROS: reactive oxygen species; VCAM-1: vascular cell adhesion molecule 1.</p>
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<p>The ROCK inhibitor study was conducted independently by two reviewers according to the screening criteria. The studies were selected in the following order: (1) Duplicate articles were excluded. (2) Those with unrelated titles were excluded. Subsequently, (3) those with unrelated abstracts were excluded, and the exclusion considerations for the abstracts were as follows: only traditional medicine, herb extracts, alkaloids, and flavonoids. No dementia model animal or patient. No cognition assessment. No ROCK inhibitors. No clinical trials. (4) The following text was checked to exclude articles that did not study the effect of ROCK inhibitors on behavioral experiments. (5) Studies included in the quantitative synthesis. (6) Finally, only studies using the Morris water maze were left.</p>
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<p>The phosphodiesterase-5 inhibitor study was performed independently along with the flow chart by two reviewers: (1) Duplicate articles were excluded. (2) Publications with unrelated titles and abstracts were excluded. (3) According to the eligibility, publications that had no therapeutic effect in the behavioral experiment were excluded. (4) Full-text articles that were not available for meta-analysis, that had no animal, cognition, or Morris water maze test, were excluded.</p>
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<p>This forest plot shows an analysis of the difference between the experimental group and the control group of each subgroup by standardized mean difference (SMD). Because of the high heterogeneity, each group was analyzed by dividing it into subgroups. SD: standard deviation.</p>
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<p>In the forest plot, the difference between the control group and the experimental group of each subgroup was analyzed by mean difference (MD). Each subgroup is located at the top and bottom with PDE-5 inhibitors and ROCK inhibitors. Because of the high heterogeneity, each group was analyzed by dividing it into subgroups.</p>
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20 pages, 1804 KiB  
Article
Physical Activity Rewires the Human Brain against Neurodegeneration
by Jose A. Santiago, James P. Quinn and Judith A. Potashkin
Int. J. Mol. Sci. 2022, 23(11), 6223; https://doi.org/10.3390/ijms23116223 - 2 Jun 2022
Cited by 21 | Viewed by 4420
Abstract
Physical activity may offset cognitive decline and dementia, but the molecular mechanisms by which it promotes neuroprotection remain elusive. In the absence of disease-modifying therapies, understanding the molecular effects of physical activity in the brain may be useful for identifying novel targets for [...] Read more.
Physical activity may offset cognitive decline and dementia, but the molecular mechanisms by which it promotes neuroprotection remain elusive. In the absence of disease-modifying therapies, understanding the molecular effects of physical activity in the brain may be useful for identifying novel targets for disease management. Here we employed several bioinformatic methods to dissect the molecular underpinnings of physical activity in brain health. Network analysis identified ‘switch genes’ associated with drastic hippocampal transcriptional changes in aged cognitively intact individuals. Switch genes are key genes associated with dramatic transcriptional changes and thus may play a fundamental role in disease pathogenesis. Switch genes are associated with protein processing pathways and the metabolic control of glucose, lipids, and fatty acids. Correlation analysis showed that transcriptional patterns associated with physical activity significantly overlapped and negatively correlated with those of neurodegenerative diseases. Functional analysis revealed that physical activity might confer neuroprotection in Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s (HD) diseases via the upregulation of synaptic signaling pathways. In contrast, in frontotemporal dementia (FTD) its effects are mediated by restoring mitochondrial function and energy precursors. Additionally, physical activity is associated with the downregulation of genes involved in inflammation in AD, neurogenesis in FTD, regulation of growth and transcriptional repression in PD, and glial cell differentiation in HD. Collectively, these findings suggest that physical activity directs transcriptional changes in the brain through different pathways across the broad spectrum of neurodegenerative diseases. These results provide new evidence on the unique and shared mechanisms between physical activity and neurodegenerative diseases. Full article
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<p>Overall study design. (<b>A</b>) We searched GEO and BaseSpace Correlation Engine (BSCE) databases for brain transcriptomic studies from subjects who engaged in different levels of physical activity and individuals with neurodegenerative diseases, including Alzheimer’s (AD), Parkinson’s (PD), Huntington’s diseases (HD), and frontotemporal dementia (FTD). Studies that met our inclusion/exclusion criteria were analyzed further. (<b>B</b>) Dataset GSE110298 containing brain transcriptomic data from aged cognitively intact individuals who engaged in different physical activity levels were analyzed by SWIM software to identify switch genes. Pathway and transcription factor analyses were performed in NetworkAnalyst. Gene ontology associations of switch genes were retrieved from HUGO database. Transcriptomic hippocampal data from subjects engaged in high and moderate physical activity were compared to those engaged in low physical activity using the meta-analysis and correlation engine tool in BSCE. Using this tool, we compared the transcriptome of physically active subjects with patients with neurodegenerative diseases to identify shared and unique pathways affected by physical activity.</p>
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<p>Swim analysis. SWIM analysis of post-mortem hippocampus brain region from cognitively intact, aged subjects engaged in various physical activity levels (GSE110298). (<b>A</b>) Distribution of log2 fold change values where the red bars are selected for further analysis. (<b>B</b>) Heat Cartography Map with nodes colored by their average Pearson Correlation Coefficient. Region R4 represents the switch genes. (<b>C</b>) Dendrogram and heat map for switch genes. The red markers indicate samples from subjects who engaged in low physical activity. (<b>D</b>) Robustness of the correlation network.</p>
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<p>Genetic overlap and correlation analysis between subjects who engaged in high and moderate physical activity and patients with neurodegenerative diseases. Venn diagram and correlation analyses of shared genes between physically active (PA) subjects in GSE110298 and (<b>A</b>) AD GSE48350, (<b>B</b>) AD GSE84422, (<b>C</b>) AD GSE36980, (<b>D</b>) FTD-GRN (+) GSE13162, (<b>E</b>) PD GSE8397, (<b>F</b>) HD GSE3790. Vertical bars represent the significance of the overlap and the correlations between the datasets. Red and green arrows denote up and downregulation, respectively. <span class="html-italic">p</span>-value is expressed as the –log10 of the <span class="html-italic">p</span>-value. Statistical significances regarding the genetic overlap and the directionality of the fold changes were derived from the non-parametric ranking method used by Base Space Correlation Engine (Illumina, Inc., San Diego, CA, USA).</p>
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<p>Genetic overlap and correlation analysis between subjects who engaged in low physical activity and patients with neurodegenerative diseases. Venn diagram and correlation analyses of shared genes between physically active (PA) subjects in GSE110298 and (<b>A</b>) AD GSE48350, (<b>B</b>) AD GSE84422, (<b>C</b>) AD GSE36980, (<b>D</b>) FTD-GRN(+) GSE13162, (<b>E</b>) PD GSE8397, (<b>F</b>) HD GSE3790. Vertical bars represent the significance of the overlap and the correlations between the datasets. Red and green arrows denote up and downregulation, respectively. <span class="html-italic">p</span>-value is expressed as the –log10 of the <span class="html-italic">p</span>-value. Statistical significances regarding the genetic overlap and the directionality of the fold changes were derived from the non-parametric ranking method used by Base Space Correlation Engine (Illumina, Inc., San Diego, CA, USA).</p>
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<p>Correlation analysis of pathways shared between physical activity and neurodegenerative diseases. Genes upregulated in physical activity (PA) and downregulated in neurodegenerative diseases were analyzed using the meta-analysis and correlation in BSCE. The analysis was performed for each neurodegenerative disease separately. Gene ontology (GO) association terms were used for functional analysis. GO groups were filtered using a minimum size of 10 genes and a maximum of 500 genes per group. Each correlated biogroup has an associated score matrix. The height of each vertical bar represents the score of the correlation between the queried dataset and the biogroup (GO group). The orange bar above the midline represents the overlap, or enrichment, between the dataset and the upregulated genes in the dataset. Likewise, the green bar below the midline represents the overlap, or enrichment, between the dataset and the downregulated genes in the dataset. The absence of a colored bar means that the correlation is insignificant. The datasets analyzed are represented on the x-axis. High and moderate physical activity datasets (1–4) compared to (<b>A</b>) Alzheimer’s disease (AD) datasets (5–7), (<b>B</b>) Frontotemporal dementia (FTD) datasets (5–6), (<b>C</b>) Parkinson’s disease (PD) datasets (5–7), (<b>D</b>) Huntington’s disease (HD) datasets (5 = HD grade 1, 6 = HD grade 2, 7 = HD grade 3, 8 = HD grade 4).</p>
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<p>Correlation analysis of pathways shared between physical activity and neurodegenerative diseases. Genes downregulated in physical activity (PA) and upregulated in neurodegenerative diseases were analyzed using the meta-analysis and correlation in BSCE. The analysis was performed for each neurodegenerative disease separately. Gene ontology (GO) association terms were used for functional analysis. GO groups were filtered using a minimum size of 10 genes and a maximum of 500 genes per group. Each correlated biogroup has an associated score matrix. The height of each vertical bar represents the score of the correlation between the queried dataset and the biogroup (GO group). The orange bar above the midline represents the overlap, or enrichment, between the dataset and the upregulated genes in the dataset. Likewise, the green bar below the midline represents the overlap, or enrichment, between the dataset and the downregulated genes in the dataset. The absence of a colored bar means that the correlation is insignificant. The datasets analyzed are represented on the x-axis. High and moderate physical activity datasets (1–4) compared to (<b>A</b>) Alzheimer’s disease (AD) datasets (5–7), (<b>B</b>) Frontotemporal dementia (FTD) datasets (5–6), (<b>C</b>) Parkinson’s disease (PD) datasets (5–7), (<b>D</b>)Huntington’s disease (HD) datasets (5 = HD grade 1, 6 = HD grade 2, 7 = HD grade 3, 8 = HD grade 4).</p>
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25 pages, 2358 KiB  
Review
Therapeutic Targeting of Rab GTPases: Relevance for Alzheimer’s Disease
by Kate L. Jordan, David J. Koss, Tiago F. Outeiro and Flaviano Giorgini
Biomedicines 2022, 10(5), 1141; https://doi.org/10.3390/biomedicines10051141 - 16 May 2022
Cited by 12 | Viewed by 4572
Abstract
Rab GTPases (Rabs) are small proteins that play crucial roles in vesicle transport and membrane trafficking. Owing to their widespread functions in several steps of vesicle trafficking, Rabs have been implicated in the pathogenesis of several disorders, including cancer, diabetes, and multiple neurodegenerative [...] Read more.
Rab GTPases (Rabs) are small proteins that play crucial roles in vesicle transport and membrane trafficking. Owing to their widespread functions in several steps of vesicle trafficking, Rabs have been implicated in the pathogenesis of several disorders, including cancer, diabetes, and multiple neurodegenerative diseases. As treatments for neurodegenerative conditions are currently rather limited, the identification and validation of novel therapeutic targets, such as Rabs, is of great importance. This review summarises proof-of-concept studies, demonstrating that modulation of Rab GTPases in the context of Alzheimer’s disease (AD) can ameliorate disease-related phenotypes, and provides an overview of the current state of the art for the pharmacological targeting of Rabs. Finally, we also discuss the barriers and challenges of therapeutically targeting these small proteins in humans, especially in the context of AD. Full article
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<p>The Intracellular pathways and a selected number of Rab GTPases associated with the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN), and endosomal pathways. Rab1 and Rab2 are localised to the ER and Golgi, and play a role in the ER to Golgi apparatus trafficking pathway, via the endoplasmic reticulum to Golgi intermediate compartment (ERGIC). Rab3 is localised to synaptic secretory vesicles (SV) and the plasma membrane and is involved in exocytosis and neurotransmitter release. Rab4 has a role in protein recycling and transport to the plasma membrane and is localised to early endosomes (EE). Rab5 is localised to the EE and aids its fusion and formation. Rab6 is involved with regulating intra-Golgi trafficking. Rab7 is localised to the late endosome (LE), lysosome (L), and autophagosomes (AP) and is involved in the maturation and transport between these vesicles. Rab8 is associated with exocytosis from the TGN to the plasma membrane, with localisation to the plasma membrane and SV. Rab10 is localised to the ER, Golgi, endosomes, and GLUT4 vesicles and is involved in ER dynamics, endocytosis, and trafficking to the plasma membrane. Rab11 is also localised to the Golgi, as well as the recycling endosome (RE) and EE. Rab13 is involved in the TGN and RE to plasma membrane transport pathway. Rab19 has been shown to localise to the Golgi, however there is little known about its role. Rab27 is involved in exocytosis, localising to SV. Rab29 and Rab39 are both localised to the Golgi. Rab32 localises to the ER and mitochondria, with a role in mitochondrial dynamics and autophagy. Rab35 localises to the plasma membrane, and is involved in endocytic recycling. Rabs more strongly associated with secretory pathways are shaded in blue while those more strongly associated with endosomal pathways are shown in red. Adapted with permission from Hutagalung et al. 2022, American Physiological Society [<a href="#B7-biomedicines-10-01141" class="html-bibr">7</a>].</p>
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<p>The cycle of Rab GTPase activation. Newly synthesised de novo Rab GTPases interact with Rab escort protein (REP), which enables prenylation via geranylgeranyltransferase (GGTase). When active, Rab GTPases are bound to GTP and associated with their target membrane. Following hydrolysis of GTP to GDP, they become inactive and reside in the cytosol. The Rab GTPase activation cycle is aided by a number of effectors. GTPase activating proteins (GAPs) catalyse the hydrolysis of GTP to GDP to inactivate the Rab. GDP dissociation inhibitors (GDIs) retrieve the inactive Rab from the membrane and solubilises it in the cytosol. However, guanine exchange factors (GEFs) catalyse the exchange of GDP with GTP, thus reactivating the Rab [<a href="#B12-biomedicines-10-01141" class="html-bibr">12</a>].</p>
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<p>Rab-mediated amyloid precursor protein metabolism. De novo amyloid precursor protein (APP) is produced with the endoplasmic reticulum (ER) and transported to Golgi for protein maturation via a Rab1 trafficking pathway. Upon passage through the Golgi and trans-Golgi network (TGN), APP resides with the plasma membranes. APP may be processed via the non-amyloidogenic route via α-secretase producing soluble APPα which is released into the extracellular environment. Alternatively, APP along with β-secretases may be internalised via Rab5-dependent endocytosis. Once in the acidic internal compartments of early and late endosomes (EE/LE) the cleavage of APP via β-secretases generates the β-C-terminal fragment (β-CTF). β-secretases is in turn recycled to the plasma membrane either directly via Rab4-mediated traffic, via recycling endosomes (RE) dependent on Rab11 trafficking or trafficking alongside the β-CTF to the Golgi. Within the Golgi and TGN, the γ-secretase complex facilitated by its association with Rab6 processes the β-CTF into β-amyloid (Aβ) which is trafficking into secretary vesicles via Rab10 and released via Rab27/Rab3-dependent process, alongside post Golgi trafficking APP and β-secretase. Also shown is the faciliatory role of Ras and Rab interactor 3 (RIN3) and adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) on Rab5-mediated endocytosis as well as the association of GDP dissociation inhibitors (GDIα/β) with the γ-secretase component presenilin-1. The alternative processing of β-CTF into Aβ within the lysosomes is not shown in this schematic. Rabs more strongly associated with secretory pathways are shaded in blue while those more strongly associated with endosomal pathways are shown in red.</p>
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15 pages, 3684 KiB  
Article
Mitochondrial Proteins Unveil the Mechanism by Which Physical Exercise Ameliorates Memory, Learning and Motor Activity in Hypoxic Ischemic Encephalopathy Rat Model
by Fred Gendi, Feifei Pei, Yuan Wang, Haoye Li, Jia Fu and Cheng Chang
Int. J. Mol. Sci. 2022, 23(8), 4235; https://doi.org/10.3390/ijms23084235 - 11 Apr 2022
Cited by 6 | Viewed by 4396
Abstract
Background: Physical exercise has been shown to improve cognitive and motor functions, promoting neurogenesis and demonstrating therapeutic benefits in neurodegenerative disorders. Nonetheless, it is crucial to investigate the cellular and molecular mechanisms by which this occurs. The study aimed to investigate and evaluate [...] Read more.
