BACE1 Overexpression Research Articles

Neuroprotective effect of Kurarinone against corticosterone-induced cytotoxicity on rat hippocampal neurons by targeting BACE1 to activate P13K-AKT signaling - A potential treatment in insomnia disorder.

The hippocampus has been implicated in the pathogenesis of insomnia disorder (ID) and the purpose of this study was to investigate the neuroprotective mechanism of the natural flavone Kurarinone (Kur) on hippocampal neurotoxicity as a potential treatment of ID. The effect of Kur on hippocampal neuronal cell (HNC) viability and apoptosis were assessed by Cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Then, the effect of Kur on β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), brain-derived neurotrophic factor (BDNF), and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) phosphorylation level were measured by Western blot. Further, SwissTargetPrediction analysis and molecular docking experiments were used to detect a potential target of Kur. Then, the p-chlorophenylalanine (PCPA) model was established in vivo to further study the effect of BACE1 expression on Kur and HNC. As a result, HNC viability was only significantly decreased by 2 μM of Kur. Kur reversed the impacts of corticosterone upon inhibiting viability (0.25-1 μM), PI3K (0.5-1 μM)/AKT phosphorylation, and BDNF (1 μM) level, and enhancing the apoptosis (0.25-1 μM) and BACE1 expression (1 μM) in HNCs. BACE1 was a potential target of Kur. Notably, Kur (150 mg/kg) attenuated PCPA-induced upregulation of BACE1 expression in rat hippocampal tissues as ZRAS (0.8 g/kg). The effects of Kur (1 μM) on corticosterone-treated HNCs were reversed by BACE1 overexpression. Collectively, Kur downregulates BACE1 level to activate PI3K/AKT, thereby attenuating corticosterone-induced toxicity in HNCs, indicating that Kur possibly exerted a neuroprotective effect, which providing a new perspective for the treatment of insomnia disorders.

β-secretase 1 overexpression by AAV-mediated gene delivery prevents retina degeneration in a mouse model of age-related macular degeneration

We reported previously that β-site amyloid precursor protein cleaving enzyme (BACE1) is strongly expressed in the normal retina and that BACE1-/- mice develop pathological phenotypes associated with age-related macular degeneration (AMD). BACE1 expression is increased within the neural retina and retinal pigment epithelium (RPE) in AMD donor eyes suggesting that increased BACE1 is compensatory. We observed that AAV-mediated BACE1 overexpression in the RPE was maintained up to 6months after AAV1-BACE1 administration. No significant changes in normal mouse visual function or retinal morphology were observed with low-dose vector while the high-dose vector demonstrated some early pathology which regressed with time. No increase in β-amyloid was observed. BACE1 overexpression in the RPE of the superoxide dismutase 2 knockdown (SOD2 KD) mouse, which exhibits an AMD-like phenotype, prevented loss of retinal function and retinal pathology, and this was sustained out to 6months. Furthermore, BACE1 overexpression was able to inhibit oxidative stress, microglial changes, and loss of RPE tight junction integrity (all features of AMD) in SOD2 KD mice. In conclusion, BACE1 plays a key role in retina/RPE homeostasis, and BACE1 overexpression offers a novel therapeutic target in the treatment of AMD.

Potential drugs for the treatment of Alzheimer's disease.

It is well known that amyloid precursor protein (APP), the enzyme β-secretase 1 (BACE1), cyclooxygenase 2 (COX-2), nicastrin (NCT), and hyperphosphorylated tau protein (p-tau) are closely related to the development of Alzheimer's disease (AD). In addition, recent evidence shows that neuroinflammation also contributes to the pathogenesis of AD. Although the mechanism is not clearly known, such inflammation could alter the activity of the aforementioned molecules. Therefore, the use of anti-inflammatory agents could slow the progression of the disease. Nimesulide, resveratrol, and citalopram are three anti-inflammatory agents that could contribute to a decrease in neuroinflammation and consequently to a decrease in the overexpression of APP, BACE1, COX-2, NCT, and p-Tau, as they possess anti-inflammatory effects that could regulate the expression of APP, BACE1, COX-2, NCT, and p-Tau of potent pro-inflammatory markers indirectly involved in the expression of APP, BACE1, NCT, COX-2, and p-Tau; therefore, their use could be beneficial as preventive treatment as well as in the early stages of AD.

