Dopamine-dependent Neurotoxicity of Alpha-synuclein: a Mechanism for Selective Neurodegeneration in Parkinson Disease

Nature Medicine. Jun, 2002  |  Pubmed ID: 12042811

The mechanism by which dopaminergic neurons are selectively lost in Parkinson disease (PD) is unknown. Here we show that accumulation of alpha-synuclein in cultured human dopaminergic neurons results in apoptosis that requires endogenous dopamine production and is mediated by reactive oxygen species. In contrast, alpha-synuclein is not toxic in non-dopaminergic human cortical neurons, but rather exhibits neuroprotective activity. Dopamine-dependent neurotoxicity is mediated by 54 83-kD soluble protein complexes that contain alpha-synuclein and 14-3-3 protein, which are elevated selectively in the substantia nigra in PD. Thus, accumulation of soluble alpha-synuclein protein complexes can render endogenous dopamine toxic, suggesting a potential mechanism for the selectivity of neuronal loss in PD.

Transcriptional Regulation of BACE1, the Beta-amyloid Precursor Protein Beta-secretase, by Sp1

Molecular and Cellular Biology. Jan, 2004  |  Pubmed ID: 14701757

Proteolytic processing of the beta-amyloid precursor protein (APP) at the beta site is essential to generate Abeta. BACE1, the major beta-secretase involved in cleaving APP, has been identified as a type 1 membrane-associated aspartyl protease. We have cloned a 2.1-kb fragment upstream of the human BACE1 gene and identified key regions necessary for promoter activity. BACE1 gene expression is controlled by a TATA-less promoter. The region of bp -619 to +46 is the minimal promoter to control the transcription of the BACE1 gene. Several putative cis-acting elements, such as a GC box, HSF-1, a PU box, AP1, AP2, and lymphokine response element, are found in the 5' flanking region of the BACE1 gene. Transcriptional activation and gel shift assays demonstrated that the BACE1 promoter contains a functional Sp1 response element, and overexpression of the transcription factor Sp1 potentiates BACE gene expression and APP processing to generate Abeta. Furthermore, Sp1 knockout reduced BACE1 expression. These results suggest that BACE1 gene expression is tightly regulated at the transcriptional level and that the transcription factor Sp1 plays an important role in regulation of BACE1 to process APP generating Abeta in Alzheimer's disease.

Degradation of BACE by the Ubiquitin-proteasome Pathway

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Oct, 2004  |  Pubmed ID: 15289451

The amyloid beta protein (Abeta) is derived from beta-amyloid precursor protein (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APPCTFbeta or C99), which is subsequently cleaved by gamma-secretase to produce Abeta. BACE (or BACE1), the major beta-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 beta-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 Abeta generation. Our data demonstrate that the degradation of BACE proteins and APP processing are regulated by the ubiquitin-proteasome pathway.

Mechanism of Promoter Activity of the Beta-amyloid Precursor Protein Gene in Different Cell Lines: Identification of a Specific 30 Bp Fragment in the Proximal Promoter Region

Journal of Neurochemistry. Sep, 2004  |  Pubmed ID: 15341527

The amyloid beta-protein (Abeta) deposited in brains of Alzheimer's disease (AD) patients is proteolytically derived from a large Abeta precursor protein (APP). APP gene expression patterns in the AD brain region indicate that abnormalities of gene regulation may be important in AD pathology. To understand the contribution of different cell types to APP gene expression, we studied it at four levels: promoter activity (by reporter gene assay of transfected cells), DNA-nuclear protein interaction (by electrophoretic mobility shift assay), RNA message and protein (by northern and western blotting, respectively). APP mRNA and protein expression levels were greater in neuroblastoma and PC12 cells than in glial or cervix epithelial cells. Relative activity among 12 different promoter regions and within single regions varied according to cell type/cell line. An upstream regulatory region containing a GATA-1 site is necessary for activity in PC12 and glial cells but not in neuroblastoma cells. DNA-protein interactions were examined in three distal and one proximal promoter elements in nuclear extracts belonging to neuronal and non-neuronal cells. The proximal promoter region is important for cell line-specific APP gene expression. Characterization of the APP regulatory region's interaction with cell type-specific nuclear factor(s) is important to understand tissue-specific expression of APP seen in AD subjects.

