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Published: 11 February 2016

Tackling amyloidogenesis in Alzheimer’s disease with A2V variants of Amyloid-β

Giuseppe Di Fede¹,
Marcella Catania¹,
Emanuela Maderna¹,
Michela Morbin¹,
Fabio Moda¹,
Laura Colombo³,
Alessandro Rossi³,
Alfredo Cagnotto³,
Tommaso Virgilio¹,
Luisa Palamara¹,
Margherita Ruggerone¹,
Giorgio Giaccone¹,
Ilaria Campagnani¹,
Massimo Costanza²,
Rosetta Pedotti²,
Matteo Salvalaglio⁴,
Mario Salmona³ &
…
Fabrizio Tagliavini¹

Scientific Reports volume 6, Article number: 20949 (2016) Cite this article

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Abstract

We developed a novel therapeutic strategy for Alzheimer’s disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6_A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6_A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6_A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the Aβ_A2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6_A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.

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Introduction

Alzheimer’s disease (AD) is the most common form of dementia in the elderly. Its clinical course is slow but irreversible since no disease-modifying treatments are currently available. As a result, this illness has a huge socio-sanitary impact and designing of effective therapies is considered a public health priority.

A central pathological feature of AD is the accumulation of misfolded Amyloid-beta (Aβ) peptides in the form of oligomers and amyloid fibrils in the brain^1,2,3. It has been advanced that aggregated Aβ species, particularly oligomeric assemblies, trigger a cascade of events that lead to hyperphosphorylation, misfolding and assembly of the tau protein with formation of neurofibrillary tangles and disruption of the neuronal cytoskeleton, widespread synaptic loss and neurodegeneration. According to this view, altered Aβ species are the primary cause of AD and the primary target for therapeutic intervention^3,4.

Aβ peptides derive from proteolytic processing of a large (695/770 amino acids) type 1 transmembrane glycoprotein known as amyloid beta precursor protein (APP). APP is cleaved at the N-terminus of the Aβ domain by β-secretase, forming a large, soluble ectodomain (sAPPβ) and a 99-residue, membrane-retained C-terminal fragment (C99). Subsequently, γ-secretase cleaves C99 to release Aβ with different carboxyl termini, including Aβ40, Aβ42 and other minor species⁵. APP may undergo an alternative, non-amyloidogenic processing where the protein is cleaved within the Aβ domain by α-secretase, forming a soluble ectodomain (sAPPα) and an 83-residue C-terminal fragment (C83)^5,6.

We identified a novel mutation in the APP gene resulting in A-to-V substitution at codon 673, corresponding to position 2 in the Aβ sequence⁷. Studies on biological samples from an A673V homozygous carrier and cellular and C. elegans models indicated that this mutation shifts APP processing towards the amyloidogenic pathway with increased production of amyloidogenic peptides. Furthermore, the A2V substitution in the Aβ sequence (Aβ_A2V) increases the propensity of the full-length peptides (i.e., Aβ1-40 and Aβ1-42) to adopt a β-sheet structure, boosts the formation of oligomers both in vitro and in vivo and enhances their neurotoxicity^8,9,10. Following the observation that humans carrying the mutation in the heterozygous state do not develop AD, we carried out in vitro studies with synthetic peptides that revealed the extraordinary ability of Aβ_A2V to interact with wild-type Aβ (Aβ_WT), interfering with its nucleation or nucleation-dependent polymerization⁷. This provides grounds for developing a disease-modifying therapy for AD based on modified Aβ_A2V peptides retaining the key functional properties of parental full-length Aβ_A2V.

Following this approach, we generated a mutated six-mer peptide (Aβ1-6_A2V), constructed entirely by D-amino acids [Aβ1-6_A2V(D)] to increase its stability in vivo, whose interaction with full-length Aβ_WT hinders oligomer production and prevents amyloid fibril formation⁸.

These results prompted us to develop a prototypic compound by linking Aβ1-6_A2V(D) to an all-D form of TAT sequence [TAT(D)], a peptide derived from HIV that powerfully increases virus transmission to neighbour cells¹¹ and is widely used for brain delivery of drugs^12,13,14. Here we report that this compound [Aβ1-6_A2VTAT(D)] has strong anti-amyloidogenic effects in vitro, leading to inhibition of oligomer, amyloid fibril formation and of Aβ-dependent neurotoxicity. Preclinical studies showed that a short-term treatment with this peptide in an AD mouse model prevents Aβ aggregation and amyloid deposition in the brain but longer treatment unexpectedly increases amyloid burden, most likely due to the TAT intrinsic attitude to enhance Aβ production and to avidly bind amyloid and boost its seeding activity, warning against the use of this carrier in therapeutic approaches for AD.

Results

In silico molecular modeling of Aβ_A2V peptide variants

To predict the structural basis of the anti-amyloidogenic effect of Aβ1-6_A2V(D), a comparative conformation analysis of WT and mutated Aβ1-6 was carried out with all-atom classical MD simulations in explicit solvent. Both Aβ1-6_WT and Aβ1-6_A2V are intrinsically disordered peptides characterized by high flexibility. Nevertheless, the substitution of Ala2 with a Val residue induces significant changes in the appearance of the peptide in solution, resulting in an increase of the apolar character of the solvent accessible surface (SAS) (Fig. 1A) and in a modification of the gyration radius distribution in the Aβ1-6_A2V. Figure 1B shows that the probability distribution of the gyration radius is characterized by a global shift to smaller values and by the appearance of a shoulder in the distribution corresponding to gyration radii of 0.5 nm.

An analysis of the secondary structure content displayed by the peptides (Fig. 1C) shows that, while both Aβ1-6_A2V and Aβ1-6_WT display a predominant coil configuration, Aβ1-6_A2V shows a slightly higher propensity to form secondary structure motifs involving two to three residues. Aβ1-6_A2V in fact displays a propensity to form a turn involving the Glu3, Phe4 and Arg5 residues (Fig. 1D). The most populated structural clusters¹⁵ (Fig. 1E), in Aβ1-6_WT are characterized by an elongated coil structure accounting for 52.6% of the configurations, while the compact “turn” state is only the third most probable cluster, with a population of around 9%. Conversely, in the Aβ1-6_A2V, while the most populated structure is still an elongated coil (32%), the “turn” configuration is the second most populated structural cluster (31%).