MiR-29c-3p Attenuates β-Amyloid-Induced Neurotoxicity in Alzheimer’s Disease Through Regulating β-Site Amyloid Precursor Protein-Cleaving Enzyme 1

The aberrantly expressed microRNAs (miRNAs) including miR-29c-3p have been reported in the brains of Alzheimer's disease (AD) patients in recent researches. Nevertheless, the functional role and underlying molecular mechanism of miR-29c-3p in AD pathogenesis are still not well elucidated. The purpose of this study was to examine whether miR-29c-3p regulated β-Ameyloid (Aβ)-induced neurotoxicity by targeting β-site amyloid precursor protein-cleaving enzyme 1 (BACE1). The expressions of miR 29c 3p and BACE1 mRNA and protein levels in Aβ-treated PC12 cellular AD model were examined by qRT-PCR and western blot analyses. Luciferase reporter assay verified the potential target of miR 29c 3p. Cell viability, apoptosis, and caspase-3 activity in PC12 cells were detected by the MTT assay, flow cytometry, and caspase-3 activity assay, respectively. Our results indicated that miR-29c-3p downregulation and BACE1 upregulation existed in the cellular AD model of PC12 cells. Moreover, miR-29c-3p directly inhibited BACE1 expression. miR-29c-3p overexpression and BACE1 knockdown strengthened Aβ-induced cell apoptosis, and caspase-3 activity in PC12 cells, which was partially eliminated by over-expression of BACE1. Conversely, BACE1 knockdown reversed the miR-29c-3p inhibition- mediated inhibitory effect on Aβ-induced cell toxicity, apoptosis, and caspase-3 activity in PC12 cells. Considering, miR-29c-3p attenuated Aβ-induced neurotoxicity through targeting BACE1 in an cellular AD model of PC12, providing a potential therapeutic target for AD treatment.

Inactivation of BACE1 increases expression of endothelial nitric oxide synthase in cerebrovascular endothelium

Cerebrovascular effects of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) inactivation have not been systematically studied. In the present study we employed cultured human brain microvascular endothelial cells (BMECs), BACE1-knockout (BACE1−/−) mice and conditional (tamoxifen-induced) endothelium-specific BACE1-knockout (eBACE1−/−) mice to determine effect of BACE1 inhibition on expression and function of endothelial nitric oxide synthase (eNOS). Deletion of BACE1 caused upregulation of eNOS and glypican-1 (GPC1) in human BMECs treated with BACE1-siRNA, and cerebral microvessels of male BACE1−/− mice and male eBACE1−/− mice. In addition, BACE1siRNA treatment increased NO production in human BMECs. These effects appeared to be independent of amyloid β-peptide production. Furthermore, adenoviral-mediated overexpression of BACE1 in human BMECs down-regulated GPC1 and eNOS. Treatment of human BMECs with GPC1siRNA suppressed mRNA and protein levels of eNOS. In basilar arteries of male eBACE1−/− mice, endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to NO donor, DEA-NONOate, were not affected, consistent with unchanged expression of eNOS and phosphorylation of eNOS at Ser1177 in large cerebral arteries. In aggregate, our findings suggest that under physiological conditions, inactivation of endothelial BACE1 increases expression of eNOS in cerebral microvessels but not in large brain arteries. This effect appears to be mediated by increased GPC1 expression.

