O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

Discovery of aryl aminothiazole γ-secretase modulators with novel effects on amyloid ß-peptide production.

A series of analogs based on a prototype aryl aminothiazole γ-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid ß-peptides (Aß), generated through processive proteolysis of precursor protein substrate by γ-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of Aß production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in Aß37 and Aß38 production without parallel decreases in their precursors Aß40 and Aß42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of Aß peptides ≥ 43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by γ-secretase.

Synthesis of human amyloid restricted to liver results in an Alzheimer disease-like neurodegenerative phenotype.

Several lines of study suggest that peripheral metabolism of amyloid beta (Aß) is associated with risk for Alzheimer disease (AD). In blood, greater than 90% of Aß is complexed as an apolipoprotein, raising the possibility of a lipoprotein-mediated axis for AD risk. In this study, we report that genetic modification of C57BL/6J mice engineered to synthesise human Aß only in liver (hepatocyte-specific human amyloid (HSHA) strain) has marked neurodegeneration concomitant with capillary dysfunction, parenchymal extravasation of lipoprotein-Aß, and neurovascular inflammation. Moreover, the HSHA mice showed impaired performance in the passive avoidance test, suggesting impairment in hippocampal-dependent learning. Transmission electron microscopy shows marked neurovascular disruption in HSHA mice. This study provides causal evidence of a lipoprotein-Aß /capillary axis for onset and progression of a neurodegenerative process.

APOE4-carrying human astrocytes oversupply cholesterol to promote neuronal lipid raft expansion and Aß generation.

The ε4 allele of APOE-encoding apolipoprotein (ApoE) is one of the strongest genetic risk factors for Alzheimer's disease (AD). One of the overarching questions is whether and how this astrocyte-enriched risk factor initiates AD-associated pathology in neurons such as amyloid-ß (Aß) accumulation. Here, we generate neurons and astrocytes from isogenic human induced pluripotent stem cells (hiPSCs) carrying either APOE ε3 or APOE ε4 allele and investigate the effect of astrocytic ApoE4 on neuronal Aß production. Secretory factors in conditioned media from ApoE4 astrocytes significantly increased amyloid precursor protein (APP) levels and Aß secretion in neurons. We further found that increased cholesterol secretion from ApoE4 astrocytes was necessary and sufficient to induce the formation of lipid rafts that potentially provide a physical platform for APP localization and facilitate its processing. Our study reveals the contribution of ApoE4 astrocytes to amyloidosis in neurons by expanding lipid rafts and facilitating Aß production through an oversupply of cholesterol.

Suppression of amyloid-ß secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid.

Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-ß (Aß) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aß in mouse neurons. CLA treatment did not affect the level of ß-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aß protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aß generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aß in neurons.

Polymorphic SERPINA3-R124C reduces pathogenesis of its wild type by shortening the lifetime of oligomeric Aß.

Amyloid beta (Aß) 42 peptide accumulated in Alzheimer disease (AD) patients' brain, often colocalized with serine protease inhibitor family A member 3 (SERPINA3). Being a chaperon, SERPINA3 accelerated Aß42 fibrillization. While analyzing chaperon activity of human SERPINA3 polymorphisms, we found SERPINA3-R124C played a role in protecting cells from Aß42 cytotoxicity. SH-SY5Y cells exposed to Aß42 preincubated with wild-type SERPINA3 (SERPINA3-WT) resulted in extended toxicity leading cell death whereas Aß42 with SERPINA3-R124C resulted in less cytotoxicity. Transmission electron microscope and thioflavin T assay revealed that SERPINA3-R124C shortened lifetime of small soluble oligomer and maintained ß-sheet rich protofibril-like aggregates for longer time compared to that of with SERPINA3-WT. Western blot assay confirmed that SERPINA3-R124C converted Aß42 mostly into high molecular aggregates. Here, we demonstrate first time that polymorphic SERPINA3 acts as a benign chaperon by modulating the transition states of Aß42, which may contribute to the reduction of AD risk.

MiR-216a-5p ameliorates learning-memory deficits and neuroinflammatory response of Alzheimer's disease mice via regulation of HMGB1/NF-κB signaling.

