BACE1: More than just a β‐secretase

Summary β‐site amyloid precursor protein cleaving enzyme‐1 (BACE1) research has historically focused on its actions as the β‐secretase responsible for the production of β‐amyloid beta, observed in Alzheimer's disease. Although the greatest expression of BACE1 is found in the brain, BACE1 mRNA and protein is also found in many cell types including pancreatic β‐cells, adipocytes, hepatocytes, and vascular cells. Pathologically elevated BACE1 expression in these cells has been implicated in the development of metabolic diseases, including type 2 diabetes, obesity, and cardiovascular disease. In this review, we examine key questions surrounding the BACE1 literature, including how is BACE1 regulated and how dysregulation may occur in disease, and understand how BACE1 regulates metabolism via cleavage of a myriad of substrates. The phenotype of the BACE1 knockout mice models, including reduced weight gain, increased energy expenditure, and enhanced leptin signaling, proposes a physiological role of BACE1 in regulating energy metabolism and homeostasis. Taken together with the weight loss observed with BACE1 inhibitors in clinical trials, these data highlight a novel role for BACE1 in regulation of metabolic physiology. Finally, this review aims to examine the possibility that BACE1 inhibitors could provide a innovative treatment for obesity and its comorbidities.

BACE1 gene is transcribed as a 501 amino acid preprotein, containing five key domains, a signal peptide and pro-, catalytic, transmembrane, and cytoplasmic domains ( Figure 1A). The signal peptide traffics the BACE1 preprotein to the endoplasmic reticulum (ER), where furin cleavage of the pro-domain produces a mature BACE1 protein. 6 The transmembrane domain determines BACE1 localization to the late Golgi, where in the trans-Golgi network BACE1 is post-translationally activated. The protease activity of BACE1 is dependent on two aspartyl active sites at D93 and D289, as well as the position of the regulatory antiparallel-hairpin flap relative to the substrate binding site ( Figure 1B). 8 The activated BACE1 protein subsequently operates at the plasma membrane in endosomes and the Golgi apparatus, functioning at an optimal pH of 4.5. 8

| ESTABLISHED BACE1 SUBSTRATES AND FUNCTIONS
Most knowledge and research around BACE1 is focused on its role in the amyloidogenic pathway, where it is responsible for the initial rate limiting cleavage of the APP protein. Sequential cleavage by BACE1 and γ-secretase produces Aβ-40 and Aβ-42 peptides. Despite having around 64% homology to BACE1, BACE2 cleaves the APP protein at an alternative site and therefore does not elicit the same β-secretase action and Aβ production. 9 BACE2 is also mainly found in peripheral tissues, in contrast to BACE1 which, by comparison, is highly expressed in the brain. 10 BACE1 production of Aβ-42 is associated with regulation of memory, synaptic function, myelin repair, and AD. 5 AD is the most common form of dementia and presents with several physiological changes in addition to the accumulation of Aβ-42 into extracellular amyloid plaques, including neurofibrillary tangles (NFTs), chronic inflammation, synapse loss, neuronal death, and hypometabolism. 11,12 The accumulation Aβ-plaques disrupts neuronal and synaptic functions leading to detrimental cognitive effects. 5 However, there is increasing evidence for non-neuronal functions of Aβ and consequences of altered BACE1 activity. The increased production of Aβ has been linked to cerebrovascular impairments including capillary degradation, impairments in the blood brain barrier (BBB), and response to vascular injury. 13,14 Some of these effects may be linked to the antimicrobial role of Aβ and its release into the blood from activated platelets in response to inflammation and immune responses. 15 Furthermore, Aβ has been shown to regulate transcription of APP and insulin-like growth factor receptors, 16 and BACE1 has been implicated in a range of metabolic functions. [17][18][19] This suggests various important physiological roles of BACE1 in different cells and organelles, in addition to its role in AD. Although the expression of BACE1 is highest in the brain, it is found widely expressed at lower levels in other tissues including endocrine tissue, the pancreas, muscle tissue, respiratory tissue, bone marrow, and lymphoid tissue. 20,21 As APP is also widely expressed, the BACE1-mediated production of Aβ could have an effect on many cells and tissues. 20,21 It is well acknowledged that APP is a poor substrate for BACE1 and, through secretome enrichment experiments, nearly 70 BACE1 substrates have been identified to date. [22][23][24][25] Many BACE1 substrates, like APP, are type 1 membrane proteins whose function can be enhanced or reduced following BACE1-mediated shedding.
