A novel immunotherapy for Alzheimer's disease: antibodies against the beta-secretase cleavage site of APP

One of the main neuropathological lesions observed at brain autopsy of Alzheimer's disease (AD) patients are the extracellular senile plaques mainly composed of amyloid-beta (Abeta) peptides. Abeta is generated by proteolytic processing of amyloid precursor protein (APP) via beta and gamma-secretases. The beta-secretase APP cleaving enzyme 1 (BACE1) has become a target of intense research aimed at blocking the enzyme activity. Recent studies showed that BACE1 is involved in processing other non-APP substrates, and that other proteases are involved in APP processing. We have recently established a novel approach to inhibit Abeta production via antibodies against the beta-secretase cleavage site of APP. These antibodies bind wild type and Swedish mutated APP expressed in transgenic mice brain tissues. The isolated antibodies do not bind any form of Abeta peptides. Antibody up-take experiments, using Chinese hamster ovary cells expressing wild-type APP, suggest that antibody internalization and trafficking are mediated via the endocytic pathway. Administration of antibodies to the cells growing media resulted in a considerable decrease in intracellular Abeta levels, as well as in the levels of the corresponding C-terminal fragment (C99). The relevance of intra-neuronal accumulation of mainly Abeta42 as an early event in AD pathogenesis suggests that this approach may be applicable as a novel therapeutic strategy in AD treatment.     

Polymorphism in the BACE gene influences the risk for Alzheimer's disease

Pathological characteristics of Alzheimer's disease (AD) are neurofibrillary tangles and amyloid-beta (Abeta) plaques. Abeta is generated by cleavage of the amyloid precursor protein by beta- and gamma-secretases. BACE (beta-site APP cleaving enzyme) was identified as the beta-secretase. Variations of the BACE gene might influence activity and function of the protein and, thus, might influence the pathogenesis of AD. Consequently, we investigated the association of different BACE polymorphisms with AD. BACE exon 5 polymorphism influenced the risk of AD. This effect was most pronounced in apolipoprotein E4 allele carriers. Furthermore, Abeta(42) CSF levels were influenced by BACE genotype. It appears that BACE polymorphism plays a more important role in the development of AD than previously assumed, possibly by influencing Abeta(42) levels.     

GGA1 acts as a spatial switch altering amyloid precursor protein trafficking and processing

The beta-amyloid (Abeta) precursor protein (APP) is cleaved sequentially by beta-site of APP-cleaving enzyme (BACE) and gamma-secretase to release the Abeta peptides that accumulate in plaques in Alzheimer's disease (AD). GGA1, a member of the Golgi-localized gamma-ear-containing ARF-binding (GGA) protein family, interacts with BACE and influences its subcellular distribution. We now report that overexpression of GGA1 in cells increased the APP C-terminal fragment resulting from beta-cleavage but surprisingly reduced Abeta. GGA1 confined APP to the Golgi, in which fluorescence resonance energy transfer analyses suggest that the proteins come into close proximity. GGA1 blunted only APP but not notch intracellular domain release. These results suggest that GGA1 prevented APP beta-cleavage products from becoming substrates for gamma-secretase. Direct binding of GGA1 to BACE was not required for these effects, but the integrity of the GAT (GGA1 and TOM) domain of GGA1 was. GGA1 may act as a specific spatial switch influencing APP trafficking and processing, so that APP-GGA1 interactions may have pathophysiological relevance in AD.     

Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer disease and normal brain

beta-Amyloid peptide (Abeta) is generated by two cleavages of amyloid precursor protein (APP). The initial cleavage by BACE is followed by gamma-secretase cleavage of the C-terminal APP fragment. Presenilin-1 (PS-1) is intimately related to gamma-secretase. Once formed, Abeta is mainly broken down by neprilysin. To estimate vulnerability to Abeta senile plaque formation, we measured the relative mRNA levels of APP695, APP751, APP770, BACE, presenilin-1 (PS-1) and neprilysin in nine brain areas and in heart, liver, spleen and kidney in a series of Alzheimer disease (AD) and control cases. Each of the mRNAs was expressed in every tissue examined. APP695 was the dominant APP isoform in brain. Compared with controls, APP695 and PS-1 mRNA levels were significantly elevated in high plaque areas of AD brain, while neprilysin mRNA levels were significantly reduced. BACE levels were not significantly different in AD compared with control brain. In peripheral organs, there were no significant differences in any of the mRNAs between AD and control cases. APP isoforms were differently expressed in the periphery than in brain, with APP 751>770>695. Neprilysin mRNA levels were much higher, while APP695 and PS-1 mRNA levels were much lower in the periphery than in brain. The data suggest that, in the periphery, the capacity to degrade Abeta is srong, accounting for the failure of Abeta deposits to form. In plaque prone areas of AD brain, the capacity to degrade Abeta is weak, while the capacity to generate Ab is upregulated. In plaque resistant areas of brain, a closer balance exists, but there is some tendency towards lower degrading and higher synthesizing capacity in AD brain compared with control brain. Overall, the data indicate that effectiveness of degradation by neprilysin may be a key factor in determining whether Abeta deposits develop.     

Platelet amyloid precursor protein processing: a bio-marker for Alzheimer's disease

The amyloid precursor protein (APP) in brain is processed either by an amyloidogenic pathway by beta-secretase and gamma-secretase to yield Abeta (beta-amyloid 4 kDa) peptide or by alpha-secretase within the beta-amyloid domain to yield non-amyloidogenic products. We have studied blood platelet levels of a 22-kDa fragment containing the Abeta (beta-amyloid 4 kDa) peptide, beta-secretase (BACE1), alpha-secretase (ADAM10), and APP isoform ratios of the 120-130 kDa to 110 kDa peptides from 31 Alzheimer's disease (AD) patients and 10 age-matched healthy control subjects. We found increased levels of Abeta4, increased activation of beta-secretase (BACE1), decreased activation of alpha-secretase (ADAM10) and decreased APP ratios in AD patients compared to normal control subjects. These observations indicate that the blood platelet APP is processed by the same amyloidogenic and non-amyloidogenic pathways as utilized in brain and that APP processing in AD patients is altered compared to control subjects and may be a useful bio-marker for the diagnosis of AD, the progression of disease and for monitoring drug responses in clinical trials.     

BACE1 (beta-secretase) knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time

The formation of Alzheimer's Abeta peptide is initiated when the amyloid precursor protein (APP) is cleaved by the enzyme beta-secretase (BACE1); inhibition of this cleavage has been proposed as a means of treating Alzheimer's disease. (AD) We have previously shown that young BACE1 knockout mice (BACE1 KO) do not generate Abeta but in other respects appear normal. Here we have extended this analysis to include both gene expression profiling and phenotypic assessment of older BACE1 KO animals to evaluate the impact of chronic Abeta deficiency. We did not detect global compensatory changes in neural gene expression in young BACE1 KO mice. In particular, expression of the beta-secretase homolog BACE2 was not upregulated. Furthermore, we found no structural alterations in any organ, including all central and peripheral neural tissues, of BACE1 KO mice up to 14 months of age. Aged BACE1 KO mice engineered to overexpress human APP (BACE1 KO/APPtg) did not develop amyloid plaques. These data provide evidence that neither beta-secretase nor Abeta plays a vital role in mouse physiology and that chronic beta-secretase inhibition could be a useful approach in treating AD.     

