Systemic vaccination with anti‐oligomeric monoclonal antibodies improves cognitive function by reducing Aβ deposition and tau pathology in 3xTg‐AD mice

Alzheimer's disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid‐beta (Aβ) peptide plays a pivotal role in the pathogenesis of AD. In AD, the conversion of Aβ from a physiological soluble monomeric form into insoluble fibrillar conformation is an important event. The most toxic form of Aβ is oligomers, which is the intermediate step during the conversion of monomeric form to fibrillar form. There are at least two types of oligomers: oligomers that are immunologically related to fibrils and those that are not. In transgenic AD animal models, both active and passive anti‐Aβ immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. In this report we studied effect of immunotherapy of two sequence‐independent non‐fibrillar oligomer specific monoclonal antibodies on the cognitive function, amyloid load and tau pathology in 3xTg‐AD mice. Anti‐oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduced tau hyperphosphorylation and improvement in cognition. These results demonstrate that systemic immunotherapy using oligomer‐specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in 3xTg‐AD mice. These findings demonstrate the potential of using oligomer specific monoclonal antibodies as a therapeutic approach to prevent and treat Alzheimer's disease.     

Calpain cleavage and inactivation of the sodium calcium exchanger‐3 occur downstream of Aβ in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β‐amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase‐3, activity is a prominent feature of AD brain. In addition, we observe increased calpain‐mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1–42. We also show that exposure of primary cortical neurons to oligomeric Aβ1–42 results in calpain‐dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.     

Antibodies bound to Aβ oligomers potentiate the neurotoxicity of Aβ by activating microglia

Beta amyloid (Aβ) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aβ immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aβ would prevent the neurotoxicity of Aβ oligomers in primary neuronal‐glial cultures. However, surprisingly, the antibodies dramatically increased the neurotoxicity of Aβ. Antibodies bound to monomeric Aβ fragments were non‐toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody‐oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aβ oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal‐glial cultures via Fc‐dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aβ or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.

     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Revisiting the peripheral sink hypothesis: inhibiting BACE1 activity in the periphery does not alter β‐amyloid levels in the CNS

Aggregation of amyloid beta (Aβ) peptides and the subsequent neural plaque formation is a central aspect of Alzheimer's disease. Various strategies to reduce Aβ load in the brain are therefore intensely pursued. It has been hypothesized that reducing Aβ peptides in the periphery, that is in organs outside the brain, would be a way to diminish Aβ levels and plaque load in the brain. In this report, we put this peripheral sink hypothesis to test by investigating how selective inhibition of Aβ production in the periphery using a β‐secretase (BACE)1 inhibitor or reduced BACE1 gene dosage affects Aβ load in the brain. Selective inhibition of peripheral BACE1 activity in wild‐type mice or mice over‐expressing amyloid precursor protein (APPswe transgenic mice; Tg2576) reduced Aβ levels in the periphery but not in the brain, not even after chronic treatment over several months. In contrast, a BACE1 inhibitor with improved brain disposition reduced Aβ levels in both brain and periphery already after acute dosing. Mice heterozygous for BACE1, displayed a 62% reduction in plasma Aβ40, whereas brain Aβ40 was only lowered by 11%. These data suggest that reduction of Aβ in the periphery is not sufficient to reduce brain Aβ levels and that BACE1 is not the rate‐limiting enzyme for Aβ processing in the brain. This provides evidence against the peripheral sink hypothesis and suggests that a decrease in Aβ via BACE1 inhibition would need to be carried out in the brain.

     

Ibrutinib modulates Aβ/tau pathology,neuroinflammation, and cognitive function in mouse models of Alzheimer's disease

We previously demonstrated that ibrutinib modulates LPS‐induced neuroinflammation in vitro and in vivo, but its effects on the pathology of Alzheimer''s disease (AD) and cognitive function have not been investigated. Here, we investigated the effects of ibrutinib in two mouse models of AD. In 5xFAD mice, ibrutinib injection significantly reduced Aβ plaque levels by promoting the non‐amyloidogenic pathway of APP cleavage, decreased Aβ‐induced neuroinflammatory responses, and significantly downregulated phosphorylation of tau by reducing levels of phosphorylated cyclin‐dependent kinase‐5 (p‐CDK5). Importantly, tau‐mediated neuroinflammation and tau phosphorylation were also alleviated by ibrutinib injection in PS19 mice. In 5xFAD mice, ibrutinib improved long‐term memory and dendritic spine number, whereas in PS19 mice, ibrutinib did not alter short‐ and long‐term memory but promoted dendritic spinogenesis. Interestingly, the induction of dendritic spinogenesis by ibrutinib was dependent on the phosphorylation of phosphoinositide 3‐kinase (PI3K). Overall, our results suggest that ibrutinib modulates AD‐associated pathology and cognitive function and may be a potential therapy for AD.     

