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1.
Cell ; 186(16): 3427-3442.e22, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37421949

RESUMO

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Ligação Proteica , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo
2.
Sci Transl Med ; 15(703): eabq5923, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37406134

RESUMO

Proteomic studies for Alzheimer's disease (AD) are instrumental in identifying AD pathways but often focus on single tissues and sporadic AD cases. Here, we present a proteomic study analyzing 1305 proteins in brain tissue, cerebrospinal fluid (CSF), and plasma from patients with sporadic AD, TREM2 risk variant carriers, patients with autosomal dominant AD (ADAD), and healthy individuals. We identified 8 brain, 40 CSF, and 9 plasma proteins that were altered in individuals with sporadic AD, and we replicated these findings in several external datasets. We identified a proteomic signature that differentiated TREM2 variant carriers from both individuals with sporadic AD and healthy individuals. The proteins associated with sporadic AD were also altered in patients with ADAD, but with a greater effect size. Brain-derived proteins associated with ADAD were also replicated in additional CSF samples. Enrichment analyses highlighted several pathways, including those implicated in AD (calcineurin and Apo E), Parkinson's disease (α-synuclein and LRRK2), and innate immune responses (SHC1, ERK-1, and SPP1). Our findings suggest that combined proteomics across brain tissue, CSF, and plasma can be used to identify markers for sporadic and genetically defined AD.


Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/metabolismo , Proteômica , Encéfalo/metabolismo , Imunidade Inata , Heterozigoto , Biomarcadores/metabolismo , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/metabolismo
3.
Genome Med ; 14(1): 140, 2022 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-36510323

RESUMO

BACKGROUND: Human proteins are widely used as drug targets. Integration of large-scale protein-level genome-wide association studies (GWAS) and disease-related GWAS has thus connected genetic variation to disease mechanisms via protein. Previous proteome-by-phenome-wide Mendelian randomization (MR) studies have been mainly focused on plasma proteomes. Previous MR studies using the brain proteome only reported protein effects on a set of pre-selected tissue-specific diseases. No studies, however, have used high-throughput proteomics from multiple tissues to perform MR on hundreds of phenotypes. METHODS: Here, we performed MR and colocalization analysis using multi-tissue (cerebrospinal fluid (CSF), plasma, and brain from pre- and post-meta-analysis of several disease-focus cohorts including Alzheimer disease (AD)) protein quantitative trait loci (pQTLs) as instrumental variables to infer protein effects on 211 phenotypes, covering seven broad categories: biological traits, blood traits, cancer types, neurological diseases, other diseases, personality traits, and other risk factors. We first implemented these analyses with cis pQTLs, as cis pQTLs are known for being less prone to horizontal pleiotropy. Next, we included both cis and trans conditionally independent pQTLs that passed the genome-wide significance threshold keeping only variants associated with fewer than five proteins to minimize pleiotropic effects. We compared the tissue-specific protein effects on phenotypes across different categories. Finally, we integrated the MR-prioritized proteins with the druggable genome to identify new potential targets. RESULTS: In the MR and colocalization analysis including study-wide significant cis pQTLs as instrumental variables, we identified 33 CSF, 13 plasma, and five brain proteins to be putative causal for 37, 18, and eight phenotypes, respectively. After expanding the instrumental variables by including genome-wide significant cis and trans pQTLs, we identified a total of 58 CSF, 32 plasma, and nine brain proteins associated with 58, 44, and 16 phenotypes, respectively. For those protein-phenotype associations that were found in more than one tissue, the directions of the associations for 13 (87%) pairs were consistent across tissues. As we were unable to use methods correcting for horizontal pleiotropy given most of the proteins were only associated with one valid instrumental variable after clumping, we found that the observations of protein-phenotype associations were consistent with a causal role or horizontal pleiotropy. Between 66.7 and 86.3% of the disease-causing proteins overlapped with the druggable genome. Finally, between one and three proteins, depending on the tissue, were connected with at least one drug compound for one phenotype from both DrugBank and ChEMBL databases. CONCLUSIONS: Integrating multi-tissue pQTLs with MR and the druggable genome may open doors to pinpoint novel interventions for complex traits with no effective treatments, such as ovarian and lung cancers.