BACE1 Gene Expression and Protein Degradation

Annals of the New York Academy of Sciences. Dec, 2004  |  Pubmed ID: 15681800

Deposition of amyloid beta protein in the brain is the major pathological feature of Alzheimer's disease. Amyloid beta protein is generated from beta-amyloid precursor protein by beta-secretase and gamma-secretase. Proteolytic processing of amyloid precursor protein at the beta site by BACE1 is essential to generate amyloid beta protein. BACE1, the major beta-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 beta 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.

Distinct Transcriptional Regulation and Function of the Human BACE2 and BACE1 Genes

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. May, 2005  |  Pubmed ID: 15857888

Amyloid beta protein (Abeta) is the principal component of neuritic plaques in Alzheimer's disease (AD). Abeta is derived from beta amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-site APP cleaving enzyme 1 (BACE1) has been identified as the major beta-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 Abeta generation is controversial. Some studies have shown that BACE2 cleaved APP at the beta-site whereas other studies showed it cleaved around the alpha-secretase site. To elucidate the involvement of BACE2 in AD pathogenesis, we compared BACE2 and BACE1 gene regulation and their functions in Abeta 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 beta-site and Abeta production whereas BACE2 did not. Overexpression of BACE2 significantly increased sAPP levels in conditioned media but markedly reduced Abeta 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 beta-secretase, but processes APP within the Abeta domain at a site downstream of the alpha-secretase cleavage site. Our data argue against BACE2 being involved in the formation of neuritic plaques in AD.

Control of APP Processing and Abeta Generation Level by BACE1 Enzymatic Activity and Transcription

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Feb, 2006  |  Pubmed ID: 16449801

Deposition of amyloid beta protein (Abeta) is one of the characteristic features of Alzheimer's disease (AD) neuropathology. Beta-secretase, a beta-site APP cleaving enzyme 1 (BACE1), is essential for Abeta biosynthesis. Although inhibition of BACE1 is considered a valid therapeutic target for AD, the enzymatic dynamics of BACE1 in regulating APP processing and Abeta 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 CTFbeta, the beta-secretase product, increased proportionally with the level of BACE1 protein expression. However, Abeta40/42 production sharply increased to the plateau level with a relatively small increase in BACE1 expression. Although further increasing BACE1 expression increased beta-secretase activity, it had no additional effect on Abeta 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 Abeta production in the normal conditions, and that a slight increase in BACE1 can induce a dramatic elevation in Abeta production, indicating that the increase in BACE1 can potentially increase neuritic plaque formation in the pathological condition.

Leaky Scanning and Reinitiation Regulate BACE1 Gene Expression

Molecular and Cellular Biology. May, 2006  |  Pubmed ID: 16611980

beta-Site beta-amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is the beta-secretase in vivo for processing APP to generate amyloid beta protein (Abeta). Abeta deposition in the brain is the hallmark of Alzheimer's disease (AD) neuropathology. Inhibition of BACE1 activity has major pharmaceutical potential for AD treatment. The expression of the BACE1 gene is relatively low in vivo. The control of BACE1 expression has not been well defined. There are six upstream AUGs (uAUGs) in the 5' leader sequence of the human BACE1 mRNA. We investigated the role of the promoter and the uATGs in the 5' untranslated region (UTR) of the human BACE1 gene in BACE1 gene transcription and translation initiation. Our results show that the first and second uATGs are the integral part of the core minimal promoter of the human BACE1 gene, while the third uAUG is skipped over by ribosomal scanning. The fourth uAUG can function as a translation initiation codon, and deletion or mutation of this uAUG increases downstream gene expression. The fourth uAUG of the BACE1 5'UTR is responsible for inhibiting the expression of BACE1. Translation initiation by the BACE1 uAUGs and physiological AUG requires intact eIF4G. Our results demonstrate that during human BACE1 gene expression, ribosomes skipped some uAUGs by leaky scanning and translated an upstream open reading frame, initiated efficiently at the fourth uAUG, and subsequently reinitiated BACE1 translation at the physiological AUG site. Such leaky scanning and reinitiation resulted in weak expression of BACE1 under normal conditions. Alterations of the leaky scanning and reinitiation in BACE1 gene expression could play an important role in AD pathogenesis.

