Abeta Oligomers Research Articles

The multifactorial role of vanillin in amelioration of aluminium chloride and D-galactose induced Alzheimer's disease in mice

The onset and progression of Alzheimer's disease (AD) are influenced by a variety of factors. These include oxidative stress, overexpression of acetylcholinesterase (AChE), depletion of acetylcholine levels, increased beta-secretase mediated conversion of Amyloid Precursor Protein (APP) to Amyloid Beta (Abeta), accumulation of Abeta oligomers, decrease in Brain Derived Neurotrophic factor (BDNF) and accelerated neuronal apoptosis due to elevated levels of caspase-3. The currently available therapeutic approaches are inadequate in affecting these pathological processes except maybe the overexpression of AChE (AChE inhibitors like donepezil, rivastigmine). There is an urgent need to develop disease modifying pharmacotherapeutic interventions which have appreciable safety and cost effectiveness. From previously reported in vitro studies and a preliminary assessment of neuroprotective effect in scopolamine induced dementia-like cognitive impairment in mice, vanillin has been used as the compound of interest in the present study. Vanillin, a phytoconstituent, has been used in humans, safely, in the form of a flavouring agent for various foods, beverages, and cosmetics. Owing to its chemical nature i.e. being a phenolic aldehyde, it has an additional antioxidant property that is congruent to the desirable characteristics that are sought in a suitable novel anti-AD agent. In our study, vanillin proved to have a nootropic effect in healthy Swiss albino mice as well as an ameliorative effect in aluminium chloride and D-galactose induced AD model in mice. Apart from tackling oxidative stress, vanillin was found to reduce the levels of AChE, beta secretase, caspase-3, enhance degradation of Abeta plaques and elevate the levels of BDNF, in cortical and hippocampal regions. Vanillin is a promising candidate for being incorporated into the search for safe and effective anti-AD molecules. However, further research might be needed to warrant its application clinically.

Rational design of a vaccine for Alzheimer’s disease using computationally-derived conformational B cell epitopes to selectively target toxic amyloid-beta oligomers (S26.004)

<h3>Objective:</h3> Design an optimal amyloid-beta (Abeta) vaccine to elicit a robust and durable antibody response against toxic Abeta oligomers (ABO) without inducing potentially detrimental B or T cell responses against plaque or normal Abeta. <h3>Background:</h3> Abeta vaccines have the potential to protect against disease but also carry the risk of eliciting proinflammatory T cell responses causing meningoencephalitis, and plaque-reactive antibodies that can increase the risk of brain edema (ARIA-E). To circumvent these issues and induce an antibody response that selectively targets soluble toxic ABO, we designed a vaccine consisting of a computationally-derived conformational B cell epitope of ABO, coupled to KLH as a carrier protein to provide T cell help. <h3>Design/Methods:</h3> Mice received 3 immunizations, 4 weeks apart, with vaccine conjugate in alum or QS-21 as adjuvants. Serum titers and IgG subtypes of antibodies to the peptide epitope were measured by ELISA. The selectivity of serum antibodies for toxic ABO versus monomers or plaque was assessed by SPR and immunohistochemistry, respectively. T helper responses to the peptide and to KLH were evaluated by ELISPOT analysis of splenic lymphocytes. <h3>Results:</h3> A robust antibody response against the ABO epitope was observed with both adjuvants and was remarkably maintained unabated out to 6 months after the last immunization. The serum antibodies reacted with ABO only, not monomers or plaque. ELISPOT analysis showed T helper cytokine production in response to stimulation with KLH but not the ABO epitope thereby confirming that the peptide only contains a B cell epitope. <h3>Conclusions:</h3> A vaccine consisting of an ABO-restricted conformational B cell epitope conjugated to KLH produced a strong Abeta antibody response with no measurable pro-inflammatory T cell response against Abeta. In addition, the oligomer selectivity of the antibodies focused the response on pathogenic ABO, potentially reducing the risk of ARIA-E associated with binding to plaque and vascular deposits of Abeta. <b>Disclosure:</b> Dr. Kaplan has received personal compensation for serving as an employee of ProMIS Neurosciences. Dr. Kaplan has stock in ProMIS Neurosciences. Dr. Kaplan has received intellectual property interests from a discovery or technology relating to health care. The institution of Dr. Napper has received research support from Weston Family Foundation. Ms. Scruten has nothing to disclose. Dr. Gibbs has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Promis Neuroscinces. Ms. Coutts has nothing to disclose. Dr. Cashman has received personal compensation for serving as an employee of ProMIS Neurosciences. Dr. Cashman has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Mitsubishi-Tanabe. Dr. Cashman has stock in ProMIS Neurosciences. The institution of Dr. Cashman has received research support from ProMIS Neurosciences. Dr. Cashman has received intellectual property interests from a discovery or technology relating to health care. Dr. Cashman has a non-compensated relationship as a BoD memeber with ALS Society of BC that is relevant to AAN interests or activities.

