Aβ Generation Research Articles

ArhGAP11A mediates amyloid-β generation and neuropathology in an Alzheimer’s disease-like mouse model

Amyloid-β (Aβ) plays an important role in the neuropathology of Alzheimer's disease (AD), but some factors promoting Aβ generation and Aβ oligomer (Aβo) neurotoxicity remain unclear. We here find that the levels of ArhGAP11A, a Ras homology GTPase-activating protein, significantly increase in patients with AD and amyloid precursor protein (APP)/presenilin-1 (PS1) mice. Reducing the ArhGAP11A level in neurons not only inhibits Aβ generation by decreasing the expression of APP, PS1, and β-secretase (BACE1) through the RhoA/ROCK/Erk signaling pathway but also reduces Aβo neurotoxicity by decreasing the expressions of apoptosis-related p53 target genes. In APP/PS1 mice, specific reduction of the ArhGAP11A level in neurons significantly reduces Aβ production and plaque deposition and ameliorates neuronal damage, neuroinflammation, and cognitive deficits. Moreover, Aβos enhance ArhGAP11A expression in neurons by activating E2F1, which thus forms a deleterious cycle. Our results demonstrate that ArhGAP11A may be involved in AD pathogenesis and that decreasing ArhGAP11A expression may be a promising therapeutic strategy for AD treatment.

G protein‐biased GPR3 signaling induces glial activation and ameliorates amyloid pathology in a preclinical Alzheimer’s disease mouse model

AbstractBackgroundBiased G protein‐coupled receptor (GPCR) ligands preferentially activate G protein‐ or β‐arrestin signaling pathways and are leading to the development of drugs with superior efficacy and reduced side‐effects in heart disease, pain management, and neuropsychiatric disorders. Although GPCRs are implicated in the pathophysiology of Alzheimer’s disease (AD), biased GPCR signaling is an unexplored area of investigation in AD. Previous work demonstrated that Gpr3 deficiency ameliorates amyloid‐β (Aβ) pathology. However, Gpr3‐deficient mice display several adverse phenotypes, including elevated anxiety‐like behavior, reduced fertility, and memory impairment, which are potentially associated with impaired G protein‐signaling. We hypothesized that generation of a G protein‐biased GPR3 mouse model, which maintains G protein‐signaling while eliminating β‐arrestin signaling, would attenuate AD‐associated phenotypes while preserving the beneficial effects of GPR3‐mediated G protein‐signaling.MethodsWe used a CRISPR/Cas9 genome‐editing strategy to develop two novel G protein‐biased GPR3 mouse models. We utilized a broad range of molecular, cellular, and biochemical methodologies, including mass spectrometry, immunoblot and immunohistochemical analyses, RNA profiling, and behavioral testing to determine the physiological relevance of G protein‐biased GPR3 signaling in naïve mice and in the preclinical AppNL‐G‐F AD mouse model.ResultsWe determine that, in contrast to Gpr3‐deficient mice, G protein‐biased GPR3 mice do not display elevated anxiety levels, reduced fertility, or cognitive impairment. We then established that GPR3 is expressed in neurons, microglia, and astrocytes and is associated with cell‐type‐specific effects on AD pathogenesis. Accordingly, we determine that G protein‐biased signaling leads to a reduction in Aβ generation in neurons, the activation and hypertrophy of disease‐associated‐microglia (DAM) and pan‐reactive astrocytes, and to an increase in amyloid plaque compaction in AppNL‐G‐F mice.ConclusionsThese studies provide the first demonstration of G protein‐biased signaling in neuronal regulation of Aβ generation and microglial and astrocytic regulation of neuroinflammation in a preclinical AD mouse model and open an exciting area of investigation on biased agonism as a therapeutic intervention strategy for AD.

BACE1 regulates expression of Clusterin in astrocytes for enhancing clearance of β-amyloid peptides