OBJECTIVE: The objective of this study was to explore whether miR-216a-5p could affect the learning-memory ability and inflammatory response of Alzheimer's disease (AD) mice via regulation of the HMGB1/NF-κB pathway. METHODS: Mice were divided into the normal (wild-type C57BL/6 mice), AD (APP/PS1 double-transgenic mice), AD + miR-216a-5p, and AD + vector groups. The Morris water maze test was used to examine learning and memory ability. Nissl staining and TUNEL staining were performed to observe the survival and apoptosis of hippocampal neurons. In addition, Aß deposition and the expression of inflammatory cytokines were determined, while miR-216a-5p expression and HMGB1/NF-κB pathway-related proteins were detected by qRT-PCR and Western blotting, respectively. RESULTS: AD mice exhibited decreased miR-216a-5p expression but increased HMGB-1 protein expression in the hippocampus, and these mice had a prolonged escape latency, fewer number of times crossing the platform location and shortened time in the target quadrant compared to those in normal mice. AD mice also had an elevated number of TUNEL-positive cells, increased deposition of Aß, increased expression of inflammatory cytokines and decreased number of Nissl-positive cells. In addition, AD mice presented with downregulated expression of cytoplasmic NF-κB p65 protein but upregulated expression of nuclear NF-κB p65 protein. However, AD mice treated with miR-216a-5p exhibited significant improvements of the abovementioned parameters. The dual-luciferase reporter assay confirmed that HMGB1 is a target gene of miR-216a-5p. CONCLUSION: MiR-216a-5p can improve learning-memory ability and attenuate the inflammatory response of AD mice through targeted inhibition of the HMGB1/NF-κB pathway.

C subunit of the ATP synthase is an amyloidogenic calcium dependent channel-forming peptide with possible implications in mitochondrial permeability transition.

The c subunit is an inner mitochondrial membrane (IMM) protein encoded by three nuclear genes. Best known as an integral part of the F0 complex of the ATP synthase, the c subunit is also present in other cytoplasmic compartments in ceroid lipofuscinoses. Under physiological conditions, this 75 residue-long peptide folds into an α-helical hairpin and forms oligomers spanning the lipid bilayer. In addition to its physiological role, the c subunit has been proposed as a key participant in stress-induced IMM permeabilization by the mechanism of calcium-induced permeability transition. However, the molecular mechanism of the c subunit participation in IMM permeabilization is not completely understood. Here we used fluorescence spectroscopy, atomic force microscopy and black lipid membrane methods to gain insights into the structural and functional properties of unmodified c subunit protein that might make it relevant to mitochondrial toxicity. We discovered that c subunit is an amyloidogenic peptide that can spontaneously fold into ß-sheets and self-assemble into fibrils and oligomers in a Ca2+-dependent manner. C subunit oligomers exhibited ion channel activity in lipid membranes. We propose that the toxic effects of c subunit might be linked to its amyloidogenic properties and are driven by mechanisms similar to those of neurodegenerative polypeptides such as Aß and α-synuclein.

Oral Treponema denticola Infection Induces Aß1-40 and Aß1-42 Accumulation in the Hippocampus of C57BL/6 Mice.

Accumulation of amyloid-ß (Aß) in the brain is a central component of pathology in Alzheimer's disease. A growing volume of evidence demonstrates close associations between periodontal pathogens including Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola) and AD. However, the effect and mechanisms of T. denticola on accumulation of Aß remain to be unclear. In this study, we demonstrated that T. denticola was able to enter the brain and act directly on nerve cells resulting in intra- and extracellular Aß1-40 and Aß1-42 accumulation in the hippocampus of C57BL/6 mice by selectively activating both ß-secretase and γ-secretase. Furthermore, both KMI1303, an inhibitor of ß-secretase, as well as DAPT, an inhibitor of γ- secretase, were found to be able to inhibit the effect of T. denticola on Aß accumulation in N2a neuronal cells. Overall, it is concluded that T. denticola increases the expression of Aß1-42 and Aß1-40 by its regulation on beta-site amyloid precursor protein cleaving enzyme-1 and presenilin 1.

Impairment of visual cortical plasticity by amyloid-beta species.