Background: Physical exercise has been shown to improve cognitive and motor functions, promoting neurogenesis and demonstrating therapeutic benefits in neurodegenerative disorders. Nonetheless, it is crucial to investigate the cellular and molecular mechanisms by which this occurs. The study aimed to investigate and evaluate the effect of swimming exercise on the changes of mitochondrial proteins in the brains of rats with hypoxic ischemic encephalopathy (HIE). Methods: the vertical pole and Morris water maze tests were used to assess the animals’ motor and cognitive functions, and western blot and immunofluorescence of brain tissue were used to assess the biomarkers of mitochondrial apoptosis and cristae stability in response to exercise training. Four groups of rats were used: (1) sham sedentary group (SHAM, NT), (2) sham exercise training group (SHAM, T) (3) hypoxic ischemic encephalopathy sedentary group (HIE, NT), and (4) hypoxic ischemic encephalopathy exercise training group (HIE, T). Results: animals with HIE showed motor and cognitive deficits, as well as increased apoptotic protein expression. Exercise, on the other hand, improved motor and cognitive functions while also suppressing the expression of apoptotic proteins. Conclusions: By stabilizing the mitochondrial cristae and suppressing the apoptotic cascade, physical exercise provided neuroprotection in hypoxic ischemia-induced brain injury. Full article
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<p>Effect of swimming on motor and cognitive performance. (<b>A</b>) Vertical pole test; exercise training animals (SHAM, T and HIE, T) spent longer time on the vertical pole than sedentary animals (SHAM, NT and HIE, NT), with HIE, NT showing a statistically significant decrease; findings are expressed as Mean ± SEM, * <span class="html-italic">p</span> &lt; 0.05 (one-way ANOVA followed by Dunnett’s post-test). (<b>B</b>) Escape latency; swimming exercise enhanced learning and memory; exercising animals showed a significantly reduced escape latency, or the time (seconds) required to escape. (<b>C</b>) Distance covered; in the exercising groups, there was an obvious increase in distance covered, but it was not statistically significant at <span class="html-italic">p</span> &lt; 0.05 (one-way ANOVA followed by Dunnett’s post-test). (<b>D</b>) Mean swimming speed; animal groups that were subjected to exercise showed a higher swimming speed compared to the sedentary groups indicating that their motor behavior was positively influenced by exercise. (<b>E</b>) Target crossing; this shows the number of times the animals crossed the site at which the platform had been positioned which as expected improved with exercise. The results are expressed as Mean ± SEM. The number of animals used in each group is 7, but the difference was not statistically significant at <span class="html-italic">p</span> &lt; 0.05 (one-way ANOVA followed by Dunnett’s post-test).</p>
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<p>Protein changes in the cytoplasm of the hippocampus. Examples of Western blot bands normalized to GAPDH are presented (with the matching wells). Units are presented relative to GAPDH content. (<b>A</b>) AIF, apoptosis-inducing factor, (<b>B</b>) cytochrome c, (<b>C</b>) cleaved caspase-3, (<b>D</b>) Smac/Diablo, and (<b>E</b>) OPA1. Asterisks denote statistically significant differences between groups (*, vs. SHAM, NT). One asterisk (*) denotes <span class="html-italic">p</span> &lt; 0.05 (**) denote <span class="html-italic">p</span> &lt; 0.01. F-values after running one-way ANOVAs with Dunnett’s post hoc multiple comparison test were: F = 2.264 (AIF), F = 3.775 (cytochrome c), F = 6.125 (cleaved caspase-3), F = 5.140 (Smac/Diablo) and F = 7.096 (OPA1). HIE, NT denotes animals with hypoxic ischemic encephalopathy that did not undergo exercise training; HIE, T denotes animals with hypoxic ischemic encephalopathy that received exercise training; SHAM, NT denotes normal animals that did not receive exercise training; and SHAM, T denotes normal animals that received exercise training.</p>
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<p>Protein changes in the nuclei of the hippocampus. Examples of Western blot bands normalized to histone 3 (H3) are presented (with the matching wells). Units are presented relative to H3 content. (<b>A</b>) AIF, apoptosis-inducing factor, (<b>B</b>) cytochrome c, (<b>C</b>) cleaved caspase-3, (<b>D</b>) Smac/Diablo, and (<b>E</b>) OPA1. Asterisks denote statistically significant differences between groups (*, vs. SHAM, NT). One asterisk (*) denotes <span class="html-italic">p</span> &lt; 0.05, (**) denote <span class="html-italic">p</span> &lt; 0.01 F values were; F = 32.870 (AIF), F = 3.510 (cytochrome c), F = 4.733 (cleaved caspase 3), F = 6.650 (Smac/Diablo) and F = 7.896 (OPA1). HIE, NT denotes animals with hypoxic ischemic encephalopathy that did not undergo exercise training; HIE, T denotes animals with hypoxic ischemic encephalopathy that were subjected to exercise training; SHAM, NT denotes normal animals that did not receive exercise training, and SHAM, T denotes normal animals that received exercise training.</p>
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<p>Protein changes in the cytoplasm of the cerebral cortex. Examples of Western blot bands normalized to GAPDH are presented (with the matching wells). Units are presented relative to GAPDH content. (<b>A</b>) AIF, apoptosis-inducing factor, (<b>B</b>) cytochrome c, (<b>C</b>) cleaved caspase-3, (<b>D</b>) Smac/Diablo, and (<b>E</b>) OPA1. Asterisks denote statistically significant differences between groups (*, vs. SHAM, NT). One asterisk (*) denotes <span class="html-italic">p</span> &lt; 0.05, (**) denote <span class="html-italic">p</span> &lt; 0.01.F values were; F = 0.6974 (AIF), F = 3.666 (cytochrome c), F = 8.992 (cleaved caspase 3), F = 5.643 (Smac/Diablo), and F = 8.257 (OPA1). HIE, NT denotes animals with hypoxic ischemic encephalopathy that did not receive exercise training, HIE, T denotes animals with hypoxic ischemic encephalopathy that received exercise training; SHAM, NT denotes normal animals that did not undergo exercise training; and SHAM, T denotes normal animals that received exercise training.</p>
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<p>Protein changes in the nuclei of the cerebral cortex. Examples of Western blot bands normalized to H3 are presented (with the matching wells). Units are presented relative to H3 content. (<b>A</b>) AIF, apoptosis-inducing factor, (<b>B</b>) cytochrome c, (<b>C</b>) cleaved caspase-3, (<b>D</b>) Smac/Diablo, and (<b>E</b>) OPA1. Asterisks denote statistically significant differences between groups (*, vs. SHAM, NT). One asterisk denotes <span class="html-italic">p</span> &lt; 0.05, two asterisk (**) denote <span class="html-italic">p</span> &lt; 0.01.F values were; F = 16.03 (AIF), F = 6.991 (cytochrome c), F = 14.36 (cleaved caspase 3), F = 6.903 (Smac/Diablo) and F = 5.895 (OPA1). HIE, NT denotes animals with hypoxic ischemic encephalopathy that did not undergo exercise training; HIE, T denotes animals with hypoxic ischemic encephalopathy that underwent exercise training; SHAM, NT denotes normal animals that did not undergo exercise training; and SHAM, T denotes normal animals that received exercise training.</p>
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<p>Immunofluorescence images and mean fluorescent intensities of the proteins in the motor cortex. (<b>A</b>) Representative immunofluorescence images and MFI of AIF in the motor cortex of each group. AIF molecules are green. AIF molecules were analyzed in the motor cortex. Results are presented as the Mean ± SEM of 3 rats from each group. (** <span class="html-italic">p</span> &lt; 0.001 and *** <span class="html-italic">p</span> &lt; 0.0001) compared with the SHAM, NT group; (<sup>##</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the HIE, NT group; each group, <span class="html-italic">n</span> = 3). Scale bar, 100 µm. (<b>B</b>) Representative immunofluorescence images and MFI of Cytochrome C in the motor cortex of each group. Cytochrome C molecules (green) were analyzed in the motor cortex. Data are presented as the Mean ± SEM of 3 rats from each group. (** <span class="html-italic">p</span> &lt; 0.001 and *** <span class="html-italic">p</span> &lt; 0.0001) compared with the SHAM, NT group; (<sup>##</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the HIE, NT group); each group, <span class="html-italic">n</span> = 3. Scale bar, 100 µm. (<b>C</b>) Representative immunofluorescence images and MFI of Cleaved caspase-3 in the motor cortex of each group. Cleaved caspase-3 molecules (green) were analyzed in the motor cortex. Results are presented as the Mean ± SEM of 3 rats from each group. (* <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.0001) compared with the SHAM, NT group); each group, <span class="html-italic">n</span> = 3. Scale bar, 100 µm. (<b>D</b>) Representative immunofluorescence images and MFI of SMAC in the motor cortex of each group. SMAC molecules (green) were analyzed in the motor cortex. Results are presented as the Mean ± SEM of 3 rats of each group. (** <span class="html-italic">p</span> &lt; 0.001 and *** <span class="html-italic">p</span> &lt; 0.0001) compared with the SHAM, NT group); each group, <span class="html-italic">n</span> = 3. Scale bar, 100 µm. (<b>E</b>) Representative immunofluorescence images and MFI of OPA1 in the motor cortex of each group. OPA1 molecules (green) were analyzed in the motor cortex. Individual data are presented as the Mean ± SEM from 3 rats in each group. (*** <span class="html-italic">p</span> &lt; 0.0001) compared with the SHAM-NT group; (<sup>##</sup> <span class="html-italic">p</span> &lt; 0.001 compared with the HIE, NT group); each group, <span class="html-italic">n</span> = 3. Scale bar, 100 µm.</p>
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<p>A schematic summary of the effect of exercise on mitochondrial proteins in the brain. Exercise downregulates the translocation of the proteins to the cytosol and nuclei of the hippocampus and cortex. This stabilizes the mitochondria and mediates exercise’s positive effects on learning, memory, and motor function.</p>
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<p>Experimental design.</p>
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16 pages, 2078 KiB  
Article
Melatonin-Induced Postconditioning Suppresses NMDA Receptor through Opening of the Mitochondrial Permeability Transition Pore via Melatonin Receptor in Mouse Neurons
by Takanori Furuta, Ichiro Nakagawa, Shohei Yokoyama, Yudai Morisaki, Yasuhiko Saito and Hiroyuki Nakase
Int. J. Mol. Sci. 2022, 23(7), 3822; https://doi.org/10.3390/ijms23073822 - 30 Mar 2022
Cited by 5 | Viewed by 2900
Abstract
Mitochondrial membrane potential regulation through the mitochondrial permeability transition pore (mPTP) is reportedly involved in the ischemic postconditioning (PostC) phenomenon. Melatonin is an endogenous hormone that regulates circadian rhythms. Its neuroprotective effects via mitochondrial melatonin receptors (MTs) have recently attracted attention. However, details [...] Read more.
