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Regulation and Function of Adult Neurogenesis
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Regulation and Function of Adult Neurogenesis

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Neurobiology".

Deadline for manuscript submissions: closed (31 December 2023) | Viewed by 10338

Special Issue Editor

Dr. Gadi Turgeman

E-Mail Website
Guest Editor
Department of Molecular Biology, Ariel University, Ariel 40700, Israel
Interests: mesenchymal stem cells; neurogenesis; neurobehavioral disorders; neuroinflammation; brain injury

Special Issue Information

Dear Colleagues,

Behavioral neuroscience has evolved greatly over the past two decades. The growing understanding of the molecular and cellular mechanisms involved in regulating affective and cognitive behavior has opened multiple avenues for future study and intervention. Among the studied mechanisms, adult hippocampal neurogenesis has gained increased attention as it was found to be involved in cognitive behavior, spatial learning, and memory. Furthermore, impaired neurogenesis is involved in neurobehavioral disorders such as Alzheimer’s disease, schizophrenia, depression, and post-traumatic stress disorder, indicating a role of hippocampal neurogenesis in affective behavior as well.  In this Special Issue, we invite papers exploring the mechanisms of neurogenesis, affective and cognitive behavior. Additionally, we welcome functional studies exploring the potential effect of MSC or their products on various behavioral aspects or neurobehavioral disorders. Further studies will increase our understanding of the molecular mechanisms involved in affective and cognitive behavior and may suggest novel therapeutic strategies for adult neurogenesis.

Dr. Gadi Turgeman
Guest Editor

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Published Papers (4 papers)

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25 pages, 4116 KiB  
Article
Parental Preconception and Pre-Hatch Exposure to a Developmental Insult Alters Offspring’s Gene Expression and Epigenetic Regulations: An Avian Model
by Issam Rimawi, Gadi Turgeman, Nataly Avital-Cohen, Israel Rozenboim and Joseph Yanai
Int. J. Mol. Sci. 2023, 24(5), 5047; https://doi.org/10.3390/ijms24055047 - 6 Mar 2023
Cited by 2 | Viewed by 2409
Abstract
Parental exposure to insults was initially considered safe if stopped before conception. In the present investigation, paternal or maternal preconception exposure to the neuroteratogen chlorpyrifos was investigated in a well-controlled avian model (Fayoumi) and compared to pre-hatch exposure focusing on molecular [...] Read more.
Parental exposure to insults was initially considered safe if stopped before conception. In the present investigation, paternal or maternal preconception exposure to the neuroteratogen chlorpyrifos was investigated in a well-controlled avian model (Fayoumi) and compared to pre-hatch exposure focusing on molecular alterations. The investigation included the analysis of several neurogenesis, neurotransmission, epigenetic and microRNA genes. A significant decrease in the vesicular acetylcholine transporter (SLC18A3) expression was detected in the female offspring in the three investigated models: paternal (57.7%, p < 0.05), maternal (36%, p < 0.05) and pre-hatch (35.6%, p < 0.05). Paternal exposure to chlorpyrifos also led to a significant increase in brain-derived neurotrophic factor (BDNF) gene expression mainly in the female offspring (27.6%, p < 0.005), while its targeting microRNA, miR-10a, was similarly decreased in both female (50.5%, p < 0.05) and male (56%, p < 0.05) offspring. Doublecortin’s (DCX) targeting microRNA, miR-29a, was decreased in the offspring after maternal preconception exposure to chlorpyrifos (39.8%, p < 0.05). Finally, pre-hatch exposure to chlorpyrifos led to a significant increase in protein kinase C beta (PKCß; 44.1%, p < 0.05), methyl-CpG-binding domain protein 2 (MBD2; 44%, p < 0.01) and 3 (MBD3; 33%, p < 0.05) genes expression in the offspring. Although extensive studies are required to establish a mechanism–phenotype relationship, it should be noted that the current investigation does not include phenotype assessment in the offspring. Full article
(This article belongs to the Special Issue Regulation and Function of Adult Neurogenesis)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Effects of paternal exposure to chlorpyrifos (CPF) on the offspring’s gene expression. Relative gene expression results obtained in the offspring after paternal exposure to chlorpyrifos. M: male offspring, F: female offspring. Number of samples (n) is presented inside each column. Results are presented as the mean ± SEM. #: PKCß, which is related to both neurogenesis and neurotransmission genes, is presented in the neurotransmission section. *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.005 and ***: <span class="html-italic">p</span> &lt; 0.0005. PKCß: protein kinase C beta, BDNF: brain-derived neurotrophic factor, FOS: C-Fos, DCX: doublecortin, CHRM2 and CHRM3: muscarinic receptors 2 and 3, SLC18A3: solute carrier family 18 member A3, SLC6A4: solute carrier family 6 member 4, MeCP2: methyl CpG binding protein 2, MBD2 and MBD3: methyl-CpG-binding domain proteins 2 and 3, SETDB1 and SETDB2: SET domain bifurcated histone lysine methyltransferase 1 and 2, CREB: cAMP-response element binding protein, REST: RE1 silencing transcription factor, miR-221: microRNA 221, miR-29a: microRNA 29a, miR-6612: microRNA 6612 and miR-10a: microRNA 10a.</p> Full article ">Figure 1 Cont.