BackgroundAbnormal accumulation of amyloid beta peptide (Aβ) in the brain induces a cascade of pathological changes in Alzheimer’s disease (AD), and inhibiting BACE1, which is required for Aβ generation, is therefore being explored for the treatment of AD by reducing Aβ accumulation. As Bace1 knockout mice exhibit increased number of reactive astrocytes and AD brains have reactive astrocytes that surround amyloid plaques, we investigated the role of BACE1 in astrocytes and determined whether BACE1 regulates astrocytic functions.MethodsWe conducted unbiased single cell RNA-seq (scRNA-seq) using purified astrocytes from Bace1 KO mice and wild type control littermates. Similar scRNA-seq was also conducted using AD mice with conditional deletion of Bace1 in the adult stage (5xFAD;Bace1fl/fl;UBC-creER compared to 5xFAD;Bace1fl/fl controls). We compared the transcriptomes of astrocyte and reactive astrocyte clusters and identified several differentially expressed genes, which were further validated using Bace1 KO astrocyte cultures. Mice with astrocyte-specific Bace1 knockout in 5xFAD background were used to compare amyloid deposition. Mechanistic studies using cultured astrocytes were used to identify BACE1 substrates for changes in gene expression and signaling activity. ResultsAmong altered genes, Clusterin (Clu) and Cxcl14 were significantly upregulated and validated by measuring protein levels. Moreover, BACE1 deficiency enhanced both astrocytic Aβ uptake and degradation, and this effect was significantly attenuated by siRNA knockdown of Clu. Mechanistic study suggests that BACE1 deficiency abolishes cleavage of astrocytic insulin receptors (IR), and this may enhance expression of Clu and Cxcl14. Acutely isolated astrocytes from astrocyte-specific knockout of Bace1 mice (Bace1 fl/fl;Gfap-cre) show similar increases in CLU and IR. Furthermore, astrocyte-specific knockout of Bace1 in a 5xFAD background resulted in a significant attenuation in cortical Aβ plaque load through enhanced clearance.ConclusionTogether, our study suggests that BACE1 in astrocytes regulates expression of Clu and Cxcl14, likely via the control of insulin receptor pathway, and inhibition of astrocytic BACE1 is a potential alternative strategy for enhancing Aβ clearance.

Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.

Octadecaneuropeptide Ameliorates Cognitive Impairments Through Inhibiting Oxidative Stress in Alzheimer's Disease Models.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by amyloid-β peptide (Aβ) deposition. Aβ accumulation induces oxidative stress, leading to mitochondrial dysfunction, apoptosis, and so forth. Octadecaneuropeptide (ODN), a diazepam-binding inhibitor (DBI)-derived peptide, has been reported to have antioxidant properties. However, it is unclear whether ODN has neuroprotective effects in AD. To profile the potential effects of ODN on AD. We established a mouse model of AD via microinjection of Aβ in the lateral ventricle. Utilizing a combination of western blotting assays, electrophysiological recordings, and behavioral tests, we investigated the neuroprotective effects of ODN on AD. DBI expression was decreased in AD model mice and cells. Meanwhile, ODN decreased Aβ generation by downregulating amyloidogenic AβPP processing in HEK-293 cells stably expressing human Swedish mutant APP695 and BACE1 (2EB2). Moreover, ODN could inhibit Aβ-induced oxidative stress in primary cultured cells and mice, as reflected by a dramatic increase in antioxidants and a decrease in pro-oxidants. We also found that ODN could reduce oxidative stress-induced apoptosis by restoring mitochondrial membrane potential, intracellular Ca2+ and cleaved caspase-3 levels in Aβ-treated primary cultured cells and mice. More importantly, intracerebroventricular injection of ODN attenuated cognitive impairments as well as long-term potentiation in Aβ-treated mice. These results suggest that ODN may exert a potent neuroprotective effect against Aβ-induced neurotoxicity and memory decline via its antioxidant effects, indicating that ODN may be a potential therapeutic agent for AD.

Inhibition of NLRP1 inflammasome improves autophagy dysfunction and Aβ disposition in APP/PS1 mice