Increased BACE1 Maturation Contributes to the Pathogenesis of Alzheimer's Disease in Down Syndrome

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jul, 2006  |  Pubmed ID: 16816111

Almost all Down syndrome (DS) patients develop characteristic Alzheimer's disease (AD) neuropathology, including neuritic plaques and neurofibrillary tangles, after middle age. The mechanism underlying AD neuropathology in DS has been unknown. Abeta is the central component of neuritic plaques and is generated from APP by cleavage by the beta- and gamma-secretases. Here we show that beta-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 Abeta 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.

BACE2, As a Novel APP Theta-secretase, is Not Responsible for the Pathogenesis of Alzheimer's Disease in Down Syndrome

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jul, 2006  |  Pubmed ID: 16816112

Amyloid beta protein (Abeta), the major component of neuritic plaques in Alzheimer's disease (AD), is derived from APP by sequential cleavages of beta- and gamma-secretases. Beta-site APP cleaving enzyme 1 (BACE1) is the major beta-secretase in vivo. Beta-site APP cleaving enzyme 2 (BACE2) is the homologue of BACE1. The majority of people with Down syndrome (DS), also called Trisomy 21 syndrome, will develop AD neuropathology after middle age. We and others have shown that APP C99, the major beta-secretase product, and Abeta are markedly increased in DS. Since BACE2 is located on chromosome 21, it is speculated that BACE2 may play a role in AD pathogenesis in DS. In this report we found that BACE2 cleaves APP at a novel theta site downstream of the alpha site, abolishing Abeta production. Overexpression of BACE2 by lentivirus markedly reduced Abeta production in primary neurons derived from Swedish mutant APP transgenic mice. Despite an extra copy of the BACE2 gene in DS and the increase of its transcription, BACE2 protein levels are unchanged. Our data clearly demonstrate that BACE2, as a novel theta-secretase to cleave APP within the Abeta domain, is not involved in the AD pathogenesis of DS patients; instead, therapeutic interventions that potentiate BACE2 may prevent AD pathogenesis.

Ubiquitin-proteasome Pathway Mediates Degradation of APH-1

Journal of Neurochemistry. Dec, 2006  |  Pubmed ID: 17059559

Gamma-secretase catalyzes intramembraneous proteolysis of several type I transmembrane proteins, including beta-amyloid precursor protein (APP), to generate amyloid beta protein (Abeta), a key player in the pathogenesis of Alzheimer's disease (AD). The critical components of the gamma-secretase complex include presenilin (PS), nicastrin (NCT), presenilin enhancer-2 (PEN-2) and anterior pharynx defective-1 (APH-1). Abnormalities of the ubiquitin-proteasome pathway have been implicated in the pathogenesis of AD; while PS and PEN-2 turnover is regulated by this pathway, it is unknown whether the ubiquitin-proteasome pathway is also involved in the degradation of APH-1 protein. In this study, we found that the expression of endogenous and exogenous APH-1 significantly increased in cells treated with proteasome-specific inhibitors. The effect of the proteasome inhibitors on APH-1 was dose- and time-dependent. APH-1 protein was ubiquitinated. Pulse-chase metabolic labeling experiments showed that the degradation of newly synthesized radiolabeled APH-1 proteins was inhibited by lactacystin. Disruption of the PS1 and PS2 genes did not affect the degradation of APH-1 by the ubiquitin-proteasome pathway. Furthermore, over-expression of APH-1 and inhibition of proteasomal APH-1 degradation facilitated gamma-secretase cleavage of APP to generate Abeta. These results demonstrate that the degradation of APH-1 protein is mediated by the ubiquitin-proteasome pathway.

