Aβ Treatment Research Articles

Aβ42 treatment of the brain side reduced the level of flotillin from endothelial cells on the blood side via FGF-2 signaling in a blood–brain barrier model

Our previous study showed that the flotillin level is decreased in the blood of patients with Alzheimer’s disease (AD) when compared to that of patients with non-AD and vascular dementia; however, the molecular mechanism remains to be determined. In this study, to elucidate whether Aβ accumulation in the brain has an effect on the blood flotillin level, we used our previously established blood–brain barrier (BBB) culture model using microvascular endothelial cells obtained from human induced pluripotent stem cells (iBMECs) and astrocytes prepared from rat cortex. In this BBB model with iBMECs plated on the upper compartment (blood side) and astrocytes plated on the lower compartment (brain side), the trans-endothelial electrical resistance values are high (over 1500 Ωm2) and stable during experiments. We found that the addition of Aβ42 (0.5 and 2 µM) to the brain side significantly reduced the level of flotillin secreted by iBMECs on the blood side. The level of basic fibroblast growth factor (FGF-2) in the brain side was significantly reduced by Aβ42 treatment, and was accompanied by a reduction in the level of phosphorylation of the fibroblast growth factor receptor in iBMECs. The brain-side Aβ42 treatment-induced reduction of flotillin secretion into the blood side was restored in a dose-dependent manner by the addition of FGF-2 into the brain side. These results indicated that Aβ accumulation in the brain side reduced FGF-2 release from astrocytes, which attenuated FGF-2-mediated iBMECs signaling via the FGF-2 receptor, and thereby reduced flotillin secretion from iBMECs on the blood side. Our findings revealed a novel signaling pathway crossing the BBB from the brain side to the blood side, which is different from the classical intramural periarterial drainage or lymphatic-system-to-blood pathway.

Amyloid Beta Alters the Expression of microRNAs Regulating HMGCR and ABCA1 Genes in Astrocytes of C57BL/6J Mice.

Dysregulation of brain cholesterol homeostasis causes the accumulation of extracellular protein deposits called amyloid plaques in the hippocampus which eventually leads to neuronal death, memory and learning deficits. The aim of the present study was to investigate the effect of beta amyloid on miRNAs regulating HMGCR and ABCA1 as cholesterol synthesis and homeostasis genes. Primary astrocytes were isolated from C57BL/6J mice, and were treated with 0.5 μM amyloid beta (Aβ). Expression levels of genes and miRNAs were measured by real-time PCR. In comparison to control, Aβ treatment resulted in a significant decrease in miR-96-5p expression as a positive and negative regulator of HMGCR and ABCA1, respectively. There was no significant increase in miR-27a-3p expression as a negative regulator of HMGCR. miR- 106b- 5p and miR-143-3p expressions were also dramatically decreased as ABCA1 negative regulators. Amyloid beta can alter the expression of major genes in the cholesterol homeostasis pathway via their regulatory miRNAs.

Role of Fragile X Mental Retardation protein in Amyloid Beta 1‐42 Induced Translational Suppression

AbstractBackgroundAlzheimer’s Disease (AD) is the most common cause of dementia where the accumulation of the peptide Amyloid‐Beta (Aβ) is the main theory linked to the pathogenesis of the disease. Fragile X Mental Retardation Protein (FMRP) is an RNA‐binding protein that functions to suppress global translation. Because Aβ is known to be a toxic peptide capable of inducing the Endoplasmic Reticulum (ER) stress response and because augmented translational suppression is one of the main molecular phenotypes in ER stress response, we ask whether and how FMRP is involved in Aß induced translational suppression.MethodPrimary mouse cortical neuronal cultures from wild type (WT) or Fmr1 KO pups were treated with Aβ (Aβ1–42) during 24 hours. For protein expression identification and quantification we used western blot and ImageJ software respectively. Protein translation was assessed by inhibition with puromycin. Neuronal networking assessment was made with multielectrode array system (MEA).ResultIn this study, we observed that treatments of synthetic Aβ peptides in culture primary mouse cortical neurons induces the expression of FMRP and FMRP‐dependent ER stress response and subsequent translational suppression. Also Aβ induces FMRP‐dependent neuronal hyposynchronicity . We further characterized that two translational regulators, eukaryotic translation initiation factor 2α (eIF2α) and eukaryotic translation elongation factor 2 (eEF2), are involved in Aβ induced translational suppression and are dysregulated in the absence of FMRP. Mechanistically we found that FMRP is responsible for regulating the levels of Protein Phosphatase 2 (PP2A) and Protein Phosphatase 1 (PP1) that differentially modulate the phosphorylation status and thereby activity of eIF2α and eEF2 following treatment of Aβ. Using a pharmacological approach to inhibit PP2A and PP1 with Okadaic Acid, we were able to rescue the impairment of eIF2α and eEF2 as well as the translational suppression and lack of neuronal hyposynchrony in the absence of FMRP.ConclusionThese results suggest a novel function of FMRP in regulating translational suppression and neuronal networking function following Aβ treatment by fine‐tuning two distinct signaling pathways involved in global translation.

