Effects of peptides derived from BACE1 catalytic domain on APP processing

One of the hallmarks of Alzheimer's disease (AD) is the deposition of beta-amyloid (Abeta) peptides in neuritic plaques. Abeta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. beta-APP cleaving enzyme-1 (BACE1) has been shown to be the major beta-secretase and is a primary therapeutic target for AD. We report here novel BACE1 inhibitory peptidomimetics, which are derived from catalytic domains of BACE1 themselves, instead of APP cleavage sites and are structurally modified by myristoylation in N-terminus for efficient cell permeability. The peptides not only inhibited the formation of APPbeta (a soluble N-terminal fragment of APP cleaved by beta-secretase), but also significantly reduced Abeta40 production. Our results suggest a new approach for identifying inhibitory agents for the treatment of AD.     

BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease

beta-site APP cleaving enzyme 1 (BACE1) is the beta-secretase enzyme required for generating pathogenic beta-amyloid (Abeta) peptides in Alzheimer's disease (AD). BACE1 knockout mice lack Abeta and are phenotypically normal, suggesting that therapeutic inhibition of BACE1 may be free of mechanism-based side effects. However, direct evidence that BACE1 inhibition would improve cognition is lacking. Here we show that BACE1 null mice engineered to overexpress human APP (BACE1(-/-).Tg2576(+)) are rescued from Abeta-dependent hippocampal memory deficits. Moreover, impaired hippocampal cholinergic regulation of neuronal excitability found in the Tg2576 AD model is ameliorated in BACE1(-/-).Tg2576(+) bigenic mice. The behavioral and electrophysiological rescue of deficits in BACE1(-/-).Tg2576(+) mice is correlated with a dramatic reduction of cerebral Abeta40 and Abeta42 levels and occurs before amyloid deposition in Tg2576 mice. Our gene-based approach demonstrates that lower Abeta levels are beneficial for AD-associated memory impairments, validating BACE1 as a therapeutic target for AD.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

Hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/gamma-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-alpha-converting enzyme (TACE, an enzyme known to cleave APP at the alpha-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1alpha increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1alpha reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1alpha overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1alpha. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1alpha conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1alpha in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.     

Inhibition of amyloid beta-peptide production by blockage of beta-secretase cleavage site of amyloid precursor protein

Amyloid beta-peptide (Abeta) is implicated as the major causative agent in Alzheimer's disease (AD). Abeta is produced by the processing of the amyloid precursor protein (APP) by BACE1 (beta-secretase) and gamma-secretase. Many inhibitors have been developed for the secretases. However, the inhibitors will interfere with the processing of not only APP but also of other secretase substrates. In this study, we describe the development of inhibitors that prevent production of Abeta by specific binding to the beta-cleavage site of APP. We used the hydropathic complementarity (HC) approach for the design of short peptide inhibitors. Some of the HC peptides were bound to the substrate peptide (Sub W) corresponding to the beta-cleavage site of APP and blocked its cleavage by recombinant human BACE1 (rhBACE1) in vitro. In addition, HC peptides specifically inhibited the cleavage of Sub W, and not affecting other BACE1 substrates. Chemical modification allowed an HC peptide (CIQIHF) to inhibit the processing of APP as well as the production of Abeta in the treated cells. Such novel APP-specific inhibitors will provide opportunity for the development of drugs that can be used for the prevention and treatment of AD with minimal side effects.     

Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice

The aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid beta (Abeta) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of Abeta-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(-/-) mice demonstrated that BACE1 is absolutely required for both Abeta generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for Abeta generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in Abeta levels in young mice, it nonetheless resulted in a dramatic reduction in Abeta plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain Abeta levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant Abeta levels lead to dramatic inhibition of Abeta-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.     

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Abeta generation in vivo

The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotide-dependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of beta-secretase (BACE) from DRMs to APP-containing membrane fractions, increased beta-cleavage of APP and high levels of Abeta peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Abeta formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Abeta levels. Pharmacological enhancement of seladin-1 activity may be a novel Abeta-lowering approach for the treatment of AD.     

Secretion and intracellular generation of truncated Abeta in beta-site amyloid-beta precursor protein-cleaving enzyme expressing human neurons

Insoluble pools of the amyloid-beta peptide (Abeta) in brains of Alzheimer's disease patients exhibit considerable N- and C-terminal heterogeneity. Mounting evidence suggests that both C-terminal extensions and N-terminal truncations help precipitate amyloid plaque formation. Although mechanisms underlying the increased generation of C-terminally extended peptides have been extensively studied, relatively little is known about the cellular mechanisms underlying production of N-terminally truncated Abeta. Thus, we used human NT2N neurons to investigate the production of Abeta11-40/42 from amyloid-beta precursor protein (APP) by beta-site APP-cleaving enzyme (BACE). When comparing undifferentiated human embryonal carcinoma NT2- cells and differentiated NT2N neurons, the secretion of sAPP and Abeta correlated with BACE expression. To study the effects of BACE expression on endogenous APP metabolism in human cells, we overexpressed BACE in undifferentiated NT2- cells and NT2N neurons. Whereas NT2N neurons produced both full-length and truncated Abeta as a result of normal processing of endogenous APP, BACE overexpression increased the secretion of Abeta1-40/42 and Abeta11-40/42 in both NT2- cells and NT2N neurons. Furthermore, BACE overexpression resulted in increased intracellular Abeta1-40/42 and Abeta11-40/42. Therefore, we conclude that Abeta11-40/42 is generated prior to deposition in senile plaques and that N-terminally truncated Abeta peptides may contribute to the downstream effects of amyloid accumulation in Alzheimer's disease.     

Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity

The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.     

Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production

We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both Abeta40 and Abeta42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of beta-cleaved amyloid precursor protein (APP) carboxyl-terminal fragments (betaCTF), the rate-limiting proteolytic intermediate in Abeta generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in beta-cleaved soluble APP relative to alpha-cleaved soluble APP was also observed following Rab5 overexpression. BetaCTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, betaCTF generation, and Abeta production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a beta-amyloidogenic pathway.     

PAR-4 is involved in regulation of beta-secretase cleavage of the Alzheimer amyloid precursor protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.     

Targeting BACE with small inhibitory nucleic acids - a future for Alzheimer's disease therapy?

beta-Secretase, a beta-site amyloid precursor protein (APP) cleaving enzyme (BACE), participates in the secretion of beta-amyloid peptides (Abeta), the major components of the toxic amyloid plaques found in the brains of patients with Alzheimer's disease (AD). According to the amyloid hypothesis, accumulation of Abeta is the primary influence driving AD pathogenesis. Lowering of Abeta secretion can be achieved by decreasing BACE activity rather than by down-regulation of the APP substrate protein. Therefore, beta-secretase is a primary target for anti-amyloid therapeutic drug design. Several approaches have been undertaken to find an effective inhibitor of human beta-secretase activity, mostly in the field of peptidomimetic, non-cleavable substrate analogues. This review describes strategies targeting BACE mRNA recognition and its down-regulation based on the antisense action of small inhibitory nucleic acids (siNAs). These include antisense oligonucleotides, catalytic nucleic acids - ribozymes and deoxyribozymes - as well as small interfering RNAs (siRNAs). While antisense oligonucleotides were first used to identify an aspartyl protease with beta-secretase activity, all the strategies now demonstrate that siNAs are able to inhibit BACE gene expression in a sequence-specific manner, measured both at the level of its mRNA and at the level of protein. Moreover, knock-down of BACE reduces the intra- and extracellular population of Abeta40 and Abeta42 peptides. An anti-amyloid effect of siNAs is observed in a wide spectrum of cell lines as well as in primary cortical neurons. Thus targeting BACE with small inhibitory nucleic acids may be beneficial for the treatment of Alzheimer's disease and for future drug design.     

Alternative pathways for production of beta-amyloid peptides of Alzheimer's disease

This highlight article describes three Alzheimer's disease (AD) studies presented at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms for producing neurotoxic beta-amyloid (Abeta) peptides. One group described the poor kinetics of BACE 1 for cleaving the wild-type (WT) beta-secretase site of APP found in most AD patients. They showed that cathepsin D displays BACE 1-like specificity and cathepsin D is 280-fold more abundant in human brain than BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the WT beta-secretase site, they suggested continuing the search for additional beta-secretase(s). The second group reported cathepsin B as an alternative beta-secretase possessing excellent kinetic efficiency and specificity for the WT beta-secretase site. Significantly, inhibitors of cathepsin B improved memory, with reduced amyloid plaques and decreased Abeta(40/42) in brains of AD animal models expressing amyloid precursor protein containing the WT beta-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Abeta. Results showed that cathepsin B, but not BACE 1, efficiently cleaves the WT beta-secretase isoaspartate site. Furthermore, cyclization of N-terminal Glu by glutaminyl cyclase generates highly amyloidogenic pGluAbeta(3-40/42). These presentations suggest cathepsin B and glutaminyl cyclase as potential new AD therapeutic targets.     

BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild-type mice

Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimer's disease (AD). Inhibition of beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE1), the enzyme that initiates Abeta production, and other Abeta-lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta-lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non-transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full-length Abeta. A newly developed ELISA detected a significant reduction of full-length soluble Abeta 1-40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x-40 Abeta was moderately reduced due to detection of non-full-length Abeta and compensatory activation of alpha-secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non-transgenic mice provide more accurate evaluation of Abeta-reducing strategies than was previously feasible.     

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for beta- and beta'-cleavage sites by human and murine BACE1

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.     

