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1.
Nat Commun ; 12(1): 5071, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417470

RESUMO

Identification of causal variants and genes underlying genome-wide association study (GWAS) loci is essential to understand the biology of alcohol use disorder (AUD) and drinks per week (DPW). Multi-omics integration approaches have shown potential for fine mapping complex loci to obtain biological insights to disease mechanisms. In this study, we use multi-omics approaches, to fine-map AUD and DPW associations at single SNP resolution to demonstrate that rs56030824 on chromosome 11 significantly reduces SPI1 mRNA expression in myeloid cells and lowers risk for AUD and DPW. Our analysis also identifies MAPT as a candidate causal gene specifically associated with DPW. Genes prioritized in this study show overlap with causal genes associated with neurodegenerative disorders. Multi-omics integration analyses highlight, genetic similarities and differences between alcohol intake and disordered drinking, suggesting molecular heterogeneity that might inform future targeted functional and cross-species studies.


Assuntos
Alcoolismo/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genômica , Doenças Neurodegenerativas/genética , Encéfalo/metabolismo , Epigênese Genética , Feto/metabolismo , Redes Reguladoras de Genes , Loci Gênicos , Marcadores Genéticos , Humanos , Desequilíbrio de Ligação/genética , Análise da Randomização Mendeliana , Mapeamento Físico do Cromossomo , Regiões Promotoras Genéticas/genética , Locos de Características Quantitativas/genética
2.
Mol Neurodegener ; 16(1): 27, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33882988

RESUMO

Alzheimer's disease (AD) is the most common type of dementia, affecting millions of people worldwide; however, no disease-modifying treatments are currently available. Genome-wide association studies (GWASs) have identified more than 40 loci associated with AD risk. However, most of the disease-associated variants reside in non-coding regions of the genome, making it difficult to elucidate how they affect disease susceptibility. Nonetheless, identification of the regulatory elements, genes, pathways and cell type/tissue(s) impacted by these variants to modulate AD risk is critical to our understanding of disease pathogenesis and ability to develop effective therapeutics. In this review, we provide an overview of the methods and approaches used in the field to identify the functional effects of AD risk variants in the causal path to disease risk modification as well as describe the most recent findings. We first discuss efforts in cell type/tissue prioritization followed by recent progress in candidate causal variant and gene nomination. We discuss statistical methods for fine-mapping as well as approaches that integrate multiple levels of evidence, such as epigenomic and transcriptomic data, to identify causal variants and risk mechanisms of AD-associated loci. Additionally, we discuss experimental approaches and data resources that will be needed to validate and further elucidate the effects of these variants and genes on biological pathways, cellular phenotypes and disease risk. Finally, we discuss future steps that need to be taken to ensure that AD GWAS functional mapping efforts lead to novel findings and bring us closer to finding effective treatments for this devastating disease.

3.
Nat Commun ; 12(1): 1610, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712570

RESUMO

Genome-wide association studies (GWAS) have identified more than 40 loci associated with Alzheimer's disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown, impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS with myeloid epigenomic and transcriptomic datasets using analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We identify AD risk enhancers and nominate candidate causal genes among their likely targets (including AP4E1, AP4M1, APBB3, BIN1, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, TP53INP1, and ZYX) in twenty loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify AD risk by regulating gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it in human induced pluripotent stem cell (hiPSC)-derived microglia and brain. Taken together, this study integrates AD GWAS with multiple myeloid genomic datasets to investigate the mechanisms of AD risk alleles and nominates candidate functional variants, regulatory elements and genes that likely modulate disease susceptibility.


Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença/genética , Genômica , Células Mieloides , Sequências Reguladoras de Ácido Nucleico/genética , Alelos , Doença de Alzheimer/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macrófagos , Microglia/metabolismo , Transcriptoma
4.
Genome Biol ; 22(1): 32, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441155

RESUMO

GWAS summary statistics are fundamental for a variety of research applications yet no common storage format has been widely adopted. Existing tabular formats ambiguously or incompletely store information about genetic variants and associations, lack essential metadata and are typically not indexed yielding poor query performance and increasing the possibility of errors in data interpretation and post-GWAS analyses. To address these issues, we adapted the variant call format to store GWAS summary statistics (GWAS-VCF) and developed open-source tools to use this format in downstream analyses. We provide open access to over 10,000 complete GWAS summary datasets converted to this format ( https://gwas.mrcieu.ac.uk ).