Increasing evidence has shown that the NOD-like receptor protein 1 (NLRP1) inflammasome is associated with Aβ generation and deposition, which contributes to neuronal damage and neuronal-inflammation in Alzheimer's disease (AD). However, the specific mechanism of NLRP1 inflammasome in the pathogenesis of AD is still unclear. It has been reported that autophagy dysfunction can aggravate the pathological symptoms of AD and plays an important role in regulating Aβ generation and clearance. We hypothesized that NLRP1 inflammasome activation may induce autophagy dysfunction contributing to the progression of AD. In the present study, we observed the relationship between Aβ generation and NLRP1 inflammasome activation, as well as AMPK/mTOR mediated-autophagy dysfunction in WT 9-month-old (M) mice, APP/PS1 6 M and APP/PS1 9 M mice. Additionally, we further studied the effect of NLRP1 knockdown on cognitive function, Aβ generation, neuroinflammation and AMPK/mTOR mediated autophagy in APP/PS1 9 M mice. Our results indicated that NLRP1 inflammasome activation and AMPK/mTOR mediated-autophagy dysfunction are closely implicated in Aβ generation and deposition in APP/PS1 9 M mice, but not in APP/PS1 6 M mice. Meanwhile, we found that knockdown of NLRP1 significantly improved learning and memory impairments, decreased the expressions of NLRP1, ASC, caspase-1, p-NF-κB, IL-1β, APP, CTF-β, BACE1 and Aβ1-42, and decreased the level of p-AMPK, Beclin 1 and LC3 II, and increased the level of p-mTOR and P62 in APP/PS1 9 M mice. Our study suggested that inhibition of NLRP1 inflammasome activation improves AMPK/mTOR mediated-autophagy dysfunction, resulting in the decrease of Aβ generation, and NLRP1 and autophagy might be important targets to delay the progression of AD.

Cellular cholesterol loss by DHCR24 knockdown leads to Aβ production by changing APP intracellular localization

For the past 20 years, the majority of cell culture studies reported that increasing cholesterol level increases amyloid-β (Aβ) production. Conversely, other studies and genetic evidences support that cellular cholesterol loss leads to Aβ generation. As a highly controversial issue in Alzheimer’s disease pathogenesis, the apparent contradiction prompted us to again explore the role of cellular cholesterol in Aβ production. Here, we adopted new neuronal and astrocytic cell models induced by 3β-hydroxysterol-Δ24 reductase (DHCR24), which obviously differ from the widely used cell models with overexpressing amyloid precursor protein (APP) in the majority of previous studies. In neuronal and astrocytic cell model, we found that deficiency of cellular cholesterol by DHCR24 knockdown obviously increased intracellular and extracellular Aβ generation. Importantly, in cell models with overexpressing APP, we found that APP overexpression could disrupt cellular cholesterol homeostasis and affect function of cells, coupled with the increase of APP β-cleavage product, 99-residue transmembrane C-terminal domain. Therefore, we suppose the results derived from the APP knockin models will need to be re-evaluated. One rational explanation for the discrepancy between our outcomes and the previous studies could be attributed to the two different cell models. Mechanistically, we showed that cellular cholesterol loss obviously altered APP intracellular localization by affecting cholesterol-related trafficking protein of APP. Therefore, our outcomes strongly support cellular cholesterol loss by DHCR24 knockdown leads to Aβ production.

Chronic basal forebrain activation improves spatial memory, boosts neurotrophin receptor expression, and lowers BACE1 and Aβ42 levels in the cerebral cortex in mice.

The etiology of Alzheimer's dementia has been hypothesized in terms of basal forebrain cholinergic decline, and in terms of reflecting beta-amyloid neuropathology. To study these different biological elements, we activated the basal forebrain in 5xFAD Alzheimer's model mice and littermates. Mice received 5 months of 1 h per day intermittent stimulation of the basal forebrain, which includes cholinergic projections to the cortical mantle. Then, mice were behaviorally tested followed by tissue analysis. The 5xFAD mice performed worse in water-maze testing than littermates. Stimulated groups learned the water maze better than unstimulated groups. Stimulated groups had 2-3-fold increases in frontal cortex immunoblot measures of the neurotrophin receptors for nerve growth factor and brain-derived neurotrophic factor, and a more than 50% decrease in the expression of amyloid cleavage enzyme BACE1. Stimulation also led to lower Aβ42 in 5xFAD mice. These data support a causal relationship between basal forebrain activation and both neurotrophin activation and reduced Aβ42 generation and accumulation. The observation that basal forebrain activation suppresses Aβ42 accumulation, combined with the known high-affinity antagonism of nicotinic receptors by Aβ42, documents bidirectional antagonism between acetylcholine and Aβ42.

BACE-1 Inhibitors Targeting Alzheimer's Disease.