Neuregulin 1 (NRG1) is a signaling protein involved in various cellular functions, including cell growth and differentiation. BACE1 cleavage of NRG1 regulates myelination, 26,27 and increased BACE1 cleavage of NRG1 has been implicated in the development of schizophrenia. 28 BACE1 cleavage of Jagged 1 (Jag1) regulates the Jag1-Notch signaling pathway important in the control of astrogenesis and neurogenesis. 29 Seizure protein 6 (SEZ6) and seizure-like protein 6 (SEZ6L) proteins influence ER functions in neurons, control synaptic connectivity, and motor coordination and therefore may likely be responsible for the seizures, motor deficits, and reduced spinal deficiency observed in BACE1 null mice. 24 Cache domain containing F I G U R E 1 The structure and domains of BACE1. (A) The primary structure of BACE1 with functional domains labeled. There are five domains: signal peptide (1-23), propeptide , catalytic domain , transmembrane domain (454-478), and the cytosolic domain (478-501). Created with BioRender.com. (B) BACE1 crystal structure made using structure 3TPR on RSCB Protein DataBank (https://www.rcsb.org/). The unannotated structure (left) and inhibitor C3 binding (red), the two aspartyl protease active sites (blue) at D93 and D289, and the antiparallel-hairpin flap (green) between Y128-G138, are shown (right). The structure is missing 62 residues protein-1 (CACHD1) and neural cell adhesion molecules (NCAM1 and 2) are involved in synapse formation, maturation, and maintenance. 22,30 L1 cell adhesion molecule (L1CAM) and neural cell adhesion molecule L1-like protein (CHL1) are BACE1 substrates involved in axon guidance. 31 Substrates SEZ6, L1CAM, leucine rich repeat neuronal 1 (LRRN1), neurotrimin, and CHL1 are all involved in neurite outgrowth. 23 BACE1 has also been implicated in regulation of sodium channel metabolism in neuronal cells, via its cleavage of voltage-gated sodium channel β2 subunit (Navβ2). 32 To date, the BACE1 substrates identified are primarily associated with neurological function and the central nervous system. However, this might be more representative of experimental designs. Therefore, the true extent of non-CNS functions may not yet be fully known.

| NON-NEURONAL BACE1 SUBSTRATES
The phenotype of the BACE1 knockout mice suggests BACE1 plays additional roles to regulation of neuronal function. Accordingly, nonneuronal physiological effects of BACE1 have come to light recently. P-selectin glycoprotein ligand-1 (PSGL-1) is a BACE1 substrate that plays an important role in immune defenses by recruiting white blood cells to the site of infection. 25 Another BACE1 substrate, interleukin-1 receptor II (IL-1R2), an interleukin-1 decoy receptor, presents a mechanism for abnormal inflammation in response to changes in BACE1 activity. 33 It is therefore evident that BACE1 is involved in many pathways and processes, and its effects on a single protein can have a complex cascade effect on other functions of the body. Through these additional substrates, BACE1 has been implicated in inflammation, cardiovascular function, glucose homeostasis, and insulin signaling 18,[34][35][36][37][38] (Table 1). This highlights the importance of investigating all substrates and pathways BACE1 is involved in, both to understand implications of its dysfunction and to benefit the development of effective therapeutics.