Both Aβ1-6_WT and Aβ1-6_A2V under physiological conditions are characterized by intramolecular salt bridges such as those between Asp1 and Arg5 or Glu3-Arg5. In the extended coil configuration (Fig. 1E), salt bridges can be dynamically formed and dissociated without requiring a specific rearrangement of the peptide backbone. However, in the turn configuration salt bridges are typically dissociated; the interaction of the apolar Val2 sidechain with the Arg5 sidechain stabilizes such a dissociated state. The additional sterical hindrance to the rearrangement induced by the Val2 sidechain also contributes to the stabilization of the turn configuration of the A2V peptide.

The propensity of the A2V mutant to adopt a Glu3-Arg5 turn configuration characterized by a significant lifetime can be interpreted as the probable source of the heterotypic interaction of the Aβ1-6_A2V with full-length Aβ, which results in hindering its assembly.

Aβ1-6_A2V retains the in vitro anti-amyloidogenic features of the parental full-length peptide

We previously showed that Aβ1-6_A2V(D) destabilizes the secondary structure of Aβ1-42_WT⁸ and is even more effective than the WT peptide [Aβ1-6_WT(D)] and the A2V-mutated L-isomer [Aβ1-6_A2V(L)] at preventing the aggregation of full-length Aβ_WT⁸.

Treatment of SH-SY5Y cells with Aβ1-6_WT(D) or Aβ1-6_A2V(D) showed that neither is toxic for living cells even at high concentrations (20 μM) (Fig. 2A,B) and that both peptides are able to reduce the toxicity induced by Aβ1-42_WT (Fig. 2C,D). However, Aβ1-6_A2V(D) showed a stronger effect in counteracting the reduction of cell viability caused by Aβ1-42_WT (Fig. 2D), suggesting that the A-to-V substitution actually amplifies the protective effects of the six-mer peptide.

Aβ1-6_A2VTAT(D) maintains the in vitro anti-amyloidogenic properties of Aβ1-6_A2V(D)

Aβ1-6_A2V(D) alone does not efficiently cross either the blood brain barrier (BBB) or cell membranes (data not shown). This is an important feature that would deeply limit its use as an in vivo anti-amyloidogenic drug. So, we linked this peptide to the all-D TAT sequence to improve the translocation of Aβ1-6_A2V(D) across the BBB and cell membranes, minimize the degradation of the peptide and reduce the immune response elicited by the molecule. The resulting compound [Aβ1-6_A2VTAT(D)] destabilizes the secondary structure of Aβ1-42_WT. Indeed, CD spectroscopy studies showed that Aβ1-6_A2VTAT(D) inhibits the acquisition of β-sheet conformation by Aβ1-42_WT (data not shown), thus affecting the folding of the full-length peptide.

We tested the ability of Aβ1-6_A2VTAT(D) to inhibit the fibrillogenic properties of the full-length Aβ in vitro and found that the compound hindered Aβ1-42_WT aggregation (Fig. 3). Polarized light and electron microscopy studies on aggregates of Aβ1-42_WT formed after 20 days incubation with or without Aβ1-6_A2VTAT(D) revealed that the mutated peptide hinders the formation of amyloid structures (Fig. 3B) and reduces the amount of fibrils generated by the full-length peptide (Fig. 3D). Moreover, AFM analysis (Fig. 3E,H) showed that Aβ1-6_A2VTAT(D) actually interferes with the oligomerization process of Aβ1-42_WT. Indeed, monomeric Aβ1-42_WT, incubated alone at a final concentration of 100 μM, formed a family of small oligomers of different size within a range of 6-20 nm in diameter (~ 70%) (Fig. 3E,G). Conversely, the co-incubation with Aβ1-6_A2VTAT(D) resulted in the formation of very small globular structures with a range of 5-8 nm in diameter and height of 200-400 pm (~ 70%), large and thin structures, apparently very rich in water (width: 500–700 nm; height: 200–500 pm). Notably, only rare oligomeric structures were detected (Fig. 3F,H).

These effects were observed by incubating Aβ1-42_WT and Aβ1-6_A2VTAT(D) at a 1:4 molar ratio, but they were also evident at equimolar concentrations of the two peptides.

Moreover, treatment of differentiated SH-SY5Y cells with Aβ1-6_A2VTAT(D) showed that the peptide is not toxic when administered at concentrations ranging between 1 and 5 μM (Fig. 2E). When co-incubated with Aβ1-42_WT, Aβ1-6_A2VTAT(D) displayed a significant dose-dependent reduction of the toxicity induced by full-length Aβ (Fig. 2F).

All these findings indicated that the designed Aβ1-6_A2VTAT(D) peptide is particularly efficient at inhibiting Aβ polymerization and toxicity in vitro and identified it as our lead compound for the subsequent in vivo studies.

Aβ1-6_A2VTAT(D) is able to cross the BBB after its intraperitoneal administration

The stability of Aβ1-6_A2VTAT(D) and Aβ1-6_A2VTAT(L) peptides to serum proteases was determined using MALDI-TOF. The D-peptide remained stable for 48 h, while the L-peptide was rapidly degraded in about 1 h (Supplementary Fig. 3 S). To check the efficacy of TAT peptide in delivering Aβ1-6_A2V to the brain, we measured the levels of Aβ1-6_A2VTAT(D) in brain tissue of mice treated with the compound i.p. once a week at the dose of 10 mg/kg for five months, 1 or 24 h after the last treatment. The analysis showed that Aβ1-6_A2VTAT(D) brain levels were 145.03 ± 38.26 pg/mg tissue (mean ± SEM) and 62.02 ± 28.68 after 1 or 24 h, respectively (Supplementary Table 3S and Fig. 4 S).

Aβ1-6_A2VTAT(D) shows in vivo anti-amyloidogenic effects in the huAPP^Swe/moAPP^0/0 mouse model of AD

A pilot preclinical study with Aβ1-6_A2VTAT(D) was performed on a limited number (n = 3) of transgenic mice - APP23 mice expressing the Swedish double mutation in the human APP gene and knock-out for endogenous APP (huAPP^Swe/moAPP^0/0), chosen to avoid the interference of murine APP in the anti-amyloidogenic properties of Aβ1-6_A2VTAT(D). These mice usually begin to develop amyloid deposits at 10 months. The animals were treated i.p. at 13 months with 0.24 mg/kg of Aβ1-6_A2VTAT(D) once a day for 3 weeks. The treatment resulted in a reduction of Aβ production and aggregation (Fig. 4A) and in a decrease of intracerebral amyloid deposits (Fig. 4B,C).

Aβ1-6_A2VTAT(D) reduces Aβ levels and amyloid in APPswe/PS1dE9 mice in a short treatment schedule

Ultimately, we decided to treat APPswe/PS1dE9 mice (n = 20 x group) i.p. with Aβ1-6_A2VTAT(D). The treatment started at the age of 4 months, when the deposition of amyloid in the brain of these animals usually begins, becoming consistent by 6 months. The schedule of treatment was established based on the results of a pilot study with the same mouse model, showing that the best anti-amyloidogenic effects are obtained by treating animals i.p. once a week with 10 mg/kg peptide (data not shown).

The short-term treatment (2.5 months) of APPswe/PS1dE9 mice with Aβ1-6_A2VTAT(D) resulted in the exciting reduction of Aβ production and aggregation (Fig. 5B) and prevention of amyloid deposition in the brain (Fig. 5C,D) of the group treated once a week with Aβ1-6_A2VTAT(D). This group showed an increase of Aβ1-42 in the soluble fraction and a concomitant decrease in the insoluble fraction, suggesting that the treatment induces a transfer of Aβ1-42 from the insoluble to soluble compartment of brain tissue, which may reflect a disaggregation of amyloid deposits. This view is corroborated by the fact that aggregated Aβ was reduced in both compartments. Moreover, the decrease of aggregated Aβ in the soluble fraction is associated with an increase of monomeric Aβ1-42 levels, suggesting that A2V-based therapy changes the dynamic equilibrium between Aβ aggregates and Aβ monomers, resulting in a reduction of the most toxic Aβ species (i.e., soluble Aβ assemblies).

The reduction of amyloid deposition involved all brain areas, including the hippocampus and was more prominent in the frontal and entorhinal cortices and olfactory bulbs (Fig. 5D).

These findings were paralleled by a slight effect on cognitive function (Fig. 5A), as suggested by performances of mice treated with Aβ1-6_A2VTAT(D) in the Novel Object Recognition test (NOR). The results of the study showed a trend towards cognitive impairment in the control group and an opposite tendency of mice treated with Aβ1-6_A2VTAT(D), whose cognitive performances were no worse than at the beginning of the treatment with Aβ1-6_A2VTAT(D). In fact, the NOR test scores showed a small, albeit not statistically significant, improvement.

However, the final outcome of treatment (after 5 months) was an unexpected substantial increase in amyloid burden (Fig. 6C) and attenuation of the effects on Aβ production (Fig. 6B), while the results of the behavioral assessment by NOR suggested preserved cognitive function in the group treated with Aβ1-6_A2VTAT(D) compared with the saline-treated mice, as indicated by significant differences in the scores for recognition and discrimination indexes obtained for the two experimental group of mice at the end of treatment (Fig. 6A).

Aβ1-6_A2VTAT(D) induces a shift in APP processing toward the amyloidogenic pathway in a chronic treatment

The unexpected outcome of the prolonged treatment with Aβ1-6_A2VTAT(D) spurred us to search for the causes underlying the removal of the anti-amyloidogenic effects previously observed after 2.5 months of treatment with the test compound.

To this end, we investigated the effects of Aβ1-6_A2VTAT(D) on APP processing and found that treatment with Aβ1-6_A2VTAT(D) resulted in a shift of APP processing towards the amyloidogenic pathway, leading to an increased C99:C83 ratio in the brain of APPswe/PS1dE9 mice (Fig. 7A–D), which presumably paves the way for overproduction of Aβ. Interestingly, the effects on APP processing were detected only after prolonged treatment with Aβ1-6_A2VTAT(D) and were not observed after short-term treatment schedules, i.e., after 3 weeks in the huAPP^Swe/moAPP^0/0 or after 2.5 months in the APPswe/PS1dE9 mice.

TAT peptide alone binds amyloid vigorously

Concomitant studies performed in our labs showed that Aβ1-6_A2VTAT(D) has a special propensity to target β-amyloid deposits (Fig. 7E–H) and that this is a specific attribute of the TAT(D) sequence.

To address this point, we prepared Aβ1-6_A2VTAT(D), Aβ1-6_WTTAT(D), Aβ1-6_A2V(D) and TAT(D) peptides containing a biotinylated residue at the C-terminus (Biot-peptide) to visualize their binding to amyloid plaques. Both Biot-Aβ1-6_WTTAT(D) and Biot-Aβ1-6_A2VTAT(D) were unable to produce any staining in brain slices of control animals (non-transgenic mice) devoid of amyloid plaques (data not shown), but they were able to bind and therefore stain the plaques in transgenic mouse brains (Fig. 7E,F respectively). Interestingly, the biotinylated form of TAT(D) [(Biot-TAT(D)] strongly stained amyloid deposits (Fig. 7G), but no immunostaining of amyloid was detected in slices incubated with Biot-Aβ1-6_A2V(D) (Fig. 7H).

These data suggest that the anti-amyloidogenic effects of Aβ1-6_A2V(D) are overcome in the chronic treatment by the TAT intrinsic attitude to promote amyloidogenic APP processing, bind to amyloid and boost its seeding activity, leading to an increase of amyloid burden in vivo.

Aβ1-6_A2VTAT(D) induces an immune response after peripheral administration in mice

Finally, we tested the Aβ1-6_A2VTAT(D) immunogenic properties and found that the chronic treatment with this compound results in the production of not negligible levels of serum IgG against Aβ1-6_A2VTAT(D) as well as against full-length Aβ (Fig. 8A,B). Interestingly, the treatment with Aβ1-6_A2VTAT(D) did not induce production of IgG against Aβ1-6_A2V peptide (Fig. 8C), while 3 out of 9 mice displayed slightly increased levels of IgG against TAT(D) (Fig. 8D). These data suggest that both Aβ1-6_A2V and TAT(D) are not per se strongly immunogenic, while the combination of the two peptides might confer strong immunogenicity to Aβ1-6_A2VTAT(D) compound that, however, did not result in any evident side effect on brain, as revealed by neuropathologic studies (data not shown).

Discussion

During the last few decades, huge efforts have been made to develop disease-modifying therapies for Alzheimer, but the results of these attempts have been frustrating. The anticipated increase of AD patients in the next few decades makes the development of efficient treatments an urgent issue¹⁶. In order to prevent the disease and radically change its irreversible course, a long series of experimental strategies against the main molecular actors of the disease (Aβ and tau)¹⁷ or novel therapeutic targets¹⁸ have been designed based on purely theoretical grounds¹⁹ as well as on evidence mainly deriving from preclinical observations in AD animal models²⁰. However, few strategies proved suitable for application in human clinical trials and none proved to be really effective²¹.Our approach differs from previous strategies - mainly those involving modified Aβ peptides that have been found to inhibit amyloidogenesis^19,22 - since it is based on a natural genetic variant of amyloid-β (Aβ_A2V) that occurs in humans and prevents the development of the disease when present in the heterozygous state⁷.