Altered amyloid-beta metabolism and deposition in genomic-based beta-secretase transgenic mice

Amyloid-beta (Abeta) the primary component of the senile plaques found in Alzheimer's disease (AD) is generated by the rate-limiting cleavage of amyloid precursor protein (APP) by beta-secretase followed by gamma-secretase cleavage. Identification of the primary beta-secretase gene, BACE1, provides a unique opportunity to examine the role this unique aspartyl protease plays in altering Abeta metabolism and deposition that occurs in AD. The current experiments seek to examine how modulating beta-secretase expression and activity alters APP processing and Abeta metabolism in vivo. Genomic-based BACE1 transgenic mice were generated that overexpress human BACE1 mRNA and protein. The highest expressing BACE1 transgenic line was mated to transgenic mice containing human APP transgenes. Our biochemical and histochemical studies demonstrate that mice overexpressing both BACE1 and APP show specific alterations in APP processing and age-dependent Abeta deposition. We observed elevated levels of Abeta isoforms as well as significant increases of Abeta deposits in these double transgenic animals. In particular, the double transgenics exhibited a unique cortical deposition profile, which is consistent with a significant increase of BACE1 expression in the cortex relative to other brain regions. Elevated BACE1 expression coupled with increased deposition provides functional evidence for beta-secretase as a primary effector in regional amyloid deposition in the AD brain. Our studies demonstrate, for the first time, that modulation of BACE1 activity may play a significant role in AD pathogenesis in vivo.     

A role for 12/15 lipoxygenase in the amyloid beta precursor protein metabolism

12/15 Lipoxygenase (12/15LO) protein levels and activity are increased in pathologically affected regions of Alzheimer's disease (AD) brains, compared with controls. Its metabolic products are elevated in cerebrospinal fluid of patients with AD and individuals with mild cognitive impairment, suggesting that this enzyme may be involved early in AD pathogenesis. Herein, we investigate the effect of pharmacologic inhibition of 12/15LO on the amyloid beta precursor protein (APP) metabolism. To this end, we used CHO and N2A cells stably expressing human APP with the Swedish mutant, and two structurally distinct and selective 12/15LO inhibitors, PD146176 and CDC. Our results demonstrated that both drugs dose-dependently reduced Abeta formation without affecting total APP levels. Interestingly, in the same cells we observed a significant reduction in secreted (s)APPbeta and beta-secretase (BACE), but not sAPPalpha and ADAM10 protein levels. Together, these data show for the first time that this enzymatic pathway influences Abeta formation whereby modulating the BACE proteolytic cascade. We conclude that specific pharmacologic inhibition of 12/15LO could represent a novel therapeutic target for treating or preventing AD pathology in humans.     

Alzheimer's disease beta-secretase BACE1 is not a neuron-specific enzyme

The brains of Alzheimer's disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of beta-amyloid peptides. The generation of beta-amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the beta-site APP cleaving enzyme 1 (BACE1). The expression of this enzyme has been localized to the brain, in particular to neurons, indicating that neurons are the major source of beta-amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for beta-amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between beta-amyloid deposits and neurons. However, under certain conditions related to chronic stress, the role of astrocytes may not be beneficial. Here we present evidence demonstrating that astrocytes are an alternative source of BACE1 and therefore may contribute to beta-amyloid plaque formation. While resting astroyctes in brain do not express BACE1 at detectable levels, cultured astrocytes display BACE1 promoter activity and express BACE1 mRNA and enzymatically active BACE1 protein. Additionally, in animal models of chronic gliosis and in brains of AD patients, there is BACE1 expression in reactive astrocytes. This would suggest that the mechanism for astrocyte activation plays a role in the development of AD and that therapeutic strategies that target astrocyte activation in brain may be beneficial for the treatment of AD. Also, there are differences in responses to chronic versus acute stress, suggesting that one consequence of chronic stress is an incremental shift to different phenotypic cellular states.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

Morphological and functional abnormalities in neuromuscular junctions of Drosophila melanogaster induced by the expression of human APP gene

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the loss of neurocortical and hippocampal synapses that precedes amyloidosis and neurodegeneration and closely correlates with memory impairment. Mutations in the amyloid precursor protein (APP) cause familial AD and result in the increased production of amyloid-beta-protein (Abeta). To gain insights into synaptic effects of APP, we expressed APP, mutant form APP-Swedish and BACE in the motor neurons of fly larvae. We have shown that targeted expression of APP (APP-Swedish) in Drosophila larval motor neurons causes significant morphological and functional changes in neuromuscular junctions (NMJs): a dramatic increase in the number of synaptic buttons and changes in exocytosis as revealed by incorporation of the styryl dye FM4-64. Analysis of the number and distribution of mitochondria showed that motor neurons overexpressing APP (APP-Swedish) had a significant reduction of functional mitochondria in the presynaptic terminal. Significant synaptic abnormalities were observed for APP (APP-Swedish) and human beta-secretase (BACE) resulting in secretion of amyloid beta protein (Abeta). We suggest that APP participates in regulation of synaptic functions and its elevated expression leads to synaptic pathology independently from neurotoxic effects of Abeta.