| CONSEQUENCES OF CHANGES IN BACE1 EXPRESSION AND ACTIVITY
Numerous physiological effects are observed in response to changes in BACE1 expression and activity in disease models, and potential therapeutics. In a variety of pathological conditions, including AD, cerebral amyloid angiopathy, and metabolic diseases such as type 2 diabetes and obesity, BACE1 expression and activity are increased and drive disease progression. 3,34,39,40 Accordingly, BACE1 knock-in mice display an AD-like pathology including elevated levels of Aβ plaques, synaptic impairments, decreased cognitive function, 41 and systemic diabetes. 34 Conversely, BACE1 knockout mice display reduced birth weight, hypomyelination, memory deficits, behavioral alterations, axon guidance impairment, impaired midbrain dopaminergic signaling, seizures, and abnormal electroencephalograms (EEGs). 42,43 Some of the observed phenotypes, such as impaired axon guidance, are more severe when BACE1 is deficient in the developmental stages. 43 In addition to its neuronal roles, changes in BACE1 activity have been associated with physiological functions including maintenance of the blood-brain barrier (BBB), angiogenesis, protection against obesity, immune and antimicrobial properties, inflammatory response, and tumor suppression. [44][45][46][47] This proposes a metabolic function of BACE1, and further research into BACE1 and its substrates is unveiling further physiological functions, which will be further discussed below. activity, and cyclic adenosine monophosphate (cAMP) levels. 50 Although not well characterized, the presence of a CREB binding site within the BACE1 promoter suggests that BACE1 regulation of the cAMP/PKA/CREB pathway, significant in glucose metabolism and lipid homeostasis, may feedback to regulation of BACE1 gene expression. 51 Inflammation is closely associated with metabolic disease, and increases in BACE1 expression in response to inflammation have been attributed to transcriptional regulation. The pro-inflammatory cytokine interferon-gamma (IFNy) causes an increase in BACE1 through subsequent Janus Kinase 2 (JAK2) and mitogen activated protein (MAP) kinase signaling causing phosphorylation of STAT1. 52 The phosphorylation of STAT1 leads to its binding of the BACE1 promoter, increasing BACE1 gene expression. 52 This action of STAT1 can be inhibited by suppressor of cytokine signaling (SOCS) 1 and 3, which prevent phosphorylation of STAT1 on the Tyrosine 701 residue. 52 Additionally, studies showed that NSAIDs, used to decrease inflammation, lower BACE1 activity, likely by reducing PPARy levels. 53,54 PPARy is a transcriptional regulator, which acts upon the BACE1 promoter increasing gene expression. Regulation of expression by transcription factors is therefore an important factor in the physiological function of BACE1, as well as its dysregulation in disease.
In addition to regulation by transcription factors, BACE1 gene expression is positively regulated at the transcriptional level by the BACE1 antisense transcript (BACE1-AS). 55 BACE1-AS is reported to stabilize BACE1 through competitive binding of miR-485-5p which would otherwise repress BACE1 mRNA translation. 56 It is therefore possible that, by acting as a sponge, BACE1-AS regulates other miRNAs in a similar manner, significant as numerous miRNAs are reported to repress BACE1. [57][58][59][60][61][62][63] The action of BACE1-AS has been implicated in AD pathology, with increased BACE-AS in turn increasing BACE1 and Aβ-42. 64 Dysregulation of the BACE1/BACE1-AS axis T A B L E 1 BACE1 substrates involved in non-neuronal physiological functions also has been implicated in both cardiac dysfunction and epilepsy. 38,65 The BACE1 mRNA transcript is further regulated by alternative splic- This is true for BACE1, with various PTMs strongly influencing activity. In order for the mature BACE1 protein to be produced, glycosylation and transient acetylation in the ER are required. This promotes trafficking of the immature protein to the trans-golgi network, preventing its degradation. 73 The BACE1 protein has four N-linked glycosylation sites, which are targeted in the Golgi and facilitate propeptide processing, maturation, and transportation. 74 Missing glycosylation sites in alternatively spliced isoforms of BACE1 are reported to contribute to reduced secretase activity. 75 Glycosylation has also been implicated in the pathological increases in BACE1 stability seen in response to oxidative stress, with bisecting N-acetylglucosamine (GlcNAc), catalyzed by GlcNAc transferase Gnt-III having been shown to prevent lysosomal degradation of BACE1. 76,77 BACE1 is also phosphorylated, with phosphorylation at threonine 252 via p25/Cdk5 pathways found to stimulate protease activity. 