Exploring Combination Therapies in AD: Additive Effects of the QPCT Inhibitor Varoglutamstat and Aducanumab on Aβ Pathology and Biomarkers In Vivo

AbstractBackgroundPyroglutamate‐3Aβ (N3pE), a toxic Aβ variant formed by the activity of glutaminyl gyclase (QPCT) has been shown to play a pivotal role in the development and progression of Alzheimer’s disease (AD). Recently, we demonstrated that a combination of the QPCT inhibitor varoglutamstat (PQ912) with the N3pE‐specific antibody m6 has an additive effect on lowering of N3pE in vivo. Here, we analyzed whether a similar additive effect could be achieved when combining varoglutamstat with the Aβ‐aggregate‐specific antibody aducanumab.MethodNine‐months old APPxhQC mice were treated with either varoglutamstat (1.6 g/kg chow, ad libitum), chimeric aducanumab (chAdu, ≈10 mg/kg i.p. weekly) or a combination of both for 16 weeks. Brain Aβ accumulation (ELISA and immunohistochemistry) and AD biomarkers in the water‐soluble brain fractions (neurogranin, BACE‐1, YKL‐40; ELISA) were analyzed and compared to a vehicle‐treated group.ResultTreatment with either varoglutamstat or chAdu significantly reduced the accumulation of both total Aβ and N3pE in the brain. The effect of chAdu was more pronounced for total Aβ (‐35% vs. ‐21% for varoglutamstat), while varoglutamstat treatment resulted in a stronger decrease of N3pE (‐28% vs. ‐19% for chAdu). The combination treatment generally led to a stronger decrease of Aβ vs. single agent treatment (Bliss combination index (CI) = 0.88 for soluble Aβ42 and 1.23 for insoluble Aβ42). For insoluble N3pE, the observed effect was nearly additive (CI = 1.09). Treatment with chAdu, varoglutamstat or both had different effects on the analyzed AD biomarkers. chAdu treatment significantly decreased BACE‐1 protein levels (‐30%), while varoglutamstat treatment significantly reduced levels of the brain inflammation marker YKL‐40 (‐27%), which is in line with earlier clinical Phase 2a study results.ConclusionOur data indicate that a combination of agents with different modes of action such as aducanumab, an Aβ aggregate‐specific antibody and varoglutamstat, a small molecule designed to block formation of toxic, aggregation‐prone N3pE, can act additively to decrease total Aβ and N3pE levels in the brain. Our results also provide a rationale for investigating different potential combination regimens, including an initial antibody‐mediated Aβ clearance followed by long‐term suppression of N3pE formation and inflammation by varoglutamstat (“treat and maintain”).

TMEM176B regulates phagosome maturation of glial cells and its increased activity contributes to the therapy of Alzheimer’s disease

AbstractBackgroundGiven recent interest in using amyloid b (Aβ)‐targeted antibodies as a therapy for patients with Alzheimer’s disease (AD), removal of Aβ by phagocytosis likely protective early in AD. Impaired phagocytic function of glial cells surrounding Aβ plaques during later stages in AD likely contributes to worsened disease outcomes, therefore enhancing phagocytic activity of glial cells could be a potent strategy for AD therapy.MethodChanges in TMEM176B expression according to the progression of AD pathology was assessed by measuring TMEM176B protein in human AD brain lysates and TMEM176B transcripts in Ab‐treated primary rat astrocytes. The roles of TMEM176B in phagosome maturation of glial cells was investigated by measuring Rab5, Rab7, LAMP1, LAMP2, cathepsin D, and LC3B in presence of Aβ. Intracellular pH of glial cells was detected using fluorescent dye‐based pHrodo Red AM intracellular pH indicator (Invitrogen, Carlsbad, CA). In 5xFAD and 3xTg AD mice model, therapeutic effects of AAV‐TMEM176B was examined. AAV (adenovirus‐associated virus)‐TMEM176B (1 – 2×1010 GC) was intracerebroventricularly injected, and Aβ plaque and p‐tau in brains were assessed 3 months later. Behavioral studies such as Y‐maze, Morris water maze, and novel‐object‐recognition were conducted to exam the effect of TMEM176B overexpression in alleviation of cognition decline.ResultIn human AD brains, Tmem176B expression was increased in moderate AD (Braak III‐IV) was decreased in severe AD (Braak V‐VI). TMEM176B was upregulated by acute Aβ treatment in primary rat astrocytes, and was gradually downregulated concomitantly with impairment of Aβ phagocytosis activity by chronic exposure to Aβ. The TMEM176B agonistic compound augmented phagosome maturation and phagosomal acidification, which led to increased Aβ phagocytosis and lysosomal degradation. Studies confirmed that intracerebroventrical administration of AAV carrying TMEM176B in AD mice results in enhanced glial phagocytosis followed by lowered Aβ burden in brain. Moreover, overexpression of TMEM176B by AAV injection significantly improved cognition impairment of AD mice.ConclusionData from these studies suggest that TMEM176B plays a direct and critical role in phagolysosmal function of glial cells and AD pathogenesis and highlight this ion channel as a potential therapeutic target for treating AD.