Mitochondrial membrane potential regulation through the mitochondrial permeability transition pore (mPTP) is reportedly involved in the ischemic postconditioning (PostC) phenomenon. Melatonin is an endogenous hormone that regulates circadian rhythms. Its neuroprotective effects via mitochondrial melatonin receptors (MTs) have recently attracted attention. However, details of the neuroprotective mechanisms associated with PostC have not been clarified. Using hippocampal CA1 pyramidal cells from C57BL mice, we studied the involvement of MTs and the mPTP in melatonin-induced PostC mechanisms similar to those of ischemic PostC. We measured changes in spontaneous excitatory postsynaptic currents (sEPSCs), intracellular calcium concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents after ischemic challenge, using the whole-cell patch-clamp technique. Melatonin significantly suppressed increases in sEPSCs and intracellular calcium concentrations. The NMDAR currents were significantly suppressed by melatonin and the MT agonist, ramelteon. However, this suppressive effect was abolished by the mPTP inhibitor, cyclosporine A, and the MT antagonist, luzindole. Furthermore, both melatonin and ramelteon potentiated depolarization of mitochondrial membrane potentials, and luzindole suppressed depolarization of mitochondrial membrane potentials. This study suggests that melatonin-induced PostC via MTs suppressed the NMDAR that was induced by partial depolarization of mitochondrial membrane potential by opening the mPTP, reducing excessive release of glutamate and inducing neuroprotection against ischemia-reperfusion injury. Full article
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<p>Diagram showing time schedules for ischemia and drug administration in each perfusion protocol. In each protocol, electrophysiological recording started collecting data 5 min before the start of ischemia and lasted up to 20 min after the reperfusion. The black band indicates the perfusion period during ischemia. White bands indicate perfusion with artificial cerebrospinal fluid. Red, blue, green, and yellow bands indicate administrations of melatonin, ramelteon, luzindole, and cyclosporine A in artificial cerebrospinal fluid, respectively. Con—control; Mel—melatonin; Ram—ramelteon; Luz—luzindole; CsA—cyclosporine A.</p>
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<p>(<b>A</b>) Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) for control (upper) and melatonin (lower) groups (100 µM) during pre-ischemic, ischemic and reperfusion periods. In each trace, sEPSCs caused by synaptic glutamate releases are seen as transient downward deflections (inward currents). For both control and melatonin groups, occurrences of sEPSCs began to increase approximately 7 min after ischemic perfusion. In traces of the control group, an explosive increase in frequencies of sEPSCs were observed 2 min after reperfusion. In contrast, for the melatonin group, increased occurrences of sEPSCs quickly receded to pre-ischemic levels after reperfusion. (<b>B</b>) Time course of cumulative sEPSCs that occurred in control and melatonin-induced PostC groups. Cumulative sEPSCs that occurred were expressed as a percentage of the total number of sEPSCs occurring in the 5 min prior to ischemia under low (10 µM), medium (100 µM), and high (1 mM) concentrations of melatonin perfusion. In each group, the majority of sEPSCs occurred in the first 5 min after reperfusion. The timeline in the graph set to 0 min at the start for the ischemic load. In each group, the cumulative sEPSCs at 0 min of timeline were set as 100%. (<b>C</b>) Each vertical rectangle and error bar indicate percent cumulative sEPSCs that occurred at 20 min after onset of ischemic perfusion (12.5 min after reperfusion) and standard error of the mean (SEM), respectively. Asterisks indicate significant difference in <span class="html-italic">t</span>-test (** <span class="html-italic">p</span> &lt; 0.01). Con—control; Mel—melatonin.</p>
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<p>Comparison between control and melatonin-induced PostC groups for the number of dead neurons due to ischemic injury in the hippocampal CA1 region. (<b>A</b>) Microscopic view of the CA1 region shows the nuclei of dead cells in the control and melatonin-induced PostC groups, respectively. Magenta cells stained with both propidium iodide and SYTOX-blue were considered dead before electrophysiological recordings; blue cells stained with SYTOX-blue alone were considered dead due to ischemia-reperfusion injury. Scale bars = 50 μm. (<b>B</b>) The number of dead neurons per 1 mm of CA1 region. The number of dead neurons was significantly lower in the melatonin-induced PostC group according to <span class="html-italic">t</span>-testing (** <span class="html-italic">p</span> &lt; 0.01). Con—control; Mel—melatonin.</p>
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<p>Effect of post-ischemic treatment and post-anoxic melatonin administration on N-methyl-D-aspartate (NMDA)-induced currents recorded from voltage-clamped hippocampal pyramidal neurons. (<b>A</b>) Typical traces of NMDA-induced currents prior to anoxia, at the end of anoxia, and after 5 min of anoxia in the Control, Mel, Ram, Mel + Luz, and Mel + CsA groups. Inward currents are represented by downward deflection. In the Mel group, NMDA-induced currents decreased and no change in waveform was seen after 5 min of anoxia. The control group showed no obvious change in NMDA-induced current. (<b>B</b>) Bar graph showing the change in mean peak amplitude of NMDA-induced current from 10 min to 20 min after anoxia in the control group, Mel group, Ram group, Mel + Luz group, and Mel + CsA group. Values are shown as currents in multiples of Amps units change relative to mean peak amplitude during the 5 min prior to anoxia. Asterisks indicate significant differences in Tukey–Kramer multiple comparisons test (** <span class="html-italic">p</span> &lt; 0.01). NMDA—N-methyl-D-aspartate; Con—control; Mel—melatonin; Ram—ramelteon; Luz—luzindole; CsA—cyclosporine A.</p>
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<p>Effect of melatonin-induced PostC on cytosolic Ca<sup>2+</sup> concentration after ischemia-reperfusion. (<b>A</b>) Representative microphotographs showing changes in Fura-2 emissions resulting from excitation at 340 and 380 nm for the control group. The elevation in the Fura-2 ratio (340/380 ratio) represents an increase in cytosolic Ca<sup>2+</sup> concentration. Scale bars = 10 µm. (<b>B</b>) Course of changes in the Fura-2 ratio during pre-anoxic, anoxic, and reperfusion periods. Percentages are relative to the mean value observed during the 5 min of the pre-anoxic period. The red horizontal bar indicates the ischemic period. The timeline in the graph is set to 0 min at the start for the ischemic load. The increase in intracellular Ca<sup>2+</sup> concentration after reperfusion is significantly inhibited by melatonin-induced PostC (<span class="html-italic">p</span> &lt; 0.05). The yellow band represents the period used for statistical analysis. (<b>C</b>) Each vertical rectangle and error bar indicate percentage change in the Fura-2 ratio during 5–10 min after 7.5 min of ischemia (yellow band in (<b>B</b>)) and SEM, respectively. Asterisks indicate significant difference in <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05). Con—control; Mel—melatonin.</p>
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<p>Changes in mitochondrial membrane potential as estimated from JC1 fluorescence during pre-ischemia, ischemic, and reperfusion periods. (<b>A</b>) Representative microphotographs of JC1 fluorescence in a slice of hippocampus: Left: infrared differential interference contrast image; Middle: green fluorescent image excited at 477 nm; Right: red fluorescent image excited at 548 nm. Scale bars = 10 µm. (<b>B</b>) Course of changes in mitochondrial membrane potential estimated with JC1 fluorescence during pre-anoxia, anoxia, and reperfusion periods. Percentages are relative to the mean value observed during the 5 min pre-anoxic period. The red horizontal bar indicates the ischemic period. The timeline in the graph set to 0 min at the start for the ischemic load. The yellow band represents the period used for statistical analysis. (<b>C</b>) Bar graph of percentage change in the JC1 green/red ratio, median data from the 7.5–12.5 min reperfusion period (yellow band in (<b>B</b>)). Asterisks indicate significant differences in Games–Howell multiple comparisons test (* <span class="html-italic">p</span> &lt; 0.05). Con—control; Mel—melatonin; Ram—ramelteon; Luz—luzindole.</p>
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11 pages, 457 KiB  
Review
Thymoquinone: Review of Its Potential in the Treatment of Neurological Diseases
by Faheem Hyder Pottoo, Abdallah Mohammad Ibrahim, Ali Alammar, Rida Alsinan, Mahdi Aleid, Ali Alshehhi, Muruj Alshehri, Supriya Mishra and Noora Alhajri
Pharmaceuticals 2022, 15(4), 408; https://doi.org/10.3390/ph15040408 - 27 Mar 2022
Cited by 34 | Viewed by 14479
Abstract
Thymoquinone (TQ) possesses anticonvulsant, antianxiety, antidepressant, and antipsychotic properties. It could be utilized to treat drug misuse or dependence, and those with memory and cognitive impairment. TQ protects brain cells from oxidative stress, which is especially pronounced in memory-related regions. TQ exhibits antineurotoxin [...] Read more.
Thymoquinone (TQ) possesses anticonvulsant, antianxiety, antidepressant, and antipsychotic properties. It could be utilized to treat drug misuse or dependence, and those with memory and cognitive impairment. TQ protects brain cells from oxidative stress, which is especially pronounced in memory-related regions. TQ exhibits antineurotoxin characteristics, implying its role in preventing neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. TQ’s antioxidant and anti-inflammatory properties protect brain cells from damage and inflammation. Glutamate can trigger cell death by causing mitochondrial malfunction and the formation of reactive oxygen species (ROS). Reduction in ROS production can explain TQ effects in neuroinflammation. TQ can help prevent glutamate-induced apoptosis by suppressing mitochondrial malfunction. Several studies have demonstrated TQ’s role in inhibiting Toll-like receptors (TLRs) and some inflammatory mediators, leading to reduced inflammation and neurotoxicity. Several studies did not show any signs of dopaminergic neuron loss after TQ treatment in various animals. TQ has been shown in clinical studies to block acetylcholinesterase (AChE) activity, which increases acetylcholine (ACh). As a result, fresh memories are programmed to preserve the effects. Treatment with TQ has been linked to better outcomes and decreased side effects than other drugs. Full article
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<p>Thymoquinone chemical structure.</p>
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18 pages, 11668 KiB  
Article
IGF-1 as a Potential Therapy for Spinocerebellar Ataxia Type 3
by Yong-Shiou Lin, Wen-Ling Cheng, Jui-Chih Chang, Ta-Tsung Lin, Yi-Chun Chao and Chin-San Liu
Biomedicines 2022, 10(2), 505; https://doi.org/10.3390/biomedicines10020505 - 21 Feb 2022
Cited by 9 | Viewed by 3552
Abstract
Although the effects of growth hormone (GH) therapy on spinocerebellar ataxia type 3 (SCA3) have been examined in transgenic SCA3 mice, it still poses a nonnegligible risk of cancer when used for a long term. This study investigated the efficacy of IGF-1, a [...] Read more.