Circular RNA AXL increases neuron injury and inflammation through targeting microRNA-328 mediated BACE1 in Alzheimer’s disease

ObjectiveOur previous study observed that circular RNA AXL (circ-AXL, access number circ_0002945) was closely correlated with disease risk and severity of Alzheimer’s Disease (AD) by microarray and RT-qPCR validation. Then this current study aimed to further investigate the effect of circ-AXL on regulating neuron injury and inflammation in cellular AD models and its underlying molecular mechanism. MethodsSK-N-SH and SK-SY5Y cell lines were treated by amyloid β to construct cellular AD models. Then control or circ-AXL overexpression, control or circ-AXL knock-down, microRNA-328 (miR-328) knock-down with or without circ-AXL knock-down, as well as BACE1 overexpression with or without miR-328 overexpression plasmids were transfected into cellular AD models. Furthermore, neuron injury and inflammation were detected. ResultsCirc-AXL overexpression increased apoptosis rate and declined neurite outgrowth, as well as elevated inflammatory cytokines in cellular AD models; but circ-AXL knockdown exhibited opposite effects. Additionally, circ-AXL negatively regulated miR-328 but positively modulated BACE1; besides, miR-328 negatively regulated BACE1; further luciferase reporter gene assay presented that circ-AXL directly bound miR-328, and miR-328 directly bound BACE1. Furthermore, miR-328 overexpression decreased apoptosis rate, elevated neurite outgrowth, and declined inflammatory cytokines in cellular AD models; but miR-328 knockdown presented opposite effects. Notably, miR-328 knockdown attenuated the effect of circ-AXL knockdown on cellular AD models. Moreover, BACE1 overexpression aggravated neuron injury and inflammation, as well as attenuated the effect of miR-328 overexpression on these functions in cellular AD models. ConclusionCirc-AXL may serve as a potential treatment target via miR-328 mediated BACE1 in AD.

MiR-29c-3p May Promote the Progression of Alzheimer's Disease through BACE1.

The aim of this study was to explore the specific role of miR-29c-3p in Alzheimer's disease (AD). Animal models of AD were established by injecting streptozotocin (STZ) into mice through the lateral ventricle, while cell models of AD were induced by 10 μM β-amyloid (Aβ). We detected miR-29c-3p and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) contents and measured AD cell proliferation and apoptosis. A low miR-29c-3p level and a high BACE1 level were detected in the brain tissue of AD animal models and AD cell models. Aβ-processed cells had markedly lower proliferation activity, higher apoptosis, increased phosphorylation of tau protein was over phosphorylated, but the overexpression of miR-29c-3p or the silencing of BACE1 significantly enhanced the cell proliferation activity and reduced cell apoptosis by regulating the contents of related proteins. Inhibition of miR-29c-3p or overexpression of BACE1 aggravated Aβ-induced side effects. We used Targetscan7.2 to predict the downstream target genes of miR-29c-3p. Then, we detected that there were target binding sites between miR-29c-3p and BACE1. The rescue experiment identified BACE1 as a functional target for miR-29c-3p. AD leads to decreased miR-29c-3p level and increased BACE1 level. MiR-29c-3p has specific binding sites with the 3′-untranslated region (3′-UTR) of BACE1 and thus negatively regulates the BACE1 level, thereby affecting the progression of AD.

Long non-coding RNA BACE1-AS plays an oncogenic role in hepatocellular carcinoma cells through miR-214-3p/APLN axis.

BACE1 antisense RNA (BACE1-AS) is implicated in promoting cell proliferation in different types of tumors. However, the function and mechanism of BACE1-AS in hepatocellular carcinoma (HCC) are still unclear. In the present study, we found that the relative expression of BACE1-AS in HCC cell lines, HCC tissues, and serum samples of HCC patients was significantly increased, and its high expression was correlated with the poor prognosis of HCC patients. In addition, overexpression of BACE1 promoted HCC cell proliferation, cell cycle progression, migration, and invasion, but inhibited cell apoptosis, while knockdown of BACE1 exerted the opposite role. Furthermore, BACE1-AS sponged miR-214-3p and inhibited its expression, thus promoting Apelin (APLN) expression. Overexpression or knockdown of miR-214-3p could partially reverse the abnormal proliferation, cell cycle progression, migration, invasion, and apoptosis caused by overexpression or knockdown of BACE1. These findings suggest that the BACE1-AS/miR-214-3p/APLN axis is a novel signaling pathway that facilitates HCC.