The Study of Neuronal Hyperactivity in the Early Stage of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disease, characterized by memory loss and cognitive impairment. The pathology of AD includes the accumulation of beta‐amyloid (Aβ) in the extracellular space and hyper‐phosphorylation of tau in neurons. Aβ pathology has been shown to begin approximately 20 years before tau pathology and mild cognitive impairment develop in AD patients. Aβ deposits cause alterations in brain function, leading to the loss of neurons in the cerebral cortex and hippocampus in the later stages of AD. However, the cellular mechanisms by which Aβ induces AD pathology remain largely elusive. Traditionally, lower neuronal activity has been thought to contribute to the synaptic failure and cognitive impairments observed in AD. However, recent studies suggest that Aβ triggers neuronal hyperactivity during the early stages of AD. Consistent with this observation, epileptic seizures have been detected in both mouse models of AD and human patients with Alzheimer’s. Uncontrolled or severe seizures can lead to further brain damage and exacerbate the cognitive decline seen in AD. Thus, neuronal hyperactivity could be one of the contributing factors to the neuronal loss induced by Aβ. However, it is unclear how Aβ can induce neuronal hyperactivity and seizures.MethodTo evaluate seizure susceptibility, we employed kainic acid‐induced seizures in a well‐established AD mouse model, the APP/PS1 mice. To measure neuronal activity, we employed primary cortical neuron cultures treated with a scrambled or Aβ 1‐42 peptide and recorded with a multi‐electrode array (MEA) recording system. Biochemistry and molecular biology assays were performed to study molecular players associated with Aβ‐induced neuronal hyperactivity.ResultOur data showed that acute Aβ treatment induces hyperexcitability in wild‐type (WT) cortical neurons. We also confirmed a higher susceptibility to seizures in APP/PS1 mice. Lastly, we identified a postsynaptic protein that is up‐regulated and potential involved in Aβ‐triggered neuronal hyperexcitability.ConclusionIn summary, our data are expected to provide insights into the etiology of AD‐associated brain hyperactivity and potentially suggest therapeutic targets for reducing seizures during early stage of AD.

The effect of ghrelin on antioxidant status in the rat's model of Alzheimer's disease induced by amyloid-beta.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with amyloid-beta (Aβ) plaque formation and oxidative stress in the brain. Ghrelin has been proven to exert antioxidant activity and neuroprotection in different neurological diseases. This study is going on to examine the effect of ghrelin on antioxidant status in the rat's model of AD induced by Aβ. Cognitive impairment was induced by intra-hippocampal administration of Aβ (10 μg) in Wistar rats and ghrelin (80 μg/kg) was administrated intraperitoneal for ten consecutive days. Behavior was assessed with Morris water maze and passive avoidance tests. Malondialdehyde (MDA) level as a marker of lipid peroxidation was assessed using the thiobarbituric acid. Catalase activity was assayed by the decomposition of H2O2. Antioxidant capacity was determined using the FRAP method. Treatment with ghrelin decreased the hippocampus and serum MDA levels in wild-type rodents and prevented an increase in hippocampal and serum MDA levels in animals receiving Aβ. There was no significant change in the serum catalase activity between the studied groups. Hippocampus catalase activity was reduced in the Aβ group and treatment with ghrelin increased it. The antioxidant capacity of the hippocampus and serum increased in the ghrelin-receiving control group. The hippocampus antioxidant capacity level decreased in the Aβ group, and treatment with ghrelin increased it, but there were no significant changes in the serum antioxidant capacity of animals receiving Aβ. These results provide evidence that the administration of ghrelin has antioxidant properties and protects against hippocampal lipid peroxidation in a rat model of AD.

