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1.
FASEB J ; 33(2): 2241-2251, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30332298

RESUMO

Diabetes mellitus prevalence is increasing rapidly and is a major cause of mortality and morbidity worldwide. In addition to the known severe complications associated with the disease, in recent years diabetes has been recognized as a major risk factor for cancer. Patients with diabetes experience significantly higher incidence of and higher mortality rates from many types of cancer. However, to date there are no conclusive data on the pathophysiology underlying the association between these two diseases. We previously reported that insulin regulates skin proliferation and differentiation, while IGF1 had different sometimes contrasting effects to those of insulin, suggesting direct involvement of insulin in transformation. To this end, we developed an epidermal skin-specific insulin receptor knockout (SIRKO) mouse, in which the insulin receptor (IR) is inactivated only in skin, with no other metabolic consequences. We found that IR inactivation by itself resulted in a marked decrease in skin tumorigenesis. In the control group 100% of the mice developed tumors, but in the SIRKO group tumor incidence was over 60% lower, and 25% of the SIRKO mice did not develop tumors at all, and the tumors that did develop were smaller and benign in their appearance. Furthermore, IR inactivation in vitro not only prevented cell transformation but also reversed the keratinocyte-transformed phenotype. We found that IR inactivation led to a striking abnormality in the major keratin cytoskeleton filaments structure in both in vivo and in vitro, a change that we were able to link to the decreased transformation potential in IR-null cells. In summary, we identified a unique pathway in which IR regulates cytoskeletal assembly, thus affecting skin transformation, opening a new potential target for cancer treatment and prevention.-Weingarten, G., Ben Yaakov, A., Dror, E., Russ, J., Magin, T. M., Kahn, C. R., Wertheimer, E. Insulin receptor plays a central role in skin carcinogenesis by regulating cytoskeleton assembly.


Assuntos
Citoesqueleto/fisiologia , Receptor de Insulina/fisiologia , Fenômenos Fisiológicos da Pele , Pele/fisiopatologia , Animais , Humanos , Queratinas/genética , Camundongos
2.
Hum Mol Genet ; 25(8): 1600-18, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26908611

RESUMO

Caspase-6 (CASP6) has emerged as an important player in Huntington disease (HD), Alzheimer disease (AD) and cerebral ischemia, where it is activated early in the disease process. CASP6 also plays a key role in axonal degeneration, further underscoring the importance of this protease in neurodegenerative pathways. As a protein's function is modulated by its protein-protein interactions, we performed a high-throughput yeast-2-hybrid (Y2H) screen against ∼17,000 human proteins to gain further insight into the function of CASP6. We identified a high-confidence list of 87 potential CASP6 interactors. From this list, 61% are predicted to contain a CASP6 recognition site. Of nine candidate substrates assessed, six are cleaved by CASP6. Proteins that did not contain a predicted CASP6 recognition site were assessed using a LUMIER assay approach, and 51% were further validated as interactors by this method. Of note, 54% of the high-confidence interactors identified show alterations in human HD brain at the mRNA level, and there is a significant enrichment for previously validated huntingtin (HTT) interactors. One protein of interest, STK3, a pro-apoptotic kinase, was validated biochemically to be a CASP6 substrate. Furthermore, our results demonstrate that in striatal cells expressing mutant huntingtin (mHTT), an increase in full length and fragment levels of STK3 are observed. We further show that caspase-3 is not essential for the endogenous cleavage of STK3. Characterization of the interaction network provides important new information regarding key pathways of interactors of CASP6 and highlights potential novel therapeutic targets for HD, AD and cerebral ischemia.


Assuntos
Caspase 6/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Proteína Huntingtina/genética , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Serina-Treonina Quinase 3 , Técnicas do Sistema de Duplo-Híbrido
3.
Genome Res ; 25(5): 701-13, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25908449

RESUMO

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.


Assuntos
Algoritmos , Proteínas do Tecido Nervoso/metabolismo , Agregação Patológica de Proteínas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Drosophila/genética , Drosophila/metabolismo , Proteína Huntingtina , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Células PC12 , Ligação Proteica , Ratos
4.
Proc Natl Acad Sci U S A ; 111(33): 12085-90, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25092318

RESUMO

Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.