78 Another important phosphorylation site is located as serine 498, by casein kinase 1. Phosphorylation is important in intracellular trafficking, recognition by Golgi-localized γ-ear-containing ARF-binding (GGAs), and retention in acidic compartments when activated, enabling substrate interactions. [79][80][81] Furthermore obesity increases cdk5 and casein kinase 1 activity is enhanced. The BACE1 protein is also acetylated at seven different lysine residues mediated by CoA:lysine acetyltransferase 1 and 2 (ATase 1 and ATase 2), and ubiquitinated at K501 which causes translocation to the lysosomes for degradation. 6 The ubiquitination site at 501 is also competitively SUMOylated, which stabilizes the protein and promotes its activity. 72  action as a potential link between AD and T2D. 83 BACE1 has been implicated in T2D via Aβ-dependent and independent processes, which will be reviewed below.

| BACE1 REGULATION OF INSULIN PATHWAYS
BACE1 is implicated in the pathogenesis of T2D through the insulin pathway. Ordinarily, high blood glucose levels stimulate insulin production in the pancreas and release into the bloodstream, where it then travels to insulin responsive tissues such as skeletal muscle, liver, and adipose tissue. 84 Cells detect increased levels of insulin through insulin receptors, which then stimulates a phosphorylation cascade. 85 This leads to tissue-specific effects such as prevention of lipolysis, glucose uptake, stimulation of glycogenesis and lipogenesis, promotion of protein synthesis, and upregulation of genes such as fatty acid synthase and malic enzyme genes. 84  through Aβ production, in skeletal muscle. This is supported by the findings that APP processing in C2C12 myotubes directly affects glucose uptake and GLUT4 translocation. 103 Therefore, as APP and BACE1 are widely expressed, local Aβ production could impact signaling in many cells and tissues.
Taken together, this presents a strong physiological role of BACE1 in insulin signaling. This may have important implications in the context of T2D and obesity, as well as providing a potential mechanistic explanation for the increased risk of AD in T2D patients.

| LEPTIN PATHWAY
In addition to insulin signaling, other metabolic changes associated with BACE1 have been observed, including lowered plasma leptin and restored hypothalamic leptin sensitivity in obese mice. 19 Leptin is a hormone released by adipocytes, with the principal role to suppress hunger and increase fat breakdown through β-oxidation. 104 The leptin receptor (LepR) is localized to the cell membrane, and when bound by leptin, dimerizes, and initiates JAK/STAT signaling ( Figure 5). As leptin functions via binding of the leptin receptor, changes in its expression and splicing can reduce metabolism at a given concentration of leptin. 104 The Janus Kinases (JAK) associated with the LepR phosphorylate the receptor, which in turn phosphorylates signal transducer and activator of transcription 3 (STAT3) proteins. Subsequently, two phosphorylated STAT3 proteins dimerize and bind target genes in the nucleus, leading to a feeling of satiety. Generally, a greater fat deposit in adipocytes results in greater leptin release, and therefore, theoretically, increased lipids should equate to decreased hunger. [104][105][106] Leptin signaling can also increase lipid breakdown via thermogenesis using β-oxidation, through innervation of adipocytes by  Figure 5B). 106 This hyperleptinemia is associated with inflammation, hyperglycemia, hyperinsulinemia, insulin resistance and high circulating triglycerides, and an increased risk of atherosclerosis. 111 In addition to hyperleptinemia, there is evidence that impairments to leptin sensitivity can result from inflammation, including ER stress. 112,113 There is evidence of an inverse relationship between BACE1 and leptin signaling. BACE1 expression is suppressed by leptin signaling, whereas BACE1 levels are increased by both obesity and T2D. 114 Therefore, BACE1 may play a role in the mechanisms behind changes in leptin levels and sensitivity. 4 Elevated levels of circulating fatty acids in the setting of obesity and leptin resistance increase palmitoylation of proteins. 117 The palmitoylation of BACE1, a modification that has been linked to metabolic dysfunction, namely fatty acid and cholesterol levels, may cause increased BACE1 activity in response to increases in circulating triglycerides. BACE1 is palmitoylated at four cysteines found in Cterminal cytosolic and transmembrane domains, which consequently stabilizes and increases protein levels. 6 When palmitoylated, BACE1 localization to lipid rafts is enhanced, promoting Aβ production. 118 Aberrant palmitoylation in response to metabolic dysfunction has been suggested to extend BACE1 half life, a theory supported by the colocalization of BACE1 and cholesterol in hypercholesterolemia. [119][120][121] This suggests that altered palmitoylation in response to metabolic dysfunction could be an important mechanism behind pathological increases in BACE1 activity.