The accumulation of amyloid-β (Aβ) is the main event related to Alzheimer's disease (AD) progression. Over the years, several disease-modulating approaches have been reported, but without clinical success. The amyloid cascade hypothesis evolved and proposed essential targets such as tau protein aggregation and modulation of β-secretase (β-site amyloid precursor protein cleaving enzyme 1 - BACE-1) and γ-secretase proteases. BACE-1 cuts the amyloid precursor protein (APP) to release the C99 fragment, giving rise to several Aβ peptide species during the subsequent γ-secretase cleavage. In this way, BACE-1 has emerged as a clinically validated and attractive target in medicinal chemistry, as it plays a crucial role in the rate of Aβ generation. In this review, we report the main results of candidates in clinical trials such as E2609, MK8931, and AZD-3293, in addition to highlighting the pharmacokinetic and pharmacodynamic-related effects of the inhibitors already reported. The current status of developing new peptidomimetic, non-peptidomimetic, naturally occurring, and other class inhibitors are demonstrated, considering their main limitations and lessons learned. The goal is to provide a broad and complete approach to the subject, exploring new chemical classes and perspectives.

Endosome and Lysosome Membrane Properties Functionally Link to γ-Secretase in Live/Intact Cells.

Our unique multiplexed imaging assays employing FRET biosensors have previously detected that γ-secretase processes APP C99 primarily in late endosomes and lysosomes in live/intact neurons. Moreover we have shown that Aβ peptides are enriched in the same subcellular loci. Given that γ-secretase is integrated into the membrane bilayer and functionally links to lipid membrane properties in vitro, it is presumable that γ-secretase function correlates with endosome and lysosome membrane properties in live/intact cells. In the present study, we show using unique live-cell imaging and biochemical assays that the endo-lysosomal membrane in primary neurons is more disordered and, as a result, more permeable than in CHO cells. Interestingly, γ-secretase processivity is decreased in primary neurons, resulting in the predominant production of long Aβ42 instead of short Aβ38. In contrast, CHO cells favor Aβ38 over the Aβ42 generation. Our findings are consistent with the previous in vitro studies, demonstrating the functional interaction between lipid membrane properties and γ-secretase and provide further evidence that γ-secretase acts in late endosomes and lysosomes in live/intact cells.

Depletion of Paraoxonase 1 (Pon1) Dysregulates mTOR, Autophagy, and Accelerates Amyloid Beta Accumulation in Mice.

Paraoxonase 1 (PON1), a homocysteine (Hcy)-thiolactone detoxifying enzyme, has been associated with Alzheimer's disease (AD), suggesting that PON1 plays an important protective role in the brain. To study the involvement of PON1 in the development of AD and to elucidate the mechanism involved, we generated a new mouse model of AD, the Pon1-/-xFAD mouse, and examined how Pon1 depletion affects mTOR signaling, autophagy, and amyloid beta (Aβ) accumulation. To elucidate the mechanism involved, we examined these processes in N2a-APPswe cells. We found that Pon1 depletion significantly downregulated Phf8 and upregulated H4K20me1; mTOR, phospho-mTOR, and App were upregulated while autophagy markers Bcln1, Atg5, and Atg7 were downregulated at the protein and mRNA levels in the brains of Pon1─/─5xFAD vs. Pon1+/+5xFAD mice. Pon1 depletion in N2a-APPswe cells by RNA interference led to downregulation of Phf8 and upregulation of mTOR due to increased H4K20me1-mTOR promoter binding. This led to autophagy downregulation and significantly increased APP and Aβ levels. Phf8 depletion by RNA interference or treatments with Hcy-thiolactone or N-Hcy-protein metabolites similarly increased Aβ levels in N2a-APPswe cells. Taken together, our findings define a neuroprotective mechanism by which Pon1 prevents Aβ generation.

Membrane lipid remodeling modulates γ-secretase processivity

Imbalances in the amounts of amyloid-β peptides (Aβ) generated by the membrane proteases β- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aβ generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aβ37 and/or Aβ38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aβ secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.

Trpc6 knockout improves behavioral dysfunction and reduces Aβ production by inhibiting CN-NFAT1 signaling in T2DM mice