Effects of Donepezil Treatment on Brain Metabolites, Gut Microbiota, and Gut Metabolites in an Amyloid Beta-Induced Cognitive Impairment Mouse Pilot Model.

Accumulated clinical and biomedical evidence indicates that the gut microbiota and their metabolites affect brain function and behavior in various central nervous system disorders. This study was performed to investigate the changes in brain metabolites and composition of the fecal microbial community following injection of amyloid β (Aβ) and donepezil treatment of Aβ-injected mice using metataxonomics and metabolomics. Aβ treatment caused cognitive dysfunction, while donepezil resulted in the successful recovery of memory impairment. The Aβ + donepezil group showed a significantly higher relative abundance of Verrucomicrobia than the Aβ group. The relative abundance of 12 taxa, including Blautia and Akkermansia, differed significantly between the groups. The Aβ + donepezil group had higher levels of oxalate, glycerol, xylose, and palmitoleate in feces and oxalate, pyroglutamic acid, hypoxanthine, and inosine in brain tissues than the Aβ group. The levels of pyroglutamic acid, glutamic acid, and phenylalanine showed similar changes in vivo and in vitro using HT-22 cells. The major metabolic pathways in the brain tissues and gut microbiota affected by Aβ or donepezil treatment of Aβ-injected mice were related to amino acid pathways and sugar metabolism, respectively. These findings suggest that alterations in the gut microbiota might influence the induction and amelioration of Aβ-induced cognitive dysfunction via the gut–brain axis. This study could provide basic data on the effects of Aβ and donepezil on gut microbiota and metabolites in an Aβ-induced cognitive impairment mouse model.

TRAIL-R Deficient Mice Are Protected from Neurotoxic Effects of Amyloid-β.

TRAIL, a member of TNF superfamily, is a potent inducer of neuronal death. Neurotoxic effects of TRAIL appear mediated by its death receptor TRAIL-R2/DR5. To assess the role of TRAIL/TRAIL-R2 pathway in AD-related neurodegeneration, we studied the impact of the treatment with amyloid-β (Aβ) upon cell viability and inflammation in TRAIL-R-deficient mice (TRAIL-R−/−). Here, we demonstrate that the lack of TRAIL-R2 protects from death cultured TRAIL-R−/− mouse embryonic hippocampal cells after treatment with either Aβ1-42 or TRAIL. Consistently, stereotaxic injection of Aβ1-42 resulted in blunted caspase activation, as well as in reduction of JNK phosphorylation and increased AKT phosphorylation in TRAIL-R−/− mice. Moreover, the lack of TRAIL-R2 was associated with blunted constitutive p53 expression in mice that have undergone Aβ1-42 treatment, as well as in decrease of phosphorylated forms of tau and GSK3β proteins. Likewise, TRAIL-R2 appears essential to both TRAIL and Aβ-mediated neurotoxicity and inflammation. Indeed, hippocampi of TRAIL-R−/− mice challenged with Aβ1-42, showed a slight expression of microglial (Iba-1) and astrocytic (GFAP) markers along with attenuated levels of IL-1β, TNF-α, NOS2 and COX2. In conclusion, the bulk of these results demonstrate that the constitutive lack of TRAIL-R2 is associated with a substantial reduction of noxious effects of Aβ1-42, providing further evidence on the prominent role played by TRAIL in course of Aβ-related neurodegeneration and confirming that the TRAIL system represents a potential target for innovative AD therapy.

Beta-amyloid Deposition in Biliary Atresia Reduces Liver Regeneration by Inhibiting Energy Metabolism and Mammalian Target of Rapamycin Signaling.

Biliary atresia (BA) is a devastating obstructive bile duct disease found in newborns. This study aims to investigate the roles and involved mechanisms of beta-amyloid (Aβ) in the pathogenesis of BA. We examined the distribution of Aβ protein and its precursor in the livers of patients with BA. A murine liver organoid and a zebrafish model were established to investigate the exact roles of Aβ in liver regeneration for BA. Both Aβ mRNA and protein significantly increased in livers of infants with BA and deposited around the central vein. In the plasma, Aβ elevated significantly in patients with BA and positively correlated with liver injury progression. In vitro , Aβ treatment induced abnormal morphology and caused impaired growth in liver organoids. Energy metabolism analysis demonstrated Aβ increased aerobic glycolysis and reduced ATP synthase in organoids, in which the mammalian target of rapamycin signaling was suppressed. In vivo , Aβ42 exposure caused liver degeneration in zebrafish larvae. Aβ depositing in livers of infants with BA reduced the liver regeneration through attenuating mitochondrial respiration and mammalian target of rapamycin signaling.