Assuntos
Estudo de Associação Genômica Ampla , Proteoma , Humanos , Estudo de Associação Genômica Ampla/métodos , Proteoma/genética , Fenótipo , Locos de Características Quantitativas , Proteômica , Polimorfismo de Nucleotídeo Único
4.
Trends Pharmacol Sci ; 43(8): 641-652, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35039149

RESUMO

Progranulin (PGRN, encoded by the GRN gene) plays a key role in the development, survival, function, and maintenance of neurons and microglia in the mammalian brain. It regulates lysosomal biogenesis, inflammation, repair, stress response, and aging. GRN loss-of-function mutations cause neuronal ceroid lipofuscinosis or frontotemporal dementia-GRN (FTD-GRN) in a gene dosage-dependent manner. Mutations that reduce PGRN levels increase the risk for developing Alzheimer's disease, Parkinson's disease, and limbic-predominant age-related transactivation response DNA-binding protein 43 encephalopathy, as well as exacerbate the progression of amyotrophic lateral sclerosis (ALS) and FTD caused by the hexanucleotide repeat expansion in the C9orf72 gene. Elevating and/or restoring PGRN levels is an attractive therapeutic strategy and is being investigated for neurodegenerative diseases through multiple mechanisms of action.


Assuntos
Demência Frontotemporal , Doenças Neurodegenerativas , Progranulinas , Demência Frontotemporal/tratamento farmacológico , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Microglia , Mutação , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Progranulinas/genética , Progranulinas/metabolismo
5.
Nat Neurosci ; 24(9): 1302-1312, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34239129

RESUMO

Understanding the tissue-specific genetic controls of protein levels is essential to uncover mechanisms of post-transcriptional gene regulation. In this study, we generated a genomic atlas of protein levels in three tissues relevant to neurological disorders (brain, cerebrospinal fluid and plasma) by profiling thousands of proteins from participants with and without Alzheimer's disease. We identified 274, 127 and 32 protein quantitative trait loci (pQTLs) for cerebrospinal fluid, plasma and brain, respectively. cis-pQTLs were more likely to be tissue shared, but trans-pQTLs tended to be tissue specific. Between 48.0% and 76.6% of pQTLs did not co-localize with expression, splicing, DNA methylation or histone acetylation QTLs. Using Mendelian randomization, we nominated proteins implicated in neurological diseases, including Alzheimer's disease, Parkinson's disease and stroke. This first multi-tissue study will be instrumental to map signals from genome-wide association studies onto functional genes, to discover pathways and to identify drug targets for neurological diseases.


Assuntos
Doença de Alzheimer , Encéfalo/metabolismo , Líquido Cefalorraquidiano/metabolismo , Plasma/metabolismo , Locos de Características Quantitativas , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Feminino , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma , Proteômica/métodos
6.
Acta Neuropathol Commun ; 8(1): 196, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33213513

RESUMO

Alpha-synuclein is the main protein component of Lewy bodies, the pathological hallmark of Parkinson's disease. However, genetic modifiers of cerebrospinal fluid (CSF) alpha-synuclein levels remain unknown. The use of CSF levels of amyloid beta1-42, total tau, and phosphorylated tau181 as quantitative traits in genetic studies have provided novel insights into Alzheimer's disease pathophysiology. A systematic study of the genomic architecture of CSF biomarkers in Parkinson's disease has not yet been conducted. Here, genome-wide association studies of CSF biomarker levels in a cohort of individuals with Parkinson's disease and controls (N = 1960) were performed. PD cases exhibited significantly lower CSF biomarker levels compared to controls. A SNP, proxy for APOE ε4, was associated with CSF amyloid beta1-42 levels (effect = - 0.5, p = 9.2 × 10-19). No genome-wide loci associated with CSF alpha-synuclein, total tau, or phosphorylated tau181 levels were identified in PD cohorts. Polygenic risk score constructed using the latest Parkinson's disease risk meta-analysis were associated with Parkinson's disease status (p = 0.035) and the genomic architecture of CSF amyloid beta1-42 (R2 = 2.29%; p = 2.5 × 10-11). Individuals with higher polygenic risk scores for PD risk presented with lower CSF amyloid beta1-42 levels (p = 7.3 × 10-04). Two-sample Mendelian Randomization revealed that CSF amyloid beta1-42 plays a role in Parkinson's disease (p = 1.4 × 10-05) and age at onset (p = 7.6 × 10-06), an effect mainly mediated by variants in the APOE locus. In a subset of PD samples, the APOE ε4 allele was associated with significantly lower levels of CSF amyloid beta1-42 (p = 3.8 × 10-06), higher mean cortical binding potentials (p = 5.8 × 10-08), and higher Braak amyloid beta score (p = 4.4 × 10-04). Together these results from high-throughput and hypothesis-free approaches converge on a genetic link between Parkinson's disease, CSF amyloid beta1-42, and APOE.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/genética , Encéfalo/metabolismo , Doença de Parkinson/genética , Fragmentos de Peptídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Apolipoproteína E4/genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Doença de Parkinson/metabolismo , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fosforilação , alfa-Sinucleína/líquido cefalorraquidiano , alfa-Sinucleína/metabolismo , Proteínas tau/líquido cefalorraquidiano , Proteínas tau/metabolismo
7.
J Exp Med ; 217(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32579671