Hypoxia Facilitates Alzheimer's Disease Pathogenesis by Up-regulating BACE1 Gene Expression

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2006  |  Pubmed ID: 17121991

The molecular mechanism underlying the pathogenesis of the majority of cases of sporadic Alzheimer's disease (AD) is unknown. A history of stroke was found to be associated with development of some AD cases, especially in the presence of vascular risk factors. Reduced cerebral perfusion is a common vascular component among AD risk factors, and hypoxia is a direct consequence of hypoperfusion. Previously we showed that expression of the beta-site beta-amyloid precursor protein (APP) cleavage enzyme 1 (BACE1) gene BACE1 is tightly controlled at both the transcriptional and translational levels and that increased BACE1 maturation contributes to the AD pathogenesis in Down's syndrome. Here we have identified a functional hypoxia-responsive element in the BACE1 gene promoter. Hypoxia up-regulated beta-secretase cleavage of APP and amyloid-beta protein (Abeta) production by increasing BACE1 gene transcription and expression both in vitro and in vivo. Hypoxia treatment markedly increased Abeta deposition and neuritic plaque formation and potentiated the memory deficit in Swedish mutant APP transgenic mice. Taken together, our results clearly demonstrate that hypoxia can facilitate AD pathogenesis, and they provide a molecular mechanism linking vascular factors to AD. Our study suggests that interventions to improve cerebral perfusion may benefit AD patients.

Recent Advances in Basic Neurosciences and Brain Disease: from Synapses to Behavior

Molecular Pain. 2006  |  Pubmed ID: 17196111

Understanding basic neuronal mechanisms hold the hope for future treatment of brain disease. The 1st international conference on synapse, memory, drug addiction and pain was held in beautiful downtown Toronto, Canada on August 21-23, 2006. Unlike other traditional conferences, this new meeting focused on three major aims: (1) to promote new and cutting edge research in neuroscience; (2) to encourage international information exchange and scientific collaborations; and (3) to provide a platform for active scientists to discuss new findings. Up to 64 investigators presented their recent discoveries, from basic synaptic mechanisms to genes related to human brain disease. This meeting was in part sponsored by Molecular Pain, together with University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain). Our goal for this meeting is to promote future active scientific collaborations and improve human health through fundamental basic neuroscience researches. The second international meeting on Neurons and Brain Disease will be held in Toronto (August 29-31, 2007).

The Cholesterol Transporter ABCG1 Modulates the Subcellular Distribution and Proteolytic Processing of Beta-amyloid Precursor Protein

Journal of Lipid Research. May, 2007  |  Pubmed ID: 17293612

Although intracellular cholesterol levels are known to influence the proteolysis of beta-amyloid precursor protein (APP), the effect of specific genes that regulate cholesterol metabolism on APP processing remains poorly understood. The cholesterol transporter ABCG1 facilitates cholesterol efflux to HDL and is expressed in brain. Notably, the human ABCG1 gene maps to chromosome 21q22.3, and individuals with Down syndrome (DS) typically manifest with Alzheimer's disease (AD) neuropathology in their 30s. Here, we demonstrate that expression of ABCG1 enhances amyloid-beta protein (Abeta) production in transfected HEK cells in a manner that requires functional cholesterol transporter activity. ABCG1-expressing cells also exhibit increased secreted APP (sAPP)alpha and sAPPbeta secretion and display increased cell surface-associated APP. These results suggest that ABCG1 increases the availability of APP as a secretase substrate for both the amyloidogenic and nonamyloidogenic pathways. In vivo, ABCG1 mRNA levels are 2-fold more abundant in DS brain compared with age- and sex-matched normal controls. Finally, both Abeta and sAPPalpha levels are increased in DS cortex relative to normal controls. These findings suggest that altered cholesterol metabolism and APP trafficking mediated by ABCG1 may contribute to the accelerated onset of AD neuropathology in DS.

Degradation of Nicastrin Involves Both Proteasome and Lysosome

Journal of Neurochemistry. May, 2007  |  Pubmed ID: 17326768

The glycoprotein nicastrin (NCT) is an essential component of the gamma-secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph-1 and Pen-2. The gamma-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 gamma-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.