Assuntos
Éxons , Proteínas do Tecido Nervoso/genética , Príons/fisiologia , Proteínas Ligadas por GPI/fisiologia , Humanos , Proteína Huntingtina , Microscopia Confocal
5.
Acta Neuropathol ; 129(1): 39-52, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25388784

RESUMO

C9orf72 promoter hypermethylation inhibits the accumulation of pathologies which have been postulated to be neurotoxic. We tested here whether C9orf72 hypermethylation is associated with prolonged disease in C9orf72 mutation carriers. C9orf72 methylation was quantified from brain or blood using methylation-sensitive restriction enzyme digest-qPCR in a cross-sectional cohort of 118 C9orf72 repeat expansion carriers and 19 non-carrier family members. Multivariate regression models were used to determine whether C9orf72 hypermethylation was associated with age at onset, disease duration, age at death, or hexanucleotide repeat expansion size. Permutation analysis was performed to determine whether C9orf72 methylation is heritable. We observed a high correlation between C9orf72 methylation across tissues including cerebellum, frontal cortex, spinal cord and peripheral blood. While C9orf72 methylation was not significantly different between ALS and FTD and did not predict age at onset, brain and blood C9orf72 hypermethylation was associated with later age at death in FTD (brain: ß = 0.18, p = 0.006; blood: ß = 0.15, p < 0.001), and blood C9orf72 hypermethylation was associated with longer disease duration in FTD (ß = 0.03, p = 0.007). Furthermore, C9orf72 hypermethylation was associated with smaller hexanucleotide repeat length (ß = -16.69, p = 0.033). Finally, analysis of pedigrees with multiple mutation carriers demonstrated a significant association between C9orf72 methylation and family relatedness (p < 0.0001). C9orf72 hypermethylation is associated with prolonged disease in C9orf72 repeat expansion carriers with FTD. The attenuated clinical phenotype associated with C9orf72 hypermethylation suggests that slower clinical progression in FTD is associated with reduced expression of mutant C9orf72. These results support the hypothesis that expression of the hexanucleotide repeat expansion is associated with a toxic gain of function.


Assuntos
Metilação de DNA , Expansão das Repetições de DNA , Mutação , Proteínas/genética , Idoso , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Proteína C9orf72 , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/genética , Transtornos Cognitivos/metabolismo , Estudos de Coortes , Estudos Transversais , Feminino , Degeneração Lobar Frontotemporal/diagnóstico , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Regiões Promotoras Genéticas , Proteínas/metabolismo
6.
Nucleic Acids Res ; 41(3): 1496-507, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23275563

RESUMO

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein-protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Técnicas do Sistema de Duplo-Híbrido , Ataxina-1 , Ataxinas , Humanos , Proteína Huntingtina , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Mapas de Interação de Proteínas , Leveduras/genética
7.
PLoS Genet ; 8(8): e1002897, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916034

RESUMO

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.


Assuntos
Complexo Mediador/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Peptídeos/química , Proteínas de Ligação a RNA/química , Animais , Ataxina-1 , Ataxinas , Células COS , Chlorocebus aethiops , Escherichia coli/genética , Humanos , Complexo Mediador/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Plasmídeos , Polimerização , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , Transfecção
8.
Acta Neuropathol ; 128(4): 525-41, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24806409

RESUMO

Hexanucleotide repeat expansions of C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal degeneration. The mutation is associated with reduced C9orf72 expression and the accumulation of potentially toxic RNA and protein aggregates. CpG methylation is known to protect the genome against unstable DNA elements and to stably silence inappropriate gene expression. Using bisulfite cloning and restriction enzyme-based methylation assays on DNA from human brain and peripheral blood, we observed CpG hypermethylation involving the C9orf72 promoter in cis to the repeat expansion mutation in approximately one-third of C9orf72 repeat expansion mutation carriers. Promoter hypermethylation of mutant C9orf72 was associated with transcriptional silencing of C9orf72 in patient-derived lymphoblast cell lines, resulting in reduced accumulation of intronic C9orf72 RNA and reduced numbers of RNA foci. Furthermore, demethylation of mutant C9orf72 with 5-aza-deoxycytidine resulted in increased vulnerability of mutant cells to oxidative and autophagic stress. Promoter hypermethylation of repeat expansion carriers was also associated with reduced accumulation of RNA foci and dipeptide repeat protein aggregates in human brains. These results indicate that C9orf72 promoter hypermethylation prevents downstream molecular aberrations associated with the hexanucleotide repeat expansion, suggesting that epigenetic silencing of the mutant C9orf72 allele may represent a protective counter-regulatory response to hexanucleotide repeat expansion.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Metilação de DNA/genética , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Proteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Proteína C9orf72 , Linhagem Celular Transformada , Ilhas de CpG/genética , Análise Mutacional de DNA , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Regiões Promotoras Genéticas/genética , Proteínas/genética , RNA Mensageiro/metabolismo
9.
Proc Natl Acad Sci U S A ; 107(17): 7710-5, 2010 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-20385841

RESUMO

Protein misfolding and formation of beta-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer's and Parkinson's disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits alpha-synuclein and amyloid-beta fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature alpha-synuclein and amyloid-beta fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to beta-sheet-rich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils.


