Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770⁺ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770⁺ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App^NL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770⁺:App^NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.     

Amyloid β-peptide directly induces spontaneous calcium transients,delayed intercellular calcium waves and gliosis in rat cortical astrocytes

The contribution of astrocytes to the pathophysiology of AD (Alzheimer''s disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer''s transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer''s brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.     

A third-generation mouse model of Alzheimer's disease shows early and increased cored plaque pathology composed of wild-type human amyloid β peptide

We previously developed single App knock-in mouse models of Alzheimer''s disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (App^NL-G-F and App^NL-F mice, respectively). These models showed Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer''s disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the App^NL-F line with the Psen1^P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new third-generation mouse model showed more cored plaque pathology and neuroinflammation than App^NL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.     

β-Arrestin1 regulates γ-secretase complex assembly and modulates amyloid-β pathology

Alzheimer''s disease (AD) is a progressive and complex neurodegenerative disease in which the γ-secretase-mediated amyloid-β (Aβ) pathology plays an important role. We found that a multifunctional protein, β-arrestin1, facilitated the formation of NCT/APH-1 (anterior pharynx-defective phenotype 1) precomplex and mature γ-secretase complex through its functional interaction with APH-1. Deficiency of β-arrestin1 or inhibition of binding of β-arrestin1 with APH-1 by small peptides reduced Aβ production without affecting Notch processing. Genetic ablation of β-arrestin1 diminished Aβ pathology and behavioral deficits in transgenic AD mice. Moreover, in brains of sporadic AD patients and transgenic AD mice, the expression of β-arrestin1 was upregulated and correlated well with neuropathological severity and senile Aβ plaques. Thus, our study identifies a regulatory mechanism underlying both γ-secretase assembly and AD pathogenesis, and indicates that specific reduction of Aβ pathology can be achieved by regulation of the γ-secretase assembly.     

Gain of PITRM1 peptidase in cortical neurons affords protection of mitochondrial and synaptic function in an advanced age mouse model of Alzheimer’s disease

Mitochondrial dysfunction is one of the early pathological features of Alzheimer''s disease (AD). Accumulation of cerebral and mitochondrial Aβ links to mitochondrial and synaptic toxicity. We have previously demonstrated the mechanism by which presequence peptidase (PITRM1)‐mediated clearance of mitochondrial Aβ contributes to mitochondrial and cerebral amyloid pathology and mitochondrial and synaptic stress in adult transgenic AD mice overexpressing Aβ up to 12 months old. Here, we investigate the effect of PITRM1 in an advanced age AD mouse model (up to 19–24 months) to address the fundamental unexplored question of whether restoration/gain of PITRM1 function protects against mitochondrial and synaptic dysfunction associated with Aβ accumulation and whether this protection is maintained even at later ages featuring profound amyloid pathology and synaptic failure. Using newly developed aged PITRM1/Aβ‐producing AD mice, we first uncovered reduction in PITRM1 expression in AD‐affected cortex of AD mice at 19–24 months of age. Increasing neuronal PITRM1 activity/expression re‐established mitochondrial respiration, suppressed reactive oxygen species, improved synaptic function, and reduced loss of synapses even at advanced ages (up to 19–24 months). Notably, loss of PITRM1 proteolytic activity resulted in Aβ accumulation and failure to rescue mitochondrial and synaptic function, suggesting that PITRM1 activity is required for the degradation and clearance of mitochondrial Aβ and Aβ deposition. These data indicate that augmenting PITRM1 function results in persistent life‐long protection against Aβ toxicity in an AD mouse model. Therefore, augmenting PITRM1 function may enhance Aβ clearance in mitochondria, thereby maintaining mitochondrial integrity and ultimately slowing the progression of AD.     

Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples

Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-β (Aβ) levels. Evidence suggests physical interactions between apoE and Aβ are partially responsible for these functional effects. However, the apoE/Aβ complex is not a single static structure; rather, it is defined by detection methods. Thus, literature results are inconsistent and difficult to interpret. An ELISA was developed to measure soluble apoE/Aβ in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aβ and an increase in soluble Aβ, specifically oligomeric Aβ (oAβ), are associated with APOE4 and AD. Previously, soluble Aβ42 and oAβ levels were greater with APOE4 compared with APOE2/APOE3 in hippocampal homogenates from EFAD transgenic mice (expressing five familial AD mutations and human apoE isoforms). In this study, soluble apoE/Aβ levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aβ levels isoform-specifically modulate soluble oAβ clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aβ levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aβ levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aβ42 levels decreased in AD patients compared with controls, oAβ levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aβ modulates oAβ levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.     