Taken together, this suggests BACE1 is a pivotal enzyme in the development of cellular leptin resistance observed in obesity and T2D; however, the mechanism is unclear. Administration of Aβ has been shown to cause leptin resistance ( Figure 5). 122,123 While BACE1-mediated proteolysis of the leptin receptor is yet to be determined.

| THE ROLE OF BACE1 IN BAT DIFFERENTIATION
In addition to β-oxidation via leptin signaling, BACE1 has also been implicated in body weight regulation via thermogenesis and its role in BAT differentiation. 124 BAT generates heat through lipid breakdown, via UCP1-mediated mitochondrial uncoupling, a function that has become a therapeutic target for obesity. 125 The abundance of BAT is greatest in infants and hibernating animals, to protect against hypothermia, as it contains a greater amount of mitochondria than white adipose. BAT dysfunction is associated with dysregulation of glucose metabolism and is observed in aging and metabolic disease. 124,126,127 This decline in BAT function has been attributed to reduced expression of the microRNA-processing node, Dicer1. 124,128 Although the reasons for downregulation of Dicer1 have not been fully elucidated, it has been shown to occur in response to hypoxia. 128  These small non-coding RNAs regulate stability, degradation and translational ability of target mRNAs and can alter adipocyte differentiation. 129 In DIO models, Dicer1 downregulation resulted in decreased expression of miR-328. 124 This reduction is miR-328 F I G U R E 5 The role of BACE1 in the leptin pathway. BACE1 contributes to dysregulated leptin signaling directly and indirectly. Elevated BACE1 expression is associated with increased PTP1B and SOCS3, which negatively regulate JAK2/ STAT3 signaling, preventing leptinstimulated gene regulation. When BACE1 is reduced, energy expenditure is increased likely via increases in UCP1 expression and □-oxidation. Aβ-mediated inflammation can cause JAK/STAT3 signaling and ER stress, which can increase PTP1B and SOCS-3 transcription, and regulate genes important in appetite and body weight regulation and β-oxidation. Created with BioRender.com was accompanied by a decrease in genes associated with BAT function, including UCP1. 124 The action of miR-328 in promoting BAT function and differentiation is believed to occur via its silencing of BACE1, preventing BACE1 promotion of myogenesis and therefore inhibition of BAT commitment ( Figure 6). Significantly, overexpression of miR-328 was found to counteract the BAT downregulation seen in obesity. 124 In addition to the Dicer1/miR-328/BACE1 axis in BAT function and differentiation, BACE1 overexpression has also been previously linked to reduced miR-328 activity in the context of AD. 62 131 In addition to the direct action of BACE1, overexpression of Aβ also coincides with a decrease in JAG1/Notch3 signaling. 46 Although it requires further investigation, it has been suggested that Aβ interact directly with Notch genes. 132 The injection of Aβ-42 into the rat hippocampus was found to increase angiogenesis, likely via vascular endothelial factor (VEGF) and an inflammatory response. 133 However, contradictory studies have shown that both Aβ and BACE1 inhibitors can inhibit angiogenesis in tumor models. 134 Furthermore, BACE1 is also responsible for ectodomain cleavage of vascular endothelial growth factor receptor 1 (VEGFR1), a receptor important in the regulation of angiogenesis and vascular permeability. 135 Whether BACE1 can regulate adipose tissue angiogenesis in the setting of metabolic dysfunction is an interesting concept that requires further investigation.