Assuntos
Neuropatias Amiloides/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Amiloide/biossíntese , Catequina/análogos & derivados , Fármacos Neuroprotetores/farmacologia , alfa-Sinucleína/metabolismo , Amiloide/efeitos dos fármacos , Neuropatias Amiloides/tratamento farmacológico , Animais , Western Blotting , Células CHO , Catequina/farmacologia , Cromatografia de Afinidade , Dicroísmo Circular , Cricetinae , Cricetulus , Escherichia coli , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Células PC12 , Ratos
10.
Nat Commun ; 13(1): 6830, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369285

RESUMO

Current concepts regarding the biology of aging are primarily based on studies aimed at identifying factors regulating lifespan. However, lifespan as a sole proxy measure for aging can be of limited value because it may be restricted by specific pathologies. Here, we employ large-scale phenotyping to analyze hundreds of markers in aging male C57BL/6J mice. For each phenotype, we establish lifetime profiles to determine when age-dependent change is first detectable relative to the young adult baseline. We examine key lifespan regulators (putative anti-aging interventions; PAAIs) for a possible countering of aging. Importantly, unlike most previous studies, we include in our study design young treated groups of animals, subjected to PAAIs prior to the onset of detectable age-dependent phenotypic change. Many PAAI effects influence phenotypes long before the onset of detectable age-dependent change, but, importantly, do not alter the rate of phenotypic change. Hence, these PAAIs have limited effects on aging.


Assuntos
Envelhecimento , Longevidade , Camundongos , Animais , Masculino , Longevidade/genética , Camundongos Endogâmicos C57BL , Envelhecimento/fisiologia , Fenótipo
11.
Nucleic Acids Res ; 37(Database issue): D657-60, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18984619

RESUMO

Human protein interaction maps have become important tools of biomedical research for the elucidation of molecular mechanisms and the identification of new modulators of disease processes. The Unified Human Interactome database (UniHI, http://www.unihi.org) provides researchers with a comprehensive platform to query and access human protein-protein interaction (PPI) data. Since its first release, UniHI has considerably increased in size. The latest update of UniHI includes over 250,000 interactions between approximately 22,300 unique proteins collected from 14 major PPI sources. However, this wealth of data also poses new challenges for researchers due to the complexity of interaction networks retrieved from the database. We therefore developed several new tools to query, analyze and visualize human PPI networks. Most importantly, UniHI allows now the construction of tissue-specific interaction networks and focused querying of canonical pathways. This will enable researchers to target their analysis and to prioritize candidate proteins for follow-up studies.


Assuntos
Bases de Dados de Proteínas , Mapeamento de Interação de Proteínas , Gráficos por Computador , Humanos , Proteínas/genética , Proteínas/metabolismo , Software , Interface Usuário-Computador
12.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33108356

RESUMO

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.


Assuntos
Envelhecimento/metabolismo , Microglia/enzimologia , Biossíntese de Proteínas , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Envelhecimento/genética , Animais , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/genética , Serina-Treonina Quinases TOR/genética
13.
Nat Cell Biol ; 23(12): 1224-1239, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34876685

RESUMO

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.


Assuntos
Cromatina/patologia , Proteínas Correpressoras/genética , Transtornos Leucocíticos/congênito , Chaperonas Moleculares/genética , Mielopoese/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Linfócitos B/citologia , Linhagem Celular , Cromatina/genética , Células-Tronco Hematopoéticas/citologia , Histonas/metabolismo , Humanos , Inflamação/patologia , Transtornos Leucocíticos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retroelementos/genética , Dermatopatias/genética , Dermatopatias/imunologia , Dermatopatias/patologia
14.
BMC Genomics ; 11: 305, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20465848

RESUMO

BACKGROUND: Human tissue displays a remarkable diversity in structure and function. To understand how such diversity emerges from the same DNA, systematic measurements of gene expression across different tissues in the human body are essential. Several recent studies addressed this formidable task using microarray technologies. These large tissue expression data sets have provided us an important basis for biomedical research. However, it is well known that microarray data can be compromised by high noise level and various experimental artefacts. Critical comparison of different data sets can help to reveal such errors and to avoid pitfalls in their application. RESULTS: We present here the first comparison and integration of four freely available tissue expression data sets generated using three different microarray platforms and containing a total of 377 microarray hybridizations. When assessing the tissue expression of genes, we found that the results considerably depend on the chosen data set. Nevertheless, the comparison also revealed statistically significant similarity of gene expression profiles across different platforms. This enabled us to construct consolidated lists of platform-independent tissue-specific genes using a set of complementary measures. Follow-up analyses showed that results based on consolidated data tend to be more reliable. CONCLUSIONS: Our study strongly indicates that the consolidation of the four different tissue expression data sets can increase data quality and can lead to biologically more meaningful results. The provided compendium of platform-independent gene lists should facilitate the identification of novel tissue-specific marker genes.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Encéfalo/metabolismo , Perfilação da Expressão Gênica/estatística & dados numéricos , Genômica , Humanos , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Especificidade de Órgãos , Reprodutibilidade dos Testes , Biologia de Sistemas
15.
Artigo em Inglês | MEDLINE | ID: mdl-32905541