Death-associated protein kinase 1 mediates Aβ42 aggregation-induced neuronal apoptosis and tau dysregulation in Alzheimer's disease

The aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils is a key pathological feature of Alzheimer''s disease (AD). An increasing amount of evidence suggests that oligomeric Aβ might be the major culprit responsible for various neuropathological changes in AD. Death-associated protein kinase 1 (DAPK1) is abnormally elevated in brains of AD patients and plays an important role in modulating tau homeostasis by regulating prolyl isomerase Pin1 phosphorylation. However, it remains elusive whether and how Aβ species influence the function of DAPK1, and whether this may further affect the function and phosphorylation of tau in neurons. Herein, we demonstrated that Aβ aggregates (both oligomers and fibrils) prepared from synthetic Aβ42 peptides were able to upregulate DAPK1 protein levels and thereby its function through heat shock protein 90 (HSP90)-mediated protein stabilization. DAPK1 activation not only caused neuronal apoptosis, but also phosphorylated Pin1 at the Ser71 residue, leading to tau accumulation and phosphorylation at multiple AD-related sites in primary neurons. Both DAPK1 knockout (KO) and the application of a specific DAPK1 inhibitor could effectively protect primary neurons against Aβ aggregate-induced cell death and tau dysregulation, corroborating the critical role of DAPK1 in mediating Aβ aggregation-induced neuronal damage. Our study suggests a mechanistic link between Aβ oligomerization and tau hyperphosphorylation mediated by DAPK1, and supports the role of DAPK1 as a promising target for early intervention in AD.     

Deficiency of Neuronal p38α MAPK Attenuates Amyloid Pathology in Alzheimer Disease Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.     

Activation of CREB‐mediated autophagy by thioperamide ameliorates β‐amyloid pathology and cognition in Alzheimer’s disease

Alzheimer''s disease (AD) is an age‐related neurodegenerative disease, and the imbalance between production and clearance of β‐amyloid (Aβ) is involved in its pathogenesis. Autophagy is an intracellular degradation pathway whereby leads to removal of aggregated proteins, up‐regulation of which may be a plausible therapeutic strategy for the treatment of AD. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Our previous study showed that thioperamide, as an antagonist of H3R, enhances autophagy and protects against ischemic injury. However, the effect of thioperamide on autophagic function and Aβ pathology in AD remains unknown. In this study, we found that thioperamide promoted cognitive function, ameliorated neuronal loss, and Aβ pathology in APP/PS1 transgenic (Tg) mice. Interestingly, thioperamide up‐regulated autophagic level and lysosomal function both in APP/PS1 Tg mice and in primary neurons under Aβ‐induced injury. The neuroprotection by thioperamide against AD was reversed by 3‐MA, inhibitor of autophagy, and siRNA of Atg7, key autophagic‐related gene. Furthermore, inhibition of activity of CREB, H3R downstream signaling, by H89 reversed the effect of thioperamide on promoted cell viability, activated autophagic flux, and increased autophagic‐lysosomal proteins expression, including Atg7, TFEB, and LAMP1, suggesting a CREB‐dependent autophagic activation by thioperamide in AD. Taken together, these results suggested that H3R antagonist thioperamide improved cognitive impairment in APP/PS1 Tg mice via modulation of the CREB‐mediated autophagy and lysosomal pathway, which contributed to Aβ clearance. This study uncovered a novel mechanism involving autophagic regulating behind the therapeutic effect of thioperamide in AD.     

Amyloid-β Pathology and APOE Genotype Modulate Retinoid X Receptor Agonist Activity in Vivo