In this context, we carried out in vitro and in vivo studies that revealed the extraordinary ability of Aβ_A2V to interact with Aβ_WT, interfering with its aggregation⁸. These findings were a proof of concept of the validity of therapeutic strategies based on the use of Aβ_A2V variant and prompted us to develop a new disease-modifying treatment for AD by designing a six-mer mutated D-isomer peptide [Aβ1-6_A2V(D)] linked to the short amino acid sequence derived from the HIV TAT peptide, widely used for brain delivery, to make the translocation of Aβ1-6_A2V(D) across the BBB feasible.

The use of TAT as a carrier for brain delivery of drugs has been employed in several experimental approaches for the treatment of AD-like pathology in mouse models^12,13. Recently, intraperitoneal administration of a TAT-BDNF peptide complex for 1 month was shown to improve the cognitive functions in AD rodent models²³.

A previous study showed that, following its peripheral injection, a fluorescein-labelled version of TAT is able to cross the BBB, bind amyloid plaques and activate microglia in the cerebral cortex of APPswe/PS1DE9 transgenic mice²⁴. TAT was then conjugated with a peptide inhibitor (RI-OR2, Ac-rGffvlkGr-NH2) consisting of a retro-inverted version of Aβ16–20 sequence²⁵ that was found to block the formation of Aβ aggregates in vitro and to inhibit the toxicity of Aβ on cultured cells²⁵. Daily i.p. injection of RI-OR2-TAT for 21 days into 10-month-old APPswe/PS1DE9 mice resulted in a reduction in Aβ oligomer levels and amyloid-β burden in cerebral cortex²⁴.

We followed a similar strategy and initially demonstrated that Aβ1-6_A2V(D), with or without the TAT sequence, retains in vitro the anti-amyloidogenic properties of the parental full-length mutated Aβ, since it is effective at hindering in vitro the production of oligomers and fibrils, the formation of amyloid and the toxicity induced by Aβ1-42_WT peptide on SYSH-5Y cells.

Based on these results, we then decided to test in vivo the anti-amyloidogenic ability of Aβ1-6_A2VTAT(D). The compound proved stable in serum after i.p. administration in mice, able to cross the BBB and associated with an immune response that was not found to cause any brain damage.

Short-term treatment with Aβ1-6_A2VTAT(D) in the APPswe/PS1DE9 mouse model prevented cognitive deterioration, Aβ aggregation and amyloid deposition in brain. Unexpectedly, a longer treatment schedule, while retaining the results for cognitive impairment, attenuated the effects on Aβ production and increased amyloid burden, most likely due to the intrinsic amyloidogenic properties of TAT.

Indeed, we found that TAT(D), unlike Aβ1-6_A2V(D), has a strong ability to bind amyloid deposits. This avidity for amyloid could boost the intrinsic seeding activity of amyloid plaques via a continuous and self-sustained recruitment of Aβ aggregates, leading to an exacerbation of the amyloidogenesis.

A similar effect of TAT was described in a study²⁶ reporting that HIV TAT promotes AD-like pathology in an AD mouse model co-expressing human APP bearing the Swedish mutation and TAT peptide (PSAPP/TAT mice). These mice indeed showed more Aβ deposition, neurodegeneration, neuronal apoptotic signalling and phospho-tau production than PSAPP mice.

Moreover, TAT was found to increase Aβ levels by inhibiting neprilysin²⁷ or enhancing β-secretase cleavage of APP, resulting in increased levels of the C99 APP fragment and 5.5-fold higher levels of Aβ42²⁸. The same study reported that stereotaxic injection of a lentiviral TAT expression construct into the hippocampus of APP/presenilin-1 (PS1) transgenic mice resulted in increased TAT-mediated production of Aβ in vivo as well as an increase in the number and size of Aβ plaques. This is consistent with our findings, indicating a shift in APP processing towards the amyloidogenic processing in vivo at the end of the 5-month treatment with Aβ1-6_A2VTAT(D) that was not observed in shorter treatment schedules with the same compound.

Therefore, these data suggest that the final outcome of our in vivo studies with Aβ1-6_A2VTAT(D) is the result of side effects of the TAT carrier, whose amyloidogenic intrinsic activity neutralized the anti-amyloidogenic properties of the Aβ_A2V variant. Nevertheless, we believe that the approach based on the use of Aβ_A2V variant can be successfully used in treating AD, because of its potential ability to tackle the main pathogenic events involved in the disease, as suggested by the natural protection against the disease which occurs in human heterozygous A673V carriers.

Interestingly, recent studies produced evidence in favour of a natural protection against AD in human carriers of the A2T Aβ mutation²⁹, another human Aβ variant characterized by an alanine-to-threonine substitution at the same APP codon of the A2V-Aβ mutation (APP-A673T or Aβ_A2T variant). This mutation segregates almost exclusively with individuals who never showed any sign of dementia, suggesting a possible protective role against AD in the Icelandic and Finnish populations³⁰. Additional studies tried to clarify the molecular basis of the A2T-induced protection for AD, suggesting a likely composite mechanism including effects on APP processing (with consequent decrease of Aβ production) and on Aβ structure, aggregation and neurotoxicity^31,32,33.

More recent papers reported that the A673T variant is extremely rare in other cohorts from the US and Asia and does not play a substantial role in risk for AD outside of Icelandic and Scandinavian geographic areas^34,35. However, the discovery of protective genetic variants like Aβ_A2V and Aβ_A2T, although rare, should prompt a novel vision of genetic studies, which until now has been limited to the identification of pathogenic variants, expanding the genetic research into the detection of “protective” DNA variations as a useful foundation for the design of efficient disease-modifying treatments in medicine.

Finally, we would emphasize that, regardless its optimal BBB delivery abilities and cell penetrating activity³⁶, some intrinsic properties of TAT sequence can promote amyloidogenesis in long treatment schedules and should be carefully taken into account whenever this peptide is employed in the design of therapeutic strategies for neurological diseases^37,38,39, particularly AD⁴⁰.