SP1 Regulates a Human SNAP-25 Gene Expression

Journal of Neurochemistry. Apr, 2008  |  Pubmed ID: 18194215

The synaptosomal-associated protein of 25 kDa (SNAP-25) is a pre-synaptic plasma membrane protein. SNAP-25 plays an important role in synaptic vesicle membrane docking and fusion, which is involved in the regulation of neurotransmitter release. SNAP-25 has been implicated in the pathogenesis of neuropsychiatric disorders including Schizophrenia, attention-deficit hyperactivity disorder and Alzheimer's disease. We cloned a 1584 bp segment of the 5' flanking region of the human SNAP-25 gene. A series of nested deletions of the 5' flanking region fragment were subcloned into the pGL3-basic luciferase reporter plasmid. N2A cells were transfected with the SNAP-25 promoter constructs and luciferase activity was measured as an indication of promoter activity. We identified a 188 bp fragment containing the transcription initiation site as the minimal region necessary for promoter activity. Several putative cis-acting elements including SP1, hypoxia inducible factor (HIF), cAMP-response element binding protein, T-cell factor/lymphocyte enhancer factor 1 (TCF/LEF1), AP1 and the signal transducer and activator of transcription-6 (STAT6) are found in the 5' flanking region of SNAP-25 gene. Transcriptional activation and gel shift assays showed that the human SNAP-25 gene promoter contains functional SP1 response elements. Over-expression of SP1 increased SNAP-25 gene expression and inhibition of SP1-mediated transcriptional activation reduced SNAP-25 gene expression. These results suggest that SP1 plays an important role in regulation of the human SNAP-25 gene expression.

Valproic Acid Inhibits Abeta Production, Neuritic Plaque Formation, and Behavioral Deficits in Alzheimer's Disease Mouse Models

The Journal of Experimental Medicine. Nov, 2008  |  Pubmed ID: 18955571

Neuritic plaques in the brains are one of the pathological hallmarks of Alzheimer's disease (AD). Amyloid beta-protein (Abeta), the central component of neuritic plaques, is derived from beta-amyloid precursor protein (APP) after beta- and gamma-secretase cleavage. The molecular mechanism underlying the pathogenesis of AD is not yet well defined, and there has been no effective treatment for AD. Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for treating epilepsy and bipolar disorder. We found that VPA decreased Abeta production by inhibiting GSK-3beta-mediated gamma-secretase cleavage of APP both in vitro and in vivo. VPA treatment significantly reduced neuritic plaque formation and improved memory deficits in transgenic AD model mice. We also found that early application of VPA was important for alleviating memory deficits of AD model mice. Our study suggests that VPA may be beneficial in the prevention and treatment of AD.

TMP21 Degradation is Mediated by the Ubiquitin-proteasome Pathway

The European Journal of Neuroscience. Nov, 2008  |  Pubmed ID: 19046380

The presenilin-associated complex regulates two independent intramembranous cleavage activities, i.e. gamma-secretase and epsilon-secretase activity. The gamma-secretase complex requires four critical components for its activity: presenilin 1, anterior pharynx-defective 1, nicastrin 1 and presenilin enhancer 2, all of which are degraded through the ubiquitin-proteasome pathway. Recently, TMP21, a type I transmembrane protein involved in endoplasmic reticulum/Golgi transport, was identified as a member of the presenilin complex. Knockdown of TMP21 selectively regulated pathogenic gamma-secretase activity, resulting in increased amyloid beta protein 40 and 42, without affecting the epsilon-cleavage of Notch. A further understanding of TMP21 degradation is required to examine the biological consequences of TMP21 protein level aberrations and their potential role in the pathogenesis of Alzheimer's disease and drug development. Here we show that human TMP21 has a short half-life of approximately 3 h. Treatment with proteasomal inhibitors can increase TMP21 protein levels in both a time- and dose-dependent manner, and both co-immunoprecipitation and immunofluorescent staining show that TMP21 is ubiquitinated. Inhibition of the lysosomal pathway failed to show a dose-dependent increase in TMP21 protein levels. Taken together, these results indicate that the degradation of TMP21, as with the other presenilin-associated gamma-secretase complex members, is mediated by the ubiquitin-proteasome pathway.

