Yigang Tong

Oxidative stress potentiates BACE1 gene expression and Aβ generation

Summary.Alzheimer’s Disease (AD) is the most common neurodegenerative disorder leading to dementia and its prevalence increases with age. The pathological features of AD are characterized by the β-amyloid protein (Aβ) deposits in the core of neuritic plaques and abnormal neurofibrillary tangles in the brain of AD patients. BACE1 is the major β-secretase to cleave the β-amyloid precursor protein (APP) to generate Aβ. Oxidative stress has been shown to affect Aβ generation in the AD pathogenesis and the mechanism of such effect is unknown. In this report we generated a novel promoterless enhanced green fluorescent protein (EGFP) reporter gene cloning vector and cloned a 1.9-kb BACE1 gene promoter fragment in this vector. The BACE1 promoter fragment can efficiently activate EGFP or luciferase gene transcription. Oxidative stress induced by hydrogen peroxide resulted in significant increase in the BACE1 promoter activity. Furthermore, hydrogen peroxide treatment facilitated β-secretase activity and Aβ generation. Thus, upregulation of BACE1 transcription by oxidative stress may contribute to the pathogenesis of Alzheimer’s disease.

Control of APP processing and Aβ generation level by BACE1 enzymatic activity and transcription

Deposition of amyloid β protein (Aβ)is one of the characteristic features of Alzheimers disease (AD) neuropathology. Beta‐secretase, a β‐site APP cleaving enzyme 1 (BACE1), is essential for A β biosynthesis. Although inhibition of BACE1 is considered a valid therapeutic target for AD, the enzymatic dynamics of BACE1 in regulating APP processing and A β generation has not yet been fully defined. To examine this issue, tightly controlled inducible BACE1 gene expression was established in the neuronal cell line N2ABP1 and the non‐neuronal cell line E2BP1 using an ecdysone‐inducible system. The BACE1 protein level was increased in a time‐and dosage‐dependent manner in the inducible BACE1 stable cells by treatment with inducer ponasterone A. The generation of APP CTFβ, the β‐secretase product, increased proportionally with the level of BACE1 protein expression. However, Aβ40/42 production sharply increased to the plateau level with a relatively small increase in BACE1 expression. Although further increasing BACE1 expression increased β‐secretase activity, it had no additional effect on A β production. Furthermore, we found that BACE1 mRNA levels and BACE1 promoter activity were significantly lower than APP mRNA levels and APP promoter activity. Our data demonstrate that lower BACE transcription is responsible for the minority of APP undergoing the amyloidogenic pathway and relatively lower A β production in the normal conditions, and that a slight increase in BACE1 can induce a dramatic elevation in A β production, indicating that the increase in BACE1 can potentially increase neuritic plaque formation in the pathological condition.—Li, Y., Zhou, W., Tong, Y., He, G., Song, W. Control of APP processing and A β generation level by BACE1 enzymatic activity and transcription. FASEB J. 20, 285–292 (2006)

Degradation of BACE by the ubiquitin-proteasome pathway

The amyloid β protein (Aβ) is derived from β‐amyloid precursor protein (APP). Cleavage of APP by β‐secretase generates a C‐terminal fragment (APPCTFβ or C99), which is subsequently cleaved by γ‐secretase to produce Aβ. BACE (or BACE1), the major β‐secretase involved in cleaving APP, has been identified as a Type 1 membrane‐associated aspartyl protease. In this study, we found that treatment with proteasome inhibitors resulted in an increase in APP C99 levels, suggesting that APP processing at the β‐secretase site may be affected by the ubiquitin‐proteasome pathway. To investigate whether the degradation of BACE is mediated by the proteasome pathway, cells stably transfected with BACE were treated with lactacystin. We found that BACE protein degradation was inhibited by lactacystin in a time‐ and dose‐dependent manner. Non‐proteasome protease inhibitors had no effect on BACE degradation. BACE protein is ubiquitinated. Furthermore, lactacystin increased APP C99 production and Aβ generation. Our data demonstrate that the degradation of BACE proteins and APP processing are regulated by the ubiquitin‐proteasome pathway.

Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes

Amyloid β protein (Aβ) is the principal component of neuritic plaques in Alzheimers disease (AD). Aβ is derived from β amyloid precursor protein (APP) by β‐ and γ‐secretases. Beta‐site APP cleaving enzyme 1 (BACE1) has been identified as the major β‐secretase. BACE2 is the homolog of BACE1. The BACE2 gene is on chromosome 21 and has been implicated in the pathogenesis of AD. However, the function of BACE2 in Aβ generation is controversial. Some studies have shown that BACE2 cleaved APP at the β‐site whereas other studies showed it cleaved around the α‐secretase site. To elucidate the involvement of BACE2 in AD pathogenesis, we compared BACE2 and BACE1 gene regulation and their functions in Aβ generation. We cloned and functionally characterized the human BACE2 promoter. The BACE2 gene is controlled by a TATA‐less promoter. Though Sp1 can regulate both BACE1 and BACE2 genes, comparative sequence analysis and transcription factor prediction showed little similarity between the two promoters. BACE1 increased APP cleavage at the β‐site and Aβ production whereas BACE2 did not. Overexpression of BACE2 significantly increased sAPP levels in conditioned media but markedly reduced Aβ production. Knockdown of BACE2 resulted in increased APP C83. Our data indicate that despite being homologous in amino acid sequence, BACE2 and BACE1 have distinct functions and transcriptional regulation. BACE2 is not a β‐secretase, but processes APP within the Aβ domain at a site downstream of the α‐secretase cleavage site. Our data argue against BACE2 being involved in the formation of neuritic plaques in AD.—Sun, X., Wang, Y., Qing, H., Christensen, M. A., Liu, Y., Zhou, W., Tong, Y., Xiao, C., Huang, Y., Zhang, S., Liu, X., Song, W. Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes. FASEB J. 19, 739–749 (2005)

Increased NF-κB signalling up-regulates BACE1 expression and its therapeutic potential in Alzheimer's disease.

Elevated levels of β-site APP cleaving enzyme 1 (BACE1) were found in the brain of some sporadic Alzheimers disease (AD) patients; however, the underlying mechanism is unknown. BACE1 cleaves β-amyloid precursor protein (APP) to generate amyloid β protein (Aβ), a central component of neuritic plaques in AD brains. Nuclear factor-kappa B (NF-κB) signalling plays an important role in gene regulation and is implicated in inflammation, oxidative stress and apoptosis. In this report we found that both BACE1 and NF-κB p65 levels were significantly increased in the brains of AD patients. Two functional NF-κB-binding elements were identified in the human BACE1 promoter region. We found that NF-κB p65 expression resulted in increased BACE1 promoter activity and BACE1 transcription, while disruption of NF-κB p65 decreased BACE1 gene expression in p65 knockout (RelA-knockout) cells. In addition, NF-κB p65 expression leads to up-regulated β-secretase cleavage and Aβ production, while non-steroidal anti-inflammatory drugs (NSAIDs) inhibited BACE1 transcriptional activation induced by strong NF-κB activator tumour necrosis factor-alpha (TNF-α). Taken together, our results clearly demonstrate that NF-κB signalling facilitates BACE1 gene expression and APP processing, and increased BACE1 expression mediated by NF-κB signalling in the brain could be one of the novel molecular mechanisms underlying the development of AD in some sporadic cases. Furthermore, NSAIDs could block the inflammation-induced BACE1 transcription and Aβ production. Our study suggests that inhibition of NF-κB-mediated BACE1 expression may be a valuable drug target for AD therapy.

Regulator of calcineurin 1 (RCAN1) facilitates neuronal apoptosis through caspase 3 activation

Individuals with Down syndrome (DS) will inevitably develop Alzheimer disease (AD) neuropathology sometime after middle age, which may be attributable to genes triplicated in individuals with DS. The characteristics of AD neuropathology include neuritic plaques, neurofibrillary tangles, and neuronal loss in various brain regions. The mechanism underlying neurodegeneration in AD and DS remains elusive. Regulator of calcineurin 1 (RCAN1) has been implicated in the pathogenesis of DS. Our data show that RCAN1 expression is elevated in the cortex of DS and AD patients. RCAN1 expression can be activated by the stress hormone dexamethasone. A functional glucocorticoid response element was identified in the RCAN1 isoform 1 (RCAN1-1) promoter region, which is able to mediate the up-regulation of RCAN1 expression. Here we show that overexpression of RCAN1-1 in primary neurons activates caspase-9 and caspase-3 and subsequently induces neuronal apoptosis. Furthermore, we found that the neurotoxicity of RCAN1-1 is inhibited by knock-out of caspase-3 in caspase-3−/− neurons. Our study provides a novel mechanism by which RCAN1 functions as a mediator of stress- and Aβ-induced neuronal death, and overexpression of RCAN1 due to an extra copy of the RCAN1 gene on chromosome 21 contributes to AD pathogenesis in DS.