Characterization of the interaction between Aβ 1–42 and glyceraldehyde phosphodehydrogenase

Advances in the understanding of AD pathogenesis have recently provided strong support for a modified Aβ protein cascade hypothesis, stating that several different Aβ assemblies contribute to the triggering of a complex pathological cascade leading to neurodegeneration. Both in vitro and in vivo, Aβ rapidly forms fibrils (fAβ), which are able to interact with various molecular partners, including proteins, lipids and proteoglycans. In a previous study aimed to identify some of these molecular partners of fAβ, we demonstrated that the GAPDH was specifically coprecipitated with fAβ. The aim of this study was to characterize this interaction. First, it was shown by TEM that synthetic GAPDH directly binds fAβ 1–42. Then rat synaptosomal proteins were purified and incubated with different forms of Aβ in various conditions, and the presence of GAPDH among the proteins coprecipitated with Aβ was studied by western blotting. Results showed that the interaction between GAPDH and fAβ 1–42 is nonionic, as is not impaired by increasing salt concentrations. GAPDH is coprecipitated not only by fAβ, but also by nonfibrillar forms of Aβ 1–42. The 41–42 Aβ sequence seems to be important in the interaction of GAPDH and Aβ, as more GAPDH was coprecipitated with fAβ 1–42 than with fAβ 1–40. GAPDH extracted from various subcellular fractions including mitochondria, was shown to interact with fAβ. Our data demonstrate a direct interaction between Aβ and GAPDH and support the possibility that this interaction has an important pathogenic role in AD. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

HDAC3 negatively regulates spatial memory in a mouse model of Alzheimer's disease

The accumulation and deposition of beta‐amyloid (Aβ) is a key neuropathological hallmark of Alzheimer's disease (AD). Histone deacetylases (HDACs) are promising therapeutic targets for the treatment of AD, while the specific HDAC isoforms associated with cognitive improvement are poorly understood. In this study, we investigate the role of HDAC3 in the pathogenesis of AD. Nuclear HDAC3 is significantly increased in the hippocampus of 6‐ and 9‐month‐old APPswe/PS1dE9 (APP/PS1) mice compared with that in age‐matched wild‐type C57BL/6 (B6) mice. Lentivirus ‐mediated inhibition or overexpression of HDAC3 was used in the hippocampus of APP/PS1 mice to investigate the role of HDAC3 in spatial memory, amyloid burden, dendritic spine density, glial activation and tau phosphorylation. Inhibition of HDAC3 in the hippocampus attenuates spatial memory deficits, as indicated in the Morris water maze test, and decreases amyloid plaque load and Aβ levels in the brains of APP/PS1 mice. Dendritic spine density is increased, while microglial activation is alleviated after HDAC3 inhibition in the hippocampus of 9‐month‐old APP/PS1 mice. Furthermore, HDAC3 overexpression in the hippocampus increases Aβ levels, activates microglia, and decreases dendritic spine density in 6‐month‐old APP/PS1 mice. In conclusion, our results indicate that HDAC3 negatively regulates spatial memory in APP/PS1 mice and HDAC3 inhibition might represent a potential therapy for the treatment of AD.     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.

     

Knockout of 5‐lipoxygenase prevents dexamethasone‐induced tau pathology in 3xTg mice

