Autosomal‐dominant Alzheimer's disease mutations at the same codon of amyloid precursor protein differentially alter Aβ production

Autosomal‐dominant Alzheimer's disease (ADAD) is a genetic disorder caused by mutations in Amyloid Precursor Protein (APP) or Presenilin (PSEN) genes. Studying the mechanisms underlying these mutations can provide insight into the pathways that lead to AD pathology. The majority of biochemical studies on APP mutations to‐date have focused on comparing mechanisms between mutations at different codons. It has been assumed that amino acid position is a major determinant of protein dysfunction and clinical phenotype. However, the differential effect of mutations at the same codon has not been sufficiently addressed. In the present study we compared the effects of the aggressive ADAD‐associated APP I716F mutation with I716V and I716T on APP processing in human neuroglioma and CHO‐K1 cells. All APP I716 mutations increased the ratio of Aβ42/40 and changed the product line preference of γ‐secretase towards Aβ38 production. In addition, the APP I716F mutation impaired the ε‐cleavage and the fourth cleavage of γ‐secretase and led to abnormal APP β‐CTF accumulation at the plasma membrane. Taken together, these data indicate that APP mutations at the same codon can induce diverse abnormalities in APP processing, some resembling PSEN1 mutations. These differential effects could explain the clinical differences observed among ADAD patients bearing different APP mutations at the same position.

     

Proteome characterization of splenocytes from an Aβpp/ps‐1 Alzheimer's disease model

Alzheimer's disease (AD) is the sixth leading cause of US deaths. In addition to neurodegenerative deficits in AD, changes in the immune system have also been observed. Proteomic analysis of specific immune cell populations may help gain insights into mechanisms of peripheral immunity in AD. Herein, we report proteome characterization for two subsets of splenocytes (i.e. CD90+ cells and a heterogeneous pool of CD90? cells) from a double transgenic mutant amyloid precursor protein/presenilin‐1 (Aβpp/ps‐ 1) AD mouse model. Overall, 906 proteins were identified from both cell types with 275 and 334 proteins uniquely identified as CD90+ and CD90? cells, respectively. Proteins identified in CD90+ and CD90? cells were significantly involved in 18 and 19 KEGG pathways, respectively. Amongst these, pathways associated with AD and antigen processing and presentation were identified in CD90+ and CD90? subsets, respectively. This is the first study to provide a reference proteome map for splenocyte populations in A βpp/ps‐ 1 double transgenic mice which will be helpful for future studies focused on understanding peripheral changes in this model. All MS data have been deposited in the ProteomeXchange with identifier PXD000203 ( http://proteomecentral.proteomexchange.org/dataset/PXD000203 ).     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Neuroprotective role of γ‐enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X

γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.     

Vitexin prevents Aβ proteotoxicity in transgenic Caenorhabditis elegans model of Alzheimer's disease by modulating unfolded protein response

Alzheimer's disease (AD) accounts for an estimated 60% to 80% of all dementia cases. The present study is aimed at evaluating the neuroprotective efficacy of vitexin, an apigenin flavone glycoside using transgenic Caenorhabditis elegans strain (CL2006) of AD. The neuroprotective effect of vitexin was determined using physiological assays, quantitative polymerase chain reaction, and Western blotting. The results of survival and paralysis assay indicate that vitexin (200 μM) significantly extended the lifespan of the nematodes. Vitexin‐treated nematodes showed a significant reduction in the expression of Aβ, ace‐1, and ace‐2 genes when compared to control. Further, vitexin significantly upregulated the expression of acr‐8 and dnj‐14, and increased the lifespan of the nematodes. Vitexin was also found to modulate the unfolded protein response genes (hsp‐4, pek‐1, ire‐1, and xbp‐1) and suppress the expression of Aβ. Overall, the results show that vitexin acts as a neuroprotective agent and protects transgenic C. elegans strains from Aβ proteotoxicity.     

Structural studies of the tethered N‐terminus of the Alzheimer's disease amyloid‐β peptide

Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid‐β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal‐binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1–16 fused to the N‐terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti‐Aβ N‐terminal antibody WO2. The structure demonstrates that Aβ residues 10–16, which are not in complex with the antibody, adopt a mixture of local polyproline II‐helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10–16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13‐metal‐His14 coordination in the Aβ_1–16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758. © 2013 Wiley Periodicals, Inc.     