<p>Effects of paternal exposure to chlorpyrifos (CPF) on the offspring’s gene expression. Relative gene expression results obtained in the offspring after paternal exposure to chlorpyrifos. M: male offspring, F: female offspring. Number of samples (n) is presented inside each column. Results are presented as the mean ± SEM. #: PKCß, which is related to both neurogenesis and neurotransmission genes, is presented in the neurotransmission section. *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.005 and ***: <span class="html-italic">p</span> &lt; 0.0005. PKCß: protein kinase C beta, BDNF: brain-derived neurotrophic factor, FOS: C-Fos, DCX: doublecortin, CHRM2 and CHRM3: muscarinic receptors 2 and 3, SLC18A3: solute carrier family 18 member A3, SLC6A4: solute carrier family 6 member 4, MeCP2: methyl CpG binding protein 2, MBD2 and MBD3: methyl-CpG-binding domain proteins 2 and 3, SETDB1 and SETDB2: SET domain bifurcated histone lysine methyltransferase 1 and 2, CREB: cAMP-response element binding protein, REST: RE1 silencing transcription factor, miR-221: microRNA 221, miR-29a: microRNA 29a, miR-6612: microRNA 6612 and miR-10a: microRNA 10a.</p> Full article ">Figure 2
<p>Effects of maternal preconception exposure to chlorpyrifos (CPF) on the offspring’s gene expression. Relative gene expression results obtained in the offspring after pre-hatch exposure to chlorpyrifos. M: male offspring, F: female offspring. Number of samples (n) is presented inside each column. Results are presented as the mean ± SEM. #: PKCß, which is related to both neurogenesis and neurotransmission genes, is presented in the neurotransmission section. *: <span class="html-italic">p</span> &lt; 0.05. For genes abbreviations, please refer to <a href="#ijms-24-05047-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 2 Cont.
<p>Effects of maternal preconception exposure to chlorpyrifos (CPF) on the offspring’s gene expression. Relative gene expression results obtained in the offspring after pre-hatch exposure to chlorpyrifos. M: male offspring, F: female offspring. Number of samples (n) is presented inside each column. Results are presented as the mean ± SEM. #: PKCß, which is related to both neurogenesis and neurotransmission genes, is presented in the neurotransmission section. *: <span class="html-italic">p</span> &lt; 0.05. For genes abbreviations, please refer to <a href="#ijms-24-05047-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 3
<p>Effects of pre-hatch exposure to chlorpyrifos (CPF) on the offspring’s gene expression. Relative gene expression results obtained in the offspring after pre-hatch exposure to chlorpyrifos. M: male offspring, F: female offspring. Number of samples (n) is presented inside each column. Results are presented as the mean ± SEM. #: PKCß, which is related to both neurogenesis and neutransmission genes, is presented in the neurotransmission section. *: <span class="html-italic">p</span> &lt; 0.05. For genes abbreviations, please refer to <a href="#ijms-24-05047-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 3 Cont.
<p>Effects of pre-hatch exposure to chlorpyrifos (CPF) on the offspring’s gene expression. Relative gene expression results obtained in the offspring after pre-hatch exposure to chlorpyrifos. M: male offspring, F: female offspring. Number of samples (n) is presented inside each column. Results are presented as the mean ± SEM. #: PKCß, which is related to both neurogenesis and neutransmission genes, is presented in the neurotransmission section. *: <span class="html-italic">p</span> &lt; 0.05. For genes abbreviations, please refer to <a href="#ijms-24-05047-f001" class="html-fig">Figure 1</a>.</p> Full article ">Figure 4
<p>Venn diagram representing shared and non-shared gene expression correlation pairs in the control and chlorpyrifos exposed (paternally, maternally and pre-hatch) offspring.</p> Full article ">Figure 5
<p>Gene co-expression correlation matrix and network in the offspring of the control (<b>a</b>), paternally (<b>b</b>), maternally (<b>c</b>), and pre-hatch (<b>d</b>) chlorpyrifos-exposed chickens. In the left panel, a correlation matrix between the different genes. Non-statistically significant correlations are marked in x. In the right panel, correlation networks between the genes represented as nodes and their correlation as edges. Node size represents the number of connecting edges of the network; only statistically significant correlations were considered. Detected modules (nodes communities) are stained with different colors. Module 1—red, Module 2—yellow, Module 3—light blue, Module 4—green and Module 5—purple. In both panels, edges and correlation color intensity signifies an increased correlation, with green for positive correlations and red for negative correlations.</p> Full article ">Figure 5 Cont.
<p>Gene co-expression correlation matrix and network in the offspring of the control (<b>a</b>), paternally (<b>b</b>), maternally (<b>c</b>), and pre-hatch (<b>d</b>) chlorpyrifos-exposed chickens. In the left panel, a correlation matrix between the different genes. Non-statistically significant correlations are marked in x. In the right panel, correlation networks between the genes represented as nodes and their correlation as edges. Node size represents the number of connecting edges of the network; only statistically significant correlations were considered. Detected modules (nodes communities) are stained with different colors. Module 1—red, Module 2—yellow, Module 3—light blue, Module 4—green and Module 5—purple. In both panels, edges and correlation color intensity signifies an increased correlation, with green for positive correlations and red for negative correlations.</p> Full article ">Figure 6
<p>Paternal exposure to chlorpyrifos timeline. Timeline displaying chlorpyrifos exposure doses and periods of male chickens in the paternal exposure group. CPF: chlorpyrifos.</p> Full article ">
15 pages, 2803 KiB  
Article
Polarized Anti-Inflammatory Mesenchymal Stem Cells Increase Hippocampal Neurogenesis and Improve Cognitive Function in Aged Mice
by Matanel Tfilin, Nikolai Gobshtis, David Fozailoff, Vadim E. Fraifeld and Gadi Turgeman
Int. J. Mol. Sci. 2023, 24(5), 4490; https://doi.org/10.3390/ijms24054490 - 24 Feb 2023
Cited by 5 | Viewed by 2559
Abstract
Age-related decline in cognitive functions is associated with reduced hippocampal neurogenesis caused by changes in the systemic inflammatory milieu. Mesenchymal stem cells (MSC) are known for their immunomodulatory properties. Accordingly, MSC are a leading candidate for cell therapy and can be applied to [...] Read more.