SIRT7 Deficiency Protects against Aβ42-Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells.

Alzheimer’s disease (AD) is an age-related neurodegenerative disease that is characterized by irreversible memory loss and cognitive decline. The deposition of amyloid-β (Aβ), especially aggregation-prone Aβ42, is considered to be an early event preceding neurodegeneration in AD. Sirtuins (SIRT1–7 in mammals) are nicotinamide adenine dinucleotide-dependent lysine deacetylases/deacylases, and several sirtuins play important roles in AD. However, the involvement of SIRT7 in AD pathogenesis is not known. Here, we demonstrate that SIRT7 mRNA expression is increased in the cortex, entorhinal cortex, and prefrontal cortex of AD patients. We also found that Aβ42 treatment rapidly increased NADPH oxidase 4 (NOX4) expression at the post-transcriptional level, and induced reactive oxygen species (ROS) production and apoptosis in neuronal SH-SY5Y cells. In contrast, SIRT7 knockdown inhibited Aβ42-induced ROS production and apoptosis by suppressing the upregulation of NOX4. Collectively, these findings suggest that the inhibition of SIRT7 may play a beneficial role in AD pathogenesis through the regulation of ROS production.

Melatonin Ameliorates the Progression of Alzheimer's Disease by Inducing TFEB Nuclear Translocation, Promoting Mitophagy, and Regulating NLRP3 Inflammasome Activity.

Background The NLRP3 inflammasome is overactivated in the brains of APP/PS1 transgenic mice and AD patients, and mitophagy has an obvious negative regulatory role on NLRP3 inflammasome activation. The protective effect of melatonin in AD may be related to the regulation of mitophagy and NLRP3 inflammasome activity. TFEB plays a critical role in maintaining autophagy/mitophagy. Studies have found that TFEB plays a protective role in AD. Methods APP/PS1 transgenic mice were given melatonin in their drinking water for 3 months. Compared with mice without melatonin treatment, the mice given melatonin showed changes in the following features: (1) cognitive function, (2) mitophagy-related proteins in the brain, (3) ROS, (4) NLRP3 inflammasome and related proteins and the concentrations of inflammatory cytokines, and (5) Aβ deposition. In in vitro experiments, effects of melatonin on mitophagy, NLRP3 inflammasome activity, and TFEB in SH-SY5Y cells with Aβ25-35 were observed. TFEB knockdown was implemented in combination with Aβ25-35 and melatonin treatment, and the expressions of TFEB, Parkin, p62, IL-1β, caspase-1, ROS, and IL-18 were explored. Results Melatonin improved cognitive function in APP/PS1 transgenic mice and decreased ROS and senile plaques. Melatonin promoted mitophagy in SH-SY5Y cells with Aβ25-35 and APP/PS1 transgenic mice. NLRP3 inflammasome activity was inhibited, and the concentrations of IL-18 and IL-1βwere clearly reduced. Compared with C57/BL6J mice, the amount of TFEB in the brain nucleus of APP/PS1 transgenic mice was decreased. Melatonin treatment increased the nuclear translocation of TFEB in SH-SY5Y cells. TFEB knockout was implemented in combination with Aβ25-35 and MT treatment; the expressions of Parkin, p62, caspase-1, IL-1β, IL-18, and ROS were accelerated. Conclusions Melatonin promotes mitophagy by inducing TFEB nuclear translocation, downregulates NLRP3 inflammasome activation, and exerts protective effects in SH-SY5Y cells and APP/PS1 transgenic mice.

Deletion of serine racemase reverses neuronal insulin signaling inhibition by amyloid-β oligomers.

