Biomedical subjects

Zhuohua Zhang

Publications and source records attributed to Zhuohua Zhang.

14 recordsLinked to original sources

PubMed2006

Antioxidants protect PINK1-dependent dopaminergic neurons in Drosophila.

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder. Mutations in the PINK1 gene are linked to the autosomal recessive early onset familial form of PD. The physiological function of PINK1 and pathological abnormality of PD-associated PINK1 mutants are largely unknown. We here show that inactivation of Drosophila PINK1 (dPINK1) using RNAi results in progressive loss of dopaminergic neurons and in ommatidial degeneration of the compound eye, which is rescued by expression of human PINK1 (hPINK1). Expression of human SOD1 suppresses neurodegeneration induced by dPINK1 inactivation. Moreover, treatment of dPINK1 RNAi flies with the antioxidants SOD and vitamin E significantly inhibits ommatidial degeneration. Thus, dPINK1 plays an essential role in maintaining neuronal survival by preventing neurons from undergoing oxidative stress, thereby suggesting a potential mechanism by which a reduction in PINK1 function leads to PD-associated neurodegeneration.

Animals↗

PubMed2006

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau.

BACKGROUND: Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. RESULTS: Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. CONCLUSION: Tumor suppressor PTEN can alleviate the phosphorylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity null PTEN increases the mutant tau phosphorylation at these sites. The changes of the tau phosphorylation status by ectopic expression of PTEN correlate to the alteration of the mutant tau's cellular distribution. In addition, the overexpression of the mutant PTEN can increase the level of the mutant tau aggregates and lead to the formation of visible aggregates in the cells.

Journal Article↗

PubMed2006

Tumor-suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules.

Neurofibrillary tangles (NFTs), consisting of abnormally hyperphosphorylated tau, are implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD). The molecular mechanisms underlying the regulation of tau phosphorylation are largely unknown. While the PI3K/Akt pathway has been shown to regulate multiple cellular events pertinent to AD pathogenesis, potential functions of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in AD pathogenesis have not been explored. Here, we examine the effects of PTEN on tau phosphorylation, its microtubule association and formation of aggregates, and consequentially neuronal morphology. In cultured cells, overexpression of wild-type (WT) PTEN alters tau phosphorylation at several sites, increases tau-microtubule association and decreases formation of tau aggregates. In addition, the phosphatase-null PTEN increases tau aggregation and impairs tau binding to microtubule and neurite outgrowth of neurons expressing the mutant PTEN. We also found a significant loss of PTEN in AD patient brains correlated with a dramatically increased concentration of phospho-tau at Ser-214 in NFTs. Together, our results demonstrate that PTEN regulates tau phosphorylation, binding to microtubules and formation of aggregates and neurite outgrowth. These findings suggest a link between malfunction of PTEN and tauopathy, and imply PTEN as a therapeutic target for tauopathy.

Alzheimer Disease↗

PubMed2006

Transcriptional regulation of APH-1A and increased gamma-secretase cleavage of APP and Notch by HIF-1 and hypoxia.

The proteolytic cleavage of Alzheimer beta-amyloid precursor protein (APP) and signaling receptor Notch is mediated by the PS/gamma-secretase complex, which consists of presenilins, nicastrin, APH-1, and PEN-2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. Here we characterized the 5'-flanking region of the human APH-1A gene and identified a 271-bp fragment containing the transcription initiation site for the promoter activity. Sequence analysis, mutagenesis, and gel shift studies revealed a binding of AP4 and HIF-1 to the promoter, which affects the promoter activity. Activation of HIF-1 by short-term NiCl2 treatments (a condition of chemical hypoxia) dramatically increased APH-1A mRNA and protein expression, accompanied by increased secretion of Abeta and decreased APP CTFs formation, indicative of an increase in gamma-secretase activity. NiCl2 treatments had little effect on APP and the other three components of the gamma-secretase complex. The cellular concentration of Notch intracellular domain (NICD) was also increased by the hypoxic treatment. Our results demonstrate that APH-1A expression and the gamma-secretase mediated Abeta and Notch NICD generation are regulated by HIF-1, and the specific control of APH-1A expression may imply physiological functions uniquely assigned to APH-1A.