MicroRNA-132-3p alleviates neuron apoptosis and impairments of learning and memory abilities in Alzheimer’s disease by downregulation of HNRNPU stabilized BACE1

ABSTRACT Alzheimer’s disease (AD) is a progressive neuro-degenerative disease characterized by dementia. MicroRNAs (miRNAs) are involved in many diseases, including AD. MiR-132-3p has been identified to be downregulated in AD. In this study, we explored the effects of miR-132-3p on neuron apoptosis and impairments of learning and memory abilities. Aβ1-42-stimulated SH-SY5Y cells were used as in vitro models of AD. An AD-like homocysteine (Hcy) rat model was established to evaluate the effects of miR-132-3p on AD pathogenesis in vivo. RIP, RNA pull down and luciferase reporter assays were conducted to investigate the relationship between miR-132-3p and its downstream target genes. The viability and apoptosis of SH-SY5Y cells were measured by CCK-8 and TUNEL assays. The rat spatial learning and memory abilities were accessed using Morris water maze test. Results indicated that miR-132-3p was downregulated in SH-SY5Y cells after Aβ1-42 treatment and promoted cell apoptosis. Mechanistically, miR-132-3p targeted heterogeneous nuclear ribonucleoprotein U (HNRNPU). HNRNPU acted as an RNA binding protein (RBP) to regulate the mRNA stability of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Overexpression of HNRNPU or BACE1 reversed the effects of miR-132-3p overexpression on the viability and apoptosis of Aβ1-42-treated SH-SY5Y cells. In vivo experiments revealed the downregulation of miR-132-3p in the hippocampus of Hcy-treated rats. MiR-132-3p suppressed levels of apoptotic genes in hippocampus and reduced impairments of learning and memory abilities in Hcy-treated rats. In conclusion, miR-132-3p reduces apoptosis of SH-SY5Y cells and alleviates impairments of learning and memory abilities in AD rats by modulating the HNRNPU/BACE1 axis.

Elevated β-secretase 1 expression mediates CD4+ T cell dysfunction via PGE2 signalling in Alzheimer’s disease

Circulating CD4+ T cells are dysfunctional in Alzheimer’s disease (AD), however, the underlying molecular mechanisms are not clear. In this study, we demonstrate that CD4+ T cells from AD patients and 5xFAD transgenic mice exhibit elevated levels of β-secretase 1 (BACE1). Overexpression of BACE1 in CD4+ T cells potentiated CD4+ T-cell activation and T-cell-dependent immune responses. Mechanistically, BACE1 modulates prostaglandin E2 (PGE2) synthetase—microsomal prostaglandin E synthase 2 (mPGES2)—to promote mPGES2 maturation and PGE2 production, which increases T-cell receptor (TCR) signalling. Moreover, administration of peripheral PGE2 signalling antagonists partially ameliorates CD4+ T cell overactivation and AD pathology in 5xFAD mice. Overall, our results reveal a potential role for BACE1 in mediating CD4+ T-cell dysfunction in AD.

A peptide fraction of Olive Flounder (Paralichthys olivaceus) Skin Hydrolysate Inhibits Amyloid-β Generation in SH-SY5Y Cells via Suppression of BACE1 Expression

The prevalence of degenerative neuro-diseases such as Alzheimer’s disease has increased with an increase in the life expectancy especially as seen in developed countries. According to the Amyloid Hypothesis, β-secretase leads to the onset of Alzheimer's disease though the generation of amyloid-β by proteolysis of amyloid precursor protein. Amyloid-β aggregates to form oligomers that build up to form plaques. Olive flounder (Paralichthys olivaceus) is high in protein content, with a higher protein content in skin (27.65%) compared to muscle (19.65%) according to proximate analysis results. Enzyme hydrolysates were prepared from skin and muscle using several enzymes. The neutrase skin hydrolysate showed the highest β-secretase inhibitory activity IC50 0.61 mg/mL. Purification steps were performed using ion exchange chromatography on QMA and CM-cellulose columns and SPE C18 column to produce an active fraction QMA-F1 with an IC50 value of 32.80 µg/mL. QMA-F1 was non-cytotoxic to SH-SY5Y cells, reduced BACE1 overexpression and attenuated amyloidogenesis. This suggests that the active β-secretase inhibitory peptides from olive flounder skin hydrolysates can be utilized as an ingredient in development of new pharmaceutical and nutraceutical therapeutic agents for Alzheimer’s Disease.

