Aβ Degradation Research Articles

Gardenia jasminoides J.Ellis extract GJ-4 alleviated cognitive deficits of APP/PS1 transgenic mice

BackgroundAccumulating evidence demonstrates that traditional Chinese medicines that act on multiple targets could effectively treat various multi-etiological diseases, including cerebrovascular diseases, Alzheimer's disease (AD), Parkinson's disease (PD) and so on. Previous studies have shown that crocin richments (GJ-4), Gardenia jasminoides J.Ellis extract, provide neuroprotective effects on cognitive impairments in AD mouse models. However, the mechanism how GJ-4 improves cognition remains still unclear. PurposeThe aim of this study was to uncover the protective effects and underlying mechanism of GJ-4 on PrP-hAβPPswe/PS1ΔE9 (APP/PS1) transgenic mice. MethodsAPP/PS1 mice were given GJ-4 (10, 20, and 50 mg/kg), donepezil (5 mg/kg) and memantine (5 mg/kg) orally at eight months of age for 12 consecutive weeks. Morris water maze and novel object recognition were conducted to assess the cognitive ability of mice. The release of inflammatory cytokines was determined by RT-PCR assay, and the pathological features of neurons and microglia were assayed by immunohistochemistry and immunofluorescence assay. The expression of Aβ-related proteins and signaling pathways were determined by Western blot. ResultsThe behavioral results revealed that GJ-4 ameliorated the cognitive deficits of APP/PS1 mice measured by Morris water maze and novel object recognition tests. Mechanism studies indicated that GJ-4 significantly decreased β-amyloid (Aβ) level through reducing Aβ production and promoting Aβ degradation. It has been reported that Aβ plaques trigger the hyper-phosphorylation of tau protein in APP/PS1 mice. Consistent with previous studies, hyper-phosphorylation of tau was also occurred in APP/PS1 mice in the present study, and GJ-4 inhibited Tau phosphorylation at different sites. Overwhelming evidence indicates that neuroinflammation stimulated by Aβ and hyperphosphorylated tau is involved in the pathological progression of AD. We found that GJ-4 suppressed neuroinflammatory responses in the brain through regulating phosphatidylinositide 3-kinase/AKT (PI3K/AKT) signaling pathway activation, and subsequent expression of inflammatory proteins and release of inflammatory cytokines. ConclusionAltogether, GJ-4 ameliorated cognition of APP/PS1 transgenic mice through multiple targets, including Aβ, tau and neuroinflammation. This study provides a solid research basis for further development of GJ-4 as a potential candidate for the treatment of AD.

Idebenone Decreases Aβ Pathology by Modulating RAGE/Caspase-3 Signaling and the Aβ Degradation Enzyme NEP in a Mouse Model of AD.

Simple SummaryThe present study reveals that the FDA-approved drug idebenone has therapeutic effects on the pathology of Alzheimer’s disease (AD) in a mouse model. In particular, idebenone regulates pathological progression associated with Aβ by downregulating the non-amyloidogenic pathway, inhibiting RAGE/caspase-3 signaling, and enhancing Aβ catabolism. In addition, idebenone modulates tauopathy by reducing levels of the tau kinase p-GSK3β, thereby suppressing tau hyperphosphorylation at Thr231. These data suggest that idebenone modulates Aβ and tau pathology in a mouse model of AD.The coenzyme Q10 analogue idebenone is an FDA-approved antioxidant that can cross the blood–brain barrier (BBB). The effects of idebenone on the pathology of Alzheimer’s disease (AD) and the underlying molecular mechanisms have not been comprehensively investigated. Here, we examined the impact of idebenone treatment on AD pathology in 5xFAD mice, a model of AD. Idebenone significantly downregulated Aβ plaque number via multi-directional pathways in this model. Specifically, idebenone reduced the RAGE/caspase-3 signaling pathway and increased levels of the Aβ degradation enzyme NEP and α-secretase ADAM17 in 5xFAD mice. Importantly, idebenone significantly suppressed tau kinase p-GSK3βY216 levels, thereby inhibiting tau hyperphosphorylation at Thr231 and total tau levels in 5xFAD mice. Taken together, the present study indicates that idebenone modulates amyloidopathy and tauopathy in 5xFAD mice, suggesting therapeutic potential for AD.