Preparation of PbS Nanoparticles by Phase-Transfer Method and Application to Pb-Selective Electrode Based on PVC Membrane

Analytical Letters. Oct, 2008  |  Pubmed ID: 19112518

A novel approach to prepare homogeneous PbS nanoparticles by phase-transfer method was developed. The preparatory conditions were studied in detail, and the nanoparticles were characterized by transmission electron microscopy (TEM) and UV-vis spectroscopy. Then a novel lead ion-selective electrode of polyvinyl chloride (PVC) membrane based on these lead sulfide nanoparticles was prepared, and the optimum ratio of components in the membrane was determined. The results indicated that the sensor exhibited a wide concentration range of 1.0x10(-5) to 1.0x10(-2) mol.L(-1). The response time of the electrode was about 10 s, and the optimal pH in which the electrode could be used was from 3.0 to 7.0. Selectivity coefficients indicated that the electrode was selective to the primary ion over the interfering ion. The electrode can be used for at least 3 months without any divergence in potential. It was successfully applied to directly determine lead ions in solution and used as an indicator electrode in potentiometric titration of lead ions with EDTA.

Upregulation of Macrophage Migration Inhibitory Factor Gene Expression in Stroke

Stroke; a Journal of Cerebral Circulation. Mar, 2009  |  Pubmed ID: 19131653

MIF has been implicated to function in many inflammatory processes. This study examined whether MIF expression was affected in stroke and its underlying molecular mechanism.

Degradation of Regulator of Calcineurin 1 (RCAN1) is Mediated by Both Chaperone-mediated Autophagy and Ubiquitin Proteasome Pathways

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Oct, 2009  |  Pubmed ID: 19509306

Regulator of calcineurin 1 (RCAN1), a gene identified from the critical region of Down syndrome, has been implied in pathogenesis of Alzheimer's disease (AD). RCAN1 expression was shown to be increased in AD brains; however, the mechanism of RCAN1 gene regulation is not well defined. The present study was designed to investigate the molecular mechanism of RCAN1 protein degradation. In addition to being degraded through the ubiquitin proteasome pathway, we found that lysosomal inhibition markedly increased RCAN1 protein expression in a time- and dosage-dependent manner. Inhibition of macroautophagy reduced RCAN1 expression, indicating that RCAN1 degradation is not through a macroautophagy pathway. However, disruption of chaperone-mediated autophagy (CMA) increased RCAN1 expression. Two CMA recognition motifs were identified in RCAN1 protein to mediate its degradation through a CMA-lysosome pathway. A promoter assay further demonstrated that inhibition of RCAN1 degradation in cells reduced calcineurin-NFAT activity. Dysfunctions of ubiquitin-proteasome and autophagy-lysosome pathways have been implicated in neurodegenerative diseases. Therefore, elucidation of RCAN1 degradation by a ubiquitin proteasome pathway and CMA-lysosome pathway in the present study may greatly advance our understanding of AD pathogenesis.

BACE1 Gene Promoter Single-nucleotide Polymorphisms in Alzheimer's Disease

Journal of Molecular Neuroscience : MN. Sep, 2010  |  Pubmed ID: 20455082

Alzheimer's disease (AD) is the most neurodegenerative disorder leading to dementia. Neuritic plaque formation in brains is a hallmark of AD pathogenesis. Amyloid beta protein (Abeta) is the central component of neuritic plaques. Processing beta-amyloid precursor protein (APP) at the beta-secretase site by the beta-site APP cleaving enzyme 1 (BACE1) is essential for generation of Abeta. Elevation of BACE1 activity and expression has been reported in AD brains. However, no mutation in the BACE1 coding sequence has been identified in AD cases. Human BACE1 expression is tightly regulated at the transcription and translation level. To determine whether there is any single-nucleotide polymorphisms in the BACE1 gene promoter region affecting BACE1 expression in AD pathogenesis, in this study, we screened 2.6 kb of the human BACE1 gene promoter region from late-onset AD patients and found that there was no significant association between single-nucleotide polymorphisms and AD cases.