Materials and Methods

In silico studies

The impact of the A2V mutation on the structural features of the Aβ1-6 peptide was investigated using classical molecular dynamics (MD) simulations with an all-atom approach using the AMBER ff99SB-ILDN force field⁴¹. The solvent environment was explicitly simulated using the Tip3p water model and applying three-dimensional periodic boundary conditions. A 1.0 nm cutoff for the direct calculation of non-bonded interactions was applied and long-range electrostatics were treated with the particle-mesh Ewald approach. The systems were prepared by dispersing the Aβ1-6_WT and Aβ1-6_A2V peptides in a cubic box of side 5.0 nm, filled with water molecules and were initially relaxed through energy minimization in order to avoid nonphysical contact between the solute and solvent molecules, then equilibrated with a 5 ns MD simulation at constant ambient pressure and temperature (300 K). The production runs were then extended for 1.5 μs each. Covalent bonds involving hydrogen were constrained by applying the LINCS algorithm, allowing a simulation timestep of 2 fs⁴². Temperature was controlled by applying the Bussi-Donadio-Parrinello thermostat, while pressure was controlled with an isotropic Parrinello-Rahman barostat⁴³. All simulations were carried out using GROMACS 4.6.5⁴².

In vitro studies

Peptide synthesis

All synthetic peptides were prepared as previously described⁷ via solid-phase Fmoc chemistry on an Applied Biosystems 433 A peptide synthesizer, characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI). Their purity was always above 95% (See Supplementary Information for details).

Polarized light, Electron Microscopy (EM)

Aβ1-42_WT, Aβ1-6_A2VTAT(D) were dissolved in 10 mM NaOH and then diluted in an equal volume of 100 mM Tris-HCl, pH 7.0, to final concentrations (0.250 mM). In studies with peptide mixtures [Aβ1-42_WT : Aβ1-6_A2VTAT(D)], peptide solutions were prepared as described above at a final concentration of 0.250 mM at either 1:1 or 1:4 molar ratios. The samples were incubated at 37 °C and the tinctorial and ultrastructural properties of the peptide assemblies were determined at various intervals, ranging from 1 h to 20 days, as previously described⁷. The experiments were repeated five times.

Atomic Force Microscopy (AFM)

Aβ1-42_WT oligomers were formed following incubation of Aβ1-42_WT at a final concentration of 100 μM in phosphate buffer 50 mM, pH 7.4 at 4 °C for 24 h, alone or in presence of Aβ1-6_A2VTAT(D), at 1:4 molar ratio. Sample solutions were diluted to a final concentration of 10 μM of Aβ1-42_WT with phosphate buffer and 50 μl of each solution was immediately added onto freshly cleaved mica at room temperature for 2 minutes. Samples were washed and dried under gentle nitrogen flow. AFM analyses were carried out on a Multimode AFM with a Nanoscope V system (Veeco/Digital Instruments). AFM images were analysed using the Scanning Probe Image Processor data analysis package. All the topographic patterns and SPIP characterization were verified via additional measurements on a minimum of five different, well separated areas.

Binding of Biot-Aβ1-6_WT/A2VTAT(D) to amyloid

It was tested on brain sections of transgenic and control mice of CRND8 strain⁴⁴. To unequivocally identify the fragment(s) of the peptide responsible for binding to plaques we used three separate segments: Biot-Aβ1-6_WT(D), Biot-Aβ1-6_A2V(D) and Biot-TAT(D). Seven-micrometre thick brain sections fixed in Carnoy and embedded in paraffin were used for staining. The immunoreaction was revealed by incubation with the avidin-biotin complex (Vectastatin ABC Kit, Vector Laboratories) and diaminobenzidine (DAB) as the substrate for horseradish peroxidase.

Cell models

Toxicity studies with Aβ1-6_WT(D), Aβ1-6_A2V(D) or Aβ1-6_A2VTAT(D) and the short peptides’ ability to inhibit the toxicity induced by Aβ1-42_WT in SH-SY5Y cell cultures were analyzed following previously described protocols⁷. Each experiment was performed in triplicate; the data provided were obtained as the mean of three independent assays.

In vivo studies

Animals

Animal care and experimental procedures were conducted in accordance with European Union (2010/63/EU) and Italian (D. Lgs. 26/2014) legislations and followed the applicable rules and guidelines of the Animal care surveillance Committee of the INCB. All the experiments involving animals were approved by the Animal care surveillance Committee of the INCB and by the Italian Ministry of Health. All animals were sacrificed by cervical dislocation under deep anesthesia (telazol 20 mg/kg and medetomidine 1,5 mg/kg i.p.).

In vivo assessment of translocation across BBB

APP23 mice (Harlan, Correzzana, Italy) were treated by intraperitoneal injection with Aβ1-6_A2VTAT(D) (10 mg/kg) or vehicle (saline solution) and sacrificed 1 h or 24 h after the last treatment. Aβ1-6_A2VTAT(D) was quantified in mouse brain using HPLC-MS/MS⁴⁵. The stability of Aβ1-6_A2VTAT(D) to protease digestion was also investigated by MALDI TOF MS (See Supplementary Information for details).

In vivo anti-amyloidogenic effects of Aβ1-6_A2VTAT(D) in the huAPP^Swe/moAPP^0/0 mouse model

Transgenic APP23 mice expressing the Swedish double mutation in the human APP gene⁴⁶ and knock-out for endogenous APP (huAPP^Swe/moAPP^0/0) were generated in our lab by crossing APP23 mice with APP^0/0 animals. Mice (n = 6) were treated intraperitoneally (i.p.) every day for 21 days with Aβ1-6_A2VTAT(D) (10 mg/kg) or saline solution and culled 24 h after the last treatment.

In vivo anti-amyloidogenic effects of Aβ1-6_A2VTAT(D) in the APPswe/PS1dE9 mouse model

Heterozygous four-month-old female APPswe/PS1dE9 transgenic mice were used in this study. These mice express human APP carrying the Swedish mutation together with human presenilin 1 (PS1) carrying the pathogenic dE9 mutation⁴⁷. Animals were divided in 2 experimental groups (n = 10 each), treated i.p. once a week with saline solution or Aβ1-6_A2VTAT(D) and culled 2.5 (intermediate time) and 5 months (final time) after treatment. In both mouse transgenic lines, the brain was removed and dissected into 2 hemibrains by midsagittal dissection. One half was immediately stored at 80 °C for biochemical assays, the other immediately immersed in formalin (10%) overnight for immunohistochemical studies.

