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Beta secretase 1 (BACE1) is a well-established causative molecule in Alzheimer’s Disease where it’s cleavage of the amyloid precursor protein (APP) produces amyloid-beta peptides, which can accumulate into plaques. BACE1 has also been linked to metabolic dysfunction and cardiovascular disease (CVD), where its role is less defined. Significantly, numerous clinical trials have shown that inhibiting BACE1 is safe, offering the potential for drug-repurposing. The aim of this project is to define the role of BACE1 in the vascular dysfunction frequently observed in metabolic dysfunction and dementia.A combination of methods was used to define the role of BACE1 in vascular dysfunction, including; (1) datamining and bioinformatic identification of novel BACE1 substrates, including those differentially expressed in disease; (2) RNA-sequencing of hCMEC/d3 brain endothelial cells overexpressing BACE1; (3) a UK Biobank analysis of single nucleotide polymorphisms (SNP) of BACE1 associated with plasma markers of BACE1 activity, including amyloid-beta 40 and 42, physiological measures of vascular function and incidence of disease.Datamining and bioinformatics analysis identified 533 BACE1 regulated proteins, of which, 120 were predicted as substrates. Of this list, 26 BACE1 dependent proteins were differentially expressed in vascular dementia, including the L1 family of cell adhesion molecules. Comparison with genes differentially expressed in endothelial cells in Alzheimer’s Disease, identified seven members of the protein tyrosine phosphatase receptor (PTPR) family as novel BACE1 substrates, including PTPRD and PTPRK. Experimental validation in BACE1 knockout primary isolated brain endothelial cells observed a significant 1.95 fold increase (p<0.05) in PTPRD expression when compared against wildtype cells. In hCMEC/D3 cells treated with a BACE1 inhibitor (M3), a 1.31 fold increase in expression of PTPRD was observed (p<0.05).RNA-sequencing analysis identified associations with ‘cytokine-cytokine receptor interactions’ (p<0.01), ‘insulin resistance’ (p<0.05), ‘lipids and atherosclerosis’ (p<0.05) and ‘protein tyrosine/threonine activity’ (p<0.05). It also identified MAF BZIP Transcription Factor F (MAFF) as a novel potential regulator of BACE1 activity, as well as associations with the inhibitor of DNA binding (ID) family. UK Biobank analysis of 18 BACE1 SNPs identified associations with plasma proteins and functional associations including; lipoproteins, leukocyte migration, negative regulation of gluconeogenesis and cellular response to insulin stimulus and diagnosis of diabetes (p<0.05). Of these, three as yet uncharacterised BACE1 SNPs were associated with significant differences in plasma amyloid beta 40 and/or 42 (p<0.05).and several validated and predicted substrates, including SEZ6L and L1CAM (p<0.05).This analysis shows BACE1 may be an important enzyme in the vascular dysfunction observed with metabolic dysfunction and in cerebrovascular contributions to dementia. It offers insights into novel substrates and functional associations of BACE1, offering a deeper understanding into the functionality of BACE1, for which the potential for drug-repurposing exists.Conflict of InterestN/A

Background The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L. Methods and results We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice. Conclusions This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice.

Current therapies for Alzheimer’s disease (AD) are symptomatic and do not target the underlying Aβ pathology and other important hallmarks including neuronal loss. PPARγ-coactivator-1α (PGC-1α) is a cofactor for transcription factors including the peroxisome proliferator-activated receptor-γ (PPARγ), and it is involved in the regulation of metabolic genes, oxidative phosphorylation, and mitochondrial biogenesis. We previously reported that PGC-1α also regulates the transcription of β-APP cleaving enzyme (BACE1), the main enzyme involved in Aβ generation, and its expression is decreased in AD patients. We aimed to explore the potential therapeutic effect of PGC-1α by generating a lentiviral vector to express human PGC-1α and target it by stereotaxic delivery to hippocampus and cortex of APP23 transgenic mice at the preclinical stage of the disease. Four months after injection, APP23 mice treated with hPGC-1α showed improved spatial and recognition memory concomitant with a significant reduction in Aβ deposition, associated with a decrease in BACE1 expression. hPGC-1α overexpression attenuated the levels of proinflammatory cytokines and microglial activation. This effect was accompanied by a marked preservation of pyramidal neurons in the CA3 area and increased expression of neurotrophic factors. The neuroprotective effects were secondary to a reduction in Aβ pathology and neuroinflammation, becausewild-type mice receiving the same treatment were unaffected. These results suggest that the selective induction of PGC-1α gene in specific areas of the brain is effective in targeting AD-related neurodegeneration and holds potential as therapeutic intervention for this disease.