APOE‐modulated Aβ‐induced neuroinflammation in Alzheimer's disease: current landscape,novel data,and future perspective

Chronic glial activation and neuroinflammation induced by the amyloid‐β peptide (Aβ) contribute to Alzheimer's disease (AD) pathology. APOE4 is the greatest AD‐genetic risk factor; increasing risk up to 12‐fold compared to APOE3, with APOE4‐specific neuroinflammation an important component of this risk. This editorial review discusses the role of APOE in inflammation and AD, via a literature review, presentation of novel data on Aβ‐induced neuroinflammation, and discussion of future research directions. The complexity of chronic neuroinflammation, including multiple detrimental and beneficial effects occurring in a temporal and cell‐specific manner, has resulted in conflicting functional data for virtually every inflammatory mediator. Defining a neuroinflammatory phenotype (NIP) is one way to address this issue, focusing on profiling the changes in inflammatory mediator expression during disease progression. Although many studies have shown that APOE4 induces a detrimental NIP in peripheral inflammation and Aβ‐independent neuroinflammation, data for APOE‐modulated Aβ‐induced neuroinflammation are surprisingly limited. We present data supporting the hypothesis that impaired apoE4 function modulates Aβ‐induced effects on inflammatory receptor signaling, including amplification of detrimental (toll‐like receptor 4‐p38α) and suppression of beneficial (IL‐4R‐nuclear receptor) pathways. To ultimately develop APOE genotype‐specific therapeutics, it is critical that future studies define the dynamic NIP profile and pathways that underlie APOE‐modulated chronic neuroinflammation.

     

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first‐generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD. Second‐generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD. In this review, we evaluate different APP mouse models of AD, and review recent studies using the second‐generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.     

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.     

Bepridil decreases Aβ and calcium levels in the thalamus after middle cerebral artery occlusion in rats

Alzheimer's disease (AD) and cerebral ischaemia share similar features in terms of altered amyloid precursor protein (APP) processing and β‐amyloid (Aβ) accumulation. We have previously shown that Aβ and calcium deposition, and β‐secretase activity, are robustly increased in the ipsilateral thalamus after transient middle cerebral artery occlusion (MCAO) in rats. Here, we investigated whether the non‐selective calcium channel blocker bepridil, which also inhibits β‐secretase cleavage of APP, affects thalamic accumulation of Aβ and calcium and in turn influences functional recovery in rats subjected to MCAO. A 27‐day bepridil treatment (50 mg/kg, p.o.) initiated 2 days after MCAO significantly decreased the levels of soluble Aβ40, Aβ42 and calcium in the ipsilateral thalamus, as compared with vehicle‐treated MCAO rats. Expression of seladin‐1/DHCR24 protein, which is a potential protective factor against neuronal damage, was decreased at both mRNA and protein levels in the ipsilateral thalamus of MCAO rats. Conversely, bepridil treatment restored seladin‐1/DHCR24 expression in the ipsilateral thalamus. Bepridil treatment did not significantly affect heme oxygenase‐1‐ or NAD(P)H quinone oxidoreductase‐1‐mediated oxidative stress or inflammatory responses in the ipsilateral thalamus of MCAO rats. Finally, bepridil treatment mitigated MCAO‐induced alterations in APP processing in the ipsilateral thalamus and improved contralateral forelimb use in MCAO rats. These findings suggest that bepridil is a plausible therapeutic candidate in AD or stroke owing to its multifunctional role in key cellular events that are relevant for the pathogenesis of these diseases.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

A mutation protective against Alzheimer's disease renders amyloid β precursor protein incapable of mediating neurotoxicity

Expression of a familial Alzheimer's disease (AD)‐linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild‐type (wt)‐APP causes neuronal death by activating an intracellular death signal (a APP‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP₇₇₀, corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP₆₉₅ (an AD‐protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD‐protective mutant of APP produce less Aβ than cells expressing wt‐APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐APP, but not in those expressing the AD‐protective mutant of APP. This result suggests that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP‐mediated intracellular death signal.

     

Accelerated microglial pathology is associated with Aβ plaques in mouse models of Alzheimer's disease

Microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network. Here, we used a digital tool for the quantitative morphometric characterization of fine cortical microglial structures in mice, and the changes they undergo with aging and in Alzheimer's‐like disease. We show that, compared with microglia in young mice, microglia in old mice are less ramified and possess fewer branches and fine processes along with a slightly increased proinflammatory cytokine expression. A similar microglial pathology appeared 6–12 months earlier in mouse models of Alzheimer's disease (AD), along with a significant increase in brain parenchyma lacking coverage by microglial processes. We further demonstrate that microglia near amyloid plaques acquire unique activated phenotypes with impaired process complexity. We thus show that along with a chronic proinflammatory reaction in the brain, aging causes a significant reduction in the capacity of microglia to scan their environment. This type of pathology is markedly accelerated in mouse models of AD, resulting in a severe microglial process deficiency, and possibly contributing to enhanced cognitive decline.     

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.