MicroRNA‐200a‐3p mediates neuroprotection in Alzheimer‐related deficits and attenuates amyloid‐beta overproduction and tau hyperphosphorylation via co‐regulating BACE1 and PRKACB

Alzheimer’s disease (AD) is characterized by two landmark pathologies, the overproduction of amyloid‐beta peptides (Aβ), predominated by the β‐amyloid protein precursor cleaving enzyme 1 (BACE1), and hyperphosphorylation of the microtubule protein, tau, due to an imbalance in a kinase / phosphatase system that involves the activation of the protein kinase A (PKA). Current evidence indicates that brain microRNAs participate in multiple aspects of AD pathology. Here, the role and underlying molecular mechanisms of microRNA‐200a‐3p (miR‐200a‐3p) in mediating neuroprotection against AD‐related deficits were investigated. The expression of miR‐200a‐3p was measured in the hippocampus of APP / PS1 and SAMP8 mice and in an AD cell model in vitro, as well as in blood plasma extracted from AD patients. The targets of miR‐200a‐3p were determined using bioinformatics and dual‐luciferase assay analyses. In addition, cell apoptosis was detected using flow cytometry and related protein levels were measured using Western blot and ELISA techniques. miR‐200a‐3p was confirmed to be depressed in microarray miRNA profile analysis in vitro and in vivo, suggesting that miR‐200a‐3p is a potential biomarker of AD. Subsequently, miR‐200a‐3p was demonstrated to inhibit cell apoptosis accompanied by the inactivation of the Bax / caspase‐3 axis and downregulation of Aβ1‐42 and tau phosphorylation levels in vitro. Further mechanistic studies revealed that miR‐200a‐3p reduced the production of Aβ1‐42 and decreased hyperphosphorylation of tau by regulating the protein translocation of BACE1 and the protein kinase cAMP‐activated catalytic subunit beta (PRKACB) associated with the three prime untranslated regions (3’‐UTRs), respectively. Importantly, the function of miR‐200a‐3p was reversed by overexpression of BACE1 or PRKACB in cultured cells. This resulted in an elevation in cell apoptosis, and an increase in Aβ1‐42, and tau hyperphosphorylation levels, involving the epitopes, threonine 205, serine 202, 214, 396, and 356, the favorable phosphorylated sites of PKA. In conclusion, our study suggests that miR‐200a‐3p is implicated in the pathology of AD, exerting neuroprotective effects against Aβ‐induced toxicity by two possible mechanisms: one involving the inhibition of Aβ overproduction via suppressing the expression of BACE1 and; two, synergistically, decreasing the hyperphosphorylation of tau via attenuating the expression of PKA.Support or Funding Information This study was supported by the National NaturalScience Foundation of China (No. U1803281 and 81673411), China; the Non‐profitCentral Research Institute Fund of Chinese Academy of Medical Sciences(2018RC350013), China; and Chinese Academy Medical Sciences (CAMS) InnovationFund for Medical Science (2017‐I2M‐1‐016), China.

MicroRNA-200a-3p Mediates Neuroprotection in Alzheimer-Related Deficits and Attenuates Amyloid-Beta Overproduction and Tau Hyperphosphorylation via Coregulating BACE1 and PRKACB.