Biomarker‐based development for optimized ACI‐24, a novel candidate vaccine for the treatment and prevention of Alzheimer’s disease

AbstractBackgroundAmyloid plaques and neurofibrillary tangles are the neuropathologic hallmarks of Alzheimer’s disease (AD) with the key constituents being Amyloid Beta (Abeta) and Tau protein, respectively. However, the initiation and propagation of neurodegeneration and neurotoxicity in Alzheimer’s disease remains incompletely understood despite recent important progress in clinical trials. It is apparent that small aggregates such as Abeta oligomers or fragments such as Abeta 1‐42 and pyroglutamate Abeta3‐42 (pGlu‐Abeta3‐42)) are of key importance for disease initiation with significant implications for translational medicine and success in clinical trial development.MethodACI‐24 is a vaccine targeting Abeta for the treatment and prevention of AD. Initial clinical data have demonstrated safety and an initial pharmacodynamic response, both in sporadic AD and people with Down syndrome (DS), a specifically vulnerable population predisposed to developing AD. Preclinical data have recently demonstrated that a new, optimized formulation of ACI‐24 has significantly improved immunogenicity against key toxic species, i.e., Abeta oligomers, Abeta 1‐42 and pGlu‐Abeta3‐42. Recent clinical trial results in AD, specifically with monoclonal antibodies targeting Abeta, have successfully employed translational biomarkers, including Amyloid PET, as key decision markers and predictors of clinical benefit.ResultWe present an innovative translational clinical trial design to understand the immunogenic properties of optimized ACI‐24 in sporadic AD and in people with DS. The biomarker‐based design, including multiple interim analyses, will enable (I) assessment of early safety and immunogenicity, (II) dose selection for early and meaningful readouts on translational biomarkers including Amyloid PET, (III) the safe transition into the more vulnerable DS population and (IV) informed transition into pivotal studies.ConclusionThe Alzheimer’s disease field needs new treatments and clinical trial approaches. Vaccination is a proven approach to managing diseases affecting global populations. The field of biomarker‐based translational medicine has been mobilized by the recent understanding of how to clinically evaluate targeting of toxic Abeta fragments and aggregates in trials using monoclonal antibodies. Here, we demonstrate how we utilize these advances in an innovative clinical trial design for an efficient, evidence‐based translational medicine approach to test optimized ACI‐24 as a novel Abeta vaccine candidate.

A randomized, placebo‐controlled, double‐blind, Phase 1b study to evaluate the safety, tolerability and pharmacodynamics of PRI‐002 in early AD

AbstractBackgroundPRI‐002 is an all‐D‐enantiomeric peptide developed to disassemble Abeta oligomers, which has shown to improve cognition in animal models and which has demonstrated its safety profile in healthy young volunteers (Kutzsche et al. 2020).Method20 AD patients in early disease stages fulfilling the ATN‐criteria were recruited to participate in a single center, randomized, placebo –controlled, double‐blind study. Patients received once daily oral doses of 300 mg PRI‐002 or placebo for 28 days. Safety and efficacy assessments were executed at baseline, day 14, day 28 and day 56 (follow up). Blood sampling was carried out at each time point, EEG and CSF measurements before and at the end of treatment. Imaging (MRI) and functional tests (CERAD, CDR) were performed in parallel to EEG/CSF and additionally at follow up.Result10 patients (age 76.9±3.4, MMSE 28±1.6) received placebo and 9 patients PRI‐002 (72.4±6.9, 27.2±2.9). One patient withdraw informed consent before treatment was started. 13 patients reported in total 27 adverse events while no serious adverse event was noted. There was no statistical difference regarding adverse events between treatment and placebo. EEG and MRI revealed no changes after treatment. While no significant changes were detected in p‐TAU, Abeta1‐42 and Abeta oligomers in CSF before and after treatment, patients receiving PRI‐002 significantly performed better than those receiving placebo in the CERAD word list at follow up (p≤0.05).ConclusionPRI‐002 showed an excellent safety profile in AD patients. While no biomarker changes were detected after 4 weeks of treatment interestingly, an improvement of memory function was noted at follow up. A phase 2 study is scheduled.Kutzsche J, Jürgens D, Willuweit A, Adermann K, Fuchs C, Simons S, Windisch M, Hümpel M, Rossberg W, Wolzt M, Willbold D. Safety and pharmacokinetics of the orally available antiprionic compound PRI‐002: A single and multiple ascending dose phase I study. Alzheimers Dement (N Y). 2020 Mar 20;6(1):e12001. https://doi.org/10.1002/trc2.12001.