Evidence That Gamma-secretase Mediates Oxidative Stress-induced Beta-secretase Expression in Alzheimer's Disease

Neurobiology of Aging. Jun, 2010  |  Pubmed ID: 18687504

Beta-secretase (BACE1), an enzyme responsible for the production of amyloid beta-peptide (Abeta), is increased by oxidative stress and is elevated in the brains of patients with sporadic Alzheimer's disease (AD). Here, we show that oxidative stress fails to induce BACE1 expression in presenilin-1 (gamma-secretase)-deficient cells and in normal cells treated with gamma-secretase inhibitors. Oxidative stress-induced beta-secretase activity and sAPPbeta levels were suppressed by gamma-secretase inhibitors. Levels of gamma- and beta-secretase activities were greater in brain tissue samples from AD patients compared to non-demented control subjects, and the elevated BACE1 level in the brains of 3xTgAD mice was reduced by treatment with a gamma-secretase inhibitor. Our findings suggest that gamma-secretase mediates oxidative stress-induced expression of BACE1 resulting in excessive Abeta production in AD.

NF-κB Signaling Inhibits Ubiquitin Carboxyl-terminal Hydrolase L1 Gene Expression

Journal of Neurochemistry. Mar, 2011  |  Pubmed ID: 21210816

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that plays a regulatory role in targeting proteins for proteasomal degradation. UCH-L1 is highly expressed in neurons and has been demonstrated to promote cell viability and maintain neuronal integrity. Reduced UCH-L1 levels have been observed in various neurodegenerative diseases, and expression of UCH-L1 can rescue synaptic dysfunction and memory deficits in Alzheimer's Disease model mice. However, the mechanisms regulating UCH-L1 expression have not been determined. In this study, we cloned a 1782 bp of the 5' flanking region of the human UCH-L1 gene and identified a 43 bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis revealed several putative regulatory elements including NF-κB, NFAT, CREB, NRSF, YY1, AP1, and STAT in the UCH-L1 promoter. A functional NF-κB response element was identified in the UCH-L1 promoter region. Expression of NF-κB suppressed UCH-L1 gene transcription. In the RelA knockout system where NF-κB activity is ablated, UCH-L1 expression was significantly increased. Furthermore, activation of NF-κB signaling by the inflammatory stimulator lipopolysaccharide and TNFα resulted in a decrease of UCH-L1 gene expression by inhibiting its transcription. As NF-κB is an important signaling module in inflammatory response, our study suggests a possibility that inflammation might compromise neuronal functions via the interaction of NF-κB and UCH-L1. A better understanding of the NF-κB-regulated UCH-L1 transcription will provide insights to the role of inflammatory responses in Alzheimer's disease and Parkinson's disease.

Regulator of Calcineurin 1 (RCAN1) Facilitates Neuronal Apoptosis Through Caspase-3 Activation

The Journal of Biological Chemistry. Mar, 2011  |  Pubmed ID: 21216952

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 NF-κB Signalling Up-regulates BACE1 Expression and Its Therapeutic Potential in Alzheimer's Disease

The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP). Feb, 2011  |  Pubmed ID: 21329555

Elevated levels of β-site APP cleaving enzyme 1 (BACE1) were found in the brain of some sporadic Alzheimer's 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.

Transcriptional Regulation of TMP21 by NFAT

Molecular Neurodegeneration. 2011  |  Pubmed ID: 21375783

TMP21 is a member of the p24 cargo protein family, which is involved in protein transport between the Golgi apparatus and ER. Alzheimer's Disease (AD) is the most common neurodegenerative disorder leading to dementia and deposition of amyloid β protein (Aβ) is the pathological feature of AD pathogenesis. Knockdown of TMP21 expression by siRNA causes a sharp increase in Aβ production; however the underlying mechanism by which TMP21 regulates Aβ generation is unknown, and human TMP21 gene expression regulation has not yet been studied.

