BACE1 and BACE2 in pathologic and normal human muscle

BACE1 and BACE2 are recently discovered enzymes participating in processing of amyloid beta precursor protein (AbetaPP). Their discovery is contributing importantly to understanding the mechanism of amyloid-beta generation, and hence the pathogenesis of Alzheimer's disease (AD). Sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy (h-IBM) are progressive muscle diseases in which overproduction of AbetaPP and accumulation of its presumably toxic proteolytic product amyloid-beta (Abeta) in abnormal muscle fibers appear to play an important upstream role in the pathogenic cascade. In normal human muscle AbetaPP was also shown to be present and presumably playing a role (a) at neuromuscular junctions and (b) during muscle development. To investigate whether BACE1 and BACE2 play a role in normal and diseased human muscle, we have now studied them by immunocytochemistry and immunoblotting in 35 human muscle biopsies, including: 5 s-IBM; 5 chromosome-9p1-linked quadriceps-sparing h-IBM; and 25 control muscle biopsies. In addition, expression of BACE1 and BACE2 was studied in normal cultured human muscle. Our studies demonstrate that BACE1 and BACE2 (a) are expressed in normal adult muscle at the postsynaptic domain of neuromuscular junctions, and in cultured human muscle; (b) are accumulated in the form of plaque-like inclusions in both s-IBM and h-IBM vacuolated muscle fibers; and (c) are immunoreactive in necrotizing muscle fibers. Accordingly, BACE1 and BACE2 participate in normal and abnormal processes of human muscle, suggesting that their functions are broader than previously thought.     

Genetic markers in the diagnosis of Alzheimer's disease

Abbreviations: AD, Alzheimer's disease; AbetaPP, amyloid beta protein precursor; BACE, beta-site AbetaPP cleaving enzyme; PS1, presenilin-1; PS2, presenilin-2; APOE, apolipoprotein E; LRP, low density lipoprotein receptor-related protein; SNPs, single-nucleotide polymorphisms; A2M, alpha-2-macroglobulin. Alzheimer's disease (AD) is the most common neurodegenerative disorder associated with dementia in the elderly population. Its clinical symptoms are manifest with increasing prominence during mid- to late stages of adulthood. In the absence of precise biological indicators that precede or accompany the cognitive decline, diagnostic confirmation of AD requires postmortem detection of histopathological characteristics such as amyloid plaques, neurofibrillary tangles, and extensive cortical atrophy. While the etiology of AD remains incompletely understood, it was recognized early on that the observed familial aggregation of AD implied the presence of one or more inherited susceptibility markers that could be useful in diagnosis and treatment. To date, genetic analyses of these pedigrees have resolved four independent genetic loci linked with inherited susceptibility to AD.     

Commentary: Abeta N- Terminal Isoforms: Critical contributors in the course of AD pathophysiology