Optimized ACI‐24, an amyloid beta (Abeta) vaccine that safely drives immunity to oligomers and Pyroglutamate Abeta, key pathological species of Alzheimer’s disease (AD)

AbstractBackgroundAmyloid immunotherapy has received recent clinical validation with monoclonal antibodies. Vaccines, such as optimized ACI‐24, are designed to produce a broader, robust antibody response against multiple pathologic forms of Abeta with the added advantage to reduce treatment frequency. Optimized ACI‐24 builds on the initial safety, immunogenicity and pharmacodynamic data of ACI‐24 that has been studied in AD and Down Syndrome‐related AD. Optimized ACI‐24 is formulated with the additional immune cell help via a non‐Abeta, universal T‐helper cell peptide, aiming to insure sustained antibody production, particularly against pathological Abeta species, such as oligomers and truncated pyroglutamate.MethodOptimized ACI‐24 was generated using the SupraAntigen® platform. Mice and non‐human primates were immunized and sera/plasma obtained to measure titers against Abeta1‐42, pyroglutamate (Abeta3‐42) and oligomers. Epitope mapping, immunohistochemistry on different human tissues, including AD brains were performed.ResultImmunization of mice and cynomolgus monkeys with optimized ACI‐24 resulted in potent antigen specific IgG levels. The polyclonal antibody response was observed to mature over time with higher affinity IgGs emerging as well as an increase in titers of IgGs that recognized the highly neurotoxic Abeta species including truncated pyroglutamate and pathological Abeta oligomers. Epitope mapping confirmed the induction of a broader spectrum of antibodies recognizing the N‐terminal containing and the truncated species of pathological Abeta, as compared to the previously clinically tested Abeta vaccines. In addition, target engagement was demonstrated as the immunized monkey sera labeled Abeta plaques in AD patient brain sections while did not show any binding to other human tissues.ConclusionThese preclinical data illustrate that optimized ACI‐24 generates IgGs with a broader N‐terminal Abeta sequence recognition than offered by clinically tested mAbs or Abeta vaccines. This unique binding profile for the pathogenic forms of Abeta, including truncated pyroglutamate and toxic oligomers, is encouraging as it combines the targets of mAbs, such as Aducanumab and Donanemab, recently linked to clinical benefits, and thus, the potential of enhancing plaque reduction. The presented data support the fast progression of optimized ACI‐24 into clinical development as a disease modifying treatment with potential for prevention in AD and Down Syndrome‐related AD.

Ex vivo target engagement of the Abeta oligomer disassembling compound RD2 in patient derived brain homogenates

AbstractBackgroundSoluble Aβ oligomers are highly toxic and may be responsible for development and progression of Alzheimer’s disease (AD). Elimination of Aβ oligomers is therefore a promising therapeutic strategy. The compound RD2 was developed to stabilize Aβ monomers in their native conformation, which is very close to an intrinsically disordered protein (IDP). RD2 has demonstrated already to directly eliminate soluble Aβ oligomers in vitro and in vivo and to improve cognition or decelerate neurodegeneration in four different animal models in four different laboratories 1,2. Here, we wanted to test if RD2 is able to disassemble Aβ oligomers obtained from AD patient derived brain tissue.MethodAβ oligomers in AD patient derived brain tissue homogenates were incubated with different concentrations of RD2 and control compounds. Aβ oligomer concentrations were determined longitudinally by the sFIDA assay, a single‐particle sensitive method for quantitating protein oligomers and aggregates.ResultRD2 concentration and incubation time‐dependent reduction of Aβ oligomers in brain homogenates was observed. Control compounds did not reduce Aβ oligomer concentrations.ConclusionThe incubation time and RD2 concentration dependent disassembly of Aβ oligomers from human brain homogenate suggest a very efficient ability of RD2 to fulfill its mode of action, namely the direct disassembly of Aβ oligomers into their Aβ monomer building blocks. Kinetic analysis and the mode of action resemble very much that of catalytic chaperones.