Backgroundβ-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease that is known for its role in the formation of amyloid plaques in Alzheimer’s disease. Recent research has shown that BACE1 also plays a role in vascular homeostasis and proteolytically cleaves various angiogenic signaling factors including VEGF receptor 1 (VEGFR1), NOTCH ligands, the insulin receptor, and occludin. BACE1 activity is elevated in models of type 2 diabetes, suggesting a potential role for its contribution to abnormal vessel growth characteristic of diabetes-related complications.MethodsRetinal staining and the fibrin gel angiogenesis assay were used to identify a role for BACE1 in vessel growth in vivo and in vitro, respectively. Endothelium of the developing retinal vasculature in BACE1 deficient (KO) and wild type (WT) mice was stained with IsolectinB4-Alexa488 and imaged using confocal microscopy. Sprout formation was further analysed using the fibrin gel angiogenesis assay with human umbilical vein endothelial cells (HUVECs) treated with or without a highly specific BACE1 inhibitor or transfected to over-express BACE1. Primary isolated pulmonary endothelial cells (PECs) were isolated from BACE1 KO and WT control mice prior to Western blots.ResultsBACE1 KO retinas had decreased radial outgrowth (16.82% ± 69.99, P=0.05), but increased branch points, vasculature area, and quantity of filopodia compared to WT mice. Moreover, BACE KO PECs had reduced NOTCH1 signalling (26.73% ± 14.15, P=0.05) and Jagged-1 proteolysis (28.48% ± 14.61, P=<0.05) compared to WT PECS. Also, when treated with a highly specific BACE1 inhibitor, WT PECs had increased phosphorylation of eNOS (51% ± 13.8% P=0.01).HUVECs treated with a BACE1 inhibitor had increased sprouting (18.70%± 5.92, P=<0.05) as well as increased phosphorylation of eNOS (83% ± 22, P=<0.05) compared to untreated cells. Moreover, HUVECs transfected to over-express BACE1 had decreased sprouting (35.22% ± 7.34, P=<0.01), increased NOTCH1 signalling (21.8% ± 3.76, P=0.01) and Jagged-1 proteolysis (24.90% ± 10.65 ± P=<0.05).ConclusionOur findings indicate a role of BACE1 in negatively regulating angiogenesis, possibly via Jagged1/NOTCH1 or Akt/eNOS/NO signalling. This provides a potential therapeutic purpose for BACE1 inhibitors, previously trialled to treat AD, in normalising BACE1 levels in individuals with type 2 diabetes and preventing associated microvascular complications.Conflict of InterestN/A

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood–brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB‐crossing adeno‐associated virus (AAV)‐based vectors to deliver VHH directly into the CNS. As a proof‐of‐concept, we explored the potential of AAV‐delivered VHH to inhibit BACE1, a well‐characterized target in Alzheimer’s disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH‐B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro . We further demonstrate that a single systemic dose of AAV‐VHH‐B9 produces positive long‐term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the App NL‐G‐F Alzheimer’s mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets. Synopsis VHH and blood‐brain‐barrier (BBB)‐crossing AAV‐based vectors are combined to achieve highly‐specific, long‐term BACE1 inhibition in a mouse model of Alzheimer's disease (AD). A gene transfer strategy based on BBB‐crossing AAV vectors is developed to deliver VHH single domain antibodies directly into the CNS. VHH‐B9 is generated to target BACE1, an enzyme critical to Aβ generation in AD, and incorporated into an AAV‐PHP.B‐based vector. A single dose of AAV‐VHH resulted in long‐term VHH expression in the App NL‐G‐F mouse model, with concomitant improvements in cognitive status, amyloidosis, neuroinflammation, and synaptic function. Graphical Abstract VHH and blood‐brain‐barrier (BBB)‐crossing AAV‐based vectors are combined to achieve highly‐specific, long‐term BACE1 inhibition in a mouse model of Alzheimer's disease (AD).