The assessment of protein or amino acid variations across evolution allows one to glean divergent features of disease-specific pathology. Within the Alzheimer's disease (AD) literature, extensive differences in Abeta processing across cell lines and evolution have clearly been observed. In the recent past, increased levels of amyloid beta Abeta1-42 have been heralded to be what distinguishes whether one is prone to the development of AD [59]. However, observations in naturally occurring, non-transgenic animals which display a great deal of parenchymal Abeta1-42 (Abeta found within extracellular plaque deposits) and a complete lack ofbeta1-40 within these same Abeta1-42 plaques raise the issue of whether Abetax-42 (Abeta that is truncated or modified at the N- terminus), rather than Abeta1-42, is instead the critical mediator of Abeta production and pathogenesis [47,49]. Distinct ratios of Abeta N-terminal variants (i.e. Abeta1-x, Abeta3-x, Abeta11-x, beta17-x) have been assessed in human amyloid plaques [18,21,40,41,42,47,48,49,52]. Moreover, ratios of specific Abeta N-terminal variants separate naturally occurring, non-transgenic animals which develop abundant levels of Abetax-42 and not Abetax-40 from human AD participants who harbor plaques that contain both the Abetax-42 and Abetax-40 variants [49]. Next, Teller and colleagues have demonstrated the presence of N-terminal truncated soluble 3kD (likely Abeta17-x) and 3.7kD peptides (in addition to 4kD Abeta) well before the appearance of amyloid plaques in Down Syndrome brain [51], indicating an early contribution of thebeta N-terminus to the formation of amyloid pathology. Additional critical facts concerning the major contribution of the Abeta N-terminus in AD pathogenesis include observations which support thatbeta generated by rodent neurons is predominantly truncated at Abeta11-x [13], the major form of APP C-terminal fragments in mice lacking functional PS1 is AbetaPP11-98 [9], beta11-x expression is increased as a function of BACE expression [55], and an interrelationship between presenilin-1 mutations and increased levels of N-terminally truncatedbeta [40]. This commentary highlights current understanding and potential biochemical, pathological, and cell biological contributions of Abeta N-terminal variants implicated during the course of AD pathogenesis. Although the amyloid beta protein precursor (AbetaPP) gene and Abeta are highly conserved across mammalian species, there are species-specific differences. For instance, the primate, guinea pig, canine, and polar bear share an identical Abeta sequence to that observed in human brain while the rat displays a distinct amino acid sequence with substitutions at residues 5 (Arg), 10 (Tyr), and 13 (His) [24,37]. All of these mammals generate Abeta1-42 via cleavage by at least two enzymes, beta (beta-) secretase and gamma (gamma-) secretase (Fig. 1). The enzyme that liberates the N- terminus of the Abeta peptide ('beta-secretase') is also termed BACE (beta-site AbetaPP cleaving enzyme) [55]. Cathepsin D, which accumulates within AD neurons [15], also cleaves at the N-terminal side of the first aspartate residue of amyloid beta [2].beta-secretase activity is necessary in order to initiate 4kD beta1-x formation by cleaving AbetaPP at the N-terminus and results in the release of a soluble 100kD AbetaPP N- terminal fragment and a 12kD membrane bound C-terminal fragment (C99/C100) [55]. The carboxyl-terminus of the Abetapeptide is liberated through cleavage by the enzyme termed gamma-secretase. In the past, potential AD therapeutic strategies have mainly been geared towards gamma-secretase inhibition. However, such strategies alone no longer appear sound as it is clear that the AbetaPP C99/C100 fragment itself, which requires beta-, but not gamma-, secretase cleavage for generation and includes the entire Abeta peptide, is neurotoxic when evaluated in cultured cells [12,30,34]. Thus, gamma-secretase inhibition alone would not preclude the generation of the neurotoxic C99/C100 fragment.     

Presenilin structure in mechanisms leading to Alzheimer's disease

Molecular genetic studies of familial Alzheimer's disease by 1995 had clearly implicated three proteins as critical to Alzheimer's disease (AD), the amyloid-beta protein precursor (AbetaPP) and the two homologous presenilins, PS-1 and PS-2. To account for the roles of these proteins in AD, we had proposed that as an early and critical step in the mechanisms that lead to AD, the PS on the surface of a brain cell engages in a specific receptor-ligand intercellular interaction with AbetaPP on the surface of a neighboring cell. This cell-cell interaction is required to trigger off a cascade of processes that lead to the production of amyloid-beta (Abeta) from AbetaPP, leading to AD. At about this time, however, many established AD researchers had obtained data that appeared to disagree with our proposed mechanism. Their immediate objections to our proposal were based on their conclusions that 1) The PS proteins were exclusively intracellular, and were not expressed at the cell surface, and 2) The topography of the PS proteins in intracellular membranes exhibits either 6 or 8-TM spanning domains, not 7. Here we discuss the evidence for the 6-TM, 7-TM, 8-TM and other models of PS topography and offer possibilities for the differences in interpretation of the various sets of data. We review the experimental demonstration of the cell-surface expression and the 7-TM structure of PS, the functional consequences of this structure, and the findings that PS-1 and PS-2 are members of the superfamily of 7-TM heterotrimeric G-protein coupled receptors (GPCRs).     