Distinguishing between amyloid‐beta‐directed antibodies: Ability of PMN310 to target toxic oligomers despite competing species

AbstractBackgroundA large body of evidence indicates that the most pathogenic species of Abeta in Alzheimer’s disease (AD) consist of soluble toxic oligomers as opposed to insoluble fibrils and monomers. The ability of a therapeutic antibody to target toxic Abeta oligomers without being diverted by binding to competing non‐toxic species is expected to result in greater efficacy. This is supported by clinical results to date showing poor efficacy of non‐selective antibodies but improved success with partially selective antibodies that bind oligomers and plaque. As a next generation antibody, PMN310 is a therapeutic candidate designed to more selectively target toxic oligomers. Avoiding interaction with plaque and vascular deposits has the additional advantage of potentially decreasing the incidence of amyloid‐related imaging abnormalities (ARIA). In this study, PMN310 was compared to other Abeta antibodies for selectivity and ability to maintain interaction with toxic oligomers in the presence of competing monomers.MethodsThe binding of multiple Abeta‐directed antibodies (PMN310 and biosimilars donanemab, aducanumab, lecanemab, crenezumab, solanezumab) to synthetic oligomers, with and without pre‐exposure to competing monomers, was evaluated by surface plasmon resonance (SPR). Binding of the antibodies to a toxic oligomer‐enriched low molecular fraction of brain extract from AD patients was similarly evaluated by SPR, with and without monomer competition. Binding to insoluble Abeta deposits was examined by immunohistochemistry (IHC) of AD brain sections.ResultsPMN310 showed little or no interaction with monomers and was among the least impacted by excess monomer competition in binding to synthetic oligomers or naturally occurring toxic oligomers in AD brain extract. This characteristic was shared by other Abeta antibodies that have shown positive clinical outcomes. Non‐selective antibodies that failed in the clinic were strongly inhibited by monomer competition. In contrast to other Abeta antibodies, PMN310 additionally avoided interaction with plaque and vascular deposits as determined by IHC.ConclusionsPMN310 distinguishes itself from other Abeta antibodies by its enhanced selectivity for toxic oligomers (negligible binding to monomers and plaque) along with its ability to withstand competition by abundant monomers. Additionally, the avoidance of interaction with plaque and vascular deposits by PMN310 could potentially reduce the risk of ARIA.

The ryanodine receptor-calstabin interaction stabilizer S107 protects hippocampal neurons from GABAergic synaptic alterations induced by Abeta42 oligomers.

The oligomeric form of the peptide amyloid beta 42 (Abeta42) contributes to the development of synaptic abnormalities and cognitive impairments associated with Alzheimer's disease (AD). To date, there is a gap in knowledge regarding how Abeta42 alters the elementary parameters of GABAergic synaptic function. Here we found that Abeta42 increased the frequency and amplitude of miniature GABAergic currents as well as the amplitude of evoked inhibitory postsynaptic currents. When we focused on paired pulse depression (PPD) to establish whether GABA release probability was affected by Abeta42, we did not observe any significant change. On the other hand, a more detailed investigation of the presynaptic effects induced by Abeta42 by means of multiple probability fluctuation analysis and cumulative amplitude analysis showed an increase in both the size of the readily releasable pool responsible for synchronous release and the number of release sites. We further explored whether ryanodine receptors (RyRs) contributed to exacerbating these changes by stabilizing the interaction between RyRs and the accessory protein calstabin. We observed that the RyR-calstabin interaction stabilizer S107 restored the synaptic parameters to values comparable to those measured in control conditions. In conclusion, our results clarify the mechanisms of potentiation of GABAergic synapses induced by Abeta42. We further suggest that RyRs are involved in the control of synaptic activity during the early stage of AD onset and that their stabilization could represent a new therapeutical approach for AD treatment. KEY POINTS: Accumulation of the peptide amyloid beta 42 (Abeta42) is a key characteristic of Alzheimer's disease (AD) and causes synaptic dysfunctions. To date, the effects of Abeta42 accumulation on GABAergic synapses are poorly understood. The findings reported here suggest that, similarly to what is observed on glutamatergic synapses, Abeta42 modifies GABAergic synapses by targeting ryanodine receptors and causing calcium dysregulation. The GABAergic impairments can be restored by the ryanodine receptor-calstabin interaction stabilizer S107. Based on this research, RyRs stabilization may represent a novel pharmaceutical strategy for preventing or delaying AD.