5.
Ann Neurol ; 89(1): 54-65, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32996171

RESUMO

OBJECTIVE: The purpose of this study was to infer causal relationships between 22 previously reported risk factors for Alzheimer's disease (AD) and the "AD phenome": AD, AD age of onset (AAOS), hippocampal volume, cortical surface area and thickness, cerebrospinal fluid (CSF) levels of amyloid-ß (Aß42 ), tau, and ptau181 , and the neuropathological burden of neuritic plaques, neurofibrillary tangles (NFTs), and vascular brain injury (VBI). METHODS: Polygenic risk scores (PRS) for the 22 risk factors were computed in 26,431 AD cases/controls and the association with AD was evaluated using logistic regression. Two-sample Mendelian randomization (MR) was used to infer the causal effect of risk factors on the AD phenome. RESULTS: PRS for increased education and diastolic blood pressure were associated with reduced risk for AD. MR indicated that only education was causally associated with reduced risk of AD, delayed AAOS, and increased cortical surface area and thickness. Total- and LDL-cholesterol levels were causally associated with increased neuritic plaque burden, although the effects were driven by single nucleotide polymorphisms (SNPs) within the APOE locus. Diastolic blood pressure and pulse pressure are causally associated with increased risk of VBI. Furthermore, total cholesterol was associated with decreased hippocampal volume; smoking initiation with decreased cortical thickness; type 2 diabetes with an earlier AAOS; and sleep duration with increased cortical thickness. INTERPRETATION: Our comprehensive examination of the genetic evidence for the causal relationships between previously reported risk factors in AD using PRS and MR supports a causal role for education, blood pressure, cholesterol levels, smoking, and diabetes with the AD phenome. ANN NEUROL 2021;89:54-65.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Colesterol/metabolismo , Emaranhados Neurofibrilares/genética , Fragmentos de Peptídeos/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/complicações , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Cognição/fisiologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Sono/fisiologia
6.
Neurobiol Dis ; 148: 105217, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33301878

RESUMO

More than forty loci contribute to genetic risk for Alzheimer's disease (AD). These risk alleles are enriched in myeloid cell enhancers suggesting that microglia, the brain-resident macrophages, contribute to AD risk. We have previously identified SPI1/PU.1, a master regulator of myeloid cell development in the brain and periphery, as a genetic risk factor for AD. Higher expression of SPI1 is associated with increased risk for AD, while lower expression is protective. To investigate the molecular and cellular phenotypes associated with higher and lower expression of PU.1 in microglia, we used stable overexpression and knock-down of PU.1 in BV2, an immortalized mouse microglial cell line. Transcriptome analysis suggests that reduced PU.1 expression suppresses expression of homeostatic genes similar to the disease-associated microglia response to amyloid plaques in mouse models of AD. Moreover, PU.1 knock-down resulted in activation of protein translation, antioxidant action and cholesterol/lipid metabolism pathways with a concomitant decrease of pro-inflammatory gene expression. PU.1 overexpression upregulated and knock-down downregulated phagocytic uptake in BV2 cells independent of the nature of the engulfed material. However, cells with reduced PU.1 expression retained their ability to internalize myelin similar to control albeit with a delay, which aligns with their anti-inflammatory profile. Here we identified several microglial responses that are modulated by PU.1 expression levels and propose that risk association of PU.1 to AD is driven by increased pro-inflammatory response due to increased viability of cells under cytotoxic conditions. In contrast, low expression of PU.1 leads to increased cell death under cytotoxic conditions accompanied by reduced pro-inflammatory signaling that decreased A1 reactive astrocytes signature supporting the protective effect of SPI1 genotype in AD. These findings inform future in vivo validation studies and design of small molecule screens for therapeutic discovery in AD.

7.
Trends Neurosci ; 43(12): 965-979, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33127097

RESUMO

Alzheimer's disease (AD) is a debilitating, chronic neurodegenerative disease. Genetic studies involving genome-wide association studies (GWAS) and meta-analysis have discovered numerous genomic loci associated with AD; however, the causal genes and variants remain unidentified in most loci. Integration of GWAS signals with epigenomic annotations has demonstrated that AD risk variants are enriched in myeloid-specific enhancers, implicating myeloid cells in AD etiology. AD risk variants in these regulatory elements modify disease susceptibility by regulating the expression of genes that play crucial roles in microglial phagocytosis. Several of these AD risk genes are specifically expressed in myeloid cells, whereas others are ubiquitously expressed but are regulated by AD risk variants within myeloid enhancers in a cell type-specific manner. We discuss the impact of established AD risk variants on microglial phagocytosis and debris processing via the endolysosomal system.