APOE4 expression is associated with impaired autophagy and mitophagy in astrocytes.

APOE4 expression is associated with impaired autophagy and mitophagy in astrocytes.

Impact of age, hypercholesterolemia, and the vitamin D receptor on brain endogenous β-amyloid peptide accumulation in mice.

Age, hypercholesterolemia, and vitamin D deficiency are risk factors that increase the brain accumulation of pathogenic β-amyloid peptides (40 and 42), precursors leading to Alzheimer's disease (AD) in humans. The relative changes accompanying aging, high cholesterol, and/or treatment of calcitriol, active vitamin D receptor (VDR) ligand, under normal physiology are unknown. We examined these relative changes in C57BL/6 mice of ages 2, 4-8, and more than 10 months old, which were fed a normal or high fat / high cholesterol diet and treated with calcitriol, active ligand of the vitamin D receptor (0 or 2.5μg/kg ×4, intraperitoneally, every other day to elicit cholesterol lowering in liver). Aβ40 but not Aβ42 accumulation in brain and lower P-glycoprotein (P-gp) and neprilysin protein expressions for Aβ efflux and degradation, respectively, were found to be associated with aging. But there was no trend for BACE1 (β-secretase 1, a cholesterol-sensitive enzyme) toward Aβ synthesis with age. In response to calcitriol treatment, P-gp was elevated, mitigating partially the age-related changes. Although age-dependent decreasing trends in mRNA expression levels existed for Cyp46a1, the brain cholesterol processing enzyme, whose inhibition increases BACE1 and ApoE to facilitate microglia Aβ degradation, mRNA changes for other cholesterol transporters: Acat1 and Abca1, and brain cholesterol levels remained unchanged. There was no observable change in the mRNA expression of amyloid precursor protein (APP) and the influx (RAGE) and efflux (LRP1) transporters with respect to age, diet, or calcitriol treatment. Overall, aging poses as a risk factor contributing to Aβ accumulation in brain, and VDR-mediated P-gp activation partially alleviates the outcome.

Dietary magnesium ions block the effects of nutrient deficiency on inducing the formation of LC3B autophagosomes and disrupting the proteolysis of autolysosomes to degrade β-amyloid protein by activating cyclooloxygenase-2 at tyrosine 385

• As an important mineral nutrient, Mg 2+ inhibits the activity of COX-2 via mTORC1. • By nutrient deprivation, COX-2 activation induces formation of LC3B autophagosomes. • Mg 2+ disrupts the formation of LC3B autophagosomes via deactivating the COX-2. • COX-2 disturbs hydrolysis of Aβ via suppressing activity of UCH-L1 in lysosomes. • Tyr 385 is critical for the activity of COX-2 in cognitive decline of AD. Nutrient deficiency has been recently suggested to be associated with the deposition of β-amyloid protein (Aβ) during the course of Alzheimer’s disease (AD) development and progression. We thereby aimed to reveal the roles of a deficiency in important mineral nutrient, magnesium ions (Mg 2+ ), in the deposition of Aβ via autophagy mechanisms. Specifically, we report here that Mg 2+ functions as a critical enhancer of the formation of the mammalian target of rapamycin complex 1 (mTORC1) by inhibiting the expression and enzymatic activity of cyclooxygenase-2 (COX-2), which is responsible for inducing autophagosome formation, as indicated by the microtubule-associated protein 1 light chain 3 beta (LC3B) puncta. Although autophagosome generation was strongly induced in COX-2 Tg mice, COX-2 overexpression facilitated Aβ deposition by disrupting the proteolytic function of autolysosomes through an ubiquitin carboxy terminal hydrolase L1 (UCH-L1)-depressing mechanism. More interestingly, COX-2 mutation at Tyr 385 restores the activity of UCH-L1, which results in Aβ degradation and improvements in the cognitive decline of APP/PS1 Tg mice.

Elevated Expression of MiR-17 in Microglia of Alzheimer's Disease Patients Abrogates Autophagy-Mediated Amyloid-β Degradation.

Autophagy is a proposed route of amyloid-β (Aβ) clearance by microglia that is halted in Alzheimer’s Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aβ and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aβ deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aβ degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aβ degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.