The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity

Amyloid beta 42 (Abeta42) is the principal trigger of neurodegeneration during Alzheimer’s disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, we here identify the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42‐mediated cell death. We demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co‐purification of Abeta42 with mitochondria and prevents Abeta42‐induced mitochondria‐dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.

Site specific NMR characterization of abeta-40 oligomers cross seeded by abeta-42 oligomers.

Extracellular accumulation of β amyloid peptides of 40 (Aβ40) and 42 residues (Aβ42) has been considered as one of the hallmarks in the pathology of Alzheimer's disease. In this work, we are able to prepare oligomeric aggregates of Aβ with uniform size and monomorphic structure. Our experimental design is to incubate Aβ peptides in reverse micelles (RMs) so that the peptides could aggregate only through a single nucleation process and the size of the oligomers is confined by the physical dimension of the reverse micelles. The hence obtained Aβ oligomers (AβOs) are 23 nm in diameter and they belong to the category of high molecular-weight (MW) oligomers. The solid-state NMR data revealed that Aβ40Os adopt the structural motif of β-loop-β but the chemical shifts manifested that they may be structurally different from low-MW AβOs and mature fibrils. From the thioflavin-T results, we found that high-MW Aβ42Os can accelerate the fibrillization of Aβ40 monomers. Our protocol allows performing cross-seeding experiments among oligomeric species. By comparing the chemical shifts of Aβ40Os cross seeded by Aβ42Os and those of Aβ40Os prepared in the absence of Aβ42Os, we observed that the chemical states of E11, K16, and E22 were altered, whereas the backbone conformation of the β-sheet region near the C-terminus was structurally invariant. The use of reverse micelles allows hitherto the most detailed characterization of the structural variability of Aβ40Os.

Novel Strategy of Anti-Dementia Drug Development for Mild AD Patients

Alzheimer&apos;s disease (AD) is the most common dementia associated with brain inflammation. Aducanumab is the first disease-modifying therapeutics approved by the US-FDA for mild AD patients. Improvements of neuronal plasticity and neural networks in dementia are the next stage of mild AD drug development. However, none of the improvers in neurotransmission failed to prevent the progression to AD. We here introduced the novel improver of brain function through T-type calcium channel activation. Reduced T-type calcium channel levels are reported in aged brain and its inhibition by Abeta oligomers impair spine formation in the brain. We developed T-type calcium channel enhancer SAK3 which activates calcium currents in Neuro2A cells overexpressed Cav3.1 and Cav3.3 but not Cav3.2 channels. SAK3 promoted the hippocampal long-term potentiation (LTP) through CaMKII activation. CaMKII activation in the hippocampus and cortex were also associated with improvement of spine morphology in APP knock-in mice. The proteasome activation possibly mediated CaMKII dependent phosphorylation of Rpt-6 subunit of 19S proteasome in APP knock-in mouse brain. Enhancement of T-type calcium channel in neuroprogenitor cells in the hippocampus was accounted for improvement of neurogenesis, thereby ameliorating depressive behaviors. the diagnostic technology defined early mild dementia patients is critical for promoting clinical development of disease-modifying therapeutics. In the symposium, we also discuss the novel diagnostic technology for mild AD to define brain inflammation.