Atherosclerosis is an important factor in the development of cardiovascular disease, including myocardial infarction, cerebrovascular accident, and stroke. 136 BACE1 is implicated in atherosclerotic plaque formation and is increased in the presence of increased cholesterol.
High cholesterol has been shown to affect endocytic trafficking of BACE1 and APP, resulting in increased Aβ production. 137,138 The production of Aβ is enhanced in atherosclerotic development, with APP knockout mouse models exhibiting a decrease in atherosclerotic plaque formation and increased stability in the aorta. 139-141 BACE1 cleavage of ST6Gal-1, a protein involved in the terminal step of N-glycan biosynthesis of glycoproteins, contributes to preventing monocyte transendothelial migration, a pivotal mechanism in the initial stages of atherosclerosis. 142 ST6Gal-1 increases adhesion between endothelial cells and monocytes as well as aids monocyte penetration into the endothelial tissue. 17,143 ST6Gal-1 is found at decreased levels in atherosclerosis development, this suggests a role for BACE1 in its regulation. Furthermore, the dysregulation of the BACE1/BACE1-AS/Aβ axis is associated with heart failure, with both BACE1-AS and BACE1 found upregulated. 38 This demonstrates the close relationship between BACE1 action, atherosclerosis formation, and cardiovascular disease.
Another impact BACE1 can have on the cardiovascular system is through the Aβ-induced vascular dysfunction. Aβ binds RAGE (receptor for advanced glycation endproducts) or CD36 receptors, activating NADPH oxidase, leading to production of reactive oxygen species (ROS). 35,36,144 An excess of ROS causes oxidative stress and decreased expression of endothelial nitric oxide synthase (eNOS). 145 This results in decreased production of the vasodilatory molecule nitric oxide (NO), and increased endothelin-1, which impairs the vasodilatory ability of blood vessels. 146 It has also recently been demonstrated that BACE1 is also modified by the vasodilator NO, which S-nitrosylates BACE1 at high levels inactivating the enzyme. 147 F I G U R E 6 The role of BACE1 in brown adipose fat commitment. Dicer1 cleavage of pre miRNA produces the miR-328. miR-328 binds BACE1 mRNA, regulating the stability of the transcript and therefore BACE1 expression. BACE1 promotes myogenesis and consequently inhibits brown adipose tissue (BAT) differentiation. Silencing of BACE1 by miR-328 therefore increases pre-adipocyte commitment to brown adipose tissue. Created with BioRender.com Furthermore, accumulation of Aβ in blood vessels, as seen in cerebral amyloid angiopathy (CAA), leads to arterial stiffness. 148 The action of Aβ on vascular endothelial cells can lead to impairments in tight junctions and BBB dysfunction, capillary degradation, inflammation, impaired vascular clearance, and atherosclerotic plaque formation. [148][149][150] Together, this presents an important physiological role for BACE1 within the cardiovascular system and as an important enzyme in cardiovascular disease. Nuclear factor-kappa B (NFκB) is a key regulator of the innate immune system and plays a role in gene expression, inflammation, and oxidative stress. 152,153 Increases in NFκB activation are associated with obesity, 154 T2D, 155 and AD. 152 NFκB-associated inflammation has also been experimentally linked to insulin resistance. 156 The BACE1 promotor has an NFκB binding site. When phosphorylated, NFκB increases BACE1 promotor activity and transcription. 152 Thus, inflammation induced activation of NFκB, facilitates the upregulation of BACE1 expression, and subsequently increase Aβ production. Activation of NFκB in response to inflammation is observed in macrophages, astrocytes, and microglia and has been implicated in a positive feedback loop in astrocytes whereby increased BACE1 leads to increases levels of neurotoxic Aβ, in turn increasing astrocyte activation and inflammation. 157 Furthermore, treatment with non-steroidal antiinflammatory drugs (NSAIDs) was found to decrease BACE1 transcription and subsequent production of Aβ. 152