Redox-Active Metal Ions and Amyloid-Degrading Enzymes in Alzheimer's Disease.

Redox-active metal ions, Cu(I/II) and Fe(II/III), are essential biological molecules for the normal functioning of the brain, including oxidative metabolism, synaptic plasticity, myelination, and generation of neurotransmitters. Dyshomeostasis of these redox-active metal ions in the brain could cause Alzheimer’s disease (AD). Thus, regulating the levels of Cu(I/II) and Fe(II/III) is necessary for normal brain function. To control the amounts of metal ions in the brain and understand the involvement of Cu(I/II) and Fe(II/III) in the pathogenesis of AD, many chemical agents have been developed. In addition, since toxic aggregates of amyloid-β (Aβ) have been proposed as one of the major causes of the disease, the mechanism of clearing Aβ is also required to be investigated to reveal the etiology of AD clearly. Multiple metalloenzymes (e.g., neprilysin, insulin-degrading enzyme, and ADAM10) have been reported to have an important role in the degradation of Aβ in the brain. These amyloid degrading enzymes (ADE) could interact with redox-active metal ions and affect the pathogenesis of AD. In this review, we introduce and summarize the roles, distributions, and transportations of Cu(I/II) and Fe(II/III), along with previously invented chelators, and the structures and functions of ADE in the brain, as well as their interrelationships.

Attenuation of Pb-induced Aβ generation and autophagic dysfunction via activation of SIRT1: Neuroprotective properties of resveratrol

This study examined the neuroprotective properties of resveratrol (Res) and its target sirtuin1 (SIRT1) against lead (Pb)-mediated toxicity and discovered that both resveratrol treatment and SIRT1 overexpression restored blocked autophagic flux as well as reduced β-amyloid (Aβ) contents. Four-week-old male C57BL/6 mice were employed to consumed 0.2% Pb(Ac)2 solution or deionized water for 3 months followed by 12 months of Res (50 mg/kg BW) or vehicle gavage. In in vitro study, SH-SY5Y cells were pretreated with the SIRT1 activator SRT1720 (2 μM) or the inhibitor EX527 (2 μM) for 2 h, then 25 μM of Pb(Ac)2 was added and incubated for 48 h. Western blotting, RT-qPCR, enzyme-linked immunosorbent assay (ELISA), and Lyso-Tracker Red Staining were next used to estimate the potential alterations of the autophagic pathway as well as BACE1-mediated amyloid processing in response to Pb exposure, respectively. Our data revealed that Res treatment or SIRT1 activation resisted the induction of autophagy by Pb exposure through inhibition of LC3 and Beclin-1 expression and promoted the degradation of Aβ and Tau phosphorylation. Besides, the SIRT1 activator (SRT1720) downregulated the expression of BACE1, the rate-limiting enzyme for Aβ production, by inhibiting the activation of nuclear factor-κB (NF-κB) in Pb-treated SH-SY5Y cells, which resulted in reduced Aβ production. Collectively, we verified the role of Res-SIRT1-autophagy as well as the SIRT1-NF-κB-BACE1 pathway in Pb-induced neuronal cell injury by in vivo or in vitro models. Our findings further elucidate the important role of SIRT1 and Res in counteracting Pb neurotoxicity, which may provide new interventions and targets for the subsequent treatment of neurodegenerative diseases.

Wide-Ranging Effects on the Brain Proteome in a Transgenic Mouse Model of Alzheimer's Disease Following Treatment with a Brain-Targeting Somatostatin Peptide.

Alzheimer’s disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-β (Aβ) peptide. A potential therapeutic intervention in Alzheimer’s disease is to enhance Aβ degradation by increasing the activity of Aβ-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound Aβ42 degradation in the hippocampus of mice overexpressing the Aβ-precursor protein with the Swedish mutation (APPswe). Using LC–MS, we further evaluated the anti-Alzheimer’s disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC–MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated α (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer’s disease therapies based on SST peptides.