Dysregulation of insulin signaling in the Alzheimer's disease (AD) brain has been extensively reported. Serine racemase (SR) modulates insulin secretion in pancreatic islets. This study aimed to examine whether SR regulates insulin synthesis and secretion in neurons, thereby modulating insulin signaling in the AD brain. Srr-knockout (Srr-/- ) mice generated with the CRISPR/Cas9 technique were used. Using immunofluorescence and fluorescence in situ hybridization, levels of insulin protein and insulin(ins2) mRNA were significantly increased in the hippocampal but not in hypothalamic sections of Srr-/- mice compared with WT mice. Real-time quantitative PCR revealed that ins2 mRNA from primary hippocampal neuronal cultures of Srr-/- mice was significantly increased compared with that from cultured neurons of WT mice. Notably, the secretion of proinsulin C-peptide was increased in Srr-/- neurons relative to WT neurons. By examining membrane fractional proteins with immunoblotting, Srr-/- neurons retained ATP-dependent potassium channels on plasmalemma and correspondingly contained higher levels of p-AMPK. After treatment with Aβ42, the phosphorylation levels of insulin receptor substrate at serine 616 636 (p-IRS1ser616,636 ) were significantly lower, whereas p-AKT308 and p-AKT473 were higher in Srr-/- neurons than in WT neurons, respectively. The phosphorylated form of c-Jun N-terminal kinase decreased in the cultured Srr-/- neurons relative to the WT neurons upon Aβ42 treatment. In contrast, phosphorylated protein kinase R remained at the same levels. Further, reactive oxygen species were reduced in cultured Srr-/- neurons under Aβ42 treatment relative to the WT neurons. Collectively, our study indicated that Srr deletion promoted insulin synthesis and secretion of proinsulin C-peptide, thereby reversing insulin resistance by Aβ42. This study suggests that targeting the neuronal SR may be utilized to enhance insulin signaling which is inhibited at the early stage of the AD brain.

Abeta-induced presynaptic release of UBC9 through extracellular vesicles involves SNAP23

Ubiquitin conjugating enzyme 9 (UBC9), the sole small ubiquitin-like modifier(SUMO) conjugating enzyme, is considered to be a vital regulator of the mechanism of SUMOylation and is likely to participate in the progression of Alzheimer’s disease (AD). Our previous studies found that UBC9 is highly mobile in neurons, but the underlying mechanism is still unknown. We designed to investigate the underlying mechanism of the synaptic redistribution of UBC9 in AD. We found that β-amyloid peptide (Aβ) significantly decreased presynaptic UBC9 expression and increased postsynaptic UBC9 expression but did not affect UBC9 expression in synaptosomes. Moreover, there is evidence that extracellular vesicles (EVs) may facilitate synaptic gap transmission. Immunoprecipitation assays showed that flotillin, which is specifically expressed in EVs, co-immunoprecipitated with UBC9 in cell lysates and media and that Aβ treatment enhanced this interaction. Additionally, epidermal growth factor and oleanolic acid have been found to either promote or inhibit the release of EVs, thus blocking the Aβ-induced presynaptic release of UBC9. However, knockdown of synaptosome associated protein 23 (SNAP-23) or inhibition of SNAP23 phosphorylation was found to secure Aβ-induced presynaptic release of UBC9. These results suggest that Aβ induces redistribution of UBC9 from presynaptic to postsynaptic terminals, which may mediate through EVs associated with SNAP23. Our study reveals the cellular mechanisms of EVs as an essential component of the presynaptic release of UBC9.

Microglial polarization differentially affects neuronal vulnerability to the β-amyloid protein: Modulation by melatonin

Microglial cells play a central but yet debated role in neuroinflammatory events occurring in Alzheimer’s disease (AD). We here explored how microglial features are modulated by melatonin following β-amyloid (Aβ42)-induced activation and examined the cross-talk with Aβ-challenged neuronal cells. Human microglial HMC3 cells were exposed to Aβ42 (200 nM) in the presence of melatonin (MEL; 1 μM) added since the beginning (MELco) or after a 72 h-exposure to Aβ42 (MELpost). In both conditions, MEL favored an anti-inflammatory activation and rescued SIRT1 and BDNF expression/release. Caspase-1 up-regulation and phospho-ERK induction following a prolonged exposure to Aβ42 were prevented by MEL. In addition, MEL partially restored proteasome functionality that was altered by long-term Aβ42 treatment, re-establishing both 20S and 26S chymotrypsin-like activity. Differentiated neuronal-like SH-SY5Y cells were exposed to Aβ42 (200 nM for 24 h) in basal medium or in the presence of conditioned medium (CM) collected from microglia exposed for different times to Aβ42 alone or in combination with MELco or MELpost. Aβ42 significantly reduced pre-synaptic proteins synaptophysin and VAMP2 and mean neuritic length. These effects were prevented by CM from anti-inflammatory microglia (Aβ42 for 6 h), or from MELco and MELpost microglia, but the reduction of neuritic length was not rescued when the SIRT1 inhibitor EX527 was added.In conclusion, our data add to the concept that melatonin shows a promising anti-inflammatory action on microglia that is retained even after pro-inflammatory activation, involving modulation of proteasome function and translating into neuroprotective microglial effects.