Age-related decline in cognitive functions is associated with reduced hippocampal neurogenesis caused by changes in the systemic inflammatory milieu. Mesenchymal stem cells (MSC) are known for their immunomodulatory properties. Accordingly, MSC are a leading candidate for cell therapy and can be applied to alleviate inflammatory diseases as well as aging frailty via systemic delivery. Akin to immune cells, MSC can also polarize into pro-inflammatory MSC (MSC1) and anti-inflammatory MSC (MSC2) following activation of Toll-like receptor 4 (TLR4) and TLR3, respectively. In the present study, we apply pituitary adenylate cyclase-activating peptide (PACAP) to polarize bone-marrow-derived MSC towards an MSC2 phenotype. Indeed, we found that polarized anti-inflammatory MSC were able to reduce the plasma levels of aging related chemokines in aged mice (18-months old) and increased hippocampal neurogenesis following systemic administration. Similarly, aged mice treated with polarized MSC displayed improved cognitive function in the Morris water maze and Y-maze assays compared with vehicle- and naïve-MSC-treated mice. Changes in neurogenesis and Y-maze performance were negatively and significantly correlated with sICAM, CCL2 and CCL12 serum levels. We conclude that polarized PACAP-treated MSC present anti-inflammatory properties that can mitigate age-related changes in the systemic inflammatory milieu and, as a result, ameliorate age related cognitive decline. Full article
(This article belongs to the Special Issue Regulation and Function of Adult Neurogenesis)
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Figure 1

Figure 1
<p>Polarized PACAP-treated MSC maintain mesenchymal phenotype. (<b>A</b>). Immunophenotyping of PACAP-treated MSC in flow cytometry presents positive expression of the mesenchymal markers CD106 (&gt;50%), CD29 (&gt;80%), CD44 (&gt;70%), CD73 (&gt;60%) and sca-1 (&gt;40%) but negative expression of the hematopoietic markers CD45 (&lt;8%), and CD11b (&lt;5%). Graphs represent flow cytometry histograms for the expression of the different markers. The negative control histogram is presented with the blue filled histogram. (<b>B</b>). Both naïve (N = 4) and PACAP-treated MSC (N = 3) expressed detectable levels of VPAC2 receptor mRNA, as detected in real-time PCR. Since the activation of Toll-like receptor 3 (TLR3) is an established marker of the MSC anti-inflammatory phenotype (MSC2) [<a href="#B15-ijms-24-04490" class="html-bibr">15</a>], we administered pituitary adenylate cyclase-activating peptide (PACAP), a neuropeptide with anti-inflammatory properties that is known to upregulate TLR3 and vice versa with TLR4, at 20 nM for 4 days to establish the anti-inflammatory MCS phenotype (MSC2). PACAP treatment of MSC in vitro (pMSC, N = 5) did not increase significantly the expression of TLR3 (<b>C</b>) or TLR4 (<b>D</b>) but increased the TLR3/TLR4 gene expression ratio (<b>E</b>) compared with naïve MSC (N = 4), as detected in real-time PCR. All graphs present mean ± SE. * <span class="html-italic">p</span> &lt; 0.05, Student <span class="html-italic">t</span>-test. ns = non-significant.</p> Full article ">Figure 1 Cont.
<p>Polarized PACAP-treated MSC maintain mesenchymal phenotype. (<b>A</b>). Immunophenotyping of PACAP-treated MSC in flow cytometry presents positive expression of the mesenchymal markers CD106 (&gt;50%), CD29 (&gt;80%), CD44 (&gt;70%), CD73 (&gt;60%) and sca-1 (&gt;40%) but negative expression of the hematopoietic markers CD45 (&lt;8%), and CD11b (&lt;5%). Graphs represent flow cytometry histograms for the expression of the different markers. The negative control histogram is presented with the blue filled histogram. (<b>B</b>). Both naïve (N = 4) and PACAP-treated MSC (N = 3) expressed detectable levels of VPAC2 receptor mRNA, as detected in real-time PCR. Since the activation of Toll-like receptor 3 (TLR3) is an established marker of the MSC anti-inflammatory phenotype (MSC2) [<a href="#B15-ijms-24-04490" class="html-bibr">15</a>], we administered pituitary adenylate cyclase-activating peptide (PACAP), a neuropeptide with anti-inflammatory properties that is known to upregulate TLR3 and vice versa with TLR4, at 20 nM for 4 days to establish the anti-inflammatory MCS phenotype (MSC2). PACAP treatment of MSC in vitro (pMSC, N = 5) did not increase significantly the expression of TLR3 (<b>C</b>) or TLR4 (<b>D</b>) but increased the TLR3/TLR4 gene expression ratio (<b>E</b>) compared with naïve MSC (N = 4), as detected in real-time PCR. All graphs present mean ± SE. * <span class="html-italic">p</span> &lt; 0.05, Student <span class="html-italic">t</span>-test. ns = non-significant.</p> Full article ">Figure 2
<p>PACAP polarizes MSC towards an anti-inflammatory (MSC2) phenotype. Conditioned medium from naïve and PACAP-treated MSC was analyzed for chemokines and cytokines secretion, utilizing Proteome Profiler Mouse Cytokine Array (R&amp;D Systems). The following chemokine and anti-inflammatory cytokines IL-2, IL-3, IL-4, IL-27, IP10, IL-1ra, RANTES, SDF-1, CCL2(JE), CCL1 (i-309), G-CSF, BLC were upregulated, while pro-inflammatory cytokines were downregulated (IL-6, IL-1a, IFN-ɣ and soluble ICAM-1). Analysis was performed on pooled samples (n = 3) for each culture.</p> Full article ">Figure 3
<p>Systemic administration of polarized MSC to aged mice normalizes systemic chemokine levels. Intravenous injection of polarized anti-inflammatory MSC (pMSC, N = 3–4) reduces the levels of plasma chemokines that are associated with aging and inflammation [<a href="#B4-ijms-24-04490" class="html-bibr">4</a>] to levels of young (3 months old) mice (N = 7–8) compared with vehicle-treated aged control mice (N = 3), as detected by Proteome Profiler Mouse Cytokine Array (R&amp;D Systems). Bars in the graph present mean ± SE. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. One-way ANOVA. ns = non-significant.</p> Full article ">Figure 4