Behavioral tests

The Novel Object Recognition test (NOR) was used to examine the cognitive performance of animal models involved in preclinical studies, following well consolidated protocols⁴⁸. The results of the NOR were expressed by using:

- Discrimination index (time exploring novel object - time exploring familiar object)/(time exploring novel object + time exploring familiar object).

- Recognition index (RI): time exploring novel object/(time exploring novel object + time exploring familiar object) %.

Biochemical studies

The left hemisphere of each brain was homogenized in 7 volumes of 10 mM Tris-HCl, pH 7.4, added with cOmplete Mini Protease Inhibitors cocktail (Roche), sonicated for 1 min using an ultrasonic homogenizer (SONOPULS) and centrifuged at 100,000 xg for 1 h at 4 °C. The supernatant was saved as the soluble fraction; the pellet was extracted in 70% formic acid and neutralized with 20 volumes of 1 M Tris (insoluble fraction).

Aβ40, Aβ42 and aggregated Aβ were measured in both soluble and insoluble fractions by ELISA (Invitrogen). Each experiment was performed in triplicate. The analysis of APP processing followed the protocol described above. After centrifugation, the supernatant was collected as the SDS-soluble fraction, which was analyzed by western blot with the A8717 antibody (Sigma). Signal intensity of the bands was measured by Quantity One (BioRad).

Neuropathological studies

Coronal slices of the right hemibrain were embedded in paraffin and cut (7μm); sections were de-waxed in xylene and hydrated through serial alcohols to water. After formic acid (80%) pre-treatment, sections were incubated overnight with anti-Aβ antibody (4G8, 1:4000; Covance). The primary antibody signal was detected with a biotinylated secondary antibody followed by horseradish streptavidin peroxidase and visualized with DAB. Amyloid deposition was quantified in mouse brain using Aβ immunostaining (4G8) and the staining intensity was evaluated semiquantitatively using a scale range from 0 to 5 by light microscopy. The assessment was conducted in two adjacent sections of the same brain area⁴⁹. A parallel quantification of the Aβ plaque load was performed using image analysis software (NIS-elements-Nikon)^50,51,52.

Measurement of serum antibodies against Aβ peptides

Sera were collected from mice before (T0) and at the end of the treatment (5 months) and tested using ELISA for the presence of IgG against Aβ1-6_A2VTAT(D), the full-length Aβ1-42, Aβ1-6_A2V(D) and TAT(D) following previously described protocols for antigen-specific mouse IgG detection⁵³, except blocking buffer that was PBS 3% milk. Anti-Aβ1-42 IgG antibodies from 6E10 IgG clone (Covance) and sera from mice immunized with Aβ1-6_A2VTAT(D) or with Aβ1-6_A2V emulsified in Complete Freund’s adjuvant were used as positive controls.

Statistical analysis

Mann Withney U-test was used to compare data obtained by NOR test (Discrimination Index, Preference Index, Recognition Index). Comparison of cell viability after administration of Aβ1-42, alone or together with Aβ1-6_A2VTAT(D) or Aβ1-6_WTTAT(D), was performed by Student t-test. Student t-test was also used to compare (i) amyloid burden in immunohistochemical studies, (ii) Aβ40, Aβ42 and aggregated Aβ levels obtained by ELISA tests and (iii) relative amounts of APP C-terminal fragments after Western Blot quantification. Two tailed P value equal or less than 0.05 was considered statistically significant. All calculations were performed using GraphPad Prism 5.

Additional Information

How to cite this article: Di Fede, G. et al. Tackling amyloidogenesis in Alzheimer's disease with A2V variants of Amyloid-β. Sci. Rep. 6, 20949; doi: 10.1038/srep20949 (2016).