| ACTION OF METABOLIC DRUGS ON BACE1
The proposed metabolic roles of BACE1 are supported by the common mechanisms and treatment targets between T2D, obesity, and AD, with the use of various antidiabetic and metabolic therapies in the treatment of AD showing promising results. 160 Although research has proved contradictory, the use of NSAIDs has been tested in the treatment of both AD and T2D. [161][162][163] NSAIDs function by reducing the disease associated inflammation, via inhibition of NF-κB signaling; however, they notably also reduce BACE1 transcription. 152,164 The antidiabetic drug liraglutide, in addition to use in treating T2D, has been investigated for the treatment of AD, obesity, and weight loss. 165 Liraglutide is a long-acting glucagon-like peptide-1 (GLP)-1 receptor agonist, which functions via increasing the release of insulin from the pancreas while simultaneously decreasing glucagon release. Liraglutide reduces Aβ plaque production and the severity of AD symptoms. [166][167][168] The action of liraglutide regarding both Aβ production and alleviating insulin resistance has been shown to occur via reducing BACE1 activity. 169 Improvements in AD are similarly seen with other antidiabetic drugs, for example Glimepiride, which when used on patients with AD shows improved memory and cognitive functions. 170 Lixisenatide also shows positive effects on glucose homeostasis and improvement of cognitive functions suggesting a possible therapeutic effect for both AD and T2D. 171,172 The T2D drug Pioglitazone, a PPAR-γ agonist, has demonstrated control of plasma Aβ levels, cerebral blood flow, and shown improvements in cognitive function. 173 This action of Pioglitazone is thought to be through its increasing lowdensity lipoprotein receptor-related protein 1 (LRP1) levels and in turn increasing Aβ clearance. 174 Metabolic drugs are known to downregulate BACE1, including statins and metformin, and although it requires further investigation, it is possible the desired action of these drugs occurs via their impact on BACE1 expression. The increasing evidence for insulin resistance and glucose metabolism playing an important role in dementia led to the investigation of metformin in AD treatment, where it was found to reduce neuronal insulin resistance and improve glucose uptake via activation of AMPK, IR, and PI3/Akt signaling, and attenuate production of Aβ. 175 It is important to note other studies have only reported a reduction in Aβ production in response to metformin when in combination with insulin. 176,177 Significantly, the increased risk of AD seen in patients suffering with T2D is reduced with metformin treatment. 178 In August 2020 the Metformin in Alzheimer's Dementia Prevention (MAP) study started a multicenter phase 2/3 prevention trial to evaluate the benefit of metformin treatment on AD development.
The likely mechanism behind this is the reduction in BACE1 activity in response to metformin treatment. 177 As insulin and Aβ competitively bind to the IR, reducing BACE1 activity will in turn reduce Aβ and should increase the insulin sensitivity of the IR. As well as presenting BACE1 as an important target in the mechanisms of metabolic drugs, this also highlights insulin insensitivity as an early stage of AD development. 179 This presents BACE1 as an important molecule in the potential repurposing of metabolic drugs, with the mechanism behind their success arguably dependent on BACE1.