INTRODUCTION: A variety of transgenic and knock-in mice that express mutant alleles of Amyloid precursor protein (APP) have been used to model the effects of amyloid-beta (Aß) on circuit function in Alzheimer's disease (AD); however phenotypes described in these mice may be affected by expression of mutant APP or proteolytic cleavage products independent of Aß. In addition, the effects of mutant APP expression are attributed to elevated expression of the amyloidogenic, 42-amino acid-long species of Aß (Aß42) associated with amyloid plaque accumulation in AD, though elevated concentrations of Aß40, an Aß species produced with normal synaptic activity, may also affect neural function. METHODS: To explore the effects of elevated expression of Aß on synaptic function in vivo, we assessed visual system plasticity in transgenic mice that express and secrete Aß throughout the brain in the absence of APP overexpression. Transgenic mice that express either Aß40 or Aß42 were assayed for their ability to appropriately demonstrate ocular dominance plasticity following monocular deprivation. RESULTS: Using two complementary approaches to measure the plastic response to monocular deprivation, we find that male and female mice that express either 40- or 42-amino acid-long Aß species demonstrate a plasticity defect comparable to that elicited in transgenic mice that express mutant alleles of APP and Presenilin 1 (APP/PS1 mice). CONCLUSIONS: These data support the hypothesis that mutant APP-driven plasticity impairment in mouse models of AD is mediated by production and accumulation of Aß. Moreover, these findings suggest that soluble species of Aß are capable of modulating synaptic plasticity, likely independent of any aggregation. These findings may have implications for the role of soluble species of Aß in both development and disease settings.

Treadmill Exercise Alleviates Brain Iron Dyshomeostasis Accelerating Neuronal Amyloid-ß Production, Neuronal Cell Death, and Cognitive Impairment in Transgenic Mice Model of Alzheimer's Disease.

Brain iron increases with age and abnormal brain iron metabolism is proving increasingly likely to be involved in the pathology of Alzheimer's disease (AD). The iron-regulatory effect of furin, a ubiquitously expressed proconvertase, might play an important role in AD. Therefore, there is an urgent need to study the effect of furin on iron regulation in AD. For that purpose, we aimed to determine the role of physical exercise in AD associated with brain iron dyshomeostasis. Treadmill exercise attenuated the AD-related abnormal brain iron regulation by furin in vivo, as demonstrated via experiments in aged APP-C105 mice. Next, we examined whether treadmill exercise decreases excessive iron, directly affecting amyloid-ß (Aß) production through the regulation of α-secretase-dependent processing of amyloid protein precursor (APP) involved in the modulation of furin activity. We first observed that cognitive decline and Aß-induced neuronal cell death were induced by disruption of APP processing via excess iron-induced disruption of furin activity in aged APP-C105 mice. The induced cognitive decline and cell death were attenuated by treadmill exercise. This result suggests that treadmill exercise alleviated cognitive decline and Aß-induced neuronal cell death by promoting α-secretase-dependent processing of APP through low iron-induced enhancement of furin activity. This is concomitant with decreasing levels of lipid peroxidation products and promoting antioxidant defense enzyme capacities. Therefore, iron-targeted therapeutic strategies involving treadmill exercise might be useful for patients with AD.

Astrocytic expression of the Alzheimer's disease risk allele, ApoEε4, potentiates neuronal tau pathology in multiple preclinical models.

ApoEε4 is a major genetic risk factor for Alzheimer's disease (AD), a disease hallmarked by extracellular amyloid-beta (Aß) plaques and intracellular neurofibrillary tangles (NFTs). The presence of the ApoEε4 allele is associated with increased Aß deposition and a role for ApoEε4 in the potentiation of tau pathology has recently emerged. This study focused on comparing the effects of adeno-associated virus (AAV)-mediated overexpression of the three predominant human ApoE isoforms within astrocytes. The isoform-specific effects of human ApoE were evaluated within in vitro models of tau pathology within neuron/astrocyte co-cultures, as well as in a transgenic tau mouse model. Tau aggregation, accumulation, and phosphorylation were measured to determine if the three isoforms of human ApoE had differential effects on tau. Astrocytic overexpression of the human ApoEε4 allele increased phosphorylation and misfolding of overexpressed neuronal tau in multiple models, including the aggregation and accumulation of added tau oligomers, in an isoform-specific manner. The ability of ApoEε4 to increase tau aggregation could be inhibited by an ApoEε4-specific antibody. This study indicates that astrocytic expression of ApoEε4 can potentiate tau aggregation and phosphorylation within neurons and supports a gain of toxic function hypothesis for the effect of hApoEε4 on tau.