BACE1: the beta-secretase enzyme in Alzheimer's disease

Data that have accumulated for well over a decade have implicated the beta-amyloid (Abeta) peptide as a central player in the pathogenesis of Alzheimer's disease (AD). Amyloid plaques, composed primarily of Abeta progressively form in the brains of AD patients, and mutations in three genes (amyloid precursor protein [APP] and presenilin 1 and 2 [PS1 and PS2]) cause early-onset familial AD (FAD) by directly increasing production of the toxic, plaque-promoting Abeta42 peptide. Given the strong association between Abeta and AD, it is likely that therapeutic strategies to lower the levels of Abeta in the brain should prove beneficial for the treatment of AD. One such strategy could involve inhibiting the enzymes that generate Abeta. Abeta is a product of catabolism of the large type-I membrane protein APP. Two proteases, called beta- and gamma-secretase, endoproteolyze APP to liberate the Abeta peptide. Recently, the molecules responsible for these proteolytic activities have been identified. Several lines of evidence suggest that the PS1 and PS2 proteins are gamma-secretase, and the identity of beta-secretase has been shown to be the novel transmembrane aspartic protease, beta-site APP-cleaving enzyme 1 (BACE1; also called Asp2 and memapsin 2). BACE2, a protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the functional properties of beta-secretase, and as the key enzyme that initiates the formation of Abeta, BACE1 is an attractive drug target for AD. This review discusses the identification and initial characterization of BACE1 and BACE2, and summarizes recent studies of BACE1 knockout mice that have validated BACE1 as the authentic beta-secretase in vivo.     

Novel mutations introduced at the beta-site of amyloid beta protein precursor enhance the production of amyloid beta peptide by BACE1 in vitro and in cells

Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEV" mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.     

S-adenosylmethionine/homocysteine cycle alterations modify DNA methylation status with consequent deregulation of PS1 and BACE and beta-amyloid production

Few diseases are characterized by high homocysteine (HCY) and low folate and vitamin B12 blood levels. Alzheimer disease (AD) is among these. It has already been shown that DNA methylation is involved in amyloid precursor protein (APP) processing and beta-amyloid (A beta) production through the regulation of Presenilin1 (PS1) expression and that exogenous S-adenosylmethionine (SAM) can silence the gene reducing A beta production. Here we demonstrate that BACE (beta-secretase), as well as PS1, is regulated by methylation and that the reduction of folate and vitamin B12 in culture medium can cause a reduction of SAM levels with consequent increase in presenilin1 and BACE levels and with increase in A beta production. The simultaneous administration of SAM to the deficient medium can restore the normal gene expression, thus reducing the A beta levels. The use of deprived medium was intended to mimic a mild nutritional deficit involved in the onset of AD.     

The β-secretase, BACE

Evidence suggests that the β-amyloid peptide (Aβ) is central to the pathophysiology of Alzheimer’s disease (AD). Amyloid plaques, primarily composed of Aβ, progressively develop in the brains of AD patients, and mutations in three genes (APP, PS1, and PS2) cause early onset familial AD (FAD) by directly increasing synthesis of the toxic, plaque-promoting Aβ42 peptide. Given the strong association between Aβ and AD, therapeutic strategies to lower the concentration of Aβ in the brain should prove beneficial for the treatment of AD. One such strategy would involve inhibiting the enzymes that generate Aβ. Aβ is a product of catabolism of the large TypeI membrane protein, amyloid precursor protein (APP). Two proteases, called β- and γ-secretase, mediate the endoproteolysis of APP to liberate the Aβ peptide. For over a decade, the molecular identities of these proteases were unknown. Recently, the γ-secretase has been tentatively identified as the presenilin proteins, PS1 and PS2, and the identity of the β-secretase has been shown to be the novel transmembrane aspartic protease, β-site APP cleaving enzyme 1 (BACE1; also called Asp2 and memapsin2). BACE2, a novel protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the properties of the β-secretase, and as the key rate-limiting enzyme that initiates the formation of Aβ, BACE1 is an attractive drug target for AD. Here, I review the identification and initial characterization of BACE1 and BACE2, and summarize our current understanding of BACE1 post-translational processing and intracellular trafficking. In addition, I discuss recent studies of BACE1 knockout mice and the BACE1 X-ray structure, and relate implications for BACE1 drug development.     

A genome wide analysis of ubiquitin ligases in APP processing identifies a novel regulator of BACE1 mRNA levels

Proteolysis of beta-amyloid precursor protein (APP) into amyloid beta peptide (Abeta) by beta- and gamma-secretases is a critical step in the pathogenesis of Alzheimer's Disease (AD), but the pathways regulating secretases are not fully characterized. Ubiquitinylation, which is dysregulated in AD, may affect APP processing. Here, we describe a screen for APP processing modulators using an siRNA library targeting 532 predicted ubiquitin ligases. Seven siRNA pools diminished Abeta production. Of these, siRNAs targeting PPIL2 (hCyp-60) suppressed beta-site cleavage. Knockdown of PPIL2 mRNA decreased BACE1 mRNA, while overexpression of PPIL2 cDNA enhanced BACE1 mRNA levels. Microarray analysis of PPIL2 or BACE1 knockdown indicated that genes affected by BACE1 knockdown are a subset of those dependent upon PPIL2; suggesting that BACE1 expression is downstream of PPIL2. The association of PPIL2 with BACE expression and its requirement for Abeta production suggests new approaches to discover disease modifying agents for AD.     