Alzheimer’s disease (AD) is characterized by two landmark pathologies, the overproduction of amyloid-beta peptides (Aβ), predominated by the β-amyloid protein precursor cleaving enzyme 1 (BACE1), and hyperphosphorylation of the microtubule protein, tau, because of an imbalance in a kinase/phosphatase system that involves the activation of the protein kinase A (PKA). Current evidence indicates that brain microRNAs participate in multiple aspects of AD pathology. Here, the role and underlying molecular mechanisms of microRNA-200a-3p (miR-200a-3p) in mediating neuroprotection against AD-related deficits were investigated. The expression of miR-200a-3p was measured in the hippocampus of APP/PS1 and SAMP8 mice and in an AD cell model in vitro, as well as in blood plasma extracted from AD patients. The targets of miR-200a-3p were determined using bioinformatics and dual-luciferase assay analyses. In addition, cell apoptosis was detected using flow cytometry, and related protein levels were measured using Western blot and enzyme-linked immunosorbent assay (ELISA) techniques. miR-200a-3p was confirmed to be depressed in microarray miRNA profile analysis in vitro and in vivo, suggesting that miR-200a-3p is a potential biomarker of AD. Subsequently, miR-200a-3p was demonstrated to inhibit cell apoptosis accompanied by the inactivation of the Bax/caspase-3 axis and downregulation of Aβ1-42 and tau phosphorylation levels in vitro. Further mechanistic studies revealed that miR-200a-3p reduced the production of Aβ1-42 and decreased hyperphosphorylation of tau by regulating the protein translocation of BACE1 and the protein kinase cAMP-activated catalytic subunit beta (PRKACB) associated with the three prime untranslated regions, respectively. Importantly, the function of miR-200a-3p was reversed by overexpression of BACE1 or PRKACB in cultured cells. This resulted in an elevation in cell apoptosis and increases in Aβ1-42 and tau hyperphosphorylation levels, involving the epitopes threonine 205 and serine 202, 214, 396, and 356, the favorable phosphorylated sites of PKA. In conclusion, our study suggests that miR-200a-3p is implicated in the pathology of AD, exerting neuroprotective effects against Aβ-induced toxicity by two possible mechanisms: one involving the inhibition of Aβ overproduction via suppression of the expression of BACE1 and synergistically decreasing the hyperphosphorylation of tau via attenuation of the expression of PKA.

MiR-16 attenuates β-amyloid-induced neurotoxicity through targeting β-site amyloid precursor protein-cleaving enzyme 1 in an Alzheimer's disease cell model.

The aberrant deposition of β-amyloid (Aβ) is closely linked to the pathogenesis and development of Alzheimer's disease (AD). MiR-16 was abnormally downregulated and may be related to the development of AD. However, the functional role and molecular mechanism of miR-16 in AD pathogenesis are still not well elucidated. The expressions of miR-16 and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA and protein levels in AD brain tissues and Aβ-treated PC12 cellular AD model were examined by qRT-PCR and western blot analyses. Luciferase reporter assay was used to verify the potential target of miR-16. The cell viability, apoptosis, and caspase-3 activity in PC12 cells were determined by the MTT assay, flow cytometry analysis, and caspase-3 activity assay, respectively. Downregulation of miR-16 and upregulation of BACE1 existed in AD tissues and the cellular AD model of PC12. In addition, miR-16 directly suppressed BACE1 expression. Moreover, miR-16 overexpression and BACE1 knockdown facilitated Aβ-induced cell toxicity, apoptosis, and caspase-3 activity in N2a cells, which was partially eliminated by overexpression of BACE1. In contrast, BACE1 knockdown reversed the miR-16 inhibition-mediated inhibitory effect on Aβ-induced cell toxicity, apoptosis, and caspase-3 activity in PC12 cells. Collectively, miR-16 attenuated Aβ-induced neurotoxicity through targeting BACE1 in an Aβ insult cellular AD model, providing a potential therapeutic target for AD treatment.