As the prevalence of diabetes and health awareness increase, type 2 diabetes mellitus -associated cognitive dysfunction is receiving increasing attention. However, the pathogenesis is not entirely understood. Transient receptor potential cation channel 6 (TRPC6) is highly correlated with intracellular Ca2+ concentrations, and neuronal calcium overload is an important cause of cognitive dysfunction. In the present study, we investigated the effect and mechanism of Trpc6 knockout in high-fat diet and streptozotocin-induced T2DM mice. The body weight and fasting blood glucose were recorded during the experiment. Behavioral dysfunction was detected using the open field test (OFT), elevated plus maze (EPM), hole-board test (HBT), Morris water maze (MWM) test and contextual fear conditioning (CFC) test. Nissl and H&E staining were used to examine neuronal damage. Western blot, quantitative real-time polymerase chain reaction (q-PCR), and immunofluorescence were performed to detect amyloid beta protein (Aβ) deposition and related indicators of neurological impairments in the cerebral cortex and hippocampus. The results indicated that Trpc6 knockout inhibited body weight loss and fasting blood glucose increase, improved spontaneous activity, learning and memory dysfunction, and alleviated neuroinflammation and neuronal damage in T2DM mice. The further results demonstrated that Trpc6 knockout decreased Aβ generation and deposition, and reduced the expressions of inflammasome-related proteins in T2DM mice. In addition, Trpc6 knockout inhibited intracellular calcium overload in diabetic mice and primary cultured hippocampal neurons, which in turn suppressed CN and NFAT1 expression. These data suggest that Trpc6 knockout may inhibit the CN-NFAT1 signaling pathway by decreasing intracellular calcium overload in the brain of T2DM mice, which consequently reduce Aβ deposition and neuroinflammation, and ultimately delay the development of T2DM-associated cognitive dysfunction.

Analysis of Non-Amyloidogenic Mutations in APP Supports Loss of Function Hypothesis of Alzheimer's Disease.

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in pathogenesis of Azheimer's disease (AD). Sequential cleavage of APP by β- and γ-secretases leads to generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) act as catalytic subunits of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 affect APP proteolysis by γ-secretase and influence levels of generated Aβ40 and Aβ42 peptides. The predominant idea in the field is the "amyloid hypothesis" that states that the resulting increase in Aβ42:Aβ40 ratio leads to "toxic gain of function" due to the accumulation of toxic Aβ42 plaques and oligomers. An alternative hypothesis based on analysis of PS1 conditional knockout mice is that "loss of function" of γ-secretase plays an important role in AD pathogenesis. In the present paper, we propose a mechanistic hypothesis that may potentially reconcile these divergent ideas and observations. We propose that the presence of soluble Aβ peptides in endosomal lumen (and secreted to the extracellular space) is essential for synaptic and neuronal function. Based on structural modeling of Aβ peptides, we concluded that Aβ42 peptides and Aβ40 peptides containing non-amyloidogenic FAD mutations in APP have increased the energy of association with the membranes, resulting in reduced levels of soluble Aβ in endosomal compartments. Analysis of PS1-FAD mutations also revealed that all of these mutations lead to significant reduction in both total levels of Aβ produced and in the Aβ40/Aβ42 ratio, suggesting that the concentration of soluble Aβ in the endosomal compartments is reduced as a result of these mutations. We further reasoned that similar changes in Aβ production may also occur as a result of age-related accumulation of cholesterol and lipid oxidation products in postsynaptic spines. Our analysis more easily reconciled with the "loss of γ-secretase function" hypothesis than with the "toxic gain of Aβ42 function" idea. These results may also explain why inhibitors of β- and γ- secretase failed in clinical trials, as these compounds are also expected to significantly reduce soluble Aβ levels in the endosomal compartments.

Calmodulin and Amyloid Beta as Coregulators of Critical Events during the Onset and Progression of Alzheimer's Disease.

Calmodulin (CaM) and a diversity of CaM-binding proteins (CaMBPs) are involved in the onset and progression of Alzheimer's disease (AD). In the amyloidogenic pathway, AβPP1, BACE1 and PSEN-1 are all calcium-dependent CaMBPs as are the risk factor proteins BIN1 and TREM2. Ca2+/CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are classic CaMBPs involved in memory and plasticity, two events impacted by AD. Coupled with these events is the production of amyloid beta monomers (Aβ) and oligomers (Aβo). The recent revelations that Aβ and Aβo each bind to both CaM and to a host of Aβ receptors that are also CaMBPs adds a new level of complexity to our understanding of the onset and progression of AD. Multiple Aβ receptors that are proven CaMBPs (e.g., NMDAR, PMCA) are involved in calcium homeostasis an early event in AD and other neurodegenerative diseases. Other CaMBPs that are Aβ receptors are AD risk factors while still others are involved in the amyloidogenic pathway. Aβ binding to receptors not only serves to control CaM's ability to regulate critical proteins, but it is also implicated in Aβ turnover. The complexity of the Aβ/CaM/CaMBP interactions is analyzed using two events: Aβ generation and NMDAR function. The interactions between Aβ, CaM and CaMBPs reveals a new level of complexity to critical events associated with the onset and progression of AD and may help to explain the failure to develop successful therapeutic treatments for the disease.