RESUMO

A hexanucleotide G4C2 repeat expansion in C9orf72 is the most common genetic cause of familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). The mutation is associated with a reduction of C9orf72 protein and accumulation of toxic RNA and dipeptide repeat aggregates. The accumulation of toxic RNA has been proposed to sequester RNA binding proteins thereby altering RNA processing, consistent with previous transcriptome studies that have shown that the C9orf72 repeat expansion is linked to abundant splicing alterations and transcriptome changes. Here, we used a subcellular fractionation method and FACS to enrich for neuronal nuclei from C9orf72 repeat expanded post-mortem human ALS/FTD brains, and to remove neuronal nuclei with TDP-43 pathology which are observed in nearly all symptomatic C9orf72 repeat expanded cases. We show that the C9orf72 expansion is associated with relatively mild gene expression changes. Dysregulated genes were enriched for vesicle transport pathways, which is consistent with the known functions of C9orf72 protein. Further analysis suggests that the C9orf72 transcriptome is not driven by toxic RNA but is rather shaped by the depletion of pathologic TDP-43 nuclei and the loss of C9orf72 expression. These findings argue against RNA binding protein sequestration in neurons as a major contributor to C9orf72 mediated toxicity.

16.
Front Neurol ; 11: 573560, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33329316

RESUMO

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the Huntingtin gene. As disease-modifying therapies for HD are being developed, peripheral blood cells may be used to indicate disease progression and to monitor treatment response. In order to investigate whether gene expression changes can be found in the blood of individuals with HD that distinguish them from healthy controls, we performed transcriptome analysis by next-generation sequencing (RNA-seq). We detected a gene expression signature consistent with dysregulation of immune-related functions and inflammatory response in peripheral blood from HD cases vs. controls, including induction of the interferon response genes, IFITM3, IFI6 and IRF7. Our results suggest that it is possible to detect gene expression changes in blood samples from individuals with HD, which may reflect the immune pathology associated with the disease.

17.
Cell Rep ; 32(7): 108050, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32814053

RESUMO

Interactome maps are valuable resources to elucidate protein function and disease mechanisms. Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. This network reveals interconnectivity across diseases and links many known ND-causing proteins, such as α-synuclein, TDP-43, and ATXN1, to a host of proteins previously unrelated to NDs. It facilitates the identification of interacting proteins that significantly influence mutant TDP-43 and HTT toxicity in transgenic flies, as well as of ARF-GEP100 that controls misfolding and aggregation of multiple ND-causing proteins in experimental model systems. Furthermore, it enables the prediction of ND-specific subnetworks and the identification of proteins, such as ATXN1 and MKL1, that are abnormally aggregated in postmortem brains of Alzheimer's disease patients, suggesting widespread protein aggregation in NDs.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/fisiopatologia , Doenças Neurodegenerativas/genética , Agregados Proteicos/genética , Mapeamento de Interação de Proteínas/métodos , Humanos
18.
Cell Rep ; 27(5): 1409-1421.e6, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31042469

RESUMO

Loss of the nuclear RNA binding protein TAR DNA binding protein-43 (TDP-43) into cytoplasmic aggregates is the strongest correlate to neurodegeneration in amyotrophic lateral sclerosis and frontotemporal degeneration. The molecular changes associated with the loss of nuclear TDP-43 in human tissues are not entirely known. Using subcellular fractionation and fluorescent-activated cell sorting to enrich for diseased neuronal nuclei without TDP-43 from post-mortem frontotemporal degeneration-amyotrophic lateral sclerosis (FTD-ALS) human brain, we characterized the effects of TDP-43 loss on the transcriptome and chromatin accessibility. Nuclear TDP-43 loss is associated with gene expression changes that affect RNA processing, nucleocytoplasmic transport, histone processing, and DNA damage. Loss of nuclear TDP-43 is also associated with chromatin decondensation around long interspersed nuclear elements (LINEs) and increased LINE1 DNA content. Moreover, loss of TDP-43 leads to increased retrotransposition that can be inhibited with antiretroviral drugs, suggesting that TDP-43 neuropathology is associated with altered chromatin structure including decondensation of LINEs.


Assuntos
Esclerose Lateral Amiotrófica/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Demência Frontotemporal/genética , Elementos Nucleotídeos Longos e Dispersos , Transporte Ativo do Núcleo Celular , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Núcleo Celular/metabolismo , Cromatina/química , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Demência Frontotemporal/metabolismo , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , Transcriptoma
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