Previous data demonstrate that bexarotene (Bex), retinoid X receptor (RXR) agonist, reduces soluble and insoluble amyloid-β (Aβ) in Alzheimer disease (AD)-transgenic mice either by increasing the levels of mouse apolipoprotein E (apoE) or increasing ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. However, although the mechanism of action of RXR agonists remains unclear, a major concern for their use is human (h)-APOE4, the greatest AD genetic risk factor. If APOE4 imparts a toxic gain-of-function, then increasing apoE4 may increase soluble Aβ, likely the proximal AD neurotoxin. If the APOE4 loss-of-function is lipidation of apoE4, then induction of ABCA1/ABCG1 may be beneficial. In novel EFAD-Tg mice (overexpressing h-Aβ42 with h-APOE), levels of soluble Aβ (Aβ42 and oligomeric Aβ) are highest in E4FAD hippocampus (HP) > E3FAD-HP > E4FAD cortex (CX) > E3FAD-CX, whereas levels of lipoprotein-associated/lipidated apoE have the opposite pattern (6 months). In E4FAD-HP, short-term RXR agonist treatment (Bex or {"type":"entrez-nucleotide","attrs":{"text":"LG100268","term_id":"1041422930","term_text":"LG100268"}}LG100268; 5.75–6 months) increased ABCA1, apoE4 lipoprotein-association/lipidation, and apoE4/Aβ complex, decreased soluble Aβ, and increased PSD95. In addition, hydrogel delivery, which mimics low sustained release, was equally effective as gavage for Bex and {"type":"entrez-nucleotide","attrs":{"text":"LG100268","term_id":"1041422930","term_text":"LG100268"}}LG100268. RXR agonists induced no beneficial effects in the E4FAD-HP in a prevention protocol (5–6 months) and actually increased soluble Aβ levels in E3FAD-CX and E4FAD-CX with the short-term protocol, possibly the result of systemic hepatomegaly. Thus, RXR agonists address the loss-of-function associated with APOE4 and exacerbated by Aβ pathology, i.e. low levels of apoE4 lipoprotein association/lipidation. Further studies are vital to address whether RXR agonists are an APOE4-specific AD therapeutic and the systemic side effects that limit translational application.     

High temperature promotes amyloid β-protein production and γ-secretase complex formation via Hsp90

Alzheimer''s disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aβ production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aβ production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aβ production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.     

Immunogenic properties of amyloid beta oligomers

Background

The central molecule in the pathogenesis of Alzheimer’s disease (AD) is believed to be a small-sized polypeptide – beta amyloid (Aβ) which has an ability to assemble spontaneously into oligomers. Various studies concerning therapeutic and prophylactic approaches for AD are based on the immunotherapy using antibodies against Aβ. It has been suggested that either active immunization with Aβ or passive immunization with anti-Aβ antibodies might help to prevent or reduce the symptoms of the disease. However, knowledge on the mechanisms of Aβ-induced immune response is rather limited. Previous research on Aβ1-42 oligomers in rat brain cultures showed that the neurotoxicity of these oligomers considerably depends on their size. In the current study, we evaluated the dependence of immunogenicity of Aβ1-42 oligomers on the size of oligomeric particles and identified the immunodominant epitopes of the oligomers.

Results

Mice were immunized with various Aβ1-42 oligomers. The analysis of serum antibodies revealed that small Aβ1-42 oligomers (1–2 nm in size) are highly immunogenic. They induced predominantly IgG2b and IgG2a responses. In contrast, larger Aβ1-42 oligomers and monomers induced weaker IgG response in immunized mice. The monoclonal antibody against 1–2 nm Aβ1-42 oligomers was generated and used for antigenic characterization of Aβ1-42 oligomers. Epitope mapping of both monoclonal and polyclonal antibodies demonstrated that the main immunodominant region of the 1–2 nm Aβ1-42 oligomers is located at the amino-terminus (N-terminus) of the peptide, between amino acids 1 and 19.

Conclusions

Small Aβ1-42 oligomers of size 1–2 nm induce the strongest immune response in mice. The N-terminus of Aβ1-42 oligomers represents an immunodominant region which indicates its surface localization and accessibility to the B cells. The results of the current study may be important for further development of Aβ-based vaccination and immunotherapy strategies.     

Gut inflammation triggers C/EBPβ/δ‐secretase‐dependent gut‐to‐brain propagation of Aβ and Tau fibrils in Alzheimer’s disease

Inflammation plays an important role in the pathogenesis of Alzheimer''s disease (AD). Some evidence suggests that misfolded protein aggregates found in AD brains may have originated from the gut, but the mechanism underlying this phenomenon is not fully understood. C/EBPβ/δ‐secretase signaling in the colon was investigated in a 3xTg AD mouse model in an age‐dependent manner. We applied chronic administration of 1% dextran sodium sulfate (DSS) to trigger gut leakage or colonic injection of Aβ or Tau fibrils or AD patient brain lysates in 3xTg mice and combined it with excision/cutting of the gut–brain connecting vagus nerve (vagotomy), in order to explore the role of the gut–brain axis in the development of AD‐like pathologies and to monitor C/EBPβ/δ‐secretase signaling under those conditions. We found that C/EBPβ/δ‐secretase signaling is temporally activated in the gut of AD patients and 3xTg mice, initiating formation of Aβ and Tau fibrils that spread to the brain. DSS treatment promotes gut leakage and facilitates AD‐like pathologies in both the gut and the brain of 3xTg mice in a C/EBPβ/δ‐secretase‐dependent manner. Vagotomy selectively blunts this signaling, attenuates Aβ and Tau pathologies, and restores learning and memory. Aβ or Tau fibrils or AD patient brain lysates injected into the colon propagate from the gut into the brain via the vagus nerve, triggering AD pathology and cognitive dysfunction. The results indicate that inflammation activates C/EBPβ/δ‐secretase and initiates AD‐associated pathologies in the gut, which are subsequently transmitted to the brain via the vagus nerve.     