Although the effects of growth hormone (GH) therapy on spinocerebellar ataxia type 3 (SCA3) have been examined in transgenic SCA3 mice, it still poses a nonnegligible risk of cancer when used for a long term. This study investigated the efficacy of IGF-1, a downstream mediator of GH, in vivo for SCA3 treatment. IGF-1 (50 mg/kg) or saline, once a week, was intraperitoneally injected to SCA3 84Q transgenic mice harboring a human ATXN3 gene with a pathogenic expanded 84 cytosine–adenine–guanine (CAG) repeat motif at 9 months of age. Compared with the control mice harboring a 15 CAG repeat motif, the SCA3 84Q mice treated with IGF-1 for 9 months exhibited the improvement only in locomotor function and minimized degeneration of the cerebellar cortex as indicated by the survival of more Purkinje cells with a more favorable mitochondrial function along with a decrease in oxidative stress caused by DNA damage. These findings could be attributable to the inhibition of mitochondrial fission, resulting in mitochondrial fusion, and decreased immunofluorescence staining in aggresome formation and ataxin-3 mutant protein levels, possibly through the enhancement of autophagy. The findings of this study show the therapeutic potential effect of IGF-1 injection for SCA3 to prevent the exacerbation of disease progress. Full article
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<p>IGF-1 prevented impairment of the motor function in the SCA3 mice. (<b>a</b>) Latency to fall (time in seconds for which the mice persisted on the rotarod) for the SCA3 15Q mice and the saline- and IGF-1-treated SCA3 84Q mice during the 9 months of treatment. (<b>b</b>) Within the same group, the latency to fall at pretreatment was normalized to 100%. (<b>c</b>) EthoVision XT 7.0 software was used to analyze trajectories of the mice in the behavioral test. (<b>d</b>) The distance of movement, time of movement, frequency of zone change, and average velocity were included in transformed indices. (<b>e</b>) Captured images of the single stance for each paw. (<b>f</b>) Catwalk parameters included the step cycle, stride length, stand, and average speed. The data are presented as the means ± SEM. Note: # <span class="html-italic">p</span> &lt; 0.05 denotes statistical significance in the saline-treated SCA3 84Q mice compared with the SCA3 15Q mice; * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p>
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<p>IGF-1 prevented the death of the PCs in the cerebellum of the SCA3 84Q mice. (<b>a</b>) The arrows indicate the PCs located at the PCL (right panel). The average number of the PCs per 100 μm in the posterior lobules of the cerebellum is presented in the bar graph (mean ± SEM) (left panel). SCA3 15Q, n = 6; SCA3 84Q, n = 8; SCA3 84Q + IGF-1, n = 8. (<b>b</b>) Western blot analysis of calbindin (left panel). Relative expression levels of calbindin in the cerebellum (mean ± SEM) (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. (<b>c</b>) The lines indicate the distance from the tip of the granular layer (GL) to the white matter (right panel). Histogram showing the thickness of the GL (mean ± SEM) (left panel). SCA3 15Q, n = 5; SCA3 84Q, n = 6; SCA3 84Q + IGF-1, n = 8. (<b>d</b>) The frames are the sampling area of the Figure and the lines refer to the distance from the PCL to the edge of the molecular layer (ML) (right panel). Histogram showing the thickness of the ML (mean ± SEM) (left panel). SCA3 15Q, n = 5; SCA3 84Q, n = 6; SCA3 84Q + IGF-1, n = 8. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p>
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<p>IGF-1 reduced the ataxin-3 protein level in the cerebellum of the SCA3 84Q mice. (<b>a</b>) Immunochemical staining of ataxin-3 in the cerebellum. The black arrows indicate PCs (right panel). Histograms show the means ± SEM (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (<b>b</b>) Western blot confirming ataxin-3 expression in the mouse cerebellum (left panel). Quantification of the ataxin-3 level relative to the total protein level (mean ± SEM) (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (<b>c</b>) Slices of the cerebellum of two mice in each group were selected and double-labeled using an aggresome detection kit (red) and an Alexa 488-conjugated secondary IgG against the anti-ataxin-3 antibody (green), and fluorescence intensities of 30–40 PCs in each mouse were examined using the ImageJ software. The white arrows indicate PCs. SCA3 15Q, n = 2; SCA3 84Q, n = 2; SCA3 84Q + IGF-1, n = 2. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p>
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<p>Expression of the autophagic influx in the SCA3 mice. Representative Western blots of the autophagy-related markers (right panel). Quantitative results of the autophagy-related proteins were normalized to those of total protein (mean ± SEM) (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p>
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<p>Expression of the mitochondrial function in the SCA3 mice. (<b>a</b>) Typical trace of respirometry measurements recorded using an Oroboros O2k with 2 mg/mL of the cerebellum. The blue curve indicates the oxygen concentration in the sealed chamber, whereas the red curve shows the oxygen consumption of tissue cells (left panel). Oxygen consumption of cells at different mitochondrial stages was corrected for ROX, and the respiratory capacities in the routine, OXPHOS, Max-Ox, and Max-U states were plotted as the means ± SEM (right panel). SCA3 15Q, n = 3; SCA3 84Q, n = 3; SCA3 84Q + IGF-1, n = 2. (<b>b</b>) The 8-OHdG protein expression in the cerebellum sections by IHC staining analysis; the arrows indicate PCs (left panel). Histogram shows the mean ± SEM (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 5; SCA3 84Q + IGF-1, n = 5. (<b>c</b>) Western blot was performed to analyze the expression of mitochondrial dynamics-related proteins (right panel). Quantification of mitochondrial dynamics-related proteins (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p>
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<p>Plasma concentration of Nf-L. SCA3 15Q, n = 6; SCA3 84Q, n = 8; SCA3 84Q + IGF-1, n = 8. Note: * <span class="html-italic">p</span> &lt; 0.05 was considered a statistically significant difference.</p>
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<p>Tissue sections of the liver, lung, and kidney. No significant histopathological findings of the kidneys, liver, and lungs were observed in the SCA3 15Q, saline-treated SCA3 84Q, and IGF-1-treated SCA3 84Q mice. n = 3 in all the groups.</p>
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16 pages, 2147 KiB  
Article
Expression of the Adenosine A2A-A3 Receptor Heteromer in Different Brain Regions and Marked Upregulation in the Microglia of the Transgenic APPSw,Ind Alzheimer’s Disease Model
by Alejandro Lillo, Iu Raïch, Jaume Lillo, Catalina Pérez-Olives, Gemma Navarro and Rafael Franco
Biomedicines 2022, 10(2), 214; https://doi.org/10.3390/biomedicines10020214 - 19 Jan 2022
Cited by 9 | Viewed by 3198
Abstract
Adenosine (Ado) receptors have been instrumental in the detection of heteromers and other higher-order receptor structures, mainly via interactions with other cell surface G-protein-coupled receptors. Apart from the first report of the A1 Ado receptor interacting with the A2A Ado receptor, [...] Read more.
Adenosine (Ado) receptors have been instrumental in the detection of heteromers and other higher-order receptor structures, mainly via interactions with other cell surface G-protein-coupled receptors. Apart from the first report of the A1 Ado receptor interacting with the A2A Ado receptor, there has been more recent data on the possibility that every Ado receptor type, A1, A2A, A2B, and A3, may interact with each other. The aim of this paper was to look for the expression and function of the A2A/A3 receptor heteromer (A2AA3Het) in neurons and microglia. In situ proximity ligation assays (PLA), performed in primary cells, showed that A2AA3Het expression was markedly higher in striatal than in cortical and hippocampal neurons, whereas it was similar in resting and activated microglia. Signaling assays demonstrated that the effect of the A2AR agonist, PSB 777, was reduced in the presence of the A3R agonist, 2-Cl-IB-MECA, whereas the effect of the A3R agonist was potentiated by the A2AR antagonist, SCH 58261. Interestingly, the expression of the heteromer was markedly enhanced in microglia from the APPSw,Ind model of Alzheimer’s disease. The functionality of the heteromer in primary microglia from APPSw,Ind mice was more similar to that found in resting microglia from control mice. Full article
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<p>Expression of A<sub>2A</sub>A<sub>3</sub>Hets in primary cultures of neurons isolated from the fetuses of pregnant C57BL/6J female mice. (<b>A</b>) A<sub>2A</sub>A<sub>3</sub>Hets were detected by the in situ proximity ligation assay (PLA) in primary cultures of striatal, hippocampal, and cortical neurons isolated from mouse fetuses. The negative control was obtained by omitting the primary antibody anti-A<sub>3</sub>R. Experiments were performed in samples from 6 different animals. (<b>B</b>) The number of red dots/cell was quantified using the Andy’s algorithm Fiji’s plug-in and represented versus the number of Hoechst-stained cell nuclei (blue). The number of red dots/cell was compared to those in neurons from different brain regions. The unpaired <span class="html-italic">t</span>-test was used for statistical analysis. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001. Scale bar: 10 μm.</p>
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<p>A<sub>2A</sub>A<sub>3</sub>Het-mediated Gs/Gi signaling in primary striatal, hippocampal, and cortical neurons from C57BL/6J mice. (<b>A</b>–<b>F</b>) Primary neurons were pre-treated with selective antagonists, 1 μM SCH 58261 -A<sub>2A</sub>R- or 1 μM PSB 10 -A<sub>3</sub>R-, and subsequently treated with the selective agonists, 100 nM PSB 777 -A<sub>2A</sub>R- or 100 nM 2-Cl-IB-MECA -A<sub>3</sub>R-. cAMP levels after 500 nM forskolin (FK) stimulation or vehicle treatment were detected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (<b>A</b>–<b>C</b>) or in % respect to levels obtained upon FK stimulation (<b>D</b>–<b>F</b>). The values are the mean ± S.E.M. of 6 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; versus basal levels (<b>A</b>–<b>C</b>) or versus FK treatment (<b>D</b>–<b>F</b>).</p>
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<p>Expression and function of A<sub>2A</sub>A<sub>3</sub>Hets in primary cultures of microglia from C57BL/6J mice. (<b>A</b>) A<sub>2A</sub>A<sub>3</sub>Hets were detected in primary cultures of microglia by the in situ proximity ligation assay (PLA) using specific antibodies. Cell nuclei were stained with Hoechst (blue). Samples from 5 different animals were processed and analyzed. Scale bar: 20 μm. (<b>B</b>–<b>D</b>) Primary cultures of microglia from C57BL/6J mice were pre-treated with antagonists, 1 μM SCH 58261 -for A<sub>2A</sub>R- or 1 μM PSB 10 -for A<sub>3</sub>R-, subsequently stimulated with selective agonists, 100 nM PSB 777 -for A<sub>2A</sub>R- or 100 nM 2-Cl-IB-MECA -for A<sub>3</sub>R-, individually or in combination and treated with 500 nM forskolin (FK) or vehicle. (<b>B</b>) ERK1/2 phosphorylation was analyzed using an AlphaScreen<sup>®</sup> SureFire<sup>®</sup> kit (PerkinElmer) while cAMP levels were collected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (<b>B</b>,<b>C</b>) or in % respect levels obtained upon 0.5 μM FK stimulation (<b>D</b>). Values are the mean ± S.E.M. of 10 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; versus basal (<b>B</b>,<b>C</b>) or versus FK treatment (<b>D</b>).</p>
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<p>Expression and function of A<sub>2A</sub>A<sub>3</sub>Hets in primary cultures of LPS-activated microglia from C57BL/6J mice. (<b>A</b>) A<sub>2A</sub>A<sub>3</sub>Hets were detected in primary cultures of non-activated and LPS-activated microglia by the in situ proximity ligation assay (PLA) using specific antibodies. The negative control was obtained by omitting the primary anti-A<sub>3</sub>R antibody. Experiments were performed in samples from 5 different animals. (<b>B</b>) The number of red dots/cell was quantified using the Andy’s algorithm Fiji’s plug-in and represented versus the number of Hoechst-stained cell nuclei (blue). The number of red dots/cell was compared between non-activated and LPS-activated microglia. The unpaired t-test was used for statistical analysis. Scale bar: 20 μm. (<b>C</b>–<b>E</b>) Primary cultures of LPS-activated microglia from C57BL/6J mice were pre-treated with antagonists, 1 μM SCH 58261 -for A<sub>2A</sub>R- or 1 μM PSB 10 -for A<sub>3</sub>R-, subsequently stimulated with selective agonists, 100 nM PSB 777 -for A<sub>2A</sub>R- or 100 nM 2-Cl-IB-MECA -for A<sub>3</sub>R-, individually or in combination and treated with 500 nM forskolin (FK) or vehicle. (<b>C</b>) ERK1/2 phosphorylation was analyzed using an AlphaScreen<sup>®</sup> SureFire<sup>®</sup> kit (PerkinElmer) while cAMP levels were collected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (<b>C</b>,<b>D</b>) or in % respect to levels obtained upon 0.5 μM FK stimulation (<b>E</b>). Values are the mean ± S.E.M. of 6 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. * <span class="html-italic">p</span> &lt; 0.05, **** <span class="html-italic">p</span> &lt; 0.0001; versus basal (<b>C</b>,<b>D</b>) or versus FK treatment (<b>E</b>).</p>
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<p>Expression and function of A<sub>2A</sub>A<sub>3</sub>Hets in primary cultures of microglia from the APP<sub>Sw,Ind</sub> mice model of Alzheimer’s disease. (<b>A</b>) A<sub>2A</sub>A<sub>3</sub>Hets were detected in primary cultures of APP<sub>Sw,Ind</sub> microglia by the in situ proximity ligation assay (PLA) using specific antibodies. The negative control was obtained by omitting the primary anti-A<sub>3</sub>R antibody. Experiments were performed in samples from 12 different animals. (<b>B</b>) The number of red dots/cell was quantified using the Andy’s algorithm Fiji’s plug-in and represented versus the number of Hoechst-stained cell nuclei (blue). The number of red dots/cell was compared to those in microglia from wild type (WT) mice. The unpaired t-test was used for statistical analysis. ** <span class="html-italic">p</span> &lt; 0.01, versus WT. Scale bar: 20 μm. (<b>C</b>–<b>E</b>) Primary cultures of microglia from APP<sub>Sw,Ind</sub> mice were pre-treated with antagonists, 1 μM SCH 58261 -for A<sub>2A</sub>R- or 1 μM PSB 10 -for A<sub>3</sub>R-, and subsequently stimulated with selective agonists, 100 nM PSB 777 -for A<sub>2A</sub>R- or 100 nM 2-Cl-IB-MECA -for A<sub>3</sub>R-, individually or in combination. (<b>C</b>) ERK1/2 phosphorylation was analyzed using an AlphaScreen<sup>®</sup> SureFire<sup>®</sup> kit (PerkinElmer) while cAMP levels were collected by the Lance Ultra cAMP kit and results were expressed in % respect to basal levels (<b>C</b>,<b>D</b>) or in % respect levels obtained upon 0.5 μM FK stimulation (<b>E</b>). Values are the mean ± S.E.M. of 6 different experiments performed in triplicates. One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests were used for statistical analysis. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001; versus basal (<b>C</b>,<b>D</b>) or versus FK treatment (<b>E</b>).</p>
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16 pages, 2868 KiB  
Article
Perilla frutescens Leaf Extract Attenuates Vascular Dementia-Associated Memory Deficits, Neuronal Damages, and Microglial Activation
by Hyun-Bae Kang, Shin-Hye Kim, Sun-Ho Uhm, Do-Kyung Kim, Nam-Seob Lee, Young-Gil Jeong, Nak-Yun Sung, Dong-Sub Kim, In-Jun Han, Young-Choon Yoo and Seung-Yun Han
Curr. Issues Mol. Biol. 2022, 44(1), 257-272; https://doi.org/10.3390/cimb44010019 - 8 Jan 2022
Cited by 10 | Viewed by 3215
Abstract
Vascular dementia (VaD) is characterized by a time-dependent memory deficit and essentially combined with evidence of neuroinflammation. Thus, polyphenol-rich natural plants, which possess anti-inflammatory properties, have received much scientific attention. This study investigated whether Perilla frutescens leaf extract (PFL) exerts therapeutic efficacy against [...] Read more.
Vascular dementia (VaD) is characterized by a time-dependent memory deficit and essentially combined with evidence of neuroinflammation. Thus, polyphenol-rich natural plants, which possess anti-inflammatory properties, have received much scientific attention. This study investigated whether Perilla frutescens leaf extract (PFL) exerts therapeutic efficacy against VaD. Sprague Dawley rats were divided into five groups: SO, sham-operated and vehicle treatment; OP, operated and vehicle treatment; PFL-L, operated and low-dose (30 mg/kg) PFL treatment; PFL-M, operated and medium-dose (60 mg/kg) PFL treatment; and PFL-H, operated and high-dose (90 mg/kg) PFL treatment. Two-vessel occlusion and hypovolemia (2VO/H) were employed as a surgical model of VaD, and PFL was given orally perioperatively for 23 days. The rats underwent the Y-maze, Barnes maze, and passive avoidance tests and their brains were subjected to histologic studies. The OP group showed VaD-associated memory deficits, hippocampal neuronal death, and microglial activation; however, the PFL-treated groups showed significant attenuations in all of the above parameters. Using lipopolysaccharide (LPS)-stimulated BV-2 cells, a murine microglial cell line, we measured PFL-mediated changes on the production of nitric oxide (NO), TNF-α, and IL-6, and the activities of their upstream MAP kinases (MAPKs)/NFκB/inducible NO synthase (iNOS). The LPS-induced upregulations of NO, TNF-α, and IL-6 production and MAPKs/NFκB/iNOS activities were globally and significantly reversed by 12-h pretreatment of PFL. This suggests that PFL can counteract VaD-associated structural and functional deterioration through the attenuation of neuroinflammation. Full article
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<p>HPLC chromatograms of a standard mixture of scutellarin, apigenin-7-O-glucuronide, and rosmarinic acid, and Perilla frutescens leaf extract obtained using a YMC Triart C18 column monitored at 254 nm of peak area, 30 min of run time, 1.0 mL/min of flow rate. Peaks of scutellarin, apigenin-7-O-glucuronide, and rosmarinic acid are indicated with circled numbers “1”, “2”, and “3”, respectively.</p>
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<p>Graphical illustration of the in vivo experiment setup. (<b>a</b>) Schematic overview of the in vivo experiment, (<b>b</b>) surgical procedure, (<b>c</b>) a representative Doppler flowmetry traced for the entire operation time, and (<b>d</b>) the area of interest for histologic analyses in this study are presented. In (<b>b</b>), circled “1” and “2” represent the steps of femoral artery catheterization and ligatures of both common carotid arteries in the 2VO/H operation, respectively. In (<b>d</b>), a rectangular box indicates a 300 µm-width area in hippocampal Cornu ammonis 1, which was used for histologic analyses.</p>
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<p>Effects of PFL on memory deficits in VaD rats. (<b>a</b>) Spontaneous alternation and (<b>b</b>) total arm entries of different groups were measured using the Y-maze test. Using the Barnes maze trial test session, (<b>c</b>) tracking plots made at the 3rd trial day and (<b>d</b>) time-dependent changes in the moved distance to the platform of different groups were obtained. Using the Barnes maze probe test session, (<b>e</b>) tracking plots and (<b>f</b>) time spent in the target quadrant of different groups were obtained. In the tracking plots, the locations of the platform and target quadrant are indicated as a black circle and dark area, respectively. (<b>g</b>) Trial latency and (<b>h</b>) escape latency in different groups were measured using the passive avoidance test. In all graphs, values are presented as the mean ± SEM (*** <span class="html-italic">p</span> &lt; 0.001 vs. SO; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 vs. VEH; <sup>§§</sup> <span class="html-italic">p</span> &lt; 0.01 vs. PFL-L; and NS, statistically not significant).</p>
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<p>Effects of PFL on neuronal viability and neuroinflammation in the VaD rat hippocampal CA1. After cresyl violet staining (<b>a</b>, top row) and immunohistochemistry against Iba1 (<b>a</b>, middle row) and GFAP (a, bottom row), representative photographs of hippocampal CA1 were obtained, and the number of (<b>b</b>) viable neurons, (<b>c</b>) Iba1<sup>+</sup> microglia, and (<b>d</b>) optical density of GFAP<sup>+</sup> astrocytes were quantified, respectively. The region of interest was within a 300 µm width in the hippocampal CA1 region. In (<b>a</b>), the three layers of the hippocampus, i.e., stratum oriens, stratum pyramidale, and stratum radiatum, are indicated as “S.O”, “S.P”, and “S.R”, respectively. Scale bar = 50 μm. In (<b>b</b>) to (<b>d</b>), values are presented as the mean ± SEM (*** <span class="html-italic">p</span> &lt; 0.001 vs. SO; <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 vs. VEH; <sup>§§</sup> <span class="html-italic">p</span> &lt; 0.01 vs. PFL-L; NS, statistically not significant).</p>
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<p>Effect of PFL on BV-2 microglial cell viability and NO, TNF-α, and IL-6 production. BV-2 cells were pretreated with the indicated doses of PFL for 12 h and further treated with 1 μg/mL lipopolysaccharide (LPS). After 24 h, (<b>a</b>) cell viability was determined by the methyl thiazolyl tetrazolium (MTT) assay. Using enzyme-linked immunosorbent assay (ELISA) kits, the contents of (<b>b</b>) NO, (<b>c</b>) TNF-α, and (<b>d</b>) IL-6 in culture media were determined. Values are presented as the mean ± SEM (** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. PFL-untreated; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 vs. 50 μg/mL PFL-treated).</p>
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<p>Effect of PFL on the expression of selected proteins in LPS-stimulated BV-2 cells. BV-2 cells were pretreated with the indicated doses of PFL for 12 h and further treated with 1 μg/mL lipopolysaccharide (LPS) for 24 h. By Western blot, (<b>a</b>) representative protein band images were obtained, and the amounts of phosphorylated MAPKs, including (<b>b</b>) p-p38, (<b>c</b>) p-ERK1/2, (<b>d</b>) p-JNK, (<b>e</b>) iNOS, and (<b>f</b>) nuclear NFκB, were semi-quantitatively assessed. The band intensities of p-p38, p-ERK1/2, p-JNK, and iNOS were normalized by β-actin, whereas nuclear NFκB intensity was normalized by LaminB1, each used as internal controls. Data were expressed as the fold of the controls and collected from at least three independent experiments. Values are presented as the mean ± SEM (*** <span class="html-italic">p</span> &lt; 0.001 vs. LPS-untreated; <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 vs. LPS only-treated; <sup>§</sup> <span class="html-italic">p</span> &lt; 0.05 vs. 50 μg/mL PFL-treated).</p>
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16 pages, 4635 KiB  
Article
Low p-SYN1 (Ser-553) Expression Leads to Abnormal Neurotransmitter Release of GABA Induced by Up-Regulated Cdk5 after Microwave Exposure: Insights on Protection and Treatment of Microwave-Induced Cognitive Dysfunction
by Wei-Jia Zhi, Si-Mo Qiao, Yong Zou, Rui-Yun Peng, Hai-Tao Yan, Li-Zhen Ma, Ji Dong, Li Zhao, Bin-Wei Yao, Xue-Long Zhao, Xin-Xing Feng, Xiang-Jun Hu and Li-Feng Wang
Curr. Issues Mol. Biol. 2022, 44(1), 206-221; https://doi.org/10.3390/cimb44010015 - 31 Dec 2021
Cited by 2 | Viewed by 2438
Abstract
With the wide application of microwave technology, concerns about its health impact have arisen. The signal transmission mode of the central nervous system and neurons make it particularly sensitive to electromagnetic exposure. It has been reported that abnormal release of amino acid neurotransmitters [...] Read more.
With the wide application of microwave technology, concerns about its health impact have arisen. The signal transmission mode of the central nervous system and neurons make it particularly sensitive to electromagnetic exposure. It has been reported that abnormal release of amino acid neurotransmitters is mediated by alteration of p-SYN1 after microwave exposure, which results in cognitive dysfunction. As the phosphorylation of SYN1 is regulated by different kinases, in this study we explored the regulatory mechanisms of SYN1 fluctuations following microwave exposure and its subsequent effect on GABA release, aiming to provide clues on the mechanism of cognitive impairment caused by microwave exposure. In vivo studies with Timm and H&E staining were adopted and the results showed abnormality in synapse formation and neuronal structure, explaining the previously-described deficiency in cognitive ability caused by microwave exposure. The observed alterations in SYN1 level, combined with the results of earlier studies, indicate that SYN1 and its phosphorylation status (ser-553 and ser62/67) may play a role in the abnormal release of neurotransmitters. Thus, the role of Cdk5, the upstream kinase regulating the formation of p-SYN1 (ser-553), as well as that of MEK, the regulator of p-SYN1 (ser-62/67), were investigated both in vivo and in vitro. The results showed that Cdk5 was a negative regulator of p-SYN1 (ser-553) and that its up-regulation caused a decrease in GABA release by reducing p-SYN1 (ser-553). While further exploration still needed to elaborate the role of p-SYN1 (ser-62/67) for neurotransmitter release, MEK inhibition had was no impact on p-Erk or p-SYN1 (ser-62/67) after microwave exposure. In conclusion, the decrease of p-SYN1 (ser-553) may result in abnormalities in vesicular anchoring and GABA release, which is caused by increased Cdk5 regulated through Calpain-p25 pathway after 30 mW/cm2 microwave exposure. This study provided a potential new strategy for the prevention and treatment of microwave-induced cognitive dysfunction. Full article
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<p>Mossy fiber sprouting of hippocampus after microwave exposure. (<b>A</b>) Timm stain of hippocampus after microwave exposure (scale bar = 100 μm); (<b>B</b>) Quantitative analysis of Timm stain. (* <span class="html-italic">p</span> &lt; 0.05 vs. sham group, ** <span class="html-italic">p</span> &lt; 0.01 vs. sham group).</p>
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<p>Expression of SYN1 in rat hippocampus. (<b>A</b>) In situ expression of SYN1 in hippocampus (scale bar = 50 μm); (<b>B</b>) Expression of SYN1 in hippocampus detected by western blot; (<b>C</b>) Quantitative analysis of western blot. (** <span class="html-italic">p</span> &lt; 0.01 vs. sham group) Gels cropped from different parts of the same gel.</p>
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<p>Expression of SYN1 (ser-553) and SYN1 (ser-62/67) related proteins in hippocampus. Expression of SYN1 (ser-553) related proteins such as Cdk5, Calpain, p25 and 35 were detected by western blot; (<b>A</b>–<b>D</b>) were the quantitative analysis of Cdk5, Calpain, p25 and 35 respectively; Expression of SYN1 (ser-62/67) related proteins such as Erk and p-Erk were detected by western blot; (<b>E</b>,<b>F</b>) were the quantitative analysis of Erk and p-Erk respectively. (* <span class="html-italic">p</span> &lt; 0.05 vs. sham group, ** <span class="html-italic">p</span> &lt; 0.01 vs. sham group) Gels cropped from different parts of the same gel.</p>
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<p>Alteration of GABA, SYN1 and its phosphorylated proteins in PC12 cells after microwave exposure. (<b>A</b>) Alteration of neurotransmitters released by PC12 cells after microwave exposure; (<b>B</b>) In situ expression of p-SYN1 (ser-62/67) in PC12 cells (scale bar = 50 μm); (<b>C</b>) In situ expression of p-SYN1 (ser-553) in PC12 cells (scale bar = 50 μm); (<b>D</b>) The expression of p-SYN1 (ser-62/67) was detected by western blot; (<b>E</b>) Quantitative analysis of western blot (* <span class="html-italic">p</span> &lt; 0.05 vs. sham group, ** <span class="html-italic">p</span> &lt; 0.01 vs. sham group) Gels cropped from different parts of the same gel.</p>
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<p>P-SYN1 silencing resulted in the reduction of SYN1 and p-SYN1 (ser-62/67). (<b>A</b>,<b>B</b>) Expression of SYN1 and p-SYN1 (ser-62/67) in PC12 cells and their Quantitative analysis. (** <span class="html-italic">p</span> &lt; 0.01 vs. control group) Gels cropped from different parts of the same gel.</p>
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<p>Alteration of p-SYN1 (ser-62/67) and its related kinases after intervention of MEK. (<b>A</b>) In situ expression of p-Erk and p-SYN1(ser-62/67) after intervention of MEK; (<b>B</b>) Expression of p-Erk and p-SYN1(ser-62/67) detected by western blot; (<b>C</b>,<b>D</b>) Quantitative analysis of p-Erk and p-SYN1(ser-62/67) in western blot (* <span class="html-italic">p</span> &lt; 0.05 vs. control group, ** <span class="html-italic">p</span> &lt; 0.01 vs. control group, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. exposure only group) Gels cropped from different parts of the same gel.</p>
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<p>Alteration of p-SYN1 and its related kinases after intervention of Cdk5. (<b>A</b>) In situ expression of Cdk5 and p-SYN1 (ser-553) after intervention of Cdk5; (<b>B</b>) Expression of Cdk5 and p-SYN1 (ser-553) detected by western blot; (<b>C</b>,<b>D</b>) Quantitative analysis of Cdk5 and p-SYN1 (ser-553) in western blot (* <span class="html-italic">p</span> &lt; 0.05 vs. sham group, ** <span class="html-italic">p</span> &lt; 0.01 vs. sham group, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01 vs. exposure only group) Gels cropped from different parts of the same gel.</p>
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<p>Alteration of Neurotransmitter after Intervention of MEK and Cdk5. The neurotransmitters such as GLU, GABA, ASP and GLY released by PC12 cells after intervention of MEK and Cdk5 were detected and quantized. (** <span class="html-italic">p</span> &lt; 0.01 vs. sham group, <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05 vs. inhibition only group and △ <span class="html-italic">p</span> &lt; 0.05 vs. exposure only group).</p>
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<p>Biological Pathways Impacted by Microwaves. Microwave exposure induces higher levels of Calpain, which subsequently promotes p35 hydrolysis into p25, with increased Cdk5/p25 complex formation, leading to p-SYN1 (ser-553) reduction and abnormal accumulation of VGAT vesicles.</p>
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26 pages, 24815 KiB  
Article
Biological Implications of a Stroke Therapy Based in Neuroglobin Hyaluronate Nanoparticles. Neuroprotective Role and Molecular Bases
by María Ángeles Peinado, David Ovelleiro, María Luisa del Moral, Raquel Hernández, Esther Martínez-Lara, Eva Siles, José Rafael Pedrajas, María Luisa García-Martín, Carlos Caro, Sebastián Peralta, María Encarnación Morales, María Adolfina Ruiz and Santos Blanco
Int. J. Mol. Sci. 2022, 23(1), 247; https://doi.org/10.3390/ijms23010247 - 27 Dec 2021
Cited by 4 | Viewed by 4491
Abstract
Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood–brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after [...] Read more.
Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood–brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies. Full article
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<p>Mortality rates of sham (<span class="html-italic">n</span> = 23), MCAO (<span class="html-italic">n</span> = 44), and MCAO-Ngb (<span class="html-italic">n</span> = 27) groups after 24 h of reperfusion.</p>
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<p>Results of Bederson’s score performed in sham (<span class="html-italic">n</span> = 22), MCAO (<span class="html-italic">n</span> = 22), and MCAO-Ngb (<span class="html-italic">n</span> = 20) groups after 24 h of reperfusion, and immediately before sacrifice. Data are average values of five experimental animals in each group (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Confocal microscopy images of histological sections of the infarcted zone from sham (<b>left</b>) and MCAO (<b>middle</b> and <b>right</b>) animals taken 24 h after the systemic injection of the hyaluronate NPs. Several nervous cells, whose nuclei are stained in blue with DAPI, show empty NPs grouped as cytoplasmic red vesicles due to its labelling with rhodamine fluorescent dye.</p>
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<p>Confocal microscopy images from the infarcted zone of MCAO animals taken 24 h after the systemic injection of the Ngb-NPs. (<b>A</b>) Cy2 green fluorescence represents Ngb. (<b>B</b>) Rhodamine red fluorescence detects NPs. (<b>C</b>) DAPI blue fluorescence marks cell nuclei. (<b>D</b>) Merge image showing the colocalization of Ngb and NPs inside the nervous cells. Ngb attached to NPs appears in yellow, due to green and red merge.</p>
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<p>Confocal microscopy images of the infarcted zone from a MCAO-Ngb animal after 24 h of reperfusion. Rhodamine red fluorescence detects the Ngb-NPs, whereas Cy2 green fluorescence binds to NeuN (neurons): Ngb attached to NPs appears in yellow, due to green and red merge. Cy5 dye bound to GFAP (astrocytes) is digitally shown in grey, and DAPI dye marks cell nuclei in blue. (<b>A</b>) Merge image showing neurons (NeuN) stained in green with yellow cytoplasmatic vesicles containing Ngb-NPs. Astrocytes are shown in grey. Only few red Ngb-NPs not associated with neurons are observed. (<b>B</b>–<b>D</b>) Higher magnification of the zone delimited by the white square in image (<b>A</b>).</p>
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<p>Cerebral T2 axial and coronal neuroimages of sham, MCAO, and MCAO-Ngb animals taken after 24 h of reperfusion. The infarcted area is visible in images (<b>B</b>,<b>C</b>) in white, but not in (<b>A</b>). Scale bars: 5 mm.</p>
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<p>Mean values of the volume of the infarcts in sham, MCAO, and MCAO-Ngb groups calculated from the T2 neuroimages. Data have been calculated using ImageJ software, and are expressed in arbitrary units. Data are average values of six experimental animals in each group (** <span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Determinations of oxidative (TBARS) and nitrosative (NOA) stresses in sham, MCAO, and MCAO-Ngb animals. Data are average values of five experimental animals in each group. No significative differences are found.</p>
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<p>Upper panel: percentage of cells stained by cresyl-violet for each group: sham (<b>A</b>), MCAO (<b>B</b>), and MCAO-Ngb (<b>C</b>), measured using ImageJ. Animals from MCAO group show fewer neurons than rats from sham and MCAO-Ngb groups. Data are average values of 5 repetitions in 10 sections of 5 experimental animals in each group (* <span class="html-italic">p</span> &lt; 0.05). Lower panel: representative microphotographs of the infarct zone (parietal cortex) of animals stained with cresyl-violet.</p>
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<p>Quality control plot of the two first principal components (PCA) using the top 500 variable proteins among the three groups (sham, MCAO, and MCAO-Ngb). As shown, MCAO-Ngb animals were clustered closer to the sham control group than to the MCAO non-treated group.</p>
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<p>Volcano plots for the three comparisons using <span class="html-italic">p</span> &lt; 0.02 and Log2 fold change of 0.5 as threshold. When comparing MCAO vs. sham, proteins FBXO7, WIPI2, NTRK2, A0A0G2JY03, and ITGB8 are underexpressed, whereas MAP1a and CPQ are overexpressed. In the comparison between MCAO-Ngb and sham, proteins PPP2r2c, WIPI2, and LRRC8d are shown to be underexpressed, whereas ATXN2l, MAP1a, and TBC1d10b are overexpressed. The comparison between MCAO-Ngb and MCAO involves the over expression of ATXN2l, FBXO7, and NTRK2.</p>
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<p>Hierarchical clustering using the intensities of proteins that present a <span class="html-italic">p</span> &lt; 0.02 in at least one of the three comparisons (MCAO vs. sham, MCAO-Ngb vs. MCAO, and MCAO-Ngb vs. sham). The five main aggrupation of proteins are highlighted as cluster 1 to cluster 5 at row level. The three types of samples (sham, MCAO, and MCAO-Ngb) are highlighted at column level.</p>
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<p>KEGG pathway hsa04144: endocytosis. Arrows and symbols explanation: <a href="https://www.genome.jp/kegg/document/help_pathway.html" target="_blank">https://www.genome.jp/kegg/document/help_pathway.html</a> (accessed on 25 November 2021).</p>
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15 pages, 1306 KiB  
Brief Report
High Caloric Diet Induces Memory Impairment and Disrupts Synaptic Plasticity in Aged Rats
by Sara L. Paulo, Catarina Miranda-Lourenço, Rita F. Belo, Rui S. Rodrigues, João Fonseca-Gomes, Sara R. Tanqueiro, Vera Geraldes, Isabel Rocha, Ana M. Sebastião, Sara Xapelli and Maria J. Diógenes
Curr. Issues Mol. Biol. 2021, 43(3), 2305-2319; https://doi.org/10.3390/cimb43030162 - 18 Dec 2021
Cited by 12 | Viewed by 3563
Abstract
The increasing consumption of sugar and fat seen over the last decades and the consequent overweight and obesity, were recently linked with a deleterious effect on cognition and synaptic function. A major question, which remains to be clarified, is whether obesity in the [...] Read more.
The increasing consumption of sugar and fat seen over the last decades and the consequent overweight and obesity, were recently linked with a deleterious effect on cognition and synaptic function. A major question, which remains to be clarified, is whether obesity in the elderly is an additional risk factor for cognitive impairment. We aimed at unravelling the impact of a chronic high caloric diet (HCD) on memory performance and synaptic plasticity in aged rats. Male rats were kept on an HCD or a standard diet (control) from 1 to 24 months of age. The results showed that under an HCD, aged rats were obese and displayed significant long-term recognition memory impairment when compared to age-matched controls. Ex vivo synaptic plasticity recorded from hippocampal slices from HCD-fed aged rats revealed a reduction in the magnitude of long-term potentiation, accompanied by a decrease in the levels of the brain-derived neurotrophic factor receptors TrkB full-length (TrkB-FL). No alterations in neurogenesis were observed, as quantified by the density of immature doublecortin-positive neurons in the hippocampal dentate gyrus. This study highlights that obesity induced by a chronic HCD exacerbates age-associated cognitive decline, likely due to impaired synaptic plasticity, which might be associated with deficits in TrkB-FL signaling. Full article
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<p>A chronic high caloric diet (HCD) induced obesity, enhanced long-term memory impairment and anxious-related behavior. (<b>a</b>) Experimental timeline. Rats were kept on a high caloric diet (HCD) starting at 1 month of age, until their sacrifice at 24 months of age. Behavior assessment was performed in the open field (OF) test, followed by the novel object recognition (NOR) test at 23 months of age. Body weight was measured immediately prior to behavior testing. Post-mortem tissue samples were analyzed by electrophysiological extracellular recordings, western blot (WB) and immunohistochemistry (IHC). (<b>b</b>) Significant increase in body weight, leading to obesity, was observed in 23-month-old HCD rats. *** <span class="html-italic">p</span> &lt; 0.001, unpaired Student’s <span class="html-italic">t</span>-test. (<b>c</b>–<b>g</b>) Long-term episodic memory of 23-month-old HCD rats was impaired in the NOR test. (<b>d</b>,<b>f</b>) Performance in the training phase. (<b>e</b>,<b>g</b>) Performance in the test phase. (<b>d</b>) No significant changes in the total exploration time of the two familiar objects (F, F’). <span class="html-italic">p</span> &gt; 0.05, unpaired Student’s <span class="html-italic">t</span>-test. (<b>e</b>) Significant decrease in the total exploration time of the familiar (F) plus the novel object (N). * <span class="html-italic">p</span> &lt; 0.05, unpaired Student’s <span class="html-italic">t</span>-test. (<b>f</b>) Both CTL and HCD aged rats showed no preference for any of the familiar objects (F, F’). <span class="html-italic">p</span> &gt; 0.05, paired Student’s <span class="html-italic">t</span>-test. (<b>g</b>) CTL aged rats were able to distinguish the novel (N) from the familiar (F) object, while HCD aged rats were not. * <span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">p</span> &gt; 0.05, paired Student’s <span class="html-italic">t</span>-test. (<b>g</b>–<b>j</b>) Anxious-related behavior was enhanced, despite preserved locomotor activity of 23-month-old HCD rats in the OF test. (<b>i</b>) Locomotor activity. (<b>j</b>) Anxious-related behavior. (<b>i</b>) No significant changes in distance travelled, * <span class="html-italic">p</span> &gt; 0.05, Mann–Whitney test. (<b>j</b>) Significant decrease in the time spent in the central zone (CZ) of the apparatus. ** <span class="html-italic">p</span> &lt; 0.01, unpaired Student’s <span class="html-italic">t</span>-test. (<b>b</b>–<b>j</b>) Data are expressed as means ± SEM.</p>
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<p>A chronic high caloric diet (HCD) promoted hippocampal synaptic plasticity impairment and reduced the levels of hippocampal TrkB-FL without impacting neurogenesis. (<b>a</b>,<b>b</b>) Representative scheme of electrophysiological recordings in acute hippocampal slices. (<b>a</b>) Schematic representation of an acute hippocampal slice with the electrophysiological recording configuration used to obtain field excitatory postsynaptic potentials (fEPSPs) from the CA1 area under the stimulation of Schaffer collateral/commissural fibers in the stratum radiatum of the CA1 area. (<b>b</b>) Representation of the applied long-term potentiation (LTP) protocol. After a stable baseline (10 min), LTP was induced through a weak θ-burst protocol (3 trains of 100 Hz, 3 stimuli, separated by 200 ms). (<b>c</b>–<b>e</b>) Hippocampal synaptic plasticity of 24-month-old HCD rats was impaired, as assessed by electrophysiological extracellular recordings. (<b>c</b>) Significant decrease in LTP magnitude, quantified as the fEPSP average slope (% baseline) obtained between 50 and 60 min after LTP induction (θ-burst). * <span class="html-italic">p</span> &lt; 0.05, unpaired Student’s <span class="html-italic">t</span>-test. Data are expressed as mean % ± SEM. (<b>d</b>) The averaged time-course changes in fEPSP slope (% baseline) induced by a θ-burst stimulation. Data are expressed as mean % ± SEM. (<b>e</b>) Tracings from representative experiments. For each condition, fEPSP tracings recorded at baseline (baseline, grey line) and after θ-burst-induced LTP (LTP, black line) from the same slice are shown overlaid. (<b>f</b>–<b>h</b>) Levels of TrkB full length (TrkB-FL) were reduced in the hippocampus of 24 month old HCD rats, as assessed by western blot (WB). (<b>f</b>) Representative WBs depict immunoreactive bands for TrkB-FL (~140 kDa), TrkB-ICD (~32 kDa) and β-actin (loading control, ~42 kDa). (<b>g</b>,<b>h</b>) Protein levels were quantified and normalized (100%) for the corresponding controls (% CTL). (<b>g</b>) Significant decrease in the levels of TrkB-FL, * <span class="html-italic">p</span> &lt; 0.05, unpaired Student’s <span class="html-italic">t</span>-test, which did not result in (<b>h</b>) changes in the levels of TrkB intracellular domain fragment (TrkB-ICD), <span class="html-italic">p</span> &gt; 0.05, unpaired Student’s <span class="html-italic">t</span>-test. (<b>i</b>–<b>m</b>) The density of immature neurons was not altered in the dentate gyrus (DG) of the hippocampus of 24-month-old HCD rats, as assessed by IHC. (<b>i</b>) Representative coronal sections immunostained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and doublecortin (DCX) (green). Scale bar = 50 μm. (<b>j</b>) Representative coronal sections immunostained with DAPI (white). Scale bar = 200 μm. (<b>k</b>) No significant difference in the density of DCX+ cells. * <span class="html-italic">p</span> &gt; 0.05, Mann–Whitney test. (<b>l</b>) No significant differences in the estimated volume of the DG or (<b>m</b>) in the average diameter of DAPI+ cells. <span class="html-italic">p</span> &gt; 0.05, unpaired Student’s t-test. (<b>c</b>,<b>d</b>) Data are expressed as mean % ± SEM. (<b>g</b>,<b>h</b>,<b>k</b>–<b>m</b>) Data are expressed as mean ± SEM.</p>
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15 pages, 2490 KiB  
Article
A Link between Mitochondrial Dysregulation and Idiopathic Autism Spectrum Disorder (ASD): Alterations in Mitochondrial Respiratory Capacity and Membrane Potential
by Hazirah Hassan, Fazaine Zakaria, Suzana Makpol and Norwahidah Abdul Karim
Curr. Issues Mol. Biol. 2021, 43(3), 2238-2252; https://doi.org/10.3390/cimb43030157 - 16 Dec 2021
Cited by 8 | Viewed by 3598
Abstract
Autism spectrum disorder (ASD) is a neurological disorder triggered by various factors through complex mechanisms. Research has been done to elucidate the potential etiologic mechanisms in ASD, but no single cause has been confirmed. The involvement of oxidative stress is correlated with ASD [...] Read more.