Baicalin Attenuated Aβ 1-42-Induced Apoptosis in SH-SY5Y Cells by Inhibiting the Ras-ERK Signaling Pathway.

Alzheimer's disease (AD) is a serious neurodegenerative disease. It is widely believed that the accumulation of amyloid beta (Aβ) in neurons around neurofibrillary plaques is the main pathological characteristic of AD; however, the molecular mechanism underlying these pathological changes is not clear. Baicalin is a flavonoid extracted from the dry root of Scutellaria baicalensis Georgi. Studies have shown that baicalin exerts excellent anti-inflammatory and neuroprotective effects. In this study, an AD cell model was established by exposing SH-SY5Y cells to Aβ1-42 and treating them with baicalin. Cell survival, cell cycle progression, and apoptosis were measured by MTT, flow cytometry, and immunofluorescence assays, respectively. The expression levels of Ras, ERK/ERK phosphorylation (p-ERK), and cyclin D1 were measured by Western blotting. In addition, whether the MEK activator could reverse the regulatory effect of baicalin on Ras-ERK signaling was investigated using Western blotting. We found that baicalin improved the survival, promoted the proliferation, and inhibited the apoptosis of SH-SY5Y cells after Aβ1-42 treatment. Baicalin also ameliorated Aβ1-42-induced cell cycle arrest at the S phase and induced apoptosis. Furthermore, baicalin inhibited the levels of Ras, p-ERK, and cyclin D1 induced by Aβ, and this effect could be reversed by the MEK activator. Therefore, we suggest that baicalin may regulate neuronal cell cycle progression and apoptosis in Aβ1-42-treated SH-SY5Y cells by inhibiting the Ras-ERK signaling pathway. This study suggested that baicalin might be a useful therapeutic agent for senile dementia, especially AD.

Methyltransferase-Like 3 Rescues the Amyloid-beta protein-Induced Reduction of Activity-Regulated Cytoskeleton Associated Protein Expression via YTHDF1-Dependent N6-Methyladenosine Modification.

Activity-regulated cytoskeleton-associated protein (ARC) is activated by the induction of long-term potentiation and plays an important role in the synaptic plasticity of memory consolidation. Previous studies have shown that abnormal expression of ARC in the brains of patients with Alzheimer’s Disease (AD) leads to the disturbance of synaptic plasticity. ARC expression is mainly regulated by transcriptional and post-translational modification. However, it is unclear whether N6-methyladenosine (m6A) engages in the epigenetic modification of ARC. The AlzData database was used to analyze the brain of AD patients, and Aβ-induced cell models were used. We revealed that ARC expression was reduced in AD patients and Aβ-induced cell models. There were five m6A modification sites of ARC mRNA that were predicted by the SRAMP database, and ARC mRNA was confirmed as the target gene of methyltransferase-like 3 (METTL3) by MeRIP. Amyloid-beta protein (Aβ) repressed the m6A modification. Knockdown of METTL3 decreased ARC mRNA m6A modification and reduced ARC protein expression, while overexpression of METTL3 rescued ARC expression after Aβ treatment. Knockdown of YTH domain family, member 1 (YTHDF1) decreased ARC protein expression, while the overexpression of YTHDF1 could not rescue the loss of ARC protein expression after 3-deazaadenosine treatment or knockdown of METTL3. Our findings identify that METTL3 rescues the Aβ-induced reduction of ARC expression via YTHDF1-Dependent m6A modification, which suggests an important mechanism of epigenetic alteration in AD.

Impairment of ciliary dynamics in an APP knock-in mouse model of Alzheimer's disease

The primary cilium is a specialized microtubule-based sensory organelle that extends from the cell body of nearly all cell types. Neuronal primary cilia, which have their own unique signaling repertoire, are crucial for neuronal integrity and the maintenance of neuronal connectivity throughout adulthood. Dysfunction of cilia structure and ciliary signaling is associated with a variety of genetic syndromes, termed ciliopathies. One of the characteristic features of human ciliopathies is impairment of memory and cognition, which is also observed in Alzheimer's disease (AD). Amyloid β peptide (Aβ) is produced through the proteolytic processing of amyloid precursor protein (APP), and Aβ accumulation in the brain is proposed to be an early toxic event in the pathogenesis of AD. To evaluate the effect of increased Aβ level on primary cilia, we assessed ciliary dynamics in hippocampal neurons in an APP knock-in AD model (AppNL-G-F mice) compared to that in wild-type mice. Neuronal cilia length in the CA1, CA3, and dentate gyrus (DG) of wild-type mice increased significantly with age. In AppNL-G-F mice, such elongation was detected in the DG but not in the CA1 and CA3, where more Aβ accumulation was observed. We further demonstrated that Aβ1-42 treatment decreased cilia length both in hTERT-RPE1 cells and dissociated rat hippocampal neurons. There is growing evidence that reduced cilia length is associated with perturbations of synaptic connectivity and dendrite complexity. Thus, our observations raise the important possibility that structural alterations in neuronal cilia might have a role in AD development.