BACE1 SUMOylation increases its stability and escalates the protease activity in Alzheimer’s disease

Amyloid beta (Aβ) is a major pathological marker in Alzheimer's disease (AD), which is principally regulated by the rate-limiting β-secretase (i.e., BACE1) cleavage of amyloid precursor protein (APP). However, how BACE1 activity is posttranslationally regulated remains incompletely understood. Here, we show that BACE1 is predominantly SUMOylated at K501 residue, which escalates its protease activity and stability and subsequently increases Aβ production, leading to cognitive defect seen in the AD mouse model. Compared with a non-SUMOylated K501R mutant, injection of wild-type BACE1 significantly increases Aβ production and triggers cognitive dysfunction. Furthermore, overexpression of wild-type BACE1, but not non-SUMOylated K501R mutant, facilitates senile plaque formation and aggravates the cognitive deficit seen in the APP/PS1 AD mouse model. Together, our data strongly suggest that K501 SUMOylation on BACE1 plays a critical role in mediating its stability and enzymatic activity.

Manipulation of Amyloid Precursor Protein Processing Impacts Brain Bioenergetics and Glucose Metabolism

The amyloid precursor protein (APP) and one of its terminal cleavage products, amyloid beta (Aβ) are proposed to play a central role in the pathogenesis of Alzheimer's disease (AD). These proteins have also been shown to co-localise within mitochondria with Aβ directly inhibiting the electron transport chain. Cleavage of APP occurs down distinct pathways. Initial cleavage of the full-length protein is mediated by either α- or β-secretase (BACE1). Subsequent cleavage by γ-secretase releases p3 or Aβ protein fragments into the extracellular milieu. Whilst these interactions with mitochondria have been noted, the impact of manipulating APP processing by these distinct pathways on cellular metabolism and bioenergetics has yet to be elucidated. We utilised the human SH-SY5Y neuronal cell line stably overexpressing BACE1 and cortical slices from mice bearing inducible overexpression of APP harbouring the Swedish and Indiana mutations, favouring Aβ production. Protein expression and enzyme activity were determined via western blotting and enzyme-linked immunosorbance assays (ELISA). A Seahorse extracellular flux analyser and radio-labelled substrate assays were used to determine cellular bioenergetics in real-time. All data are expressed as mean ± standard error of the mean and statistical significance determined by Student's t-test. Manipulation of APP cleavage resulted in alterations of 2-deoxyglucose uptake into cells. Chronic elevation in BACE1 resulted in impaired functioning of a key fuel-partitioning enzyme, pyruvate dehydrogenase (PDH, activity reduced to 69 ± 8%, p < 0.05, n=6). This reduced substrate delivery to the mitochondria and increased reliance upon aerobic glycolysis for ATP generation (oxygen consumption rate (OCR) reduced to 65 ± 9%, p < 0.01, n=7 and extracellular acidification rate (ECAR) increased to 165 ± 16%, p < 0.01, n=7). Thus BACE1 overexpression impairs neuronal glucose oxidation resulting in an increased reliance upon aerobic glycolysis for ATP generation. Acute cortical overexpression of APP favouring amyloidogenic processing resulted in a significant reduction in PDH protein expression (reduced to 62 ± 6%, p < 0.01, n = 9-10) and activity of key tricarboxylic acid cycle enzymes, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase (reduced to 62 ± 7%, p < 0.01, n=6 and 88 ± 2%, p < 0.001, n = 6). Therefore, manipulation of APP processing towards Aβ production in cells and tissue significantly impacts cellular metabolic pathways at the level of glucose uptake, substrate entry to the mitochondria and the TCA cycle.

Cholinergic Surveillance over Hippocampal RNA Metabolism and Alzheimer's-Like Pathology.

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aβ1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.