Distinct effects of SDC3 and FGFRL1 on selective neurodegeneration in AD and PD.

Alzheimer's disease (AD) and Parkinson's disease (PD) are age-dependent neurodegenerative disorders. There is a profound neuronal loss in the basal forebrain cholinergic system in AD and severe dopaminergic deficiency within the nigrostriatal pathway in PD. Swedish APP (APPSWE ) and SNCAA53T mutations promote Aβ generation and α-synuclein aggregation, respectively, and have been linked to the pathogenesis of AD and PD. However, the mechanisms underlying selective cholinergic and dopaminergic neurodegeneration in AD and PD are still unknown. We demonstrated that APPSWE mutation enhanced Aβ generation and increased cell susceptibility to Aβ oligomer in cholinergic SN56 cells, whereas SNCAA53T mutations promoted aggregates formation and potentiated mutant α-synuclein oligomer-induced cytotoxicity in MN9D cells. Furthermore, syndecan-3 (SDC3) and fibroblast growth factor receptor-like 1 (FGFRL1) genes were differentially expressed in SN56 and MN9D cells carrying APPSWE or SNCAA53T mutation. SDC3 and FGFRL1 proteins were preferentially expressed in the cholinergic nucleus and dopaminergic neurons of APPSWE and SNCAA53T mouse models, respectively. Finally, the knockdown of SDC3 and FGFRL1 attenuated oxidative stress-induced cell death in SN56-APPSWE and MN9D-SNCAA53T cells. The results demonstrate that SDC3 and FGFRL1 mediated the specific effects of APPSWE and SNCAA53T on cholinergic and dopaminergic neurodegeneration in AD and PD, respectively. Our study suggests that SDC3 and FGFRL1 could be potential targets to alleviate the selective neurodegeneration in AD and PD.

Upregulation of endocytic protein expression in the Alzheimer’s disease male human brain

Amyloid-beta (Aβ) is produced from amyloid precursor protein (APP) primarily after APP is internalised by endocytosis and clathrin-mediated endocytic processes are altered in Alzheimer’s disease (AD). There is also evidence that cholesterol and flotillin affect APP endocytosis. We hypothesised that endocytic protein expression would be altered in the brains of people with AD compared to non-diseased subjects which could be linked to increased Aβ generation. We compared protein expression in frontal cortex samples from men with AD compared to age-matched, non-diseased controls. Soluble and insoluble Aβ40 and Aβ42, the soluble Aβ42/Aβ40 ratio, βCTF, BACE1, presenilin-1 and the ratio of phosphorylated:total GSK3β were significantly increased while the insoluble Aβ42:Aβ40 ratio was significantly decreased in AD brains. Total and phosphorylated tau were markedly increased in AD brains. Significant increases in clathrin, AP2, PICALM isoform 4, Rab-5 and caveolin-1 and 2 were seen in AD brains but BIN1 was decreased. However, using immunohistochemistry, caveolin-1 and 2 were decreased. The results obtained here suggest an overall increase in endocytosis in the AD brain, explaining, at least in part, the increased production of Aβ during AD.

Ginsenoside Rg1 treatment protects against cognitive dysfunction via inhibiting PLC–CN–NFAT1 signaling in T2DM mice

BackgroundAs a complication of Type II Diabetes Mellitus (T2DM), the etiology, pathogenesis, and treatment of cognitive dysfunction are still undefined. Recent studies demonstrated that Ginsenoside Rg1 (Rg1) has promising neuroprotective properties, but the effect and mechanism in diabetes-associated cognitive dysfunction (DACD) deserve further investigation. MethodsAfter establishing the T2DM model with a high-fat diet and STZ intraperitoneal injection, Rg1 was given for 8 weeks. The behavior alterations and neuronal lesions were judged using the open field test (OFT) and Morris water maze (MWM), as well as HE and Nissl staining. The protein or mRNA changes of NOX2, p-PLC, TRPC6, CN, NFAT1, APP, BACE1, NCSTN, and Aβ1-42 were investigated by immunoblot, immunofluorescence or qPCR. Commercial kits were used to evaluate the levels of IP3, DAG, and calcium ion (Ca2+) in brain tissues. ResultsRg1 therapy improved memory impairment and neuronal injury, decreased ROS, IP3, and DAG levels to revert Ca2+ overload, downregulated the expressions of p-PLC, TRPC6, CN, and NFAT1 nuclear translocation, and alleviated Aβ deposition in T2DM mice. In addition, Rg1 therapy elevated the expression of PSD95 and SYN in T2DM mice, which in turn improved synaptic dysfunction. ConclusionsRg1 therapy may improve neuronal injury and DACD via mediating PLC–CN–NFAT1 signal pathway to reduce Aβ generation in T2DM mice.