The same could be said for statins, a widely prescribed cholesterol reducing drug. Statins are associated with decreased Aβ formation, attributed to the reduction in cholesterol which in turn regulates BACE1. [180][181][182][183] Statins also reduce the levels of mevalonate, which normally stimulates cholesterol transporter (apoE) secretion, a risk factor for AD. 184 A reduction in apoE secretion therefore reduces plaque formation and in turn improves cognitive function. 180  For example, Verubecestat is a BACE1 inhibitor tested in phase 3 clinical trials on patients with mild to moderate AD. 186 Despite showing a reduction in cerebrospinal fluid Aβ levels to 63%-81%, no beneficial effect on cognition was observed for Verubecestat. 186 Adverse side effects were seen including an increase in falls and injuries, sleep disturbance, suicidal thoughts, hair color change and weight loss along with decreased appetite. 187 However, the side effects of weight loss and the impact on appetite deemed negative in the treatment of AD could be hugely beneficial for the treatment of metabolic syndrome. These effects likely occur through the action of BACE1 on the leptin pathway and insulin signaling pathway, and could revolutionize the treatment of metabolic dysfunction. Similar effects were seen with Lanabecestat, another BACE1 inhibitor undergoing clinical trials, with a lack of cognitive improvement observed but with the side effect of weight loss. 188 A greater number of individuals observed a weight loss of at least 7% when taking Lanabecestat, than individuals taking the placebo, with mean weight loss at À1.9 kg for the 50-mg Lanabecestat group compared with 0 for the placebo group. 188 The potential of Lanabecestat in obesity treatment has been recognized by AstraZeneca, who have recently obtained a patent for obesity treatment. 189 In line with trial findings, the patent highlights differences in weight loss based upon body mass index (BMI), an effect supported by preclinical research. 19 In trials, weight loss was greatest in individuals with a higher BMI, suggesting Lanabecestat could treat excess weight observed in obesity, without causing extreme weight loss when BMI is reduced. Despite the promise of this approach, it is important to consider the difficulties faced with BACE1 inhibition. The broad expression and substrate profile of BACE1 may be responsible for the various adverse effects on cellular processes observed in clinical trials. However, a greater understanding on the physiological roles of BACE1 may help unravel previous failings. It is also important to note that Verubecestat shows greater selectivity for BACE2 than BACE1, with Lanabecestat having a similar selectivity for both. 190 It may therefore require further investigation into ensuring these effects are mediated by BACE1. Given the extent of research into effective BACE1 inhibitors for AD, the repurposing of BACE1 inhibitory drugs for treatment of metabolic syndromes such as diabetes and obesity, or as a dual therapy against both diseases, could be a huge advancement in the treatment of metabolic disorders.

| BACE1 AS A BIOMARKER
BACE1 protein or markers of activity have shown promise to be blood-based biomarkers for a number of diseases, including AD.
BACE1 protein is detectable in plasma and levels are significantly raised in patients with mild cognitive impairment, while also predicting conversion of MCI to AD. 191 Furthermore, plasma BACE1 protein levels are elevated in people with T2D and correlate with glycemic control, independently of cognition. 192 The lncRNA species BACE1-AS strongly correlates with BACE1 expression and is also measurable in human plasma samples, with levels increased in patients with AD 193 and autism. 194 Currently, there is a lack of consensus for plasma Aβ as a useful biomarker for AD. However, Aβ levels are elevated in patients with cardiometabolic diseases including obesity, T2D, and heart failure. 38,195,196

| FUTURE DIRECTIONS
It is clear that the BACE1-AS/BACE1/Aβ axis has important physiological functions outside the brain. While, elevated BACE1 activity is observed during the development of a number of diseases, in addition to AD. This poses the exciting concept that current BACE1 inhibitors, developed for the treatment of AD, could be repurposed for the treatment of cardiometabolic diseases.
Whether expression and activity of BACE1 could be used as potential prognostic and/or therapeutic biological marker(s) will require further research into disease specificity and sensitivity but remains a promising possibility.
Taken together, this demonstrates the underappreciated functions of an enzyme primarily investigated for its role in AD. BACE1 clearly plays a role in multiple physiological and pathological cellular processes, and future studies are needed to fully understand this