Emerging evidence suggests that dysregulation stress hormones, such as glucocorticoids, in aged persons put them at a higher risk to develop Alzheimer's disease (AD). However, the mechanisms underlying such vulnerability remain to be unraveled. Pharmacologic inhibition of 5‐lipoxygenase (5LO), an active player in AD pathogenesis whose protein level increases with aging in the human, has been shown to blunt glucocorticoid‐mediated amyloid β (Ab) formation in vitro. In this article, we investigated the role of this pathway in modulating the development of the corticosteroid‐dependent AD‐like phenotype in the triple transgenic mice (3xTg). Dexamethasone was administered for 1 week to 3xTg or 3xTg genetically deficient for 5LO (3xTg/5LO^?/?) mice, and its effect on memory, amyloid‐β and tau levels, and metabolism assessed. At the end of the treatment, we observed that dexamethasone did not induce changes in behavior. Compared with controls, treated mice did not show significant alterations in brain soluble Aβ levels. While total tau protein levels were unmodified in all groups, we found that dexamethasone significantly increased tau phosphorylation at S396, as recognized by the antibody PHF‐13, which was specifically associated with an increase in the GSK3β activity. Additionally, dexamethasone‐treated mice had a significant increase in the tau insoluble fraction and reduction in the postsynaptic protein PDS‐95. By contrast, these modifications were blunted in the 3xTg/5LO^?/? mice. Our findings highlight the functional role that 5LO plays in stress‐induced AD tau pathology and support the hypothesis that pharmacologic inhibition of this enzyme could be a useful tool for individuals with this risk factor.     

Brains of rhesus monkeys display Aβ deposits and glial pathology while lacking Aβ dimers and other Alzheimer's pathologies

Cerebral amyloid beta (Aβ) deposits are the main early pathology of Alzheimer's disease (AD). However, abundant Aβ deposits also occur spontaneously in the brains of many healthy people who are free of AD with advancing aging. A crucial unanswered question in AD prevention is why AD does not develop in some elderly people, despite the presence of Aβ deposits. The answer may lie in the composition of Aβ oligomer isoforms in the Aβ deposits of healthy brains, which are different from AD brains. However, which Aβ oligomer triggers the transformation from aging to AD pathogenesis is still under debate. Some researchers insist that the Aβ 12‐mer causes AD pathology, while others suggest that the Aβ dimer is the crucial molecule in AD pathology. Aged rhesus monkeys spontaneously develop Aβ deposits in the brain with striking similarities to those of aged humans. Thus, rhesus monkeys are an ideal natural model to study the composition of Aβ oligomer isoforms and their downstream effects on AD pathology. In this study, we found that Aβ deposits in aged monkey brains included 3‐mer, 5‐mer, 9‐mer, 10‐mer, and 12‐mer oligomers, but not 2‐mer oligomers. The Aβ deposits, which were devoid of Aβ dimers, induced glial pathology (microgliosis, abnormal microglia morphology, and astrocytosis), but not the subsequent downstream pathologies of AD, including Tau pathology, neurodegeneration, and synapse loss. Our results indicate that the Aβ dimer plays an important role in AD pathogenesis. Thus, targeting the Aβ dimer is a promising strategy for preventing AD.     

Alzheimer's disease BIN1 coding variants increase intracellular Aβ levels by interfering with BACE1 recycling

Genetic studies have identified BIN1 as the second most important risk locus associated with late-onset Alzheimer''s disease (LOAD). However, it is unclear how mutation of this locus mechanistically promotes Alzheimer’s disease (AD) pathology. Here we show the consequences of two coding variants in BIN1 (rs754834233 and rs138047593), both in terms of intracellular beta-amyloid (iAbeta) accumulation and early endosome enlargement, two interrelated early cytopathological AD phenotypes, supporting their association with LOAD risk. We previously found that Bin1 deficiency potentiates iAbeta production by enabling BACE1 cleavage of the amyloid precursor protein in enlarged early endosomes due to decreased BACE1 recycling. Here, we discovered that the expression of the two LOAD mutant forms of Bin1 does not rescue the iAbeta accumulation and early endosome enlargement induced by Bin1 knockdown and recovered by wild-type Bin1. Moreover, the overexpression of Bin1 mutants, but not wild-type Bin1, increased the iAbeta42 fragment by reducing the recycling of BACE1, which accumulated in early endosomes, recapitulating the phenotype of Bin1 knockdown. We showed that the mutations in Bin1 reduced its interaction with BACE1. The endocytic recycling of transferrin was similarly affected, indicating that Bin1 is a general regulator of endocytic recycling. These data demonstrate that the LOAD-coding variants in Bin1 lead to a loss of function in endocytic recycling, which may be an early causal mechanism of LOAD.     

Impairments of long‐term depression induction and motor coordination precede Aβ accumulation in the cerebellum of APPswe/PS1dE9 double transgenic mice

Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β‐ and γ‐secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity; cerebellar long‐term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre‐Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model.