Reticulons RTN3 and RTN4-B/C interact with BACE1 and inhibit its ability to produce amyloid beta-protein

Beta-secretase beta-site APP cleaving enzyme 1 (BACE1), is a membrane-bound aspartyl protease necessary for the generation of amyloid beta-protein (Abeta), which accumulates in the brains of individuals with Alzheimer's disease (AD). To gain insight into the mechanisms by which BACE1 activity is regulated, we used proteomic methods to search for BACE1-interacting proteins in human neuroblastoma SH-SY5Y cells, which overexpress BACE1. We identified reticulon 4-B (RTN4-B; Nogo-B) as a BACE1-associated membrane protein. Co-immunoprecipitation experiments confirmed a physical association between BACE1 and RTN4-B, RTN4-C (the shortest isoform of RTN-4), and their homologue reticulon 3 (RTN3), both in SH-SY5Y cells and in transfected human embryonic kidney (HEK) 293 cells. Overexpression of these reticulons (RTNs) resulted in a 30-50% reduction in the secretion of both Abeta40 and Abeta42 from HEK293 cells expressing the AD-associated Swedish mutant amyloid precursor protein (APP), but did not affect Abeta secretion from cells expressing the APP beta-C-terminal fragment (beta-CTF), indicating that these RTNs can inhibit BACE1 activity. Furthermore, a BACE1 mutant lacking most of the N-terminal ectodomain also interacted with these RTNs, suggesting that the transmembrane region of BACE1 is critical for the interaction. We also observed a similar interaction between these RTNs and the BACE1 homologue BACE2. Because RTN3 and RTN4-B/C are substantially expressed in neural tissues, our findings suggest that they play important roles in the regulation of BACE1 function and Abeta production in the brain.     

Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease

CONTEXT: Amyloid plaques, a major pathological feature of Alzheimer disease (AD), are composed of an internal fragment of amyloid precursor protein (APP): the 4-kd amyloid-beta protein (Abeta). The metabolic processing of APP that results in Abeta formation requires 2 enzymatic cleavage events, a gamma-secretase cleavage dependent on presenilin, and a beta-secretase cleavage by the aspartyl protease beta-site APP-cleaving enzyme (BACE). OBJECTIVE: To test the hypothesis that BACE protein and activity are increased in regions of the brain that develop amyloid plaques in AD. METHODS: We developed an antibody capture system to measure BACE protein level and BACE-specific beta-secretase activity in frontal, temporal, and cerebellar brain homogenates from 61 brains with AD and 33 control brains. RESULTS: In the brains with AD, BACE activity and protein were significantly increased (P<.001). Enzymatic activity increased by 63% in the temporal neocortex (P =.007) and 13% in the frontal neocortex (P =.003) in brains with AD, but not in the cerebellar cortex. Activity in the temporal neocortex increased with the duration of AD (P =.008) but did not correlate with enzyme-linked immunosorbent assay measures of insoluble Abeta in brains with AD. Protein level was increased by 14% in the frontal cortex of brains with AD (P =.004), with a trend toward a 15% increase in BACE protein in the temporal cortex (P =.07) and no difference in the cerebellar cortex. Immunohistochemical analysis demonstrated that BACE immunoreactivity in the brain was predominantly neuronal and was found in tangle-bearing neurons in AD. CONCLUSIONS: The BACE protein and activity levels are increased in brain regions affected by amyloid deposition and remain increased despite significant neuronal and synaptic loss in AD.     

BACE1 structure and function in health and Alzheimer's disease

Amyloid plaques, hallmark neuropathological lesions in Alzheimer's disease (AD) brain, are composed of the beta-amyloid peptide (Abeta). Much evidence suggests that Abeta is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. Given the strong correlation between Abeta and AD, therapeutic strategies to lower cerebral Abeta levels should prove beneficial for AD treatment. Abeta is derived from amyloid precursor protein (APP) via cleavage by two proteases, beta- and gamma-secretase. The beta-secretase has been identified as a novel aspartic protease named BACE1 (beta-site APP Cleaving Enzyme 1) that initiates Abeta formation. Importantly, BACE1 appears to be dysregulated in AD. As the rate-limiting enzyme in Abeta generation, BACE1, in principle, is an excellent therapeutic target for strategies to reduce the production of Abeta in AD. While BACE1 knockout (BACE1-/-) mice have been instrumental in validating BACE1 as the authentic beta-secretase in vivo, data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-/- mice. Here we review the basic biology of BACE1, focusing on the regulation, structure and function of this enzyme. We pay special attention to the putative function of BACE1 during normal conditions and discuss in detail the relationship that exists between key risk factors for AD and the pathogenic alterations in BACE1 that are observed in the diseased state.     