Loss of Activated CaMKII at the Synapse Underlies Alzheimer's Disease Memory Loss

Journal of Neurochemistry. Nov, 2011  |  Pubmed ID: 21899538

Control of BACE1 Degradation and APP Processing by Ubiquitin Carboxyl-terminal Hydrolase L1

Journal of Neurochemistry. Dec, 2011  |  Pubmed ID: 22212137

J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07644.x ABSTRACT: Deposition of amyloid β protein (Aβ) in the brain is the hallmark of Alzheimer's disease (AD) pathogenesis. Beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the β-secretase in vivo essential for generation of Aβ. Previously we demonstrated that BACE1 is ubiquitinated and the degradation of BACE1 is mediated by the ubiquitin-proteasome pathway (UPP). However the mechanism underlying regulation of BACE1 degradation by UPP remains elusive. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme highly specific to neuron, catalyzing the hydrolysis of ubiquitin conjugates from ubiquitinated substrates. UCHL1 regulates ubiquitin-dependent protein degradation. However, whether UCHL1 is particularly involved in the proteasomal degradation of BACE1 and what is the role of UCHL1 in AD pathogenesis remain elusive. To investigate the effect of UCHL1 on BACE1 degradation, HUCH cells, a UCHL1 stably over-expressed HEK293 cell line, was established. We found that inhibition of UCHL1 significantly increased BACE1 protein level in a time-dependent manner. Half life of BACE1 was reduced in HUCH cells compared with HEK. Over-expression of UCHL1 decreased APP C-terminal fragment C99 and Aβ levels in HUCH cells. Moreover, disruption of Uchl1 gene significantly elevated levels of endogenous BACE1, C99 and Aβ in the Uchl1-null gad mice. These results demonstrated that UCHL1 accelerates BACE1 degradation and affects APP processing and Aβ production. This study suggests that potentiation of UCHL1 might be able to reduce the level of BACE1 and Aβ in brain, which makes it a novel target for AD drug development.

Hypoxia Regulation of ATP13A2 (PARK9) Gene Transcription

Journal of Neurochemistry. Jan, 2012  |  Pubmed ID: 22288903

Parkinson's disease (PD) is the second most common neurodegenerative disorders with a variable combination of motor and non-motor symptoms. Mutations in several genes including ATP13A2 (PARK9) are reported to be associated with PD. The underlying mechanism of PD is not well defined, however, both genetic and environmental causes contribute to it. ATP13A2 gene locates in chromosome 1 and contains 29 exons encoding for a protein of 1180 amino acids with 10 transmembrane domains. Abnormal gene expression has been implicated in neurodegenerative disorders. The transcriptional regulation of the ATP13A2 gene is unknown. In this report we cloned and functionally characterized the human ATP13A2 gene promoter. We showed that the promoter region of the human ATP13A2 gene contains hypoxia response elements (HREs) which can bind to transcription factor hypoxia-inducible factor 1α (HIF-1α). Hypoxia upregulated ATP13A2 transcription via HIF-1α in HEK293 and dopaminergic MN9D cells. Our study indicates that hypoxia signaling plays a very important role in the regulation of human ATP13A2 gene expression. Further study is needed to determine the role of hypoxia in the pathogenesis of PD and its interaction with other PD causative genes, which will provide insights to the role of hypoxia and dysregulation of gene expression in Parkinson's disease. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Regulation of β-site APP-cleaving Enzyme 1 Gene Expression and Its Role in Alzheimer's Disease

Journal of Neurochemistry. Jan, 2012  |  Pubmed ID: 22122349

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaques are the hallmark neuropathology in AD brains. Proteolytic processing of amyloid-β precursor protein at the β site by beta-site amyloid-β precursor protein-cleaving enzyme 1 (BACE1) is essential to generate Aβ, a central component of the neuritic plaques. BACE1 is increased in some sporadic AD brains, and dysregulation of BACE1 gene expression plays an important role in AD pathogenesis. This review will focus on the regulation of BACE1 gene expression at the transcriptional, post-transcriptional, translation initiation, translational and post-translational levels, and its role in AD pathogenesis. Further studies on BACE1 gene expression regulation will greatly contribute to our understanding of AD pathogenesis and reveal potential novel approaches for AD prevention and drug development.