MiR-191-5p Attenuates Tau Phosphorylation, Aβ Generation, and Neuronal Cell Death by Regulating Death-Associated Protein Kinase 1.

Dysregulation of microRNAs has been implicated in diverse diseases, including Alzheimer's disease (AD). MiR-191-5p in plasma/serum has been identified as a novel and promising noninvasive diagnostic biomarker for AD. However, whether miR-191-5p is involved in AD pathogenesis is largely unknown, and its levels in human AD brains are undetermined. Herein, we demonstrated that miR-191-5p downregulated tau phosphorylation at multiple AD-related sites and promoted neurite outgrowth using immunoblotting, immunofluorescence, and neurite outgrowth assays. Moreover, immunoblotting and enzyme-linked immunosorbent assays indicated that miR-191-5p decreased amyloid precursor protein phosphorylation levels and beta-amyloid (Aβ) generation. Furthermore, miR-191-5p reduced ceramide-induced neuronal cell death analyzed by trypan blue staining, the in situ cell death detection kit, and Annexin V-FITC/PI flow cytometry. Next, we verified that death-associated protein kinase 1 (DAPK1) was a direct target of miR-191-5p through the dual luciferase reporter assay and confirmed that the effects of miR-191-5p were antagonized by restoration of DAPK1 expression. Finally, the hippocampal miR-191-5p level was found to be decreased in humans with AD compared with controls and was inversely correlated with the DAPK1 expression level. Collectively, these findings suggest that miR-191-5p might exert inhibitory effects on tau phosphorylation, Aβ secretion, and neuronal cell death by directly targeting DAPK1, providing an attractive therapeutic option for AD.

Differential modulation of amyloid‐beta levels by psychotropic medications in an in vitro model of Alzheimer’s disease

AbstractBackgroundPreclinical studies suggest that psychotropic drugs (e.g. antidepressants, benzodiazepines) can affect amyloid‐β (Aβ) levels, yet mechanisms remain unclear. We aimed to determine the effects of common psychiatric drugs on Aβ generation and clearance in an in vitro model of Alzheimer’s disease (AD).MethodThe effects of a range of psychotropic drugs on Aβ generation and clearance were assessed in N2a/APP695swe cells, a neuroblastoma cell line overexpressing APP with the Swedish mutation. Cells were incubated for 24h with either fluoxetine (selective serotonin reuptake inhibitor (SSRI); 1‐10μM), venlafaxine (serotonin‐norepinephrine reuptake inhibitor (SNRI); 0.1‐10μM), mirtazapine (α2‐adrenergic antagonist; 1‐25μM), phenelzine (monoamine oxidase inhibitor (MAOI); 10‐50μM) or nordiazepam (benzodiazepine (BNZ); 1‐25μM). Aβ42 levels were quantified in conditioned media using an enzyme‐linked immunosorbent assay (ELISA). Amyloid precursor protein (APP) processing was studied using western blots (WBs), including expression of different secretases, full‐length APP, C‐terminal fragments (CTFs) in membrane preps and APP soluble‐α in media. Neprilysin and insulin degrading enzyme (IDE) expression was quantified as major Aβ clearance factors.ResultsCertain drugs increased Aβ levels; in particular, Venlafaxine (SNRI) and nordiazepam (BNZ) elevated Aβ42 at 10 µM, due to increased amyloidogenic APP cleavage. Conversely, the anti‐depressant drug fluoxetine (SSRI; 10 µM) reduced Aβ42, concomitant with reduced β‐secretase (BACE1) and presenilin‐2 expressions. Mirtazapine (α2‐adrenergic antagonist) and phenelzine (MAOI), on the other hand, did not modulate Aβ levels.ConclusionCertain psychotropic medications, i.e. nordiazepam (BNZ) and venlafaxine (SNRI) can increase Aβ levels, while fluoxetine (SSRI) can reduce them. This results from the effects of these drugs on Aβ generation rather than clearance. This is important, as the administration of these drugs could affect AD‐risk.