Autism spectrum disorder (ASD) is a neurological disorder triggered by various factors through complex mechanisms. Research has been done to elucidate the potential etiologic mechanisms in ASD, but no single cause has been confirmed. The involvement of oxidative stress is correlated with ASD and possibly affects mitochondrial function. This study aimed to elucidate the link between mitochondrial dysregulation and idiopathic ASD by focusing on mitochondrial respiratory capacity and membrane potential. Our findings showed that mitochondrial function in the energy metabolism pathway was significantly dysregulated in a lymphoblastoid cell line (LCL) derived from an autistic child (ALCL). Respiratory capacities of oxidative phosphorylation (OXPHOS), electron transfer of the Complex I and Complex II linked pathways, membrane potential, and Complex IV activity of the ALCL were analyzed and compared with control cell lines derived from a developmentally normal non-autistic sibling (NALCL). All experiments were performed using high-resolution respirometry. Respiratory capacities of OXPHOS, electron transfer of the Complex I- and Complex II-linked pathways, and Complex IV activity of the ALCL were significantly higher compared to healthy controls. Mitochondrial membrane potential was also significantly higher, measured in the Complex II-linked pathway during LEAK respiration and OXPHOS. These results indicate the abnormalities in mitochondrial respiratory control linking mitochondrial function with autism. Correlating mitochondrial dysfunction and autism is important for a better understanding of ASD pathogenesis in order to produce effective interventions. Full article
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Figure 1
<p>Cytochrome <span class="html-italic">c</span> oxidase (CIV) activities of NALCL and ALCL. (<b>a</b>,<b>b</b>) are the protocols for measuring CIV activities in NALCL and ALCL, respectively. Titrations: Cell, dig (digitonin, cell permeabilization), Ama (antimycin A, CIII inhibition), CCCP (oxidative phosphorylation uncoupling), As (ascorbate, maintaining TMPD in a reduced state), TMPD (reducing cytochrome <span class="html-italic">c</span>), <span class="html-italic">c</span> (cytochrome <span class="html-italic">c</span> integrity of outer mt-membrane), and Azd (azide, CIV inhibition). Blue plots indicate O<sub>2</sub> concentration and red plots are the O<sub>2</sub> consumption expressed per cell. (<b>c</b>) The total O<sub>2</sub> consumption rate was baseline-corrected for autoxidation after inhibition of CIV. Results are expressed as means ± S.D. * denotes <span class="html-italic">p</span> &lt; 0.05 compared to NALCL as determined by <span class="html-italic">t</span>-test.</p>
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<p>Mitochondrial respiration of NALCL and ALCL. (<b>a</b>,<b>b</b>) are the protocols for measuring respiration in NALCL and ALCL, respectively. Titrations: Cell, dig (digitonin, cell permeabilization), P and M (pyruvate and malate, non-phosphorylating N-LEAK respiration, N(GM)<span class="html-italic"><sub>L</sub></span><sub>(n)</sub>), ADP (N-OXPHOS capacity, N(GM)<span class="html-italic"><sub>P</sub></span>), <span class="html-italic">c</span> (cytochrome <span class="html-italic">c</span> integrity of outer mt-membrane), G (glutamate, N-OXPHOS capacity, N(PGM)<span class="html-italic"><sub>P</sub></span>), S (succinate, NS-OXPHOS, NS<span class="html-italic"><sub>P</sub></span>), CCCP (NS-ET capacity, NS<span class="html-italic"><sub>E</sub></span>), Rot (rotenone, CI inhibition, S-ET capacity, S<span class="html-italic"><sub>E</sub></span>), and Ama (antimycin A, CIII inhibition, <span class="html-italic">Rox</span>). Oxygen consumption was corrected for <span class="html-italic">Rox</span>. Blue plots indicate O<sub>2</sub> concentration and red plots are the O<sub>2</sub> consumption expressed per cell. (<b>c</b>) The <span class="html-italic">Rox</span>-corrected respiration: ROUTINE respiration, <span class="html-italic">R</span>, was measured in non-permeabilized cells in MiR05. After plasma membrane permeabilization, five respiratory states were sequentially established to measure NADH-linked LEAK respiration with pyruvate and malate, N<span class="html-italic"><sub>L</sub></span>, OXPHOS capacity, N<span class="html-italic"><sub>P</sub></span>, NS-pathway OXPHOS capacity, NS<span class="html-italic"><sub>P</sub></span>, and NS- and S-pathway ET capacity, NS<span class="html-italic"><sub>E</sub></span> and S<span class="html-italic"><sub>E</sub></span>, where S indicates the succinate pathway. Results are expressed as means ± S.D. * denotes <span class="html-italic">p</span> &lt; 0.05 while ** denotes <span class="html-italic">p</span> &lt; 0.01 compared to NALCL as determined by <span class="html-italic">t</span>-test.</p>
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<p>Succinate-linked respiration of NALCL and ALCL measured simultaneously with mitochondrial membrane potential in the presence of safranin. (<b>a</b>,<b>b</b>) are the protocols for measuring respiration in NALCL and ALCL, respectively, while (<b>c</b>,<b>d</b>) are the protocols for measuring mitochondrial membrane potential, mtMP using safranin concentration in NALCL and ALCL, respectively. Before the cells were added, safranin was calibrated up to 2 µM. The initial drop to 1.6 µM safranin is mainly due to unspecific binding upon the addition of cells. Low safranin concentrations indicate high mtMP. Titrations: Cell, dig (digitonin, cell permeabilization), Rot (rotenone, CI inhibition), S (succinate, S-LEAK respiration, S<span class="html-italic"><sub>L</sub></span><sub>(n)</sub>), ADP (S-OXPHOS capacity, S<span class="html-italic"><sub>P</sub></span>), Omy (oligomycin, S-LEAK, S<span class="html-italic"><sub>L</sub></span><sub>(Omy)</sub>), CCCP (S-ET capacity, S<span class="html-italic"><sub>E</sub></span>), Mna (malonate, CII inhibition), and Ama (antimycin A, CIII inhibition, <span class="html-italic">Rox</span>). Oxygen consumption was corrected for <span class="html-italic">Rox</span>. Blue plots indicate O<sub>2</sub> concentration, red plots are the O<sub>2</sub> consumption expressed per cell, and neon plots indicate safranin concentrations. (<b>e</b>) The <span class="html-italic">Rox</span>-corrected respiration and (<b>f</b>) the relative safranin signal: ROUTINE state, <span class="html-italic">R</span>, and in four respiratory states in permeabilized cells: S-linked LEAK respiration in the absence of adenylates, S<span class="html-italic"><sub>L</sub></span><sub>(n)</sub>, S-linked OXPHOS capacity, S<span class="html-italic"><sub>P</sub></span>, S-linked LEAK respiration after inhibition of ATP synthase by oligomycin, S<span class="html-italic"><sub>L</sub></span><sub>(Omy)</sub>, and S-linked ET capacity, S<span class="html-italic"><sub>E</sub></span>. Results are expressed as mean ± S.D. * denotes <span class="html-italic">p</span> &lt; 0.05 while ** denotes <span class="html-italic">p</span> &lt; 0.01 compared to NALCL as determined by Student’s <span class="html-italic">t</span>-test.</p>
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<p>Succinate-linked respiration of NALCL and ALCL measured simultaneously with mitochondrial membrane potential in the presence of safranin. (<b>a</b>,<b>b</b>) are the protocols for measuring respiration in NALCL and ALCL, respectively, while (<b>c</b>,<b>d</b>) are the protocols for measuring mitochondrial membrane potential, mtMP using safranin concentration in NALCL and ALCL, respectively. Before the cells were added, safranin was calibrated up to 2 µM. The initial drop to 1.6 µM safranin is mainly due to unspecific binding upon the addition of cells. Low safranin concentrations indicate high mtMP. Titrations: Cell, dig (digitonin, cell permeabilization), Rot (rotenone, CI inhibition), S (succinate, S-LEAK respiration, S<span class="html-italic"><sub>L</sub></span><sub>(n)</sub>), ADP (S-OXPHOS capacity, S<span class="html-italic"><sub>P</sub></span>), Omy (oligomycin, S-LEAK, S<span class="html-italic"><sub>L</sub></span><sub>(Omy)</sub>), CCCP (S-ET capacity, S<span class="html-italic"><sub>E</sub></span>), Mna (malonate, CII inhibition), and Ama (antimycin A, CIII inhibition, <span class="html-italic">Rox</span>). Oxygen consumption was corrected for <span class="html-italic">Rox</span>. Blue plots indicate O<sub>2</sub> concentration, red plots are the O<sub>2</sub> consumption expressed per cell, and neon plots indicate safranin concentrations. (<b>e</b>) The <span class="html-italic">Rox</span>-corrected respiration and (<b>f</b>) the relative safranin signal: ROUTINE state, <span class="html-italic">R</span>, and in four respiratory states in permeabilized cells: S-linked LEAK respiration in the absence of adenylates, S<span class="html-italic"><sub>L</sub></span><sub>(n)</sub>, S-linked OXPHOS capacity, S<span class="html-italic"><sub>P</sub></span>, S-linked LEAK respiration after inhibition of ATP synthase by oligomycin, S<span class="html-italic"><sub>L</sub></span><sub>(Omy)</sub>, and S-linked ET capacity, S<span class="html-italic"><sub>E</sub></span>. Results are expressed as mean ± S.D. * denotes <span class="html-italic">p</span> &lt; 0.05 while ** denotes <span class="html-italic">p</span> &lt; 0.01 compared to NALCL as determined by Student’s <span class="html-italic">t</span>-test.</p>
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20 pages, 3912 KiB  
Article
The Protective Mechanism of Deuterated Linoleic Acid Involves the Activation of the Ca2+ Signaling System of Astrocytes in Ischemia In Vitro
by Egor A. Turovsky, Elena G. Varlamova, Sergey V. Gudkov and Egor Y. Plotnikov
Int. J. Mol. Sci. 2021, 22(24), 13216; https://doi.org/10.3390/ijms222413216 - 8 Dec 2021
Cited by 18 | Viewed by 2951
Abstract
Ischemia-like (oxygen-glucose deprivation, OGD) conditions followed by reoxygenation (OGD/R) cause massive death of cerebral cortex cells in culture as a result of the induction of necrosis and apoptosis. Cell death occurs as a result of an OGD-induced increase in Ca2+ ions in [...] Read more.