Promiscuous Installation of d-Amino Acids in Gene-Encoded Peptides.

d-Amino acids can have major effects on the structure, proteolytic stability, and bioactivity of peptides. Proteusin radical S-adenosyl methionine epimerases regioselectively install such residues in ribosomal peptides to generate peptides with the largest number of d-residues currently known in biomolecules. To study their utility in synthetic biology, we investigated the substrate tolerance and substrate-product relationships of the cyanobacterial model epimerase OspD using libraries of point mutants as well as distinct extended peptides that were fused to an N-terminal leader sequence. OspD was found to exhibit exceptional substrate promiscuity in E. coli, accepting 15 different amino acids and converting peptides with a broad range of compositions, secondary structures, and polarities. Diverse single and multiple epimerization patterns were identified that were dictated by the peptide sequence. The data suggest major potential in creating genetically encoded products previously inaccessible by synthetic biology.

Association of Vitamin D Receptor Polymorphisms with Amyloid-ß Transporters Expression and Risk of Mild Cognitive Impairment in a Chilean Cohort.

BACKGROUND: Amyloid-ß peptide (Aß) deposition in Alzheimer's disease (AD) is due to an imbalance in its production/clearance rate. Aß is transported across the blood-brain barrier by LRP1 and P-gp as efflux transporters and RAGE as influx transporter. Vitamin D deficit and polymorphisms of the vitamin D receptor (VDR) gene are associated with high prevalence of mild cognitive impairment (MCI) and AD. Further, vitamin D promotes the expression of LRP1 and P-gp in AD-animal model brains. OBJECTIVE: To associate VDR polymorphisms Apa I (rs7975232), Taq I (rs731236), and Fok I (rs2228570) with the risk of developing MCI in a Chilean population, and to evaluate the relationship of these polymorphisms to the expression of VDR and Aß-transporters in peripheral blood mononuclear cells (PBMCs). METHODS: VDR polymorphisms Apa I, Taq I, and Fok I were determined in 128 healthy controls (HC) and 66 MCI patients. mRNA levels of VDR and Aß-transporters were evaluated in subgroups by qPCR. RESULTS: Alleles A of Apa I and C of Taq I were associated with a lower risk of MCI. HC with the Apa I AA genotype had higher mRNA levels of P-gp and LRP1, while the expression of VDR and RAGE were higher in MCI patients and HC. For Fok I, the TC genotype was associated with lower expression levels of Aß-transporters in both groups. CONCLUSION: We propose that the response to vitamin D treatment will depend on VDR polymorphisms, being more efficient in carriers of protective alleles of Apa I polymorphism.

Pimavanserin, a 5HT2A receptor inverse agonist, rapidly suppresses Aß production and related pathology in a mouse model of Alzheimer's disease.

Amyloid-ß (Aß) peptide aggregation into soluble oligomers and insoluble plaques is a precipitating event in the pathogenesis of Alzheimer's disease (AD). Given that synaptic activity can regulate Aß generation, we postulated that 5HT2A -Rs may regulate Aß as well. We treated APP/PS1 transgenic mice with the selective 5HT2A inverse agonists M100907 or Pimavanserin systemically and measured brain interstitial fluid (ISF) Aß levels in real-time using in vivo microdialysis. Both compounds reduced ISF Aß levels by almost 50% within hours, but had no effect on Aß levels in 5HT2A -R knock-out mice. The Aß-lowering effects of Pimavanserin were blocked by extracellular-regulated kinase (ERK) and NMDA receptor inhibitors. Chronic administration of Pimavanserin by subcutaneous osmotic pump to aged APP/PS1 mice significantly reduced CSF Aß levels and Aß pathology and improved cognitive function in these mice. Pimavanserin is FDA-approved to treat Parkinson's disease psychosis, and also has been shown to reduce psychosis in a variety of other dementia subtypes including Alzheimer's disease. These data demonstrate that Pimavanserin may have disease-modifying benefits in addition to its efficacy against neuropsychiatric symptoms of Alzheimer's disease. Read the Editorial Highlight for this article on page 560.

PGC-1α reduces Amyloid-ß deposition in Alzheimer's disease: Effect of increased VDR expression.