<p>Systemic administration of polarized MSC increases neurogenesis in aged mice. To assess the therapeutic potential of anti-inflammatory MSC, we administered 2 × 10<sup>5</sup> naïve (MSC) and polarized PACAP-treated MSC (pMSC) intravenously to 18-months aged mice. Immunohistochemistry for newly formed neurons expressing doublecortin in the granular cell layer was significantly increased in pMSC-treated animals (N = 5) compared with naïve-MSC- (N = 5) and vehicle-treated aged mice (N = 5) (<b>A</b>). Immunohistochemistry for proliferating neuro-progenitors (Ki67<sup>+</sup>) in the sub-granular zone of the dentate gyrus demonstrated increased number of cells in the hippocampus of mice treated with either naïve (N = 4) or polarized MSC (N = 6) compared with vehicle-treated aged mice (N = 4) (<b>B</b>). (<b>C</b>). Representing micrographs of DCX<sup>+</sup> and Ki67<sup>+</sup> cells in the dentate gyrus of the different treatment groups. Scale bar 20 μm. Arrows indicate positive cells and nuclei.. Linear regression graphs depicting the correlation between DCX<sup>+</sup> and Ki67<sup>+</sup> cell numbers and sICAM levels (<b>D</b>,<b>E</b>), respectively. Bars in graphs represent mean ± SE. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, one-way ANOVA. Correlation was calculated using Pearson test.</p> Full article ">Figure 5
<p>Systemic administration of polarized MSC improves cognitive function in aged mice. To assess the therapeutic potential of anti-inflammatory MSC, we administered 2 * 10<sup>5</sup> naïve (MSC) and polarized PACAP-treated MSC (pMSC) intravenously to 18-months aged mice. No significant differences were observed between the different groups in general locomotion activity, as assessed in the open field test (<b>A</b>). Morris water maze assay demonstrated significant improvement in locating the hidden platform in pMSC-treated animals at days 4 and 5 compared with day 1 (<b>B</b>). The probe trial assay following the 5-day Morris water maze demonstrated significant increased localization in the platform zone in pMSC-treated animals compared with naïve-MSC-treated animals (<b>C</b>). Administration of pMSC also resulted in improved memory performance, as reflected by the increased alteration ratio in the Y-maze assay (<b>D</b>). Graphs present data as mean ± SE. N = 5 for all groups. * <span class="html-italic">p</span> &lt; 0.05, one-way ANOVA. Correlation was calculated using Pearson test.</p> Full article ">Figure 6
<p>Cognitive behavior correlates with chemokine serum levels in aged mice. Linear regression graphs depicting the correlation between CCL2 and CCL12 serum levels and behavioral performance in the Y-maze test (<b>A</b>,<b>B</b>), respectively (N = 7). Correlations were calculated using Pearson test.</p> Full article ">Figure 7
<p>MSC engraft to various organs following intravenous injection in mice. DiR labeled naïve and polarized MSC (2 × 10<sup>5</sup>) were injected into the tail vein of 3-month-old mice. Labeled cells were detected using the Maestro in vivo imaging system at days 0, 1, 4, 7, and 14 following the injection. Imaging was performed and presented for the following dissected organs: (<b>A</b>) lungs (scale bar = 1 cm), (<b>B</b>) liver (scale bar = 3 cm), and (<b>C</b>) brain (scale bar = 1.5 cm). In each organ, the negative control (vehicle injected animal) is presented in the left rectangle, naïve MSC engrafted organ in the upper right and polarized MSC (pMSC) in the lower right rectangle. In each rectangle, the upper photographs represent fluorescent detection of DiR signal in the organ and the corresponding lower photograph, a visual light image of the same organ.</p> Full article ">Figure 7 Cont.
<p>MSC engraft to various organs following intravenous injection in mice. DiR labeled naïve and polarized MSC (2 × 10<sup>5</sup>) were injected into the tail vein of 3-month-old mice. Labeled cells were detected using the Maestro in vivo imaging system at days 0, 1, 4, 7, and 14 following the injection. Imaging was performed and presented for the following dissected organs: (<b>A</b>) lungs (scale bar = 1 cm), (<b>B</b>) liver (scale bar = 3 cm), and (<b>C</b>) brain (scale bar = 1.5 cm). In each organ, the negative control (vehicle injected animal) is presented in the left rectangle, naïve MSC engrafted organ in the upper right and polarized MSC (pMSC) in the lower right rectangle. In each rectangle, the upper photographs represent fluorescent detection of DiR signal in the organ and the corresponding lower photograph, a visual light image of the same organ.</p> Full article ">
19 pages, 4939 KiB  
Article
Effects of Positive Fighting Experience and Its Subsequent Deprivation on the Expression Profile of Mouse Hippocampal Genes Associated with Neurogenesis
by Olga E. Redina, Vladimir N. Babenko, Dmitry A. Smagin, Irina L. Kovalenko, Anna G. Galyamina, Vadim M. Efimov and Natalia N. Kudryavtseva
Int. J. Mol. Sci. 2023, 24(3), 3040; https://doi.org/10.3390/ijms24033040 - 3 Feb 2023
Cited by 2 | Viewed by 2230
Abstract
The hippocampus is known as the brain region implicated in visuospatial processes and processes associated with learning and short- and long-term memory. An important functional characteristic of the hippocampus is lifelong neurogenesis. A decrease or increase in adult hippocampal neurogenesis is associated with [...] Read more.