Increased BACE1 maturation contributes to the pathogenesis of Alzheimer's disease in Down syndrome

Almost all Down syndrome (DS) patients develop characteristic Alzheimers disease (AD) neuropathology, including neuritic plaques and neurofibrillary tangles, after middle age. The mechanism underlying AD neuropathology in DS has been unknown. Aβ is the central component of neuritic plaques and is generated from APP by cleavage by the β‐ and γ‐secretases. Here we show that β‐secretase activity is markedly elevated in DS. The ratio of mature to immature forms of BACE1 is altered in DS. DS has significantly higher levels of mature BACE1 proteins in Golgi than normal controls. Time‐lapse live image analysis showed that BACE1 proteins were predominantly immobile in Golgi in DS cells, while they underwent normal trafficking in controls. Thus, overproduction of Aβ in DS is caused by abnormal BACE1 protein trafficking and maturation. Our results provide a novel molecular mechanism by which AD develops in DS and support the therapeutic potential of inhibiting BACE1 in AD and DS.—Sun, X., Tong, Y., Qing, H., Chen, C‐H., Song, W. Increased BACE1 maturation contributes to Alzheimers disease pathogenesis in Down syndrome. FASEB J. 20, 1361–1368 (2006)

Degradation of nicastrin involves both proteasome and lysosome

The glycoprotein nicastrin (NCT) is an essential component of the γ‐secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph‐1 and Pen‐2. The γ‐secretase complex is not only involved in APP processing but also in the processing of an increasing number of other type I integral membrane proteins. As the largest subunit of the γ‐secretase complex, NCT plays a crucial role in its activation. Considerable information exists on the distribution, structure and function of NCT; however, little is known of its proteolysis. The present study is aimed at exploring the molecular mechanism of NCT degradation. We found that either proteasomal or lysosomal inhibition can significantly increase the levels of both endogenous and exogenous NCT in various cell lines, and the effect of these inhibitions on NCT was time‐ and dose‐dependent. Immunofluorescent microscopic analysis revealed that NCT accumulates in the ER and Golgi apparatus after proteasomal inhibition, while lysosomal inhibition leads to the accumulation of NCT in the lysosomal apparatus. Co‐immunoprecipitation can pull down both NCT and ubiquitin. Taken together, our results demonstrate that NCT degradation involves both the proteasome and the lysosome.

BACE1 Gene Expression and Protein Degradation

Abstract: Deposition of amyloid β protein in the brain is the major pathological feature of Alzheimers disease. Amyloid β protein is generated from β‐amyloid precursor protein by β‐secretase and γ‐secretase. Proteolytic processing of amyloid precursor protein at the β site by BACE1 is essential to generate amyloid β protein. BACE1, the major β‐secretase involved in cleaving amyloid precursor protein, has been identified as a type 1 membrane‐associated aspartyl protease. In this study, we found that BACE1 gene expression is controlled by a TATA‐less promoter. BACE1 gene expression is tightly regulated at the transcriptional level and the transcription factor Sp1 plays an important role in regulation of BACE1 to process amyloid precursor protein generating amyloid β protein. Furthermore, we found that BACE1 protein is ubiquitinated, and the degradation of BACE1 proteins and amyloid precursor protein processing are regulated by the ubiquitin‐proteasome pathway.

P4-197 Oxidative stress potentiates BACE1 gene expression and Aβ generation

Alzheimer’s Disease (AD) is the most common neurodegenerative disorder leading to dementia and its prevalence increases with age. The pathological features of AD are characterized by the β-amyloid protein (Aβ) deposits in the core of neuritic plaques and abnormal neurofibrillary tangles in the brain of AD patients. BACE1 is the major β-secretase to cleave the β-amyloid precursor protein (APP) to generate Aβ. Oxidative stress has been shown to affect Aβ generation in the AD pathogenesis and the mechanism of such effect is unknown. In this report we generated a novel promoterless enhanced green fluorescent protein (EGFP) reporter gene cloning vector and cloned a 1.9-kb BACE1 gene promoter fragment in this vector. The BACE1 promoter fragment can efficiently activate EGFP or luciferase gene transcription. Oxidative stress induced by hydrogen peroxide resulted in significant increase in the BACE1 promoter activity. Furthermore, hydrogen peroxide treatment facilitated β-secretase activity and Aβ generation. Thus, upregulation of BACE1 transcription by oxidative stress may contribute to the pathogenesis of Alzheimer’s disease.