References

Duyckaerts, C., Delatour, B. & Potier, M. C. Classification and basic pathology of Alzheimer disease. Acta Neuropathol 118, 5–36, doi: 10.1007/s00401-009-0532-1 (2009).
Article CAS PubMed Google Scholar
Hyman, B. T. et al. National Institute on Aging-Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease. Alzheimers Dement 8, 1–13, doi: 10.1016/j.jalz.2011.10.007 (2012).
Article PubMed PubMed Central Google Scholar
Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353–356, doi: 10.1126/science.1072994 (2002).
Article ADS CAS PubMed Google Scholar
Shankar, G. M. et al. Amyloid-beta protein dimers isolated directly from Alzheimer’s brains impair synaptic plasticity and memory. Nat Med 14, 837–842, doi: 10.1038/nm1782 (2008).
Article CAS PubMed PubMed Central Google Scholar
Querfurth, H. W. & LaFerla, F. M. Alzheimer’s disease. N Engl J Med 362, 329–344, doi: 10.1056/NEJMra0909142 (2010).
Article CAS PubMed Google Scholar
Chow, V. W., Mattson, M. P., Wong, P. C. & Gleichmann, M. An overview of APP processing enzymes and products. Neuromolecular Med 12, 1–12, doi: 10.1007/s12017-009-8104-z (2010).
Article CAS PubMed PubMed Central Google Scholar
Di Fede, G. et al. A recessive mutation in the APP gene with dominant-negative effect on amyloidogenesis. Science 323, 1473–1477, doi: 10.1126/science.1168979 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Di Fede, G. et al. Good gene, bad gene: new APP variant may be both. Prog Neurobiol 99, 281–292, doi: 10.1016/j.pneurobio.2012.06.004 (2012).
Article CAS PubMed Google Scholar
Diomede, L. et al. Expression of A2V-mutated Aβ in Caenorhabditis elegans results in oligomer formation and toxicity. Neurobiol Dis 62, 521–532, doi: 10.1016/j.nbd.2013.10.024 (2014).
Article CAS PubMed PubMed Central Google Scholar
Messa, M. et al. The peculiar role of the A2V mutation in amyloid-β (Aβ) 1–42 molecular assembly. J Biol Chem 289, 24143–24152, doi: 10.1074/jbc.M114.576256 (2014).
Article CAS PubMed PubMed Central Google Scholar
Chauhan, A., Tikoo, A., Kapur, A. K. & Singh, M. The taming of the cell penetrating domain of the HIV Tat: myths and realities. J Control Release 117, 148–162, doi: 10.1016/j.jconrel.2006.10.031 (2007).
Article CAS PubMed Google Scholar
Borsello, T. et al. A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat Med 9, 1180–1186, doi: 10.1038/nm911 (2003).
Article CAS PubMed Google Scholar
Colombo, A. et al. The TAT-JNK inhibitor peptide interferes with beta amyloid protein stability. Cell Death Differ 14, 1845–1848, doi: 10.1038/sj.cdd.4402202 (2007).
Article CAS PubMed Google Scholar
Zhu, Y., Bu, Q., Liu, X., Hu, W. & Wang, Y. Neuroprotective effect of TAT-14-3-3ε fusion protein against cerebral ischemia/reperfusion injury in rats. PLoS One 9, e93334, doi: 10.1371/journal.pone.0093334 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Daura, X. et al. Peptide folding: when simulation meets experiment. Angew. Chem. Int. Ed. Engl. 38, 236–240 (1999).
Article CAS Google Scholar
Reitz, C., Brayne, C. & Mayeux, R. Epidemiology of Alzheimer disease. Nat Rev Neurol 7, 137–152, doi: 10.1038/nrneurol.2011.2 (2011).
Article PubMed PubMed Central Google Scholar
Galimberti, D. & Scarpini, E. Alzheimer’s disease: from pathogenesis to disease-modifying approaches. CNS Neurol Disord Drug Targets 10, 163–174 (2011).
Article CAS PubMed Google Scholar
Rampa, A., Gobbi, S., Belluti, F. & Bisi, A. Emerging targets in neurodegeneration: new opportunities for Alzheimer’s disease treatment ? Curr Top Med Chem 13, 1879–1904 (2013).
Article CAS PubMed Google Scholar
Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. L. & Beyreuther, K. Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer’s disease beta A4 peptides. J Mol Biol 228, 460–473 (1992).
Article CAS PubMed Google Scholar
Franco, R. & Cedazo-Minguez, A. Successful therapies for Alzheimer’s disease: why so many in animal models and none in humans ? Front Pharmacol 5, 146, doi: 10.3389/fphar.2014.00146 (2014).
Article CAS PubMed PubMed Central Google Scholar
Schneider, L. S. et al. Clinical trials and late-stage drug development for Alzheimer’s disease: an appraisal from 1984 to 2014. J Intern Med 275, 251–283, doi: 10.1111/joim.12191 (2014).
Article CAS PubMed PubMed Central Google Scholar
Soto, C. & Estrada, L. Amyloid inhibitors and beta-sheet breakers. Subcell Biochem 38, 351–364 (2005).
Article CAS PubMed Google Scholar
Wu, Y. et al. Intraperitoneal Administration of a Novel TAT-BDNF Peptide Ameliorates Cognitive Impairments via Modulating Multiple Pathways in Two Alzheimer’s Rodent Models. Sci Rep 5, 15032, doi: 10.1038/srep15032 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Parthsarathy, V. et al. A novel retro-inverso peptide inhibitor reduces amyloid deposition, oxidation and inflammation and stimulates neurogenesis in the APPswe/PS1ΔE9 mouse model of Alzheimer’s disease. PLoS One 8, e54769, doi: 10.1371/journal.pone.0054769 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Taylor, M. et al. Development of a proteolytically stable retro-inverso peptide inhibitor of beta-amyloid oligomerization as a potential novel treatment for Alzheimer’s disease. Biochemistry 49, 3261–3272, doi: 10.1021/bi100144m (2010).
Article CAS PubMed Google Scholar
Giunta, B. et al. HIV-1 Tat contributes to Alzheimer’s disease-like pathology in PSAPP mice. Int J Clin Exp Pathol 2, 433–443 (2009).
CAS PubMed PubMed Central Google Scholar
Rempel, H. C. & Pulliam, L. HIV-1 Tat inhibits neprilysin and elevates amyloid beta. AIDS 19, 127–135 (2005).
Article CAS PubMed Google Scholar
Kim, J., Yoon, J. H. & Kim, Y. S. HIV-1 Tat interacts with and regulates the localization and processing of amyloid precursor protein. PLoS One 8, e77972, doi: 10.1371/journal.pone.0077972 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Jonsson, T. et al. A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 488, 96–99, doi: 10.1038/nature11283 (2012).
Article ADS CAS PubMed Google Scholar
Kero, M. et al. Amyloid precursor protein (APP) A673T mutation in the elderly Finnish population. Neurobiol Aging 34, 1518.e1511–1513, doi: 10.1016/j.neurobiolaging.2012.09.017 (2013).
Article CAS Google Scholar
Hashimoto, Y. & Matsuoka, M. A mutation protective against Alzheimer’s disease renders amyloid β precursor protein incapable of mediating neurotoxicity. J Neurochem 130, 291–300, doi: 10.1111/jnc.12717 (2014).
Article CAS PubMed Google Scholar
Maloney, J. A. et al. Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein. J Biol Chem 289, 30990–31000, doi: 10.1074/jbc.M114.589069 (2014).
Article CAS PubMed PubMed Central Google Scholar
Benilova, I. et al. The Alzheimer disease protective mutation A2T modulates kinetic and thermodynamic properties of amyloid-β (Aβ) aggregation. J Biol Chem 289, 30977–30989, doi: 10.1074/jbc.M114.599027 (2014).
Article CAS PubMed PubMed Central Google Scholar
Liu, Y. W. et al. Absence of A673T variant in APP gene indicates an alternative protective mechanism contributing to longevity in Chinese individuals. Neurobiol Aging 35, 935.e911–932, doi: 10.1016/j.neurobiolaging.2013.09.023 (2014).
Article CAS Google Scholar
Wang, L. S. et al. Rarity of the Alzheimer disease-protective APP A673T variant in the United States. JAMA Neurol 72, 209–216, doi: 10.1001/jamaneurol.2014.2157 (2015).
Article CAS PubMed PubMed Central Google Scholar
Khafagy, e.-S. & Morishita, M. Oral biodrug delivery using cell-penetrating peptide. Adv Drug Deliv Rev 64, 531–539, doi: 10.1016/j.addr.2011.12.014 (2012).
Article CAS Google Scholar
Kang, W. H., Simon, M. J., Gao, S., Banta, S. & Morrison, B. Attenuation of astrocyte activation by TAT-mediated delivery of a peptide JNK inhibitor. J Neurotrauma 28, 1219–1228, doi: 10.1089/neu.2011.1879 (2011).
Article PubMed Google Scholar
Deng, B. et al. Targeted delivery of neurogenin-2 protein in the treatment for cerebral ischemia-reperfusion injury. Biomaterials 34, 8786–8797, doi: 10.1016/j.biomaterials.2013.07.076 (2013).
Article CAS PubMed Google Scholar
Arribat, Y. et al. Systemic delivery of P42 peptide: a new weapon to fight Huntington’s disease. Acta Neuropathol Commun 2, 86, doi: 10.1186/s40478-014-0086-x (2014).
Article PubMed PubMed Central Google Scholar
Lou, G. et al. Intranasal administration of TAT-haFGF(14-154) attenuates disease progression in a mouse model of Alzheimer’s disease. Neuroscience 223, 225–237, doi: 10.1016/j.neuroscience.2012.08.003 (2012).
Article ADS CAS PubMed Google Scholar
Lindorff-Larsen, K. et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 78, 1950–1958, doi: 10.1002/prot.22711 (2010).
Article CAS PubMed PubMed Central Google Scholar
Lindahl, E., Hess, B. & van der Spoel, D. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model 7, 306–317 (2001).
Article CAS Google Scholar
Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: A new molecular dynamics method. J Appl Phys 52, 7182–7190 (1981).
Article ADS CAS Google Scholar
Chishti, M. A. et al. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. J Biol Chem 276, 21562–21570, doi: 10.1074/jbc.M100710200 (2001).
Article CAS PubMed Google Scholar
Davoli, E. et al. Determination of tissue levels of a neuroprotectant drug: the cell permeable JNK inhibitor peptide. J Pharmacol Toxicol Methods 70, 55–61, doi: 10.1016/j.vascn.2014.04.001 (2014).
Article CAS PubMed Google Scholar
Sturchler-Pierrat, C. & Staufenbiel, M. Pathogenic mechanisms of Alzheimer’s disease analyzed in the APP23 transgenic mouse model. Ann N Y Acad Sci 920, 134–139 (2000).
Article ADS CAS PubMed Google Scholar
Puli, L. et al. Effects of human intravenous immunoglobulin on amyloid pathology and neuroinflammation in a mouse model of Alzheimer’s disease. J Neuroinflammation 9, 105, doi: 10.1186/1742-2094-9-105 (2012).
Article CAS PubMed PubMed Central Google Scholar
Antunes, M. & Biala, G. The novel object recognition memory: neurobiology, test procedure and its modifications. Cogn Process 13, 93–110, doi: 10.1007/s10339-011-0430-z (2012).
Article CAS PubMed Google Scholar
Capetillo-Zarate, E. et al. Selective vulnerability of different types of commissural neurons for amyloid beta-protein-induced neurodegeneration in APP23 mice correlates with dendritic tree morphology. Brain 129, 2992–3005, doi: 10.1093/brain/awl176 (2006).
Article PubMed Google Scholar
Garcia-Alloza, M. et al. Characterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease. Neurobiol Dis 24, 516–524, doi: 10.1016/j.nbd.2006.08.017 (2006).
Article CAS PubMed Google Scholar
DaRocha-Souto, B. et al. Brain oligomeric β-amyloid but not total amyloid plaque burden correlates with neuronal loss and astrocyte inflammatory response in amyloid precursor protein/tau transgenic mice. J Neuropathol Exp Neurol 70, 360–376, doi: 10.1097/NEN.0b013e318217a118 (2011).
Article CAS PubMed Google Scholar
Samaroo, H. D. et al. High throughput object-based image analysis of β-amyloid plaques in human and transgenic mouse brain. J Neurosci Methods 204, 179–188, doi: 10.1016/j.jneumeth.2011.10.003 (2012).
Article PubMed Google Scholar
Costanza, M., Musio, S., Abou-Hamdan, M., Binart, N. & Pedotti, R. Prolactin is not required for the development of severe chronic experimental autoimmune encephalomyelitis. J Immunol 191, 2082–2088, doi: 10.4049/jimmunol.1301128 (2013).
Article CAS PubMed Google Scholar