The hippocampus is known as the brain region implicated in visuospatial processes and processes associated with learning and short- and long-term memory. An important functional characteristic of the hippocampus is lifelong neurogenesis. A decrease or increase in adult hippocampal neurogenesis is associated with a wide range of neurological diseases. We have previously shown that in adult male mice with a chronic positive fighting experience in daily agonistic interactions, there is an increase in the proliferation of progenitor neurons and the production of young neurons in the dentate gyrus (in hippocampus), and these neurogenesis parameters remain modified during 2 weeks of deprivation of further fights. The aim of the present work was to identify hippocampal genes associated with neurogenesis and involved in the formation of behavioral features in mice with the chronic experience of wins in aggressive confrontations, as well as during the subsequent 2-week deprivation of agonistic interactions. Hippocampal gene expression profiles were compared among three groups of adult male mice: chronically winning for 20 days in the agonistic interactions, chronically victorious for 20 days followed by the 2-week deprivation of fights, and intact (control) mice. Neurogenesis-associated genes were identified whose transcription levels changed during the social confrontations and in the subsequent period of deprivation of fights. In the experimental males, some of these genes are associated with behavioral traits, including abnormal aggression-related behavior, an abnormal anxiety-related response, and others. Two genes encoding transcription factors (Nr1d1 and Fmr1) were likely to contribute the most to the between-group differences. It can be concluded that the chronic experience of wins in agonistic interactions alters hippocampal levels of transcription of multiple genes in adult male mice. The transcriptome changes get reversed only partially after the 2-week period of deprivation of fights. The identified differentially expressed genes associated with neurogenesis and involved in the control of a behavior/neurological phenotype can be used in further studies to identify targets for therapeutic correction of the neurological disturbances that develop in winners under the conditions of chronic social confrontations. Full article
(This article belongs to the Special Issue Regulation and Function of Adult Neurogenesis)
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Figure 1

Figure 1
<p>Differences in the transcription profile of the hippocampus between winners and control animals (principal coordinate analysis using Euclidean distances). C: Control mice without the experience of agonistic interactions; A20: males with consecutive 20 days of wins in the daily agonistic interactions; AD: A20 mice after subsequent 14 days of fighting deprivation.</p> Full article ">Figure 2
<p>KEGG pathways for the 72 genes associated with neurogenesis and found to be differentially expressed in our comparison of the hippocampal transcription profile between control male mice and males with consecutive 20 days of wins in daily agonistic interactions.</p> Full article ">Figure 3
<p>(<b>a</b>) Axes maximizing distances between control and A20 mice (males with consecutive 20 days of wins in daily agonistic interactions); (<b>b</b>) distribution of expressed genes along the axis representing the correlation between gene expression and PLS-DA Axis 1. DEGs: differentially expressed genes.</p> Full article ">Figure 4
<p>KEGG pathways related to the 31 genes associated with neurogenesis and found to be differentially expressed in our comparison of the hippocampal transcription profile between control male mice and males with consecutive 20 days of wins in daily agonistic interactions and then deprived of fighting for 14 days.</p> Full article ">Figure 5
<p>Functional enrichment analysis of the neurogenesis network constructed for the C_AD comparison. This analysis suggests that the DEGs in question may contribute to the signaling and response to stress. The functional enrichment network was constructed by means of the STRING database (<a href="https://string-db.org/" target="_blank">https://string-db.org/</a>; accessed on 7 November 2022) using the DEGs associated with neurogenesis. Each node represents all the proteins produced by a single protein-coding gene. Edges are protein–protein associations. Purple lines indicate experimentally determined interactions; blue lines denote known interactions from curated databases; dark blue lines represent gene co-occurrence; black lines indicate coexpression; and green lines represent results of text mining. Protein–protein interaction (PPI) enrichment <span class="html-italic">p</span>-value &lt; 1.0 × 10<sup>−16</sup>. FDR: false discovery rate.</p> Full article ">Figure 6
<p>Genes that significantly changed their levels of transcription during the period of agonistic interactions (C_A20 DEGs), whose expression did not get restored during the fighting deprivation (C_AD differences are statistically significant). FPKM, fragments per kilobase of transcript per million mapped reads; value C: expression in control mice (no experience of agonistic interactions); value_A20: expression in males with consecutive 20 days of wins in daily agonistic interactions; value_AD: expression in males with consecutive 20 days of wins in daily agonistic interactions deprived of fighting for 14 days.</p> Full article ">Figure 7
<p>Neurogenesis-associated C_AD DEGs whose expression changed during the fighting deprivation. * DEGs in the A20_AD comparison; <sup>#</sup> DEGs in the C_A20 comparison. FPKM, fragments per kilobase of transcript per million mapped reads; value C: expression in control mice (no experience of agonistic interactions); value_A20: expression in males with consecutive 20 days of wins in daily agonistic interactions; value_AD: expression in males with consecutive 20 days of wins in daily agonistic interactions and then deprived of fighting for 14 days.</p> Full article ">Figure 8
<p>(<b>a</b>) Axes maximizing the distances between control and AD mice (males with consecutive 20 days of wins in daily agonistic interactions and then deprived of fighting for 14 days). (<b>b</b>) Distribution of expressed genes along the axis representing the correlation between gene expression and PLS-DA Axis 1. DEGs: differentially expressed genes.</p> Full article ">Figure 9
<p>Graphical representation of the experiment. A20: males with consecutive 20 days of wins in daily agonistic interactions; AD: the A20 mice after subsequent 14 days of fighting deprivation; Control: mice without the experience of agonistic interactions. Control males were housed one per cage for 5 days, which enabled them to feel dominant and potentially able to demonstrate aggressive behavior in a conflict situation.</p> Full article ">
18 pages, 7492 KiB  
Article
FOXG1 Contributes Adult Hippocampal Neurogenesis in Mice
by Jia Wang, Hong-Ru Zhai, Si-Fei Ma, Hou-Zhen Shi, Wei-Jun Zhang, Qi Yun, Wen-Jun Liu, Zi-Zhong Liu and Wei-Ning Zhang
Int. J. Mol. Sci. 2022, 23(23), 14979; https://doi.org/10.3390/ijms232314979 - 29 Nov 2022
Cited by 8 | Viewed by 2338
Abstract
Strategies to enhance hippocampal precursor cells efficiently differentiate into neurons could be crucial for structural repair after neurodegenerative damage. FOXG1 has been shown to play an important role in pattern formation, cell proliferation, and cell specification during embryonic and early postnatal neurogenesis. Thus [...] Read more.