Amyloid Precursor Protein Secretases↗

PubMed2006

Association of PINK1 and DJ-1 confers digenic inheritance of early-onset Parkinson's disease.

Mutations in genes encoding both DJ-1 and pten-induced kinase 1 (PINK1) are independently linked to autosomal recessive early-onset familial forms of Parkinson's disease (PD). We here report identification of a family with PD patients harboring novel heterozygous missense mutations in both PINK1 and DJ-1 genes encoding DJ-1A39S and PINK1P399L, respectively. In transfected cells, DJ-1 interacts with PINK1. PINK1P399L is less stable than the wild-type protein and is degraded via the ubiquitin-mediated proteasomal pathway. Expression of wild-type DJ-1 increased steady-state levels of PINK1, whereas expression of DJ-1A39S reduced steady-state levels of PINK1. Furthermore, co-expression of wild-type DJ-1 and PINK1 suppresses neurotoxin 1-methyl-4-phenylpyridinium (MPP(+))-induced death of dopaminergic SH-SY5Y cells. In contrast, co-expression of PD-associated DJ-1A39S and PINK1P399L significantly potentiated susceptibility of SH-SY5Y cells to MPP(+)-induced cell death. This study reports the first case of autosomal recessive PD with digenic inheritance and demonstrates that DJ-1 and PINK1 physically associate and collaborate to protect cells against stress via complex formation.

Adult↗

PubMed2006

Transcriptional regulation of PEN-2, a key component of the gamma-secretase complex, by CREB.

Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's beta-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APPalpha and Abeta without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2.

Amyloid Precursor Protein Secretases↗

PubMed2005

Positive inter-regulation between beta-catenin/T cell factor-4 signaling and endothelin-1 signaling potentiates proliferation and survival of prostate cancer cells.

Both malignant and normal prostate epithelial cells produce endothelin-1 (ET-1), a critical factor in prostate cancer (CaP) progression. beta-Catenin (beta-cat), a key component of the Wnt signaling pathway, is also implicated in CaP progression via beta-cat/T cell factor (Tcf) signaling. We recently demonstrated that beta-cat/Tcf-4 regulates transcription of ET-1 in colon cancer cells. In the present study, we found that Tcf-4 specifically bound to and activated the ET-1 promoter in vivo in human CaP cells and mouse prostate tissue. Expression of ET-1 in DU145 CaP cells was down-regulated by knocking down endogenous beta-cat or Tcf-4. Ectopic activation of beta-cat/Tcf-4 signaling significantly elevated expression of ET-1 in LNCaP cells. In addition, genetic ablation of beta-cat significantly inhibited transcription of ET-1 in primary prostate epithelial cells. Meanwhile, exogenous ET-1 enhanced beta-cat/Tcf signaling and ET-1 expression in DU145 cells, which was blocked by both selective phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) and endothelin-A receptor antagonist cyclo(L-Leu-D-Trp-D-Asp-L-Pro-D-Val) (BQ123). Furthermore, knockdown of either beta-cat or Tcf-4 substantially reduced cell proliferation and potentiated paclitaxel-induced apoptosis in DU145 cells, which largely were rescued by treatment with exogenous ET-1. Together, our results suggest that beta-cat/Tcf-4 signaling transcriptionally activates ET-1 in CaP cells; meanwhile, ET-1 enhances beta-cat/Tcf-4 signaling and in turn further increases ET-1 expression in a PI3K-dependent manner. The positive inter-regulation between beta-cat/Tcf-4 signaling and ET-1 signaling potentiates proliferation and survival of CaP cells, thereby representing a novel mechanism that contributes to CaP progression.

Animals↗

PubMed2005

beta-Catenin activates the growth factor endothelin-1 in colon cancer cells.

Endothelin-1 (EDN1) is a growth factor that is frequently produced by cancer cells and plays a critical role in tumorigenesis. However, the molecular mechanism controlling the expression of EDN1 in cancers is unknown. Constitutive activation of beta-catenin pathway is responsible for the initiation of the vast majority of colon cancers. Here we show that the EDN1 gene is directly regulated by beta-catenin in colon cancer cells. A specific DNA element within the EDN1 promoter is required for activation, and is associated with beta-catenin's cognate DNA binding partner, TCF4, in vivo. Inhibition of beta-catenin signaling results in lowered expression of EDN1, while enhancement of beta-catenin signaling leads to further activation of the gene. Significantly elevated EDN1 expression occurs in 80% of primary human colon cancers, consistent with it being a direct target of beta-catenin. Furthermore, EDN1 is able to rescue colon cancer cells from growth arrest and apoptosis resulting from inhibition of beta-catenin signaling, implicating a key role of EDN1 in promoting the oncogenic function of beta-catenin. These results indicate EDN1 overexpression as a major cause in colon cancers and reveal further details of the genetic programs responsible for tumorigenesis of colon cancers.

Colonic Neoplasms↗

PubMed2004

Differential distribution of KChIPs mRNAs in adult mouse brain.

The K(+) channel interacting proteins (KChIPs1-4) interact with and modulate activity and trafficking of Kv4 potassium channels. We report here the distribution of KChIPs in adult mouse brain. KChIP1 was expressed in a subpopulation of neurons widely distributed in brain and enriched in Purkinje cells of the cerebellum and in the reticular thalamic and medial habenular nuclei. KChIP2 transcripts were highly expressed in layer IV of the cerebral cortex and in striatum and hippocampus, but expressed at low levels in cerebellum. KChIP3 transcripts were detected primarily in the layer V and deep layer VI of the cerebral cortex, the hippocampus, and the entire cerebellum. KChIP4 was highly expressed by neurons in layers II-IV of cortex and in hippocampus, thalamus and the Purkinje cell layer of the cerebellum. Collectively, different KChIPs appear to be expressed by selected neuronal populations in different brain regions where expression of Kv4.2 and Kv4.3 have been reported. These findings support the likelihood of functional interactions between KChIPs and Kv4 K(+) channels in brain.

Animals↗

PubMed2004

Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity.

Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S-nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

Aggregate formation and synaptic abnormality induced by DSCR1.

Aggregation of conformation-abnormal peptides probably plays a key role in the pathogenesis of many neurodegenerative diseases. DSCR1 Down syndrome (DS) critical region 1, was identified from a chromosomal region (21q22.1-q22.2) for the clinical manifestations of DS when an extra-copy is present. We report that expression of DSCR1 in several cell types, including primary neurons, causes microtubule-dependent aggresome-like inclusion body formation. Disease-associated huntingtin (Q148) and ataxin-3 (Q84) co-localize with DSCR1 aggregates. Neurons bearing DSCR1 aggregates show reduced synaptophysin staining in processes. DSCR1 residues 31-90 constitute an aggregation-prone domain that is predicted to form a hydrophobic patch on the protein surface when residues 1-30 are removed. This study identifies a novel function of DSCR1 that may underlie DS neuropathology.

Alzheimer Disease↗

PubMed2003

GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2.

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.

Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes.

Amino Acid Sequence↗

PubMed2002

[A simple and convenient method for construction of gene site-directed mutagenesis].

OBJECTIVE: To introduce a new technique for rapid construction of gene site-directed mutagenesis. METHODS: Three primers are synthesized. One is a primer with the needed mutation; the other two containing appropriate enzyme sites for construction of the PCR fragment into a suitable plasmid are located at the flanks of the mutation primer. After the amplification of the PCR fragment using the mutation primer and the reverse flanking primer, another PCR is performed using the previous PCR mutation segment as primer and the other flanking primer. The final PCR segment can be cloned into an appropriate plasmid by using the enzyme sites in the primers. RESULTS: Two site-directed mutagenesis have been successfully constructed in the Parkin gene by this method. CONCLUSION: The method is effective and simple for construction of gene site-directed mutagenesis.

Base Sequence↗