RESUMO

TREM2 is a receptor for lipids expressed in microglia. The R47H variant of human TREM2 impairs ligand binding and increases Alzheimer's disease (AD) risk. In mouse models of amyloid ß (Aß) accumulation, defective TREM2 function affects microglial response to Aß plaques, exacerbating tissue damage, whereas TREM2 overexpression attenuates pathology. Thus, AD may benefit from TREM2 activation. Here, we examined the impact of an anti-human TREM2 agonistic mAb, AL002c, in a mouse AD model expressing either the common variant (CV) or the R47H variant of TREM2. Single-cell RNA-seq of microglia after acute systemic administration of AL002c showed induction of proliferation in both CV- and R47H-transgenic mice. Prolonged administration of AL002c reduced filamentous plaques and neurite dystrophy, impacted behavior, and tempered microglial inflammatory response. We further showed that a variant of AL002c is safe and well tolerated in a first-in-human phase I clinical trial and engages TREM2 based on cerebrospinal fluid biomarkers. We conclude that AL002 is a promising candidate for AD therapy.


Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Glicoproteínas de Membrana/metabolismo , Microglia/patologia , Receptores Imunológicos/metabolismo , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Ansiedade/patologia , Biomarcadores/líquido cefalorraquidiano , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Glicoproteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/patologia , Osteopontina/metabolismo , Conformação Proteica , Receptores Imunológicos/imunologia , Transdução de Sinais , Solubilidade
8.
Neurobiol Dis ; 143: 104962, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32535152

RESUMO

Recent human genetic studies have challenged long standing hypotheses about the chain of events in Alzheimer's disease (AD), as the identification of genetic risk factors in microglial genes supports a causative role for microglia in the disease. Parallel transcriptome and histology studies at the single-cell level revealed a rich palette of microglial states affected by disease status and genetic risk factors. Taken together, those findings support microglia dysfunction as a central mechanism in AD etiology and thus the therapeutic potential of modulating microglial activity for AD treatment. Here we review how human genetic studies discovered microglial AD risk genes, such as TREM2, CD33, MS4A and APOE, and how experimental studies are beginning to decipher the cellular functions of some of these genes. Our review also focuses on recent transcriptomic studies of human microglia from postmortem tissue to critically assess areas of similarity and dissimilarity between human and mouse models currently in use in order to better understand the biology of innate immunity in AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , Microglia/patologia , Animais , Predisposição Genética para Doença , Humanos
9.
Cell Syst ; 4(4): 404-415.e5, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28330615

RESUMO

Human age-associated traits, such as cognitive decline, can be highly variable across the population, with some individuals exhibiting traits that are not expected at a given chronological age. Here we present differential aging (Δ-aging), an unbiased method that quantifies individual variability in age-associated phenotypes within a tissue of interest, and apply this approach to the analysis of existing transcriptome-wide cerebral cortex gene expression data from several cohorts totaling 1,904 autopsied human brain samples. We subsequently performed a genome-wide association study and identified the TMEM106B and GRN gene loci, previously associated with frontotemporal dementia, as determinants of Δ-aging in the cerebral cortex with genome-wide significance. TMEM106B risk variants are associated with inflammation, neuronal loss, and cognitive deficits, even in the absence of known brain disease, and their impact is highly selective for the frontal cerebral cortex of older individuals (>65 years). The methodological framework we describe can be broadly applied to the analysis of quantitative traits associated with aging or with other parameters.