Strategies to enhance hippocampal precursor cells efficiently differentiate into neurons could be crucial for structural repair after neurodegenerative damage. FOXG1 has been shown to play an important role in pattern formation, cell proliferation, and cell specification during embryonic and early postnatal neurogenesis. Thus far, the role of FOXG1 in adult hippocampal neurogenesis is largely unknown. Utilizing CAG-loxp-stop-loxp-Foxg1-IRES-EGFP (Foxg1fl/fl), a specific mouse line combined with CreAAV infusion, we successfully forced FOXG1 overexpressed in the hippocampal dentate gyrus (DG) of the genotype mice. Thereafter, we explored the function of FOXG1 on neuronal lineage progression and hippocampal neurogenesis in adult mice. By inhibiting p21cip1 expression, FOXG1-regulated activities enable the expansion of the precursor cell population. Besides, FOXG1 induced quiescent radial-glia like type I neural progenitor, giving rise to intermediate progenitor cells, neuroblasts in the hippocampal DG. Through increasing the length of G1 phase, FOXG1 promoted lineage-committed cells to exit the cell cycle and differentiate into mature neurons. The present results suggest that FOXG1 likely promotes neuronal lineage progression and thereby contributes to adult hippocampal neurogenesis. Elevating FOXG1 levels either pharmacologically or through other means could present a therapeutic strategy for disease related with neuronal loss. Full article
(This article belongs to the Special Issue Regulation and Function of Adult Neurogenesis)
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Figure 1

Figure 1
<p>rAAV2/9 forces FOXG1 overexpressed in adult hippocampal DG of the <span class="html-italic">Foxg1</span> genotype mice. (<b>A</b>) Genotypes of the <span class="html-italic">Foxg1</span><sup>fl/fl</sup> offspring were determined by PCR analysis using the primers for <span class="html-italic">Foxg1</span>. Lanes of 2–3, 6, 9–14, 17–19 represent <span class="html-italic">Foxg1</span><sup>fl/fl</sup> genotype. The 378-bp band results from amplification of the <span class="html-italic">Foxg1</span> allele. (<b>B</b>) Location of infusion sites is within the hippocampus area of genotype mice. Photomicrograph represents cresyl violet stained coronal sections from the brain of a mouse with representative placement in the dentate gyrus (DG) region of the hippocampus. (<b>C</b>) Expression patterns of FOXG1 were assessed with an antibody against EGFP in the hippocampal DG of mice. Green = EGFP-labeled FOXG1; Blue = DAPI. Scale bars = 100 μm. (<b>D</b>) Hippocampal lysates of two genotype mice were immunoblotted using an antibody against GFP. Tubulin was used as loading control. (<b>E</b>) Values are expressed as means ± S. E. M. For each group, <span class="html-italic">n =</span> 6/group. *** <span class="html-italic">p</span> &lt; 0.001 for noted differences between <span class="html-italic">Foxg1</span><sup>fl/fl</sup> and <span class="html-italic">Foxg1</span><sup>fl/fl</sup>-CreAAV groups.</p> Full article ">Figure 2
<p>FOXG1 increases the amount of neuronal stem cells (aNSCs) in adult hippocampal DG of the <span class="html-italic">Foxg1</span> genotype mice and promotes qNSCs giving rise to activated aNSCs. (<b>A</b>) Expression patterns of GFAP-expressing qNSCs in the hippocampal DG of <span class="html-italic">Foxg1</span> genotype mice were assessed with immunofluorescence. Red = GFAP; Green = EGFP-labeled FOXG1; Blue = DAPI. Scale bars = 100 μm. Arrow heads: newly generated qNSCs with FOXG1 activation. (<b>D</b>) Levels of total qNSCs in adult hippocampus are showed as %GFAP<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>E</b>) Levels of endogenous qNSCs in adult hippocampus are showed as %GFP<sup>−</sup>GFAP<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>B</b>) Expression patterns and (<b>F</b>) qualification of total qNSCs in adult hippocampus were studied with immunohischemisitry using an antibody against GFAP. Scale bars = 100 μm. (<b>C</b>) Hippocampal lysates of the two genotype mice were immunoblotted using an antibody against GFAP. (<b>G</b>) Qualification of GFAP expression is illustrated by bar graph. GAPDH was used as the loading control. (<b>H</b>) Expression patterns of proliferated qNSCs in the hippocampal DG of <span class="html-italic">Foxg1</span> genotype mice were assessed with immunofluorescence. Red = GFAP; Green = Proliferating Cell Nuclear Antigen (PCNA); Blue = DAPI. Scale bars = 100 μm. Arrow heads: proliferated qNSCs. (<b>J</b>) Levels of qNSCs in adult hippocampus are showed as %GFAP<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>K</b>) Levels of proliferated qNSCs in adult hippocampus are showed as %GFAP<sup>+</sup>PCNA<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>I</b>) Expression patterns of activated aNSCs in the hippocampal DG of <span class="html-italic">Foxg1</span> genotype mice were assessed with immunofluorescence. Red = brain lipid binding protein (BLBP); Green = GFAP; Blue = DAPI. Scale bars = 100 μm. Arrow heads: activated aNSCs. (<b>L</b>) Levels of aNSCs in adult hippocampus are showed as %GFAP<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>M</b>) Levels of activated aNSCs in adult hippocampus are showed as %GFAP<sup>+</sup>BLBP<sup>+</sup>/DAPI<sup>+</sup> cells. Values are expressed as means ± S. E. M. <span class="html-italic">n =</span> 6/group. Significant levels set at *** <span class="html-italic">p</span> &lt; 0.001 noted difference between <span class="html-italic">Foxg1</span><sup>fl/fl</sup> and <span class="html-italic">Foxg1</span><sup>fl/fl</sup>-CreAAV animals. ns—not significant.</p> Full article ">Figure 3