Stability of the novel blood‐based biomarkers under pre‐analytical sample handling conditions: Results of the SABB‐GBSC working group

AbstractBackgroundPre‐analytical sample handling might affect the results of Alzheimer’s blood‐based biomarkers. In this collaborative study of the SABB‐GBSC working group guided by the Alzheimer’s Association, we elucidate the effects of the most common sample handling variations on blood‐based amyloid and non‐amyloid biomarkers.MethodFreshly collected blood was differentially treated, to create sample sets (n=10 per condition). We investigated the effect of collection tube type, and of EDTA plasma handling variations: delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze‐thawing. Frozen aliquots were shipped to participating labs, to measure amyloid beta (Abeta) peptides (with different technologies: C2N LS‐mass spectrometry (MS), Shimadzu MALID‐MS, N4PE and N3PA Simoa, Euroimmun and Araclon ELISA), Abeta oligomerization tendency (OAβ; PeopleBio), glial fibrillary acidic protein (GFAP; Quanterix), neurofilament light (NfL; Quanterix), total tau (tTau; Quanterix) and phosphorylated tau181 (pTau181; Eli Lilly Simoa assay). Data analysis was descriptive.ResultUsing other sample types than EDTA plasma resulted in different absolute levels for most of the measured biomarkers (figures 1‐4), with generally higher levels in lithium‐heparin plasma, lower levels in sodium‐citrate plasma, and variable but comparable levels in serum. Independent of the technology used, when EDTA plasma was kept at room temperature pre‐ or post‐centrifugation, Abeta42 and Abeta40 levels remained stable for up to 3 hours, while at 24 hours levels were decreased. When kept at 2‐8°C, Abeta levels were stable for up to 24 hours delayed centrifugation or storage (figure 1 and 2). The Abeta42/40 ratio mitigated some of the variability noted in Abeta42 and Abeta40 levels for some of the technologies and conditions, but not for all (figure 3). Abeta42 and Abeta40 levels were robust to differences in tube filling, short‐term ‐20°C storage, and up to 4 freeze‐thaw cycles (figure 1 and 2). While the other biomarkers OAβ, GFAP, NfL and pTau181 were mostly resistant to pre‐analytical sample handling variations, tTau levels were affected by delayed centrifugation and by centrifugation temperature (figure 4).ConclusionBased on the current findings, we construct a standardized operating procedure for pre‐analytical blood sample handling, that will facilitate swift introduction of blood‐based biomarkers into research and clinical settings.

Plaque-Associated Oligomeric Amyloid-Beta Drives Early Synaptotoxicity in APP/PS1 Mice Hippocampus: Ultrastructural Pathology Analysis.

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by initial memory impairments that progress to dementia. In this sense, synaptic dysfunction and loss have been established as the pathological features that best correlate with the typical early cognitive decline in this disease. At the histopathological level, post mortem AD brains typically exhibit intraneuronal neurofibrillary tangles (NFTs) along with the accumulation of amyloid-beta (Abeta) peptides in the form of extracellular deposits. Specifically, the oligomeric soluble forms of Abeta are considered the most synaptotoxic species. In addition, neuritic plaques are Abeta deposits surrounded by activated microglia and astroglia cells together with abnormal swellings of neuronal processes named dystrophic neurites. These periplaque aberrant neurites are mostly presynaptic elements and represent the first pathological indicator of synaptic dysfunction. In terms of losing synaptic proteins, the hippocampus is one of the brain regions most affected in AD patients. In this work, we report an early decline in spatial memory, along with hippocampal synaptic changes, in an amyloidogenic APP/PS1 transgenic model. Quantitative electron microscopy revealed a spatial synaptotoxic pattern around neuritic plaques with significant loss of periplaque synaptic terminals, showing rising synapse loss close to the border, especially in larger plaques. Moreover, dystrophic presynapses were filled with autophagic vesicles in detriment of the presynaptic vesicular density, probably interfering with synaptic function at very early synaptopathological disease stages. Electron immunogold labeling showed that the periphery of amyloid plaques, and the associated dystrophic neurites, was enriched in Abeta oligomers supporting an extracellular location of the synaptotoxins. Finally, the incubation of primary neurons with soluble fractions derived from 6-month-old APP/PS1 hippocampus induced significant loss of synaptic proteins, but not neuronal death. Indeed, this preclinical transgenic model could serve to investigate therapies targeted at initial stages of synaptic dysfunction relevant to the prodromal and early AD.

Donepezil modulation of beta- amyloid oligomer phagocytosis in peripheral monocytes. Bed to bench observations from Alzheimer's disease patients

Beta-Amyloid (Abeta) aggregates are reputed to play a central role in AD pathogenesis, partly by triggering neuroinflammation. Peripheral monocytes can shift into the CNS, hence participating in Abeta oligomer clearance. Donepezil's anti-AD mechanism remains largely elusive; however, it can interact with monocyte α7nAChR that is involved in Abeta phagocytosis. We believe that by binding to α7nAChR, Donepezil can modulate Abeta internalization driven by both α7nAChR and TREM2.