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.     

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.     

BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice

Evidence suggests that beta-amyloid (Abeta) peptide triggers a pathogenic cascade leading to neuronal loss in Alzheimer's disease (AD). However, the causal link between Abeta and neuron death in vivo remains unclear since most animal models fail to recapitulate the dramatic cell loss observed in AD. We have recently developed transgenic mice that overexpress human APP and PS1 with five familial AD mutations (5XFAD mice) and exhibit robust neuron death. Here, we demonstrate that genetic deletion of the beta-secretase (BACE1) not only abrogates Abeta generation and blocks amyloid deposition but also prevents neuron loss found in the cerebral cortex and subiculum, brain regions manifesting the most severe amyloidosis in 5XFAD mice. Importantly, BACE1 gene deletion also rescues memory deficits in 5XFAD mice. Our findings provide strong evidence that Abeta ultimately is responsible for neuron death in AD and validate the therapeutic potential of BACE1-inhibiting approaches for the treatment of AD.     

Inhibition by KMI-574 leads to dislocalization of BACE1 from lipid rafts

BACE1 initiates processing of the amyloid precursor protein (APP) in the production of amyloid beta (Abeta) peptide. After beta-cleavage by BACE1, the C-terminal stub of the APP fragment is further processed by the gamma-secretase complex to produce Abeta. Because APP, Abeta, the gamma-secretase complex, and BACE1 are found in lipid raft membranes, Abeta production is widely accepted to occur in lipid rafts. However, whether BACE1 is activated within the rafts is unclear. To analyze the relationship between the activity and the localization of BACE1, we used a new BACE1 inhibitor, KMI-574, and separated raft membranes on sucrose density gradients. In the presence of KMI-574, the localization of BACE1 shifted from the rafts to nonraft membranes in HEK293 cells. We also analyzed the proteolytically inactive mutants, D93A, D289A, and D93A/D289A, of BACE1. These mutants also moved from rafts to nonrafts, and the D93A/D289A double-mutant localized exclusively to nonraft membranes. The mutants were defective in maturation by glynosylation and formed hyperoligomers, suggesting that the BACE1 oligomers could not exit from the ER and be transported to the Golgi apparatus. Our findings suggest that the activated conformation of BACE1 is important for protein transport and localization to lipid rafts.     

Retinoic acid normalizes nuclear receptor mediated hypo-expression of proteins involved in beta-amyloid deposits in the cerebral cortex of vitamin A deprived rats

Recent data have revealed that disruption of vitamin A signaling observed in Alzheimer's disease (AD) leads to a deposition of beta-amyloid (Abeta). The aim of this study was to precise the role of vitamin A and its nuclear receptors (RAR) in the processes leading to the Abeta deposits. Thus, the effect of vitamin A depletion and subsequent administration of retinoic acid (RA, the active metabolite of vitamin A) on the expression of RARbeta, and of proteins involved in amyloidogenic pathway, e.g., amyloid precursor protein (APP), beta-secretase enzyme (BACE), and APP carboxy-terminal fragment (APP-CTF) was examined in the whole brain, hippocampus, striatum, and cerebral cortex of rats. Rats fed a vitamin A-deprived diet for 13 weeks exhibited decreased amount of RARbeta, APP695, BACE, and of APP-CTF in the whole brain and in the cerebral cortex. Administration of RA is able to restore all expression. The results suggest that fine regulation of vitamin A mediated gene expression seems fundamental for the regulation of APP processing.     

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.