Role of Neuron and Glia in Alzheimer’s Disease and Associated Vascular Dysfunction

Amyloidogenicity and vascular dysfunction are the key players in the pathogenesis of Alzheimer’s disease (AD), involving dysregulated cellular interactions. An intricate balance between neurons, astrocytes, microglia, oligodendrocytes and vascular cells sustains the normal neuronal circuits. Conversely, cerebrovascular diseases overlap neuropathologically with AD, and glial dyshomeostasis promotes AD-associated neurodegenerative cascade. While pathological hallmarks of AD primarily include amyloid-β (Aβ) plaques and neurofibrillary tangles, microvascular disorders, altered cerebral blood flow (CBF), and blood-brain barrier (BBB) permeability induce neuronal loss and synaptic atrophy. Accordingly, microglia-mediated inflammation and astrogliosis disrupt the homeostasis of the neuro-vascular unit and stimulate infiltration of circulating leukocytes into the brain. Large-scale genetic and epidemiological studies demonstrate a critical role of cellular crosstalk for altered immune response, metabolism, and vasculature in AD. The glia associated genetic risk factors include APOE, TREM2, CD33, PGRN, CR1, and NLRP3, which correlate with the deposition and altered phagocytosis of Aβ. Moreover, aging-dependent downregulation of astrocyte and microglial Aβ-degrading enzymes limits the neurotrophic and neurogenic role of glial cells and inhibits lysosomal degradation and clearance of Aβ. Microglial cells secrete IGF-1, and neurons show a reduced responsiveness to the neurotrophic IGF-1R/IRS-2/PI3K signaling pathway, generating amyloidogenic and vascular dyshomeostasis in AD. Glial signals connect to neural stem cells, and a shift in glial phenotype over the AD trajectory even affects adult neurogenesis and the neurovascular niche. Overall, the current review informs about the interaction of neuronal and glial cell types in AD pathogenesis and its critical association with cerebrovascular dysfunction.

Lovastatin attenuates sevoflurane-induced cognitive disorder in aged rats via reducing Aβ accumulation

As a general anesthetic widely used in surgical, sevoflurane has been shown to cause cognitive and memory deficits in the elderly. It's important to find out agents that can counteract sevoflurane-induced cognitive dysfunction. This study is aimed to investigate the effect of lovastatin on sevoflurane-induced cognitive impairment in aged rats and reveal the potential mechanisms. BV-2 cells, rat hippocampal neurons or male aged rats were exposed to 2% sevoflurane for 5 h. The cells were pretreated with 10 μM lovastatin. The rats were intraperitoneally injected with 5 mg/kg/day lovastatin for three days. The results showed that lovastatin enhanced exosomal IDE secretion from sevoflurane-exposed BV-2 cells and promoted Aβ degradation. Lovastatin treatment also inhibited the increased expressions of β-secretase 1 (BACE1) and γ-secretase in hippocampal neurons under sevoflurane exposure in vitro. In animal experiments, the discrimination index in novel object recognition test and percentage of spontaneous alternation in Y-maze test were significantly elevated after lovastatin administration. In addition, Aβ plaque area and contents of soluble Aβ1-40 and Aβ1-42 in the hippocampal tissues were decreased upon lovastatin treatment. Furthermore, lovastatin reversed sevoflurane-induced Aβ accumulation via up-regulating IDE expression, and down-regulating amyloid precursor protein (APP)-related protein expression (β-C-terminal fragment (CTF), BACE1 and γ-secretase). In conclusion, lovastatin alleviates sevoflurane-induced cognitive deficient in aged rats via promoting Aβ degradation and reducing Aβ production. Lovastatin may be beneficial in preventing anesthetic-induced cognitive impairment.

Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer's Disease (Mouse Model) through Autophagy Induced by Activating Axl.