Assuntos
Envelhecimento/genética , Córtex Cerebral/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Progranulinas/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Envelhecimento/fisiologia , Córtex Cerebral/patologia , Estudos de Coortes , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imunidade Inata/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Degeneração Neural/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Fenótipo , Progranulinas/metabolismo , Progranulinas/fisiologia
11.
Nature ; 500(7460): 45-50, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23883936

RESUMO

Late-onset Alzheimer's disease (LOAD) risk is strongly influenced by genetic factors such as the presence of the apolipoprotein E ε4 allele (referred to here as APOE4), as well as non-genetic determinants including ageing. To pursue mechanisms by which these affect human brain physiology and modify LOAD risk, we initially analysed whole-transcriptome cerebral cortex gene expression data in unaffected APOE4 carriers and LOAD patients. APOE4 carrier status was associated with a consistent transcriptomic shift that broadly resembled the LOAD profile. Differential co-expression correlation network analysis of the APOE4 and LOAD transcriptomic changes identified a set of candidate core regulatory mediators. Several of these--including APBA2, FYN, RNF219 and SV2A--encode known or novel modulators of LOAD associated amyloid beta A4 precursor protein (APP) endocytosis and metabolism. Furthermore, a genetic variant within RNF219 was found to affect amyloid deposition in human brain and LOAD age-of-onset. These data implicate an APOE4 associated molecular pathway that promotes LOAD.


Assuntos
Doença de Alzheimer/genética , Apolipoproteína E4/genética , Genoma Humano/genética , Genômica , Idade de Início , Idoso , Alelos , Doença de Alzheimer/epidemiologia , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Endocitose , Epistasia Genética , Feminino , Fibroblastos , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Levetiracetam , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Piracetam/análogos & derivados , Piracetam/farmacologia , Polimorfismo Genético/genética , Proteólise/efeitos dos fármacos , Transcriptoma/genética
12.
Neuron ; 77(3): 425-39, 2013 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-23395371

RESUMO

Recent genome-wide association studies have linked common variants in the human genome to Parkinson's disease (PD) risk. Here we show that the consequences of variants at 2 such loci, PARK16 and LRRK2, are highly interrelated, both in terms of their broad impacts on human brain transcriptomes of unaffected carriers, and in terms of their associations with PD risk. Deficiency of the PARK16 locus gene RAB7L1 in primary rodent neurons, or of a RAB7L1 ortholog in Drosophila dopamine neurons, recapitulated degeneration observed with expression of a familial PD mutant form of LRRK2, whereas RAB7L1 overexpression rescued the LRRK2 mutant phenotypes. PD-associated defects in RAB7L1 or LRRK2 led to endolysosomal and Golgi apparatus sorting defects and deficiency of the VPS35 component of the retromer complex. Expression of wild-type VPS35, but not a familial PD-associated mutant form, rescued these defects. Taken together, these studies implicate retromer and lysosomal pathway alterations in PD risk.


Assuntos
Córtex Cerebral/patologia , Neurônios/metabolismo , Doença de Parkinson , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Drosophila , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Polimorfismo de Nucleotídeo Único/genética , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/genética , Ratos , Ratos Sprague-Dawley , Estatística como Assunto , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Transfecção , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Tirosina 3-Mono-Oxigenase , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
13.
Nat Commun ; 3: 1084, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23011138

RESUMO

α-Synuclein is implicated both in physiological functions at neuronal synaptic terminals as well as in pathological processes in the context of Parkinson's disease. However, the molecular mechanisms for these apparently diverse roles are unclear. Here we show that specific RNA transcript isoforms of α-synuclein with an extended 3' untranslated region, termed aSynL, appear selectively linked to pathological processes, relative to shorter α-synuclein transcripts. Common variants in the aSynL 3' untranslated region associated with Parkinson's disease risk promote the accumulation and translation of aSynL transcripts. The presence of intracellular dopamine can further enhance the relative abundance of aSynL transcripts through alternative polyadenylation site selection. We demonstrate that the presence of the extended aSynL transcript 3' untranslated region impacts accumulation of α-synuclein protein, which appears redirected away from synaptic terminals and towards mitochondria, reminiscent of Parkinson's disease pathology. Taken together, these findings identify a novel mechanism for aSyn regulation in the context of Parkinson's disease-associated genetic and environmental variations.


Assuntos
Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Dopamina/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Cell ; 146(3): 359-71, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21816272

RESUMO

Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aß, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.


Assuntos
Doença de Alzheimer/patologia , Transdiferenciação Celular , Medicina Regenerativa/métodos , Pele/citologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Células Cultivadas , Fibroblastos/citologia , Humanos , Neurônios/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo
15.
Drug Metab Lett ; 5(2): 73-84, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21457135

RESUMO

The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.


Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Flavonoides/metabolismo , Acetilcisteína/metabolismo , Animais , Indução Enzimática , Epóxido Hidrolases/fisiologia , Feminino , Hidroxilação , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo
16.
Biochim Biophys Acta ; 1790(4): 219-30, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19714863