<p>FOXG1 promotes the formation of neurosphere from cultured aNSCs and suppresses p21<sup>cip1</sup>-mediated cell-cycle exit. (<b>A</b>) Primary cultures of aNSCs at 1, 2, 3, 4 and 5 days of culture. Scale bar = 100 μm, applies to all images. (<b>B</b>) Flow cytometric analysis of p21 positive population in G<sub>1</sub> phase of cell cycle. Tested N<sub>2</sub>A cells were transfected with <span class="html-italic">vector</span> or <span class="html-italic">Foxg1</span> plasmids for 48 h. After incubation with an antibody against p21, <span class="html-italic">vector-</span> or <span class="html-italic">Foxg1-</span>transfected cells were treated with RNase A and propidium iodide mixture. Dot plots show the cell cycle distribution and FITC-A fluorescence intensity. (<b>C</b>) Column diagrams show the median fluorescent intensity of FITC-A (the strength of p21 expression) in the G<sub>1</sub>-phase of cell cycle. Values are expressed as means ± S. E. M. For each group, <span class="html-italic">n =</span> 6. Significant levels set at *** <span class="html-italic">p &lt;</span> 0.001 noted difference between <span class="html-italic">vector</span> and <span class="html-italic">Foxg1</span> transfected cells.</p> Full article ">Figure 4
<p>FOXG1 increases the amount of Tbr2-expressing IPCs and promotes their proliferation in adult hippocampal DG of the <span class="html-italic">Foxg1</span> genotype mice. (<b>A</b>) Expression patterns of Tbr2-expressing IPCs in the hippocampal DG of <span class="html-italic">Foxg1</span><sup>fl/fl</sup> mice were assessed with immunofluorescence. Red = Tbr2; Green = EGFP-labeled FOXG1; Blue = DAPI. Scale bars = 100 μm. Arrow heads: newly generated IPCs with FOXG1 activation. (<b>D</b>) Levels of total intermediate progenitor cells (IPCs) in adult hippocampus are showed as %Tbr2<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>E</b>) Levels of endogenous IPCs in adult hippocampus are showed as %GFP<sup>−</sup>Tbr2<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>B</b>) Expression patterns and (<b>F</b>) qualification of total IPCs in adult hippocampus were studied with immunohischemisitry. Scale bars = 100 μm. (<b>C</b>) Expression patterns of proliferated IPCs in the hippocampal DG of <span class="html-italic">Foxg1</span> genotype mice were assessed with immunofluorescence. Red = Tbr2; Green = PCNA. Scale bars = 100 μm. Arrow heads: proliferated IPCs. (<b>G</b>) Levels of IPCs in adult hippocampus are showed as %Tbr2<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>H</b>) Levels of proliferated IPCs in adult hippocampus are showed as %Tbr2<sup>+</sup>PCNA<sup>+</sup>/DAPI<sup>+</sup> cells. Values are expressed as means ± S. E. M. <span class="html-italic">n =</span> 6/group. Significant levels set at *** <span class="html-italic">p</span> &lt; 0.001 noted difference between <span class="html-italic">Foxg1</span><sup>fl/fl</sup> and <span class="html-italic">Foxg1</span><sup>fl/fl</sup>-CreAAV animals. ns—not significant.</p> Full article ">Figure 5
<p>FOXG1 increases the amount of doublecortin (Dcx)-expressing newborn neuroblasts in adult hippocampal DG of the <span class="html-italic">Foxg1</span> genotype mice. (<b>A</b>) Expression patterns of Dcx-expressing neuroblasts in the hippocampal DG of <span class="html-italic">Foxg1</span> genotype mice were assessed with immunofluorescence. Red = Dcx; Green = EGFP-labeled FOXG1; Blue = DAPI. Scale bars = 100 μm. Arrow heads: newly generated neuroblasts with FOXG1 activation. (<b>B</b>) Levels of total neuroblasts in adult hippocampus are showed as %Dcx<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>C</b>) Levels of endogenous neuroblasts in adult hippocampus are showed as %GFP<sup>−</sup>Dcx<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>D</b>,<b>E</b>) Hippocampal lysates of the two genotype mice were immunoblotted using an antibody against Dcx. Values are expressed as means ± S. E. M. <span class="html-italic">n =</span> 6/group. Significant levels set at *** <span class="html-italic">p</span> &lt; 0.001 noted difference between <span class="html-italic">Foxg1</span><sup>fl/fl</sup> and <span class="html-italic">Foxg1</span><sup>fl/fl</sup>-CreAAV animals. ns—not significant.</p> Full article ">Figure 6