Neuronal expression of a single‐chain variable fragment antibody against Aβ oligomers protects synapses and rescues memory in Alzheimer models

AbstractBackgroundAlzheimer's disease (AD) is the main cause of dementia in the elderly and is characterized by abnormal accumulation of the beta‐amyloid peptide (Aß) in the brain. Considerable evidence has shown that soluble Abeta oligomers (AßOs) are the main neurotoxins involved in synaptic dysfunction and the memory loss.MethodWe evaluated the therapeutic potential of NUsc1, a single chain variable Fragment (scFv) antibody isolated from a screen to identify scFv’s targeting AβOs. Using an adeno‐associated virus derived vector (AAV) we analyzed its protective effects both in vitro and in vivo models of AD. AAV‐NUsc1 capability to induce NUsc1 expression and secretion in humans was tested using human brain slices in culture.ResultWe here show that recombinant NUsc1, a single‐chain variable fragment (scFv) antibody that selectively targets a subset of neurotoxic AβOs, prevented AβO‐induced inhibition of long‐term potentiation in hippocampal slices, and blocked memory impairment induced by intracerebroventricular infusion of AβOs in mice. We next developed an adeno‐associated virus vector to drive neuronal expression of NUsc1 (AAV‐NUsc1) as a novel therapeutic approach for AD. Importantly, transduction by AAV‐NUsc1 induced NUsc1 expression and secretion in adult human brain slices. In primary hippocampal cultures, transduction by AAV‐NUsc1 reduced AβO binding to neurons and prevented AβO‐induced loss of dendritic spines. Remarkably, AAV‐NUsc1 prevented memory impairment caused by AβO infusion in mice and reversed memory deficits in APPswe/PS1ΔE9 AD mice.ConclusionAAV‐NUsc1 represents a potential tool for gene therapy in AD by using an AAV vector to induce in vivo sustained expression of a single chain variable fragment antibody to neutralize AβOs.

Age-related impairment of cerebral blood flow response to KATP channel opener in Alzheimer’s disease mice with presenilin-1 mutation

Local cerebral blood flow (CBF) responses to neuronal activity are essential for cognition and impaired CBF responses occur in Alzheimer’s disease (AD). In this study, regional CBF (rCBF) responses to the KATP channel opener diazoxide were investigated in 3xTgAD, WT and mutant Presenilin 1(PS1M146V) mice from three age groups using Laser-Doppler flowmetry. The rCBF response was reduced early in young 3xTgAD mice and almost absent in old 3xTgAD mice, up to 30%–40% reduction with altered CBF velocity and mean arterial pressure versus WT mice. The impaired rCBF response in 3xTgAD mice was associated with progression of AD pathology, characterized by deposition of intracellular and vascular amyloid-β (Aβ) oligomers, senile plaques and tau pathology. The nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine abolished rCBF response to diazoxide suggesting NO was involved in the mediation of vasorelaxation. Levels of phosphor-eNOS (Ser1177) diminished in 3xTgAD brains with age, while the rCBF response to the NO donor sodium nitroprusside remained. In PS1M146V mice, the rCBF response to dizoxide reduced and high molecular weight Abeta oligomers were increased indicating PS1M146V contributed to the dysregulation of rCBF response in AD mice. Our study revealed an Aβ oligomer-associated compromise of cerebrovascular function in rCBF response to diazoxide in AD mice with PS1M146V mutation.

Differential Inhibition of E-S Potentiation and Long-term Potentiation by Cell-Derived and Arctic Amyloid Beta

Soluble oligomers of amyloid-beta peptide (Abeta) have been implicated in the onset of memory deficits in Alzheimer's disease, perhaps due to their reported ability to impair long-term potentiation (LTP) of synaptic strength. We previously showed the effect of Abeta on LTP depends on the strength of LTP induction. Furthermore, Abeta affects EPSP-Spike (E-S) potentiation more robustly than LTP, suggesting that E-S potentiation may be equally important to learning and memory in the context of Alzheimer's disease. Here we extend our findings to two additional forms of Abeta that form higher concentrations of soluble Abeta oligomers and show that they also affect E-S potentiation at induction strengths where there is no effect on LTP in hippocampal slices.

Amyloidogenic and anti-amyloidogenic properties of presenilin 1.

Presenilin 1 (PS1) is an intramembrane protease, the active subunit of the γ-secretase complex. Its well-studied function is the amyloidogenic cleavage of the C-terminal fragment of the amyloid precursor protein, also known as C99, to produce the Abeta peptide. Recent findings from the Greengard laboratory suggest that PS1 also have anti-amyloidogenic activities, which reduce Abeta levels. First, it redirects APP-C99 toward autophagic degradation, lowering the amount that can be converted into Abeta. The protein kinase CK1γ2 phosphorylates PS1 at Ser367. Phosphorylated PS1 at this position interacts with Annexin A2, which, in turn, interacts with the lysosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vamp8. Annexin A2 facilitates the binding of Vamp8 to the autophagosomal SNARE Syntaxin 17 to modulate the fusion of autophagosomes with lysosomes. Thus, PS1 phosphorylated at Ser367 has an anti-amyloidogenic function, promoting autophagosome-lysosome fusion and increasing C99 degradation. Second, it enhances the ability of microglia to phagocyte and degrade extracellular Abeta oligomer, through regulating the expression of the lysosomal master regulator TFEB. Thus, PS1 has a role in both the production and the clearance of Abeta. Drugs designed to activate CK1γ2 and increase the level of PS1 phosphorylated at Ser367 should be useful in the treatment of Alzheimer's disease.

Amyloid Beta42 oligomers up-regulate the excitatory synapses by potentiating presynaptic release while impairing postsynaptic NMDA receptors.

NMDA receptors (NMDARs) are key molecules for controlling neuronal plasticity, learning and memory processes. Their function is impaired during Alzheimer's disease (AD) but the exact consequence on synaptic function is not yet fully identified. An important hallmark of AD onset is represented by the neuronal accumulation of Amyloid Beta42 oligomers (Abeta42) that we have recently shown to be responsible for the increased intracellular Ca2+ concentration through ryanodine receptors (RyRs). Here we characterized the effects of Abeta42 on NMDA synapses showing specific pre- and post-synaptic functional changes that lead to a potentiation of basal and synchronous NMDA synaptic transmission. These overall effects can be abolished by decreasing Ca2+ release from RyRs with specific inhibitors that we propose as new pharmacological tools for AD treatment. We have recently shown that Amyloid Beta42 oligomers (Abeta42) cause calcium dysregulation in hippocampal neurons by stimulating Ca2+ release from ryanodine receptors (RyRs) and inhibiting Ca2+ entry through NMDA receptors (NMDARs). Here, we found that Abeta42 decrease the average NMDA-activated inward current and that Ca2+ entry through NMDARs is accompanied by Ca2+ release from the stores. The overall amount of intraellular Ca2+ concentration([Ca2+ ]i ) increase during NMDA application is 50% associated with RyR opening and 50% with NMDARs activation. Addition of Abeta42 does not change this proportion. We estimated the number of NMDARs expressed in hippocampal neurons and their unitary current. We found that Abeta42 decrease the number of NMDARs without altering their unitary current. Paradoxically, the oligomer increases the size of electrically evoked eEPSCs induced by NMDARs activation. We found that this is the consequence of the increased release probability (p) of glutamate and the number of release sites (N) of NMDA synapses, while the quantal size (q) is significantly decreased as expected from the decreased number of NMDARs. An increased number of release sites induced by Abeta42 is also supported by the increased size of the ready releasable pool (RRPsyn) and by the enhanced percentage of paired pulse depression (PPD). Interestingly, the RyRs inhibitor dantrolene prevents the increase of PPD induced by Abeta42 oligomers. In conclusion, Abeta42 up-regulates NMDA synaptic responses with a mechanism involving RyRs that occurs during the early stages of Alzheimer's disease (AD) onset. This suggests that new selective modulators of RyRs may be useful for designing effective therapies to treat AD patients.

Sclerotiorin Stabilizes the Assembly of Nonfibrillar Abeta42 Oligomers with Low Toxicity, Seeding Activity, and Beta-sheet Content

The self-assembly of the 42-residue amyloid-β peptide, Aβ42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer’s disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aβ42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aβ42 oligomers. These structures exhibit low β-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, β-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aβ42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aβ42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aβ42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, β-sheet-rich Aβ42 fibrils in AD, while structures with low β-sheet content may be less relevant.