8.
Nat Commun ; 11(1): 4799, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968074

RESUMO

Genetic association studies have identified 44 common genome-wide significant risk loci for late-onset Alzheimer's disease (LOAD). However, LOAD genetic architecture and prediction are unclear. Here we estimate the optimal P-threshold (Poptimal) of a genetic risk score (GRS) for prediction of LOAD in three independent datasets comprising 676 cases and 35,675 family history proxy cases. We show that the discriminative ability of GRS in LOAD prediction is maximised when selecting a small number of SNPs. Both simulation results and direct estimation indicate that the number of causal common SNPs for LOAD may be less than 100, suggesting LOAD is more oligogenic than polygenic. The best GRS explains approximately 75% of SNP-heritability, and individuals in the top decile of GRS have ten-fold increased odds when compared to those in the bottom decile. In addition, 14 variants are identified that contribute to both LOAD risk and age at onset of LOAD.


Assuntos
Doença de Alzheimer/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Adulto , Idade de Início , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Risco
9.
Mol Psychiatry ; 25(8): 1859-1875, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-30108311

RESUMO

The Alzheimer's Disease Sequencing Project (ADSP) undertook whole exome sequencing in 5,740 late-onset Alzheimer disease (AD) cases and 5,096 cognitively normal controls primarily of European ancestry (EA), among whom 218 cases and 177 controls were Caribbean Hispanic (CH). An age-, sex- and APOE based risk score and family history were used to select cases most likely to harbor novel AD risk variants and controls least likely to develop AD by age 85 years. We tested ~1.5 million single nucleotide variants (SNVs) and 50,000 insertion-deletion polymorphisms (indels) for association to AD, using multiple models considering individual variants as well as gene-based tests aggregating rare, predicted functional, and loss of function variants. Sixteen single variants and 19 genes that met criteria for significant or suggestive associations after multiple-testing correction were evaluated for replication in four independent samples; three with whole exome sequencing (2,778 cases, 7,262 controls) and one with genome-wide genotyping imputed to the Haplotype Reference Consortium panel (9,343 cases, 11,527 controls). The top findings in the discovery sample were also followed-up in the ADSP whole-genome sequenced family-based dataset (197 members of 42 EA families and 501 members of 157 CH families). We identified novel and predicted functional genetic variants in genes previously associated with AD. We also detected associations in three novel genes: IGHG3 (p = 9.8 × 10-7), an immunoglobulin gene whose antibodies interact with ß-amyloid, a long non-coding RNA AC099552.4 (p = 1.2 × 10-7), and a zinc-finger protein ZNF655 (gene-based p = 5.0 × 10-6). The latter two suggest an important role for transcriptional regulation in AD pathogenesis.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Regulação da Expressão Gênica/genética , Imunidade/genética , Transcrição Genética/genética , Sequenciamento Completo do Exoma , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/imunologia , Apolipoproteínas E/genética , Feminino , Haplótipos/genética , Humanos , Imunoglobulina G , Fatores de Transcrição Kruppel-Like/genética , Masculino , Polimorfismo Genético/genética , RNA Longo não Codificante/genética
11.
Neurosci Lett ; 711: 134403, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31351091

RESUMO

Recent methylome-wide association studies (MWAS) in humans have solidified the concept that aberrant DNA methylation is associated with Alzheimer's disease (AD). We summarize these findings to improve the understanding of mechanisms governing DNA methylation pertinent to transcriptional regulation, with an emphasis of AD-associated promoter DNA hypermethylation, which establishes an epigenetic barrier for transcriptional activation. By considering brain cell type specific expression profiles that have been published only for non-demented individuals, we detail functional activities of selected neuron, microglia, and astrocyte-enriched genes (AGAP2, DUSP6 and GPR37L1, respectively), which are DNA hypermethylated at promoters in AD. We highlight future directions in MWAS including experimental confirmation, functional relevance to AD, cell type-specific temporal characterization, and mechanism investigation.


Assuntos
Doença de Alzheimer/genética , Metilação de DNA/genética , Animais , Humanos , Regiões Promotoras Genéticas/genética
12.
J Biol Chem ; 293(32): 12634-12646, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29794134

RESUMO

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.


Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Glicoproteínas de Membrana/química , Proteínas Mutantes/química , Receptores Imunológicos/química , Doença de Alzheimer/patologia , Cristalografia por Raios X , Células Dendríticas/química , Células Dendríticas/patologia , Variação Genética , Humanos , Ligantes , Macrófagos/química , Macrófagos/patologia , Glicoproteínas de Membrana/genética , Microglia/química , Microglia/patologia , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Osteoclastos/química , Osteoclastos/patologia , Conformação Proteica , Domínios Proteicos/genética , Receptores Imunológicos/genética
13.
Bioinformatics ; 34(16): 2724-2731, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29590295

RESUMO

Motivation: Annotation of genomic variants is an increasingly important and complex part of the analysis of sequence-based genomic analyses. Computational predictions of variant function are routinely incorporated into gene-based analyses of rare-variants, though to date most studies use limited information for assessing variant function that is often agnostic of the disease being studied. Results: In this work, we outline an annotation process motivated by the Alzheimer's Disease Sequencing Project, illustrate the impact of including tissue-specific transcript sets and sources of gene regulatory information and assess the potential impact of changing genomic builds on the annotation process. While these factors only impact a small proportion of total variant annotations (∼5%), they influence the potential analysis of a large fraction of genes (∼25%). Availability and implementation: Individual variant annotations are available via the NIAGADS GenomicsDB, at https://www.niagads.org/genomics/ tools-and-software/databases/genomics-database. Annotations are also available for bulk download at https://www.niagads.org/datasets. Annotation processing software is available at http://www.icompbio.net/resources/software-and-downloads/. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Anotação de Sequência Molecular/métodos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Software , Bases de Dados Genéticas , Genoma , Genômica , Humanos
14.
Immunity ; 47(3): 398-400, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28930654

RESUMO

Microglial cell function is implicated in the etiology of Alzheimer's disease by human genetics. In this issue of Immunity, Krasemann et al. (2017) describe a gene expression signature associated with an APOE- and TREM2-dependent response of microglia to brain tissue damage that accumulates in aging and disease, defining an axis that might be amenable to therapeutic targeting.


Assuntos
Apolipoproteínas E , Microglia/imunologia , Envelhecimento , Doença de Alzheimer , Encéfalo , Humanos
15.
Nat Neurosci ; 20(8): 1052-1061, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28628103

RESUMO

A genome-wide survival analysis of 14,406 Alzheimer's disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and 14 novel loci associated with age at onset. Linkage disequilibrium score regression of 220 cell types implicated the regulation of myeloid gene expression in AD risk. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability was enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affected the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.


Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Alelos , Animais , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Desequilíbrio de Ligação/genética , Masculino , Camundongos , Fatores de Risco , Fatores de Transcrição/genética
16.
J Huntingtons Dis ; 3(4): 333-41, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25575954

RESUMO

BACKGROUND: Phosphodiesterase 10A (PDE10A) is expressed at high levels in the striatum and has been proposed both as a biomarker for Huntington's disease pathology and as a target for intervention. OBJECTIVE: PDE10A radiotracers have been successfully used to measure changes in binding density in Huntington's disease patients, but little is known about PDE10A binding in mouse models that are used extensively to model pathology and test therapeutic interventions. METHODS: Our study investigated changes in PDE10A binding using the selective tracer 3H-7980 at specific ages of two Huntington's disease transgenic mouse models: R6/2, a short-lived model carrying exon-1 of mutant HTT and BACHD, a longer-lived model carrying full-length mutant HTT. PDE10A binding was compared to binding of known markers of striatal atrophy in Huntington's disease, e.g. dopamine transporter (DAT) and dopamine receptors D1 and D2. RESULTS: We found that in the R6/2 model at 6 weeks of age, mice showed high variability of binding, however binding of all ligands was significantly decreased at 8 and 12 weeks of age. In contrast, no changes were detectable in the BACHD model at 8, 10 or 12 month of age. CONCLUSIONS: These findings suggest that radiotracer binding of PDE10A, DAT, D1 and D2 receptor in the R6/2 model may be a good indicator of striatal pathological changes that are observed in Huntington's disease patients, and that the first 12 months in the BACHD model may be more reflective of early stages of the disease.


Assuntos
Corpo Estriado/química , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Doença de Huntington/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Receptores Dopaminérgicos/metabolismo , Animais , Gânglios da Base/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Ensaio Radioligante , Substância Negra/metabolismo
17.
Hum Mol Genet ; 19(22): 4373-84, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20739295

RESUMO

Expansion of a polyglutamine (polyQ) tract in the Huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Loss of the normal function of Htt is thought to be an important pathogenetic component of HD. However, the function of wild-type Htt is not well defined. Htt is thought to be a multifunctional protein that plays distinct roles in several biological processes, including synaptic transmission, intracellular transport and neuronal transcription. Here, we show with biochemical and live cell imaging studies that wild-type Htt stimulates the transport of nuclear factor κ light-chain-enhancer of activated B cells (NF-κB) out of dendritic spines (where NF-κB is activated by excitatory synaptic input) and supports a high level of active NF-κB in neuronal nuclei (where NF-κB stimulates the transcription of target genes). We show that this novel function of Htt is impaired by the polyQ expansion and thus may contribute to the etiology of HD.