Sodium azide and 2-deoxy-D-glucose-induced cellular stress affects phosphorylation-dependent AbetaPP processing

Production of the amyloid beta (Abeta) peptide via altered metabolism of the amyloid beta-Protein Precursor (AbetaPP) appears to be a key event in the pathology of Alzheimer Disease (AD). Accordingly, altered processing of AbetaPP was observed under conditions of abnormal cellular stress induced by sodium azide in the presence of 2-deoxy-D-glucose (2DG). As previously reported, the production of sAbetaPP (the secreted fragment of AbetaPP) was inhibited. However, our data further suggests that 2DG alone can account for most of the observed effects on AbetaPP processing in COS-1 cells and PC12 cells. It appears that 2DG interferes with the normal glycosylation of AbetaPP and its maturation process, having direct consequences on sAbetaPP production. Interestingly, PMA (phorbol 12-myristate 13-acetate)-induced sAbetaPP production was maintained under the stress conditions used, suggesting that potential non-amyloidogenic AbetaPP processing can still be favoured. This is of potential therapeutic interest, since it indicates that even under adverse stress conditions drugs such as PMA can affect AbetaPP processing, leading to increased sAbetaPP production and a concomitant reduction in Abeta production. However, the induction of sAbetaPP production was not identical when the phosphatase inhibitor OA (okadaic acid) was used. In fact, the typical OA-induced increase in sAbetaPP production could be abolished under specific conditions. This constitutes an interesting precedent for the possible dissociation of the PMA and OA responses in terms of sAbetaPP production. The involvement of protein phosphatases, which are inhibited by OA, inbetaPP processing, was reinforced by the increased co-localization of AbetaPP and PP1alpha (protein phosphatase 1alpha) at the cell surface upon exposure to OA and PMA. Overall, our results support the notion that signal transduction processes may be of particular relevance for our understanding of the molecular basis of AD, and for the design of rational signal transduction therapeutics.     

Deposition of amyloid fibrils promotes cell-surface accumulation of amyloid beta precursor protein

Amyloid beta protein (Abeta) deposition and neuronal degeneration are characteristic pathological features of Alzheimer's disease (AD). In vitro, Abeta fibrils (fAbeta) induce neuronal degeneration reminiscent to AD, but the mechanism of neurotoxicity is unknown. Here we show that amyloid fibrils increase the level of cell-surface full-length amyloid beta precursor protein (h-AbetaPP) and secreted AbetaPP (s-AbetaPP). Pulse-chase analysis indicated that fAbeta selectively inhibited the turnover of cell-surface AbetaPP, without altering its intracellular levels. FAbeta-induced AbetaPP accumulation was not abrogated by cycloheximide, suggesting that increased protein synthesis is not critically required. Abeta fibrils sequester s-AbetaPP from the culture medium and promote its accumulation at the cell surface, indicating that binding of Abeta fibrils mediates AbetaPP accumulation. A time course analysis of Abeta treatment showed that AbetaPP level is elevated before significant cell death can be detected, while other toxic insults do not augment AbetaPP level, suggesting that AbetaPP may be specifically involved in early stages of Abeta-induced neurodegeneration. Finally, Abeta fibrils promote clustering of h-AbetaPP in abnormal focal adhesion-like (FA-like) structures that mediate neuronal dystrophy, increasing its association with the cytoskeleton. These results indicate that the interaction of Abeta fibrils with AbetaPP is an early event in the mechanism of Abeta-induced neurodegeneration that may play a significant role in AD pathogenesis.     