Alzheimer’s disease (AD) is the most common age-dependent disease of dementia, and there is currently no cure available. This hallmark pathologies of AD are the presence of amyloid plaques and neurofibrillary tangles. Although the exact etiology of AD remains a mystery, studies over the past 30 have shown that abnormal generation or accumulation of β-amyloid peptides (Aβ) is likely to be a predominant early event in AD pathological development. Aβ is generated from amyloid precursor protein (APP) via proteolytic cleavage by β-site APP cleaving enzyme 1 (BACE1). Chemical inhibition of BACE1 has been shown to reduce Aβ in animal studies and in human trials. While BACE1 inhibitors are currently being tested in clinical trials to treat AD patients, it is highly important to understand whether BACE1 inhibition will significantly impact cognitive functions in AD patients. This review summarizes the recent studies on BACE1 synaptic functions. This knowledge will help to guide the proper use of BACE1 inhibitors in AD therapy.

BACE1 is the rate-limiting enzyme for amyloid-β peptides (Aβ) generation, a key event in the pathogenesis of Alzheimer’s disease (AD). By an unknown mechanism, levels of BACE1 and a BACE1 mRNA-stabilizing antisense RNA (BACE1-AS) are elevated in the brains of AD patients, implicating that dysregulation of BACE1 expression plays an important role in AD pathogenesis. We found that nuclear factor erythroid-derived 2-related factor 2 (NRF2/NFE2L2) represses the expression of BACE1 and BACE1-AS through binding to antioxidant response elements (AREs) in their promoters of mouse and human. NRF2-mediated inhibition of BACE1 and BACE1-AS expression is independent of redox regulation. NRF2 activation decreases production of BACE1 and BACE1-AS transcripts and Aβ production and ameliorates cognitive deficits in animal models of AD. Depletion of NRF2 increases BACE1 and BACE1-AS expression and Aβ production and worsens cognitive deficits. Our findings suggest that activation of NRF2 can prevent a key early pathogenic process in AD.

Cardiovascular disease is a known risk factor for dementia, associating two of the most abundant age-associated diseases. Around 7.6 million people are living with heart and circulatory diseases within the UK, presenting a substantial number of people with a potentially modifiable risk of dementia. Understanding the molecular mechanisms acting upon the cerebrovasculature could therefore provide a therapeutic target for vascular dementia and have a huge impact on public health.The beta secretase 1 (BACE1) enzyme is a clinically relevant therapeutic target for Alzheimer’s Disease, and a well-established causative molecule in the development of amyloid beta (Aβ) plaques in the brain, including in cerebrovascular vessels. BACE1 and Aβ have been shown to regulate peripheral blood vessel function, suggesting the same might be true in the brain. My project therefore aims to investigate whether increased BACE1 activity and/or Aβ deposition in the cerebral vessels leads to vascular dysfunction and may be a mechanism behind the increased risk of dementia for individuals with cardiovascular disease.Due to the low substrate specificity that BACE1 has for its perceived main substrate, amyloid precursor protein (APP), I investigated novel substrates of BACE1, which may play important roles in pathological changes in BACE1 expression and activity. I used a datamining and bioinformatics approach to identify pathways controlled by BACE1. Through stratification and comparison of publicly available proteomics and RNA sequencing datasets, I identified 533 BACE1 regulated proteins, of which, 119 were predicted as BACE1 substrates. Of this list, 26 BACE1 dependent proteins were differentially expressed in vascular dementia, including the L1 family of cell adhesion molecules. Comparison with genes differentially expressed in endothelial cells in Alzheimer’s Disease, identified seven members of the PTPR family as novel BACE1 substrates, including PTPRD. Experimental validation observed a significant 1.95 fold increase (p<0.05) in PTPRD expression in BACE1 knockout primary isolation brain endothelial cells when compared with wild type. The analysis was repeated in human brain endothelial hCMEC/D3 cells, a cell line derived from human temporal lobe microvessels. In hCMEC/D3 cells treated with a BACE1 inhibitor (M3), a 1.31 fold increase in expression of PTPRD was observed (p>0.9999). Dysregulation of receptor-type protein tyrosine phosphatase signalling is frequently observed in disease, including diabetes and cardiovascular disease, which are associated with an increased risk of dementia. The Netrin receptor DCC (DCC) protein was also found to increase in response to BACE1 knockout in endothelial cells compared to wild type by 1.53 fold (p<0.05), and in response to BACE1 inhibitor treatment (M3) in hCMEC/D3 cells by 1.11 fold (p>0.9999). The Netrin-1/DCC interaction has been shown to be involved in inducing angiogenesis via endothelial cell nitric oxide. This computational analysis has therefore identified novel BACE1 substrates which may play important roles in cerebrovascular disease, with the potential for further analysis to unveil additional substrates. Further analysis of these novel associations with BACE1 may provide a greater understanding of BACE1 in health and cerebrovascular disease and contribute towards targeting BACE1 for therapeutic benefit in the treatment and/or prevention on vascular dementia.Conflict of InterestN/A