Beta-Amyloid (Aβ1-42) Increases the Expression of NKCC1 in the Mouse Hippocampus.

Alzheimer’s disease (AD) is a neurodegenerative disorder with an increasing need for developing disease-modifying treatments as current therapies only provide marginal symptomatic relief. Recent evidence suggests the γ-aminobutyric acid (GABA) neurotransmitter system undergoes remodeling in AD, disrupting the excitatory/inhibitory (E/I) balance in the brain. Altered expression levels of K-Cl-2 (KCC2) and N-K-Cl-1 (NKCC1), which are cation–chloride cotransporters (CCCs), have been implicated in disrupting GABAergic activity by regulating GABAA receptor signaling polarity in several neurological disorders, but these have not yet been explored in AD. NKCC1 and KCC2 regulate intracellular chloride [Cl−]i by accumulating and extruding Cl−, respectively. Increased NKCC1 expression in mature neurons has been reported in these disease conditions, and bumetanide, an NKCC1 inhibitor, is suggested to show potential therapeutic benefits. This study used primary mouse hippocampal neurons to explore if KCC2 and NKCC1 expression levels are altered following beta-amyloid (Aβ1-42) treatment and the potential neuroprotective effects of bumetanide. KCC2 and NKCC1 expression levels were also examined in 18-months-old male C57BL/6 mice following bilateral hippocampal Aβ1-42 stereotaxic injection. No change in KCC2 and NKCC1 expression levels were observed in mouse hippocampal neurons treated with 1 nM Aβ1-42, but NKCC1 expression increased 30-days post-Aβ1-42-injection in the CA1 region of the mouse hippocampus. Primary mouse hippocampal cultures were treated with 1 nM Aβ1-42 alone or with various concentrations of bumetanide (1 µM, 10 µM, 100 µM, 1 mM) to investigate the effect of the drug on cell viability. Aβ1-42 produced 53.1 ± 1.4% cell death after 5 days, and the addition of bumetanide did not reduce this. However, the drug at all concentrations significantly reduced cell viability, suggesting bumetanide is highly neurotoxic. In summary, these results suggest that chronic exposure to Aβ1-42 alters the balance of KCC2 and NKCC1 expression in a region-and layer-specific manner in mouse hippocampal tissue; therefore, this process most likely contributes to altered hippocampal E/I balance in this model. Furthermore, bumetanide induces hippocampal neurotoxicity, thus questioning its suitability for AD therapy. Further investigations are required to examine the effects of Aβ1-42 on KCC2 and NKCC1 expression and whether targeting CCCs might offer a therapeutic approach for AD.

Elevated amyloid beta disrupts the nanoscale organization and function of synaptic vesicle pools in hippocampal neurons.

Alzheimer's disease is linked to increased levels of amyloid beta (Aβ) in the brain, but the mechanisms underlying neuronal dysfunction and neurodegeneration remain enigmatic. Here, we investigate whether organizational characteristics of functional presynaptic vesicle pools, key determinants of information transmission in the central nervous system, are targets for elevated Aβ. Using an optical readout method in cultured hippocampal neurons, we show that acute Aβ42 treatment significantly enlarges the fraction of functional vesicles at individual terminals. We observe the same effect in a chronically elevated Aβ transgenic model (APPSw,Ind) using an ultrastructure-function approach that provides detailed information on nanoscale vesicle pool positioning. Strikingly, elevated Aβ is correlated with excessive accumulation of recycled vesicles near putative endocytic sites, which is consistent with deficits in vesicle retrieval pathways. Using the glutamate reporter, iGluSnFR, we show that there are parallel functional consequences, where ongoing information signaling capacity is constrained. Treatment with levetiracetam, an antiepileptic that dampens synaptic hyperactivity, partially rescues these transmission defects. Our findings implicate organizational and dynamic features of functional vesicle pools as targets in Aβ-driven synaptic impairment, suggesting that interventions to relieve the overloading of vesicle retrieval pathways might have promising therapeutic value.