Alzheimer’s disease (AD) is thought to be caused by amyloid-β (Aβ) accumulation in the central nervous system due to deficient clearance. The aim of the present study was to investigate the effect of ganoderic acid A (GAA) on Aβ clearance in microglia and its anti-AD activity. Aβ degradation in BV2 microglial cells was determined using an intracellular Aβ clearance assay. GAA stimulated autophagosome formation via the Axl receptor tyrosine kinase (Axl)/RAC/CDC42-activated kinase 1 (Pak1) pathway was determined by Western blot analyses, and fluorescence-labeled Aβ42 was localized in lysosomes in confocal laser microscopy images. The in vivo anti-AD activity of GAA was evaluated by object recognition and Morris water maze (MWM) tests in an AD mouse model following intracerebroventricular injection of aggregated Aβ42. The autophagy level in the hippocampus was assayed by immunohistochemical assessment against microtubule-associated proteins 1A/1B light-chain 3B (LC3B). Intracellular Aβ42 levels were significantly reduced by GAA treatment in microglial cells. Additionally, GAA activated autophagy according to increased LC3B-II levels, with this increased autophagy stimulated by upregulating Axl and Pak1 phosphorylation. The effect of eliminating Aβ by GAA through autophagy was reversed by R428, an Axl inhibitor, or IPA-3, a Pak1 inhibitor. Consistent with the cell-based assay, GAA ameliorated cognitive deficiency and reduced Aβ42 levels in an AD mouse model. Furthermore, LC3B expression in the hippocampus was up-regulated by GAA treatment, with these GAA-specific effects abolished by R428. GAA promoted Aβ clearance by enhancing autophagy via the Axl/Pak1 signaling pathway in microglial cells and ameliorated cognitive deficiency in an AD mouse model.

A Blood-Brain Barrier Penetrant Photooxygenation Catalyst for Aβ Degradation

Key words photooxygenation - brain-penetrant - amyloid β - Alzheimer’s disease

A nanocleaner specifically penetrates the blood‒brain barrier at lesions to clean toxic proteins and regulate inflammation in Alzheimer's disease

Insurmountable blood‒brain barrier (BBB) and complex pathological features are the key factors affecting the treatment of Alzheimer's disease (AD). Poor accumulation of drugs in lesion sites and undesired effectiveness of simply reducing Aβ deposition or TAU protein need to be resolved urgently. Herein, a nanocleaner is designed with a rapamycin-loaded ROS-responsive PLGA core and surface modification with KLVFF peptide and acid-cleavable DAG peptide [R@(ox-PLGA)-KcD]. DAG can enhance the targeting and internalization effect of nanocleaner towards neurovascular unit endothelial cells in AD lesions, and subsequently detach from nanocleaner in response to acidic microenvironment of endosomes to promote the transcytosis of nanocleaner from endothelial cells into brain parenchyma. Then exposed KLVFF can capture and carry Aβ to microglia, attenuating Aβ-induced neurotoxicity. Strikingly, rapamycin, an autophagy promoter, is rapidly liberated from nanocleaner in the high ROS level of lesions to improve Aβ degradation and normalize inflammatory condition. This design altogether accelerates Aβ degradation and alleviates oxidative stress and excessive inflammatory response. Collectively, our finding offers a strategy to target the AD lesions precisely and multi-pronged therapies for clearing the toxic proteins and modulating lesion microenvironment, to achieve efficient AD therapy.

Microglial autophagy-mediated clearance of amyloid-beta plaques is dysfunctional in Alzheimer’s Disease mice

Abstract Alzheimer’s Disease (AD) is a neurodegenerative disorder with no preventative methods or cure. Deposition of aggregated amyloid-beta (Aβ) in the brain is hypothesized to initiate a vicious cascade, leading to inflammation and tissue damage. Microglia are brain-resident immune cells which help maintain homeostasis and clear protein aggregates, including Aβ. However, in AD, microglia produce inflammatory mediators and do not adequately clear Aβ. This project seeks to determine how healthy microglia clear Aβ from the brain and why diseased AD microglia do not. Autophagy is a homeostatic process providing the cell with energy during stress. Autophagy can act as a defense mechanism, by clearing foreign particles and defective proteins. Previous research on AD established that autophagy is dysfunctional in neurons, but autophagy is not well-characterized in AD microglia. Functional autophagy requires expression of multiple autophagy proteins. We hypothesized that AD microglia fail to clear Aβ due to reduced expression of autophagy proteins and that restoring expression of these proteins will enhance autophagy activity. Utilizing microglia from adult AD model mice and various autophagy mice, we demonstrated that AD microglia do not degrade Aβ due to reduced expression of autophagy proteins, which are necessary for Aβ degradation. We identified an upregulated cluster of microRNA in microglia from mouse and human AD brain tissue samples. Inhibiting these microRNA improves expression of specific autophagy proteins and restores Aβ degradation in AD mouse microglia. Our findings indicate that autophagy is dysfunctional in AD microglia and establish a mechanism behind dysfunctional autophagy which may be therapeutically targeted.