Presenilin 1 is involved in the maturation of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1)

One of the pathologic hallmarks of Alzheimer's disease is the excessive deposition of beta-amyloid peptides (Abeta) in senile plaques. Abeta is generated when beta-amyloid precursor protein (APP) is cleaved sequentially by beta-secretase, identified as beta-site APP-cleaving enzyme 1 (BACE1), and gamma-secretase, a putative enzymatic complex containing presenilin 1 (PS1). However, functional interaction between PS1 and BACE1 has never been known. In addition to this classical role in the generation of Abeta peptides, it has also been proposed that PS1 affects the intracellular trafficking and maturation of selected membrane proteins. We show that the levels of exogenous and endogenous mature BACE1 expressed in presenilin-deficient mouse embryonic fibroblasts (PS-/-MEFs) were reduced significantly compared to those in wild-type MEFs. Moreover, the levels of mature BACE1 were increased in human neuroblastoma cell line, SH-SY5Y, stably expressing wild-type PS1, compared to native cells. Conversely, the maturation of BACE1 was compromised under the stable expression of dominant-negative mutant PS1 overexpression. Immunoprecipitation assay showed that PS1 preferably interacts with proBACE1 rather than mature BACE1, indicating that PS1 can be directly involved in the maturation process of BACE1. Further, endogenous PS1 was immunoprecipitated with endogenous BACE1 in SH-SY5Y cells and mouse brain tissue. We conclude that PS1 is directly involved in the maturation of BACE1, thus possibly functioning as a regulator of both beta- and gamma-secretase in Abeta generation.     

Association studies using novel polymorphisms in BACE1 and BACE2.

The release of amyloid-beta peptide (Abeta) from beta-amyloid precursor protein (APP) requires cleavage by beta- and gamma-secretases. Several groups have identified a candidate for the beta-site APP-cleaving enzyme, BACE1, and its homologue BACE2. We sequenced the genes for BACE1 and BACE2 and found several polymorphisms in both genes. Genotyping a large cohort of AD cases and controls has shown no association between AD and the intronic polymorphism in BACE2 while there was a weak association between the BACE1 polymorphism in exon 5 and AD in those carrying the APOE epsilon4 allele.     

Von Willebrand factor promoter targets the expression of amyloid beta protein precursor to brain vascular endothelial cells of transgenic mice

Dysfunction of brain vascular endothelial cells may be associated with the pathogenesis of several diseases including cerebral amyloid angiopathy, hemorrhagic stroke and Alzheimer disease. New model systems are necessary to examine the contribution of brain endothelial cells in these disorders. The Von Willebrand factor gene promoter fragment that spans sequences -487 to +247 targets the expression of LacZ marker gene in transgenic mice specifically to brain vascular endothelial cells. Transgenic mice have been prepared that express human amyloid beta protein precursor protein (AbetaPP) isoforms 695 and 751 (wild-type and Dutch variant mutations) under the regulation of this VWF promoter sequence. These AbetaPP transgenes are specifically expressed in brain vascular endothelial cells. The VWF promoter is a valuable tool for targeting gene expression to brain vascular endothelial cells to provide a model to directly examine endothelial cell placement of genes and their contribution to cerebral vascular disease.     

BACE1 mRNA expression in Alzheimer's disease postmortem brain tissue

β-site AβPP cleaving enzyme 1 (BACE1) catalyses the rate-limiting step for production of amyloid-β (Aβ) peptides, involved in the pathological cascade underlying Alzheimer's disease (AD). Elevated BACE1 protein levels and activity have been reported in AD postmortem brains. Our study explored whether this was due to elevated BACE1 mRNA expression. RNA was prepared from five brain regions in three study groups: controls, individuals with AD, and another neurodegenerative disease group affected by either Parkinson's disease (PD) or dementia with Lewy bodies (DLB). BACE1 mRNA levels were measured using quantitative realtime PCR (qPCR) and analyzed by qbasePLUS using validated stably-expressed reference genes. Expression of glial and neuronal markers (glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), respectively) were also analyzed to quantify the changing activities of these cell populations in the tissue. BACE1 mRNA levels were significantly elevated in medial temporal and superior parietal gyri, compared to the PD/DLB and/or control groups. Superior frontal gryus BACE1 mRNA levels were significantly increased in the PD/DLB group, compared to AD and control groups. For the AD group, BACE1 mRNA changes were analyzed in the context of the reduced NSE mRNA, and strongly increased GFAP mRNA levels apparent as AD progressed (indicated by Braak stage). This analysis suggested that increased BACE1 mRNA expression in remaining neuronal cells may contribute to the increased BACE1 protein levels and activity found in brain regions affected by AD.