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Acknowledgements

This study was supported by the Telethon Foundation (Project GGP10120), the Italian Ministry of Health RF-2009-1473239 and Banca Intesa Sanpaolo 2014-2015 Grant. We thank Flamma srl (Bergamo, Italy) for the kind gift of FMOC peptides.

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Authors and Affiliations

Neurology V and Neuropathology Unit, IRCCS Foundation “Carlo Besta” Neurological Institute (INCB), Via Celoria 11, Milan, 20133, Italy
Giuseppe Di Fede, Marcella Catania, Emanuela Maderna, Michela Morbin, Fabio Moda, Tommaso Virgilio, Luisa Palamara, Margherita Ruggerone, Giorgio Giaccone, Ilaria Campagnani & Fabrizio Tagliavini
Neuroimmunology and Neuromuscular Disorder Unit, IRCCS Foundation “C. Besta” Neurological Institute (INCB), Milan, Via Celoria 11, 20133, Italy
Massimo Costanza & Rosetta Pedotti
Department of Molecular Biochemistry and Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa 19, Milan, 20158, Italy
Laura Colombo, Alessandro Rossi, Alfredo Cagnotto & Mario Salmona
Department of Chemical Engineering, University College London, London, WC1E 7JE, UK
Matteo Salvalaglio

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Giuseppe Di Fede
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Marcella Catania
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Contributions

G.D.F. was responsible for the conception of hypotheses, design of study and analysis of results. M.Salv. performed in silico studies. M.C. was involved in in vitro studies on cell lines and in vivo biochemical studies. M.M. and L.P. performed ultrastructural studies. F.M. and E.M. were responsible for behavioural assessment in animal models. E.M. and G.G. were involved in neuropahologic studies. L.C. was involved in AFM studies and analysis of binding of biotinylated peptides to amyloid. M.R., I.C. and T.V. performed treatments of animals. M.C. and R.P. performed immunological studies. A.C. and L.C. were responsible for peptide synthesis and purification. A.R. and L.C. were involved in HPLC-MS/MS analysis and MALDI TOF MS studies for the measurement of BBB cross, serum stability and brain levels of peptides. F.T. and M.S. were responsible for scientific input, coordination of the experimental plan and analysis of the results.

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G.D.F., M.M. and F.T. have a patent 0001383392 issued and a patent 08838209.8 pending, both related to this work .

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Di Fede, G., Catania, M., Maderna, E. et al. Tackling amyloidogenesis in Alzheimer’s disease with A2V variants of Amyloid-β. Sci Rep 6, 20949 (2016). https://doi.org/10.1038/srep20949

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Received: 27 October 2015
Accepted: 13 January 2016
Published: 11 February 2016
DOI: https://doi.org/10.1038/srep20949

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