BACE1: a novel player in the pathogenesis of Th17 cells in EAE

Abstract Th17 cells are implicated in autoimmune disease, including attack of the central nervous system (CNS) in multiple sclerosis. β-site APP-cleaving enzyme 1 (BACE1) is a membrane protease expressed in neurons and astrocytes. BACE1 is most well known for its role in promoting neurodegeneration in Alzheimer’s disease by cleaving amyloid precursor protein, although it also plays a role in central nervous system myelination. BACE1 has also been reported to contribute to lesion severity following brain injury, as has IL-17, although these two molecules have not previously been linked. We observed that BACE1 is upregulated in experimental autoimmune encephalomyelitis (EAE) lesions, a mouse model of multiple sclerosis, suggesting a role in autoimmune CNS damage. Surprisingly, we discovered that Bace1-deficient MOG-reactive T cells were defective in IL-17A production. In vitro differentiated Th17 cells were similarly defective in IL-17A. Conversely, overexpression of Bace1 increased IL-17A production. Transfer experiments demonstrated that BACE1 deficiency in T cells impairs their pathogenic function and confers resistance in EAE. Although the impact on IL-17A production was dramatic, there was only a mild defect in expression of RORγt, IL-23R and other prototypic Th17-associated molecules such as IL-17F. This was confirmed by RNA-Seq, which revealed other potential Bace1 targets outside the classic Th17 armory. Therefore, we propose that Bace1 is a novel modulator of the effector function of Th17 cells, particularly IL-17A production.

Long-term electromagnetic pulse exposure induces Abeta deposition and cognitive dysfunction through oxidative stress and overexpression of APP and BACE1

A progressively expanded literature has been devoted in the past years to the noxious or beneficial effects of electromagnetic field (EMF) to Alzheimer׳s disease (AD). This study concerns the relationship between electromagnetic pulse (EMP) exposure and the occurrence of AD in rats and the underlying mechanisms, focusing on the role of oxidative stress (OS). 55 healthy male Sprague Dawley (SD) rats were used and received continuous exposure for 8 months. Morris water maze (MWM) test was conducted to test the ability of cognitive and memory. The level of OS was detected by superoxide dismutase (SOD) activity and glutathione (GSH) content. We found that long-term EMP exposure induced cognitive damage in rats. The content of β-amyloid (Aβ) protein in hippocampus was increased after long-term EMP exposure. OS of hippocampal neuron was detected. Western blotting and immunohistochemistry (IHC) assay showed that the content of Aβ protein and its oligomers in EMP-exposed rats were higher than that of sham-exposed rats. The content of Beta Site App Cleaving Enzyme (BACE1) and microtubule-associated protein 1 light chain 3-II (LC3-II) in EMP-exposed rats hippocampus were also higher than that of sham-exposed rats. SOD activity and GSH content in EMP-exposed rats were lower than sham-exposed rats (p<0.05). Several mechanisms were proposed based on EMP exposure-induced OS, including increased amyloid precursor protein (APP) aberrant cleavage. Although further study is needed, the present results suggest that long-term EMP exposure is harmful to cognitive ability in rats and could induce AD-like pathological manifestation.

BACE1 activity impairs neuronal glucose oxidation: rescue by beta-hydroxybutyrate and lipoic acid

Glucose hypometabolism and impaired mitochondrial function in neurons have been suggested to play early and perhaps causative roles in Alzheimer's disease (AD) pathogenesis. Activity of the aspartic acid protease, beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), responsible for beta amyloid peptide generation, has recently been demonstrated to modify glucose metabolism. We therefore examined, using a human neuroblastoma (SH-SY5Y) cell line, whether increased BACE1 activity is responsible for a reduction in cellular glucose metabolism. Overexpression of active BACE1, but not a protease-dead mutant BACE1, protein in SH-SY5Y cells reduced glucose oxidation and the basal oxygen consumption rate, which was associated with a compensatory increase in glycolysis. Increased BACE1 activity had no effect on the mitochondrial electron transfer process but was found to diminish substrate delivery to the mitochondria by inhibition of key mitochondrial decarboxylation reaction enzymes. This BACE1 activity-dependent deficit in glucose oxidation was alleviated by the presence of beta hydroxybutyrate or α-lipoic acid. Consequently our data indicate that raised cellular BACE1 activity drives reduced glucose oxidation in a human neuronal cell line through impairments in the activity of specific tricarboxylic acid cycle enzymes. Because this bioenergetic deficit is recoverable by neutraceutical compounds we suggest that such agents, perhaps in conjunction with BACE1 inhibitors, may be an effective therapeutic strategy in the early-stage management or treatment of AD.