Ischemia-like (oxygen-glucose deprivation, OGD) conditions followed by reoxygenation (OGD/R) cause massive death of cerebral cortex cells in culture as a result of the induction of necrosis and apoptosis. Cell death occurs as a result of an OGD-induced increase in Ca2+ ions in the cytosol of neurons and astrocytes, an increase in the expression of genes encoding proapoptotic and inflammatory genes with suppression of protective genes. The deuterated form of linoleic polyunsaturated fatty acid (D4-Lnn) completely inhibits necrosis and greatly reduces apoptotic cell death with an increase in the concentration of fatty acid in the medium. It was shown for the first time that D4-Lnn, through the activation of the phosphoinositide calcium system of astrocytes, causes their reactivation, which correlates with the general cytoprotective effect on the cortical neurons and astrocytes in vitro. The mechanism of the cytoprotective action of D4-Lnn involves the inhibition of the OGD-induced calcium ions, increase in the cytosolic and reactive oxygen species (ROS) overproduction, the enhancement of the expression of protective genes, and the suppression of damaging proteins. Full article
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Figure 1
<p>Scheme of the study of the mechanisms of the cytoprotective action of D4-Lnn in the OGD and OGD/R conditions. Cells were grown up to day 9 in vitro and D4-Lnn or its non-deuterated form was added for 24 h. Then, the cells were loaded with fluorescent probes, and Ca<sup>2+</sup> dynamics in the cytosol, ROS production, and vitality tests were performed during OGD. Part of the cells after 40 min OGD was returned to the CO<sub>2</sub> incubator for 24 h, which corresponded to the conditions of reoxygenation. After OGD/R, total RNA was isolated from part of the cells and PCR analysis was performed, and part of the culture was used for immunocytochemical staining.</p>
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<p>The effect of different concentrations of D4-Lnn on induction of apoptosis and necrosis in the cortical cells after 40 min OGD and 24 h reoxygenation: (<b>A</b>) Cytogram demonstrating the viability of cortical cells after OGD/R without pre-incubation with D4-Lnn and after OGD/R with 24 h pre-incubation with different concentrations of D4-Lnn. (<b>B</b>) Comparison of the effect of 10 µg/mL D4-Lnn and Lnn on cell survival after 24 h of OGD/R. Control-cells were not exposed to OGD/R; OGD/R: cells of the cerebral cortex after 24 h OGD/R without pre-incubation with PUFAs; X-axis: the intensity of PI fluorescence; Y-axis: the intensity of Hoechst 33342 fluorescence. Cells were stained with the probes after 24 h the OGD/R. Panels A and B show cells (several hundred) in one experiment. (<b>C</b>) Effects of different D4-Lnn concentrations and 10 µM Lnn on the induction of necrosis and apoptosis after 24 h OGD/R. The percentage of healthy cells and cells with early apoptosis, apoptosis, and necrosis. Statistical significance was assessed using one-way ANOVA followed by the Tukey–Kramer test. Comparison of experimental groups relative to control: n/s—data not significant (<span class="html-italic">p</span> &gt; 0.05), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. Statistical differences indicated by red asterisks represent comparison of treatment groups relative to OGD/R. Panel C shows the average results obtained from four cell cultures. N (number of animals used for cell cultures preparation) = 4.</p>
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<p>Protective effect of 10 µg/mL D4-Lnn on mouse cortical cells: (<b>A</b>,<b>B</b>) Ca<sup>2+</sup> -signals of neurons (<b>A</b>) and astrocytes (<b>B</b>) during a 40 min OGD (red traces), 40 min OGD with D4-Lnn (in media, blue traces), after 40 min D4-Lnn (green traces), and 24 h preincubation (black traces) with D4-Lnn or after 24 h pre-incubation with Lnn (orange traces). The figure shows the averaged Ca<sup>2+</sup> signals obtained from several tens of cells for each curve. Individual Ca<sup>2+</sup> responses of neurons and astrocytes are presented in <a href="#app1-ijms-22-13216" class="html-app">Supplementary, Figure S3</a>. (<b>C</b>) Images of cortical cell culture in Propidium Iodide fluorescence detection channel before the experiment (Control) and after 40-min OGD or OGD with different time treatment with 10 µg/mL D4-Lnn. The white dots represent the nuclei of necrotic cells. For panels A and B, the averaged Ca<sup>2+</sup> signals in one experiment, obtained from several tens of astrocytes and neurons, are presented. Panel C presents typical vitality test results corresponding to experiments from panels A and C. (<b>D</b>) The percentage of PI<sup>+</sup> cortical cells that died due to OGD-induced necrosis in the control (without OGD), in the absence of D4-Lnn (OGD) and after different time incubation with 10 µg/mL D4-Lnn or Lnn (24 h preincubation). Statistical significance was assessed using one-way ANOVA followed by the Tukey–Kramer test. Comparison of experimental groups relative to OGD: n/s—data not significant (<span class="html-italic">p</span> &gt; 0.05), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001. Statistical differences based on incubation time with D4-Lnn are indicated by red asterisks. The results obtained on 4 cell cultures are presented. N (number of animals used for cell cultures preparation) = 4.</p>
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<p>ROS production in neurons (<b>A</b>) and astrocytes (<b>B</b>) under OGD conditions versus incubation time with 10 µg/mL D4-Lnn. Average kinetics of ROS production obtained from several tens of neurons and astrocytes for each curve are presented in one experiment. (<b>C</b>) The rate of ROS formation in neurons and astrocytes during OGD, depending on the time treatment with D4-Lnn. Average values obtained from three separate cell cultures are presented here. (<b>D</b>) Effect of 24 h preincubation with 10 µg/mL D4-Lnn or non-deuterated Lnn (Lnn) on baseline and OGD/R-induced expression level of genes encoding proteins regulating cell redox status. Gene expression in control cells are marked by dashed line. Statistical significance was assessed using one-way ANOVA followed by the Tukey–Kramer test. Comparison of experimental groups regarding control: n/s—data not significant (<span class="html-italic">p</span> &gt; 0.05), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. Comparison of experimental groups relative to each other is indicated in red. For panels D and E, the number of RNA samples is 5. N (number of animals used for cell cultures preparation) = 5.</p>
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<p>Effect of 24 h incubation of cells with 10 µM D4-Lnn on baseline and OGD/R induced levels of expression of genes encoding proteins regulating apoptosis, inflammatory status (<b>A</b>) and receptors (<b>B</b>). Gene expression in control cells are marked by dashed line. Statistical significance was assessed using one-way ANOVA followed by the Tukey-Kramer test. Comparison of experimental groups regarding control:n/s–data not significant (<span class="html-italic">p</span> &gt; 0.05), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. Comparison of experimental groups relative to each other is indicated in red. The number of RNA samples is 5. N (number of animals used for cell cultures preparation) = 5.</p>
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<p>Application of D4-Lnn or Lnn induces the generation of Ca<sup>2+</sup> signals in astrocytes, but not in neurons of the cerebral cortex: (<b>A</b>) Application of 30 µg/mL D4-Lnn causes the rapid generation of three types of Ca<sup>2+</sup> signals in astrocytes, and Ca<sup>2+</sup> responses occur in neurons (single, no more than 0.5% of the total population of neurons) after a lag period. Individual Ca<sup>2+</sup> responses are presented. (<b>B</b>) Short-term applications (30 s) of increasing concentrations of D4-Lnn to cell cultures of the cerebral cortex. After washing with D4-Lnn, the registration of the [Ca<sup>2+</sup>]<sub>i</sub> dynamics was suspended for 10 min. The averaged curves obtained from several tens of cells are presented. (<b>C</b>) Application of 30 µg/mL non-deuterated Lnn causes the rapid generation of biphasic Ca<sup>2+</sup> signals in astrocytes and does not cause Ca<sup>2+</sup> responses in neurons. The averaged curves obtained from several tens of cells are presented. Number of parallel coverslips with cell cultures in each analysis = 5. N (number of animals used for cell cultures preparation) = 3.</p>
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<p>Application of D4-Lnn induces the generation of Ca<sup>2+</sup> signals in astrocytes of the cerebral cortex through the phosphoinositide signaling cascade. (<b>A</b>,<b>B</b>) Addition of 30 µg/mL D4-Lnn in a calcium-free medium containing 0.5 mM EGTA (<b>A</b>) and after emptying the ER with 10 µM thapsigargin in a calcium-free medium with EGTA (<b>B</b>). (<b>C</b>,<b>D</b>) Application 30 µg/mL D4-Lnn against the background of PLC blockers (<b>C</b>, U73122, 5 µM) and IP<sub>3</sub>R (<b>D</b>, XeC, XestospongineC, 1 µM). The figure shows the Ca<sup>2+</sup> signals of individual astrocytes and their mean value (red curves). Number of parallel coverslips with cell cultures in each analysis = 4. N (number of animals used for cell cultures preparation) = 3.</p>
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<p>Pre-incubation of cortical cells with 10 µM D4-Lnn for 24 h causes an increase in GFAP protein expression. The reactivation effect persisted 24 h after OGD/R: (<b>A</b>) Immunostaining cortical cells with antibodies against GFAP in the control, after 24-h pre-incubation with 10 µg/mL D4-Lnn, 10 µg/mL Lnn, and after 40 min OGD with 24-h reoxygenation (cells 24-h pre-incubated with 10 µg/mL D4-Lnn before OGD/R). Draq5—nuclei staining. (<b>B</b>) Intensity levels of GFAP were determined by confocal imaging. We analyzed individual cells which had fluorescence of secondary antibodies. The quantitative data reflecting the level of GFAP expression are presented as fluorescence intensity values in summary bar charts (mean +/− SEM). The values were averaged by 150 cells for each column. The results obtained after immunostaining agree well with the data of fluorescent presented in panels (<b>A</b>). Each value is the mean ± SE (<span class="html-italic">n</span> ≥ 3, <span class="html-italic">p</span> &lt; 0.05). Statistical significance was assessed using one-way ANOVA followed by the Tukey–Kramer test. n/s—data not significant (<span class="html-italic">p</span> &gt; 0.05), * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. For repeats, 4 separate cell cultures were used. N (number of animals used for cell cultures preparation) = 4.</p>
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<p>The putative scheme of activation of the Ca<sup>2+</sup> signaling system of cortical astrocytes and the cytoprotective mechanism by exogenous D4-Lnn.</p>
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11 pages, 2194 KiB  
Article
Farnesol Ameliorates Demyelinating Phenotype in a Cellular and Animal Model of Charcot-Marie-Tooth Disease Type 1A
by Na-Young Park, Geon Kwak, Hyun-Myung Doo, Hye-Jin Kim, So-Young Jang, Yun-Il Lee, Byung-Ok Choi and Young-Bin Hong
Curr. Issues Mol. Biol. 2021, 43(3), 2011-2021; https://doi.org/10.3390/cimb43030138 - 13 Nov 2021
Cited by 3 | Viewed by 4283
Abstract
Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disease affecting the peripheral nervous system that is caused by either the demyelination of Schwann cells or degeneration of the peripheral axon. Currently, there are no treatment options to improve the degeneration of peripheral nerves in [...] Read more.
Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disease affecting the peripheral nervous system that is caused by either the demyelination of Schwann cells or degeneration of the peripheral axon. Currently, there are no treatment options to improve the degeneration of peripheral nerves in CMT patients. In this research, we assessed the potency of farnesol for improving the demyelinating phenotype using an animal model of CMT type 1A. In vitro treatment with farnesol facilitated myelin gene expression and ameliorated the myelination defect caused by PMP22 overexpression, the major causative gene in CMT. In vivo administration of farnesol enhanced the peripheral neuropathic phenotype, as shown by rotarod performance in a mouse model of CMT1A. Electrophysiologically, farnesol-administered CMT1A mice exhibited increased motor nerve conduction velocity and compound muscle action potential compared with control mice. The number and diameter of myelinated axons were also increased by farnesol treatment. The expression level of myelin protein zero (MPZ) was increased, while that of the demyelination marker, neural cell adhesion molecule (NCAM), was reduced by farnesol administration. These data imply that farnesol is efficacious in ameliorating the demyelinating phenotype of CMT, and further elucidation of the underlying mechanisms of farnesol’s effect on myelination might provide a potent therapeutic strategy for the demyelinating type of CMT. Full article
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Figure 1
<p>Farnesol increases cell proliferation. RT4 (1 × 10<sup>4</sup>) cells grown in 12-well plates were transfected with pCMV-myc-<span class="html-italic">PMP22</span> or pCMV-myc vector with or without 0.1 μM of farnesol. Direct cell counts after farnesol treatment for indicated times. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Changes in myelin gene expression by farnesol. After transfection of pCMV-myc-<span class="html-italic">PMP22</span> or pCMV-myc vector into RT4 cells, cells were treated with 0.01 μM and 0.1 μM of farnesol for 48 h. (<b>A</b>) <span class="html-italic">Oct6</span> mRNA levels were determined by RT-PCR. (<b>B</b>) Changes in <span class="html-italic">MPZ</span> mRNA levels were determined by RT-PCR. ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Behavioral and electrophysiological enhancements in farnesol-administered mice. (<b>A</b>) Experimental scheme of farnesol administration to C22 mice. C22 mice were fed AIN-76A diet containing 0.5% (<span class="html-italic">w</span>/<span class="html-italic">w</span>) farnesol (C22-FSN, <span class="html-italic">n</span> = 10) or AIN-76A diet (C22-Con, <span class="html-italic">n</span> = 10) from 3 weeks of age. Age-matched wild-type mice (<span class="html-italic">n</span> = 10) were used as a reference (<b>B</b>) Rotarod test was performed to evaluate locomotor function. (<b>C</b>) Representative images of electrophysiological evaluation. (<b>D</b>) motor nerve conduction velocity (MNCV). (<b>E</b>) Compound muscle action potential (CMAP). WT, wild-type mice; C22-Con, control diet fed C22 mice; C22-FNS, farnesol diet fed C22 mice; * <span class="html-italic">p</span> &lt; 0.05; and ***, <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Farnesol improves myelination in sciatic nerves of C22 mice. (<b>A</b>) Electron microscopic images of the sciatic nerve. Scale bar, 2 μm. (<b>B</b>) Distribution of myelinated axon diameters. Three hundred and twenty to three hundred and fifty axons from 3 mice in each group were counted. (<b>C</b>) g-ratio of myelinated axon. WT, wild-type mice; C22-Con, control diet fed C22 mice; C22-FNS, farnesol diet fed C22 mice.</p>
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<p>Restoration of sciatic nerve myelination by farnesol. (<b>A</b>) Immunofluorescent staining of MPZ and NCAM. Scale bar, 50 μm. (<b>B</b>) Fluorescent intensity of MPZ. (<b>C</b>) Fluorescent intensity of NCAM. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001.</p>
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