Insulin resistance is a major risk factor for Alzheimer’s disease (AD). Chenodeoxycholic acid (CDCA) and synthetic Farnesoid X receptor (FXR) ligands have shown promising outcomes in ameliorating insulin resistance associated with various medical conditions. This study aimed to investigate whether CDCA treatment has any potential in AD management through improving insulin signaling. Adult male Wistar rats were randomly allocated into three groups and treated for six consecutive weeks; control (vehicle), AD-model (AlCl3 50 mg/kg/day i.p) and CDCA-treated group (AlCl3 + CDCA 90 mg/kg/day p.o from day 15). CDCA improved cognition as assessed by Morris Water Maze and Y-maze tests and preserved normal histological features. Moreover, CDCA lowered hippocampal beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and amyloid-beta 42 (Aβ42). Although no significant difference was observed in hippocampal insulin level, CDCA reduced insulin receptor substrate-1 phosphorylation at serine-307 (pSer307-IRS1), while increased protein kinase B (Akt) activation, glucose transporter type 4 (GLUT4), peroxisome proliferator-activated receptor gamma (PPARγ) and glucagon-like peptide-1 (GLP-1). Additionally, CDCA activated cAMP response element-binding protein (CREB) and enhanced brain-derived neurotrophic factor (BDNF). Ultimately, CDCA was able to improve insulin sensitivity in the hippocampi of AlCl3-treated rats, which highlights its potential in AD management.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with no definite treatment. The expression of miR-29 family is significantly reduced in AD, suggesting a part for the family members in pathogenesis of the disease. The recent emergence of microRNA (miRNA)-based therapeutic approaches is emphasized on the efficiency of miRNA transfer to target cells. The endogenously-made secretory vesicles could provide a biological vehicle for drug delivery. Characteristics such as small sizes, the ability to cross the blood-brain barrier, the specificity in binding to the right target cells, and most importantly the capacity to be engineered as drug carriers have made exosomes desirable vehicles to deliver genetic materials to the central nervous system. Here, we transfected rat bone marrow stem cells (r-BMSC) and HEK-293T cells with recombinant expression vectors, carrying either mir-29a or mir-29b precursor sequences. A significant overexpression of miR-29 and down-regulation of their targets genes: BACE1 and BIM were confirmed in the transfected cells. Then, we confirmed the packaging of miR-29 in exosomes secreted from the transfected cells. Finally, we investigated a possible therapeutic effect of the engineered exosomes to reduce the pathological effects of Aβ in a rat model of AD. Aβ-treated model rats showed some deficits in spatial learning and memory. However, in animals injected with miR-29-containing exosomes at CA1, the aforementioned impairments were prevented. In conclusion, our findings provide a new approach for the packaging of miR-29 in exosomes and that the engineered exosomes might have a therapeutic potential in AD.