<p>FOXG1 prolongs G<sub>1</sub>-phase length and promotes cell redistribution. FUCCI cells were cultured in 35 mm dishes for 24 h. The cells were transfected with <span class="html-italic">vector</span> or <span class="html-italic">Foxg1</span> plasmid for 48 h. (<b>A</b>,<b>B</b>) Fluorescence images of mAG and mKO2 emission signals in <span class="html-italic">vector</span> or <span class="html-italic">Foxg1</span> transfected 293T cells (Green = mAG; Red = mKO2) were disaplyed. Scale bar = 50 μm. Ratio percent of (<b>C</b>) G<sub>1</sub>- and (<b>D</b>) G<sub>2</sub>-M phase cells were calculated using the formula [G<sub>1</sub> phase: mKO2<sup>+</sup> cells/(mKO2<sup>+</sup> cells + mAG<sup>+</sup> cells) × 100%; G<sub>2</sub>-M phase: mAG<sup>+</sup> cells/(mKO2<sup>+</sup> cells + mAG<sup>+</sup> cells) × 100%. (<b>E</b>) Flow cytometric analysis of cell cycle distribution. Tested N<sub>2</sub>A cells were treated with RNase A and propidium iodide mixture. Data were gated to distinguish cell cycle. (<b>F</b>) Quantitative evaluation of ratio percentage of cells in G<sub>1</sub>-phase. (<b>G</b>) N<sub>2</sub>A cells were stained with Hoechst 33342 and pyronin Y (PY). Flow cytometry technology was utilized to analyze cDNA and RNA contents after cells were transfected with <span class="html-italic">vector</span> or <span class="html-italic">Foxg1</span> plasmids. (<b>H</b>) Quantification evaluation of ratio percentage of cells in G<sub>0</sub>-phase. Values are expressed as means ± S. E. M. <span class="html-italic">n =</span> 6/group. Significant levels set at *** <span class="html-italic">p</span> &lt; 0.001 noted difference between <span class="html-italic">vector</span> and <span class="html-italic">Foxg1</span> transfected cells.</p> Full article ">Figure 7
<p>FOXG1 induces final mature of GNs in adult hippocampus DG and increases synaptic plasticity. (<b>A</b>) Expression patterns of NeuN-expressing GNs in the hippocampal DG of <span class="html-italic">Foxg1</span> genotype mice were assessed with immunofluorescence. Red = NeuN; Green = EGFP-labeled FOXG1; Blue = DAPI. Scale bars = 100 μm. Arrow heads: newly generated GNs with FOXG1 activation. (<b>B</b>) Levels of total GNs in adult hippocampus are showed as %NeuN<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>C</b>) Levels of endogenous GNs in adult hippocampus are showed as %GFP<sup>−</sup>NeuN<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>D</b>) Expression patterns and (<b>E</b>) qualification of total GNs in adult hippocampus were studied with immunohischemisitry. Scale bars = 100 μm. (<b>F</b>,<b>G</b>) Hippocampal lysates of the two genotype mice were immunoblotted using an antibody against NeuN. GAPDH was used as the loading control. Values are expressed as means ± S. E. M. For each group, <span class="html-italic">n =</span> 6. Significant levels set at * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001 noted difference between <span class="html-italic">Foxg1</span><sup>fl/fl</sup> and <span class="html-italic">Foxg1</span><sup>fl/fl</sup>-CreAAV animals. (<b>H</b>) Fluorescence microscopic analysis of differentiation-related morphological changes of N<sub>2</sub>A cells by stained the F-actin cytoskeleton with fluorescently labeled phalloidin. Green = Phalloidin; Blue = DAPI. As positive control, cells were differentiated with 10 μM RA. Scale bars = 25 μm. (<b>I</b>) Quantification of the average neurite lengths are illustrated in the column diagrams. Values are expressed as means ± S. E. M. For each group, <span class="html-italic">n =</span> 6. Different letters indicate statistical differences in mean values among groups (<span class="html-italic">p</span> &lt; 0.05). ns—not significant.</p> Full article ">Figure 8
<p>FOXG1 has no ability to induce the production of oligodendrocytes in adult hippocampal DG of the <span class="html-italic">Foxg1</span> genotype mice. (<b>A</b>) Expression patterns of oligodendrocytes in the hippocampal DG of <span class="html-italic">Foxg1</span> genotype mice were assessed with immunofluorescence. Red = Oligo2; Green = EGFP-labeled FOXG1; Blue = DAPI. Scale bars = 100 μm. (<b>B</b>) Levels of total oligodendrocytes in adult hippocampus are showed as %Oligo2<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>C</b>) Levels of endogenous oligodendrocytes in adult hippocampus are showed as %GFP<sup>−</sup>Oligo2<sup>+</sup>/DAPI<sup>+</sup> cells. (<b>D</b>) Expression patterns and (<b>E</b>) qualification of total oligodendrocytes in adult hippocampus were studied with immunohischemisitry. Scale bars = 100 μm. (<b>F</b>,<b>G</b>) Hippocampal lysates of the two genotype mice were immunoblotted using an antibody against Oligo2. Tubulin was used as the loading control. Values are expressed as means ± S. E. M. <span class="html-italic">n =</span> 6/group. ns—not significant.</p> Full article ">
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