Assuntos
Núcleo Celular/metabolismo , Mutação , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Sinapses/metabolismo , Animais , Transporte Biológico/genética , Núcleo Celular/genética , Núcleo Celular/patologia , Técnicas de Introdução de Genes , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/genética , Sinapses/patologia
18.
J Neurosci ; 28(50): 13673-83, 2008 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-19074040

RESUMO

SynGAP, a prominent Ras/Rap GTPase-activating protein in the postsynaptic density, regulates the timing of spine formation and trafficking of glutamate receptors in cultured neurons. However, the molecular mechanisms by which it does this are unknown. Here, we show that synGAP is a key regulator of spine morphology in adult mice. Heterozygous deletion of synGAP was sufficient to cause an excess of mushroom spines in adult brains, indicating that synGAP is involved in steady-state regulation of actin in mature spines. Both Ras- and Rac-GTP levels were elevated in forebrains from adult synGAP(+/-) mice. Rac is a well known regulator of actin polymerization and spine morphology. The steady-state level of phosphorylation of cofilin was also elevated in synGAP(+/-) mice. Cofilin, an F-actin severing protein that is inactivated by phosphorylation, is a downstream target of a pathway regulated by Rac. We show that transient regulation of cofilin by treatment with NMDA is also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 mum NMDA triggered transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion of synGAP lacked the transient regulation by the NMDA receptor. Depression of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices from synGAP(+/-) mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Citoesqueleto , Espinhas Dendríticas/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos , Camundongos Knockout , Microscopia Confocal , N-Metilaspartato/farmacologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Fosforilação , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas ras/metabolismo
19.
Mol Cell Neurosci ; 28(4): 727-36, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15797719

RESUMO

NeuroD/BETA2 (referred to as NeuroD hereafter) is a basic helix-loop-helix (bHLH) transcription factor that is required for the development and survival of a subset of neurons and pancreatic endocrine cells in mice. Gain-of-function analyses demonstrated that NeuroD can (i) convert epidermal fate into neuronal fate when overexpressed in Xenopus embryos, and (ii) activate the insulin promoter in pancreatic beta cell lines in response to glucose stimulation. In glucose-stimulated INS-1 pancreatic beta cells, mutations of S259, S266, and S274 to alanines inhibited the ability of NeuroD to activate the insulin promoter. Phosphorylation of those serine residues by ERK1/2 was required for NeuroD activity in that assay. To determine whether the same residues are implicated in the neurogenic activity of NeuroD, we mutated the conserved S259, S266, and S274 of Xenopus NeuroD to alanines (S259A, S266A, and S274A), and performed an ectopic neurogenesis assay in Xenopus embryos. In contrast to what has been observed in the pancreatic beta cell line, the S266A and S274A mutant forms of Xenopus NeuroD displayed significantly increased abilities to form ectopic neurons, while S259A had little effect. In addition, S266A and S274A of Xenopus NeuroD resulted in increased accumulation of protein in the injected embryos while the corresponding mutations on mouse NeuroD did not have the same effect in an insulinoma cell line. Our results demonstrate that the consequence of NeuroD protein modification is context-dependent at both the molecular and functional levels.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem Celular Tumoral , Cricetinae , Feminino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fosforilação , Serina/genética , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/genética , Xenopus laevis
20.
Proc Natl Acad Sci U S A ; 100(16): 9578-83, 2003 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-12881483

RESUMO

NeuroD (ND) is a basic helix-loop-helix transcription factor important for neuronal development and survival. By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt). Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntington's disease, a neurodegenerative disorder. However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown. Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND. Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2. To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt. We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.


Assuntos
Carbono-Oxigênio Liases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , MAP Quinase Quinase Quinases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Xenopus , Fosfatase Alcalina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sobrevivência Celular , DNA/metabolismo , DNA Complementar/metabolismo , Deleção de Genes , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Camundongos , Modelos Biológicos , Neurônios/metabolismo , Fosforilação , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Transcrição Genética , Transfecção , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Xenopus
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