The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid-β Production and Tau Hyperphosphorylation

We have previously shown that the L-type calcium channel (LCC) antagonist nilvadipine reduces brain amyloid-β (Aβ) accumulation by affecting both Aβ production and Aβ clearance across the blood-brain barrier (BBB). Nilvadipine consists of a mixture of two enantiomers, (+)-nilvadipine and (−)-nilvadipine, in equal proportion. (+)-Nilvadipine is the active enantiomer responsible for the inhibition of LCC, whereas (−)-nilvadipine is considered inactive. Both nilvadipine enantiomers inhibit Aβ production and improve the clearance of Aβ across the BBB showing that these effects are not related to LCC inhibition. In addition, treatment of P301S mutant human Tau transgenic mice (transgenic Tau P301S) with (−)-nilvadipine reduces Tau hyperphosphorylation at several Alzheimer disease (AD) pertinent epitopes. A search for the mechanism of action of (−)-nilvadipine revealed that this compound inhibits the spleen tyrosine kinase (Syk). We further validated Syk as a target-regulating Aβ by showing that pharmacological inhibition of Syk or down-regulation of Syk expression reduces Aβ production and increases the clearance of Aβ across the BBB mimicking (−)-nilvadipine effects. Moreover, treatment of transgenic mice overexpressing Aβ and transgenic Tau P301S mice with a selective Syk inhibitor respectively decreased brain Aβ accumulation and Tau hyperphosphorylation at multiple AD relevant epitopes. We show that Syk inhibition induces an increased phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase-3β, a primary Tau kinase involved in Tau phosphorylation, by activating protein kinase A, providing a mechanism explaining the reduction of Tau phosphorylation at GSK3β-dependent epitopes following Syk inhibition. Altogether our data highlight Syk as a promising target for preventing both Aβ accumulation and Tau hyperphosphorylation in AD.     

Alzheimer Presenilin-1 Mutations Dramatically Reduce Trimming of Long Amyloid β-Peptides (Aβ) by γ-Secretase to Increase 42-to-40-Residue Aβ

The presenilin-containing γ-secretase complex produces the amyloid β-peptide (Aβ) through intramembrane proteolysis, and >100 presenilin mutations are associated with familial early-onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aβ critical to pathogenesis, but other Aβs of 38–49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aβ. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aβ: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Here we show that the addition of synthetic long Aβ peptides (Aβ45–49) directly into purified preparations of γ-secretase leads to the formation of Aβ40 and Aβ42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aβ42 to Aβ40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aβ peptides to Aβ40 and Aβ42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aβ42/Aβ40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aβ42/Aβ40.     

Microglial cathepsin E plays a role in neuroinflammation and amyloid β production in Alzheimer’s disease

Regulation of neuroinflammation and β‐amyloid (Aβ) production are critical factors in the pathogenesis of Alzheimer''s disease (AD). Cathepsin E (CatE), an aspartic protease, is widely studied as an inducer of growth arrest and apoptosis in several types of cancer cells. However, the function of CatE in AD is unknown. In this study, we demonstrated that the ablation of CatE in human amyloid precursor protein knock‐in mice, called APP^NL−G−F mice, significantly reduced Aβ accumulation, neuroinflammation, and cognitive impairments. Mechanistically, microglial CatE is involved in the secretion of soluble TNF‐related apoptosis‐inducing ligand, which plays an important role in microglia‐mediated NF‐κB‐dependent neuroinflammation and neuronal Aβ production by beta‐site APP cleaving enzyme 1. Furthermore, cannula‐delivered CatE inhibitors improved memory function and reduced Aβ accumulation and neuroinflammation in AD mice. Our findings reveal that CatE as a modulator of microglial activation and neurodegeneration in AD and suggest CatE as a therapeutic target for AD by targeting neuroinflammation and Aβ pathology.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

Validation and Characterization of a Novel Peptide That Binds Monomeric and Aggregated β-Amyloid and Inhibits the Formation of Neurotoxic Oligomers

Although the formation of β-amyloid (Aβ) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aβ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble Aβ oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted Aβ and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D''Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203–214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t = 300 min, c.f. t = 30 min for the parent peptide). This candidate reduced the formation of soluble Aβ42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25–30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aβ42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aβ42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aβ42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies.