[A Simple and Convenient Method for Construction of Gene Site-directed Mutagenesis]

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Apr, 2002  |  Pubmed ID: 11941593

To introduce a new technique for rapid construction of gene site-directed mutagenesis.

Control of Beta-catenin Phosphorylation/degradation by a Dual-kinase Mechanism

Cell. Mar, 2002  |  Pubmed ID: 11955436

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.

GC-GAP, a Rho Family GTPase-activating Protein That Interacts with Signaling Adapters Gab1 and Gab2

The Journal of Biological Chemistry. Sep, 2003  |  Pubmed ID: 12819203

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.

Aggregate Formation and Synaptic Abnormality Induced by DSCR1

Journal of Neurochemistry. Mar, 2004  |  Pubmed ID: 15009650

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.

Nitrosative Stress Linked to Sporadic Parkinson's Disease: S-nitrosylation of Parkin Regulates Its E3 Ubiquitin Ligase Activity

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2004  |  Pubmed ID: 15252205

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.

Differential Distribution of KChIPs MRNAs in Adult Mouse Brain

Brain Research. Molecular Brain Research. Sep, 2004  |  Pubmed ID: 15363885

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.

Beta-Catenin Activates the Growth Factor Endothelin-1 in Colon Cancer Cells

Oncogene. Jan, 2005  |  Pubmed ID: 15558022

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.

Positive Inter-regulation Between Beta-catenin/T Cell Factor-4 Signaling and Endothelin-1 Signaling Potentiates Proliferation and Survival of Prostate Cancer Cells

Molecular Pharmacology. Feb, 2006  |  Pubmed ID: 16291872

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.

Transcriptional Regulation of PEN-2, a Key Component of the Gamma-secretase Complex, by CREB

Molecular and Cellular Biology. Feb, 2006  |  Pubmed ID: 16449647

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.

Association of PINK1 and DJ-1 Confers Digenic Inheritance of Early-onset Parkinson's Disease

Human Molecular Genetics. Jun, 2006  |  Pubmed ID: 16632486

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.

Transcriptional Regulation of APH-1A and Increased Gamma-secretase Cleavage of APP and Notch by HIF-1 and Hypoxia

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jun, 2006  |  Pubmed ID: 16645044

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.

Tumor-suppressor PTEN Affects Tau Phosphorylation, Aggregation, and Binding to Microtubules

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jun, 2006  |  Pubmed ID: 16645045

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.

Tumor Suppressor PTEN Affects Tau Phosphorylation: Deficiency in the Phosphatase Activity of PTEN Increases Aggregation of an FTDP-17 Mutant Tau

Molecular Neurodegeneration. 2006  |  Pubmed ID: 16930454

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.

Antioxidants Protect PINK1-dependent Dopaminergic Neurons in Drosophila

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2006  |  Pubmed ID: 16938835

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.

Renaming the DSCR1/Adapt78 Gene Family As RCAN: Regulators of Calcineurin

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Oct, 2007  |  Pubmed ID: 17595344

Dispensable Role of Drosophila Ortholog of LRRK2 Kinase Activity in Survival of Dopaminergic Neurons

Molecular Neurodegeneration. 2008  |  Pubmed ID: 18257932

Parkinson's disease (PD) is the most prevalent incurable neurodegenerative movement disorder. Mutations in LRRK2 are associated with both autosomal dominant familial and sporadic forms of PD. LRRK2 encodes a large putative serine/threonine kinase with GTPase activity. Increased LRRK2 kinase activity plays a critical role in pathogenic LRRK2 mutant-induced neurodegeneration in vitro. Little is known about the physiological function of LRRK2.

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.

Parkin, PINK1, and DJ-1 Form a Ubiquitin E3 Ligase Complex Promoting Unfolded Protein Degradation

The Journal of Clinical Investigation. Mar, 2009  |  Pubmed ID: 19229105

Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock-induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis.

KChIP1: a Potential Modulator to GABAergic System

Acta Biochimica Et Biophysica Sinica. Apr, 2009  |  Pubmed ID: 19352544

Compelling evidences from transgenic mice, immunoprecipitation data, gene expression analysis, and functional heterologous expression studies supported the role of Kv channel interacting proteins (KChIPs) as modulators of Kv4 (Shal) channels underlying the cardiac transient outward current and neuronal A-type current. Till now, there are four members (KChIP1-4) identified in this family. KChIP1 is expressed predominantly in brain, with relative abundance in Purkinje cells of cerebellum, the reticular thalamic nuclei, the medial habenular nuclei, the hippocampus, and striatum. Our results from in situ hybridization and immunostaining assay revealed that KChIP1 was expressed in a subpopulation of parvalbumin-positive neurons suggesting its functional relationship with the GABAergic inhibitory neurons. Moreover, results obtained from KChIP1-deficient mice showed that KChIP1 mutation did not impair survival or alter the overall brain architecture, arguing against its essential function in brain development. However, the mice bearing KChIP1 deletion showed increased susceptibility to anti-GABAergic convulsive drug pentylenetetrazole-induced seizure, indicating that KChIP1 might play pivotal roles in the GABAergic inhibitory system.

Leucine-Rich Repeat Kinase 2 Interacts with Parkin, DJ-1 and PINK-1 in a Drosophila Melanogaster Model of Parkinson's Disease

Human Molecular Genetics. Nov, 2009  |  Pubmed ID: 19692353

Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.

Genetic Screening for Mutations in the Nrdp1 Gene in Parkinson Disease Patients in a Chinese Population

Parkinsonism & Related Disorders. Mar, 2010  |  Pubmed ID: 19800834

Strong evidence has shown that a defect in the Parkin gene is known to be a common, genetic cause of Parkinson disease (PD). The E3 ubiquitin ligase Nrdp1 is shown to interact with the N terminal of Parkin (the first 76 amino acids) and catalyze degradation of Parkin via the ubiquitin-proteasome pathway, suggesting that Nrdp1 may be involved in the development of PD via the regulation of Parkin, We believe we are the first to have screened PD patients for mutations in the Nrdp1 gene to determine the association between these variants and PD. By direct sequencing, we analysed the entire coding regions and 5' UTR of Nrdp1 in 209 Chinese PD patients and 302 unrelated healthy individuals. No variant was detected in the coding regions (exons 3-7); only 2 variants (c.-206 T > A and c.-208-8 A > G) were identified in the 5' UTR (exon 2) and intron 1. Furthermore, a study of the allelic and genotypic association between patients and controls showed no significant association between the c.-206 T > A polymorphism and PD; c.-208-8 A > G was identified in one PD patient and not in controls. Our data do not support the hypothesis of a major role for the Nrdp1 gene in PD development in the Chinese population.

Roles of KChIP1 in the Regulation of GABA-mediated Transmission and Behavioral Anxiety

Molecular Brain. 2010  |  Pubmed ID: 20678225

K+ channel interacting protein 1 (KChIP1) is a neuronal calcium sensor (NCS) protein that interacts with multiple intracellular molecules. Its physiological function, however, remains largely unknown. We report that KChIP1 is predominantly expressed at GABAergic synapses of a subset of parvalbumin-positive neurons in the brain. Forced expression of KChIP1 in cultured hippocampal neurons increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), reduced paired pulse facilitation of autaptic IPSCs, and decreases potassium current density. Furthermore, genetic ablation of KChIP1 potentiated potassium current density in neurons and caused a robust enhancement of anxiety-like behavior in mice. Our study suggests that KChIP1 is a synaptic protein that regulates behavioral anxiety by modulating inhibitory synaptic transmission, and drugs that act on KChIP1 may help to treat patients with mood disorders including anxiety.

Trafficking Abnormality and ER Stress Underlie Functional Deficiency of Hearing Impairment-associated Connexin-31 Mutants

Protein & Cell. Oct, 2010  |  Pubmed ID: 21204020

Hearing impairment (HI) affects 1/1000 children and over 2% of the aged population. We have previously reported that mutations in the gene encoding gap junction protein connexin-31 (C×31) are associated with HI. The pathological mechanism of the disease mutations remains unknown. Here, we show that expression of C×31 in the mouse inner ear is developmentally regulated with a high level in adult inner hair cells and spiral ganglion neurons that are critical for the hearing process. In transfected cells, wild type C×31 protein (C×31wt) forms functional gap junction at cell-cell-contacts. In contrast, two HI-associated C×31 mutants, C×31R180X and C×31E183K resided primarily in the ER and Golgi-like intracellular punctate structures, respectively, and failed to mediate lucifer yellow transfer. Expression of C×31 mutants but not C×31wt leads to upregulation of and increased association with the ER chaperone BiP indicating ER stress induction. Together, the HI-associated C×31 mutants are impaired in trafficking, promote ER stress, and hence lose the ability to assemble functional gap junctions. The study reveals a potential pathological mechanism of HI-associated C×31 mutations.

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.

Critical Role of PINK1 in Regulating Parkin Protein Levels in Vivo

Archives of Neurology. May, 2011  |  Pubmed ID: 21555650

Interaction Between Amyloid Precursor Protein and Nogo Receptors Regulates Amyloid Deposition

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Sep, 2011  |  Pubmed ID: 21670066

Excessive production or accumulation of β-amyloid (Aβ) peptides in human brains leads to increased amyloid deposition and cognitive dysfunction, which are invariable pathological features in patients with Alzheimer's disease (AD). Many cellular factors can regulate the production of Aβ. In this study, we show that a family of proteins named Nogo receptor proteins (NgR1 to NgR3) regulates Aβ production via interaction with amyloid precursor protein (APP). Further mapping of the interacting domain indicates that a small region adjacent to the BACE1 cleavage site of APP mediates interaction of APP with Nogo receptor proteins. Our results also indicate that increased interaction between Nogo receptor and APP reduces surface expression of APP and favors processing of APP by BACE1. When NgR2 was ablated in AD transgenic mice expressing Swedish APP and PS1ΔE9, amyloid deposition was clearly reduced (0.66% of total measured area in APP(swe)/PS1ΔE9/NgR2(-/-) mice vs. 0.76% of total measured area in APP(swe)/PS1ΔE9 mice). Our results demonstrate that down-regulation of NgR expression is a potential approach for inhibiting amyloid deposition in AD patients.

Regulation of Intracellular Manganese Homeostasis by Kufor-Rakeb Syndrome-associated ATP13A2 Protein

The Journal of Biological Chemistry. Aug, 2011  |  Pubmed ID: 21724849

Mutations in the ATP13A2 gene are associated with Kufor-Rakeb syndrome (KRS) and are found also in patients with various other types of parkinsonism. ATP13A2 encodes a predicted lysosomal P5-type ATPase that plays important roles in regulating cation homeostasis. Disturbance of cation homeostasis in brains is indicated in Parkinson disease pathogenesis. In this study, we explored the biological function of ATP13A2 as well as the pathogenic mechanism of KRS pathogenic ATP13A2 mutants. The results revealed that wild-type ATP13A2, but not the KRS pathogenic ATP13A2 mutants, protected cells from Mn(2+)-induced cell death in mammalian cell lines and primary rat neuronal cultures. In addition, wild-type ATP13A2 reduced intracellular manganese concentrations and prevented cytochrome c release from mitochondria compared with the pathogenic mutants. Furthermore, endogenous ATP13A2 was up-regulated upon Mn(2+) treatment. Our results suggest that ATP13A2 plays important roles in protecting cells against manganese cytotoxicity via regulating intracellular manganese homeostasis. The study provides a potential mechanism of KRS and parkinsonism pathogenesis.