Amyloid-ß (Aß) is the core component of amyloid plaques of Alzheimer's disease (AD). Recent evidence has confirmed that Aß triggers neurodegeneration by dramatically suppressing vitamin D receptor (VDR) expression. Thus far, the onset mechanisms and means of preventing AD are largely unknown. Perioxisome proliferator-activated receptor-γ coactivator (PGC-1α), as a transcriptional coactivator of VDR could protect cells against oxidative stress. Thus, upregulation of PGC-1α is a candidate therapeutic strategy for AD. To investigate the effect of PGC-1α in AD, and to illuminate the precise involvement of VDR in the neuroprotective strategy, the varies of molecular of PGC-1α and VDR were studied in APP/PS-1 double transgenic (2xTg-AD) mice at 6 months of age, significant reduction in the expression of PGC-1α and VDR was found in their hippocampus and the cortex. Besides, a specific mouse line, Dlx5/6-Cre:PGC-1αfl/fl in which the PGC-1α deficiency was limited to the hippocampus and the cortex, was used to study the target intervention of PGC-1α, decreased expression of VDR and increased oxidative damage were observed in AD-related brain regions by PGC-1α deficiency. To explore the function and therapeutic strategy of PGC-1α in AD, an adeno-associated virus (AAV) was used to induce PGC-1α overexpressed in the hippocampus of 2xTg-AD mice. Overexpressed PGC-1α results in a remarkable increase in the levels of VDR associated with a significant reduction in the expression of Aß plaques and of 8-oxo-dG in 2xTg-AD mice. These data may have ramifications for neuroprotective strategies targeting overexpression of PGC-1α in Alzheimer's disease.

Design and synthesis of novel methoxypyridine-derived gamma-secretase modulators.

The evolution of gamma-secretase modulators (GSMs) through the introduction of novel heterocycles with the goal of aligning activity for reducing the levels of Aß42 and properties consistent with a drug-like molecule are described. The insertion of a methoxypyridine motif within the tetracyclic scaffold provided compounds with improved activity for arresting Aß42 production as well as improved properties, including solubility. In vivo pharmacokinetic analysis demonstrated that several compounds within the novel series were capable of crossing the BBB and accessing the therapeutic target. Treatment with methoxypyridine-derived compound 64 reduced Aß42 levels in the plasma of J20 mice, in addition to reducing Aß42 levels in the plasma and brain of Tg2576 mice.

Effect of tert-alcohol functional imidazolium salts on oligomerization and fibrillization of amyloid ß (1-42) peptide.

Imidazolium based IL's has gained vast interest in developing biological applications. Oligomerization and fibrillization of amyloid ß (1-42) peptide are mainly responsible for the extra-neuronal deposition of amyloid fibrils in neurodegenerative disorders like Alzheimer's disease (AD). Here, we report an effect of tert-BuOH-functional imidazolium ILs on oligomerization and fibrillization of amyloid ß (1-42) Peptide in vitro. In this study, a series of these [alkyl-tOHim][OMs] ILs with methyl sulphonate counter anion by varying alkyl chains were used. Among the seven protic ILs, four showed strong binding and inhibition activity for the formation of amyloid ß (1-42) aggregation by using Thioflavin T fluorescence binding assay. The secondary structural analysis of the peptide, pre-incubated with active ILs shows the loss of ordered ß-sheet amyloid structure. The longer alkyl chain ILs showed that an increased in amyloid binding and hence an inhibition effect on amyloid aggregation was enhanced. Thus, we propose that ILs could be presented as potential candidates for therapeutic intervention against Alzheimer's disease (AD).

ACE2 activator diminazene aceturate ameliorates Alzheimer's disease-like neuropathology and rescues cognitive impairment in SAMP8 mice.

Previously, we revealed that brain Ang-(1-7) deficiency was involved in the pathogenesis of sporadic Alzheimer's disease (AD). We speculated that restoration of brain Ang-(1-7) levels might have a therapeutic effect against AD. However, the relatively short duration of biological effect limited the application of Ang-(1-7) in animal experiments. Since Ang-(1-7) is generated by its metabolic enzyme ACE2, we then tested the efficacy of an ACE2 activator diminazene aceturate (DIZE) on AD-like neuropathology and cognitive impairment in senescence-accelerated mouse prone substrain 8 (SAMP8) mice, an animal model of sporadic AD. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or DIZE once a day for 30 consecutive days. DIZE markedly elevated brain Ang-(1-7) and MAS1 levels. Meanwhile, DIZE significantly reduced the levels of Aß1-42, hyperphosphorylated tau and pro-inflammatory cytokines in the brain. The synaptic and neuronal losses in the brain were ameliorated by DIZE. Importantly, DIZE improved spatial cognitive functions in the Morris water maze test. In conclusion, this study demonstrates that DIZE ameliorates AD-like neuropathology and rescues cognitive impairment in SAMP8 mice. These beneficial effects of DIZE may be achieved by activating brain ACE2/Ang-(1-7)/MAS1 axis. These findings highlight brain ACE2/Ang-(1-7)/MAS1 axis as a potential target for the treatment of sporadic AD.