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.

Degradation of Alzheimer’s Amyloid-β by a Catalytically Inactive Insulin-Degrading Enzyme

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer’s amyloid-β (Aβ). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aβ degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aβ(1–40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aβ(1–40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aβ fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aβ(1–40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aβ(1–40) inactivates cf-E111Q-IDE’s catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aβ degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE’s function.

Characterization of monomeric and soluble aggregated Aβ in Down’s syndrome and Alzheimer’s disease brains

The major characteristics of Alzheimer’s disease (AD) are amyloid plaques, consisting of aggregated beta amyloid (Aβ) peptides, together with tau pathology (tangles, neuropil treads and dystrophic neurites surrounding the plaques), in the brain. Down’s syndrome (DS) individuals are at increased risk to develop AD-type pathology; most DS individuals have developed substantial pathology already at the age of 40. DS individuals have an extra copy of chromosome 21, harbouring the amyloid precursor protein gene (APP). Our aim was to investigate the Aβ peptide pattern in DS and AD brains to investigate differences in their amyloid deposition and aggregation, respectively. Cortical tissue from patients with DS (with amyloid pathology), sporadic AD and controls were homogenized and fractionated into TBS (water soluble) and formic acid (water insoluble) fractions. Immunoprecipitation (IP) was performed using a variety of antibodies targeting different Aβ species including oligomeric Aβ. Mass spectrometry was then used to evaluate the presence of Aβ species in the different patient groups. A large number of Aβ peptides were identified including Aβ1-X, 2-X, 3-X, 4-X, 5-X, 11-X, and Aβ peptides extended N terminally of the BACE1 cleavage site and ending at amino 15 in the Aβ sequence APP/Aβ(-X to 15), as well as peptides post-translationally modified by pyroglutamate formation. Most Aβ peptides had higher abundance in AD and DS compared to controls, except the APP/Aβ(-X to 15) peptides which were most abundant in DS followed by controls and AD. Furthermore, the abundancies of AβX-40 and AβX-34 were increased in DS compared with AD. Aβ1-40, Aβ1-42, and Aβ4-42 were identified as the main constitutes of protofibrils (IP’d using mAb158) and higher relative Aβ1-42 signals were obtained compared with samples IP’d with 6E10 + 4G8, indicating that the protofibrils/oligomers were enriched with peptides ending at amino acid 42. All Aβ peptides found in AD were also present in DS indicating similar pathways of Aβ peptide production, degradation and accumulation, except for APP/Aβ(-X to 15). Likewise, the Aβ peptides forming protofibrils/oligomers in both AD and DS were similar, implying the possibility that treatment with clinical benefit in sporadic AD might also be beneficial for subjects with DS.

Pharmacological ablation of astrocytes reduces A\u03b2 degradation and synaptic connectivity in an ex vivo model of Alzheimer\u2019s disease

BackgroundAstrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer’s disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear.MethodsTo explore the role of astrocytes on Aβ pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aβ42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h.ResultsTreatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aβ due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control.ConclusionsAstrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

The interplay between lipid and Aβ amyloid homeostasis in Alzheimer’s Disease: risk factors and therapeutic opportunities

Alzheimer’s Diseases (AD) is characterized by the accumulation of amyloid deposits of Aβ peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aβ deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable. Recently, many studies have described the key role played by membrane bound Aβ assemblies in neurotoxicity. Moreover, it has been suggested that a derangement of the ubiquitin proteasome pathway and autophagy is significantly correlated with toxic Aβ aggregation and dysregulation of lipid levels. Thus, studies focusing on the role played by lipids in Aβ aggregation and proteostasis could represent a promising area of investigation for the design of valuable treatments. In this review we examine current knowledge concerning the effects of lipids in Aβ aggregation and degradation processes, focusing on the therapeutic opportunities that a comprehensive understanding of all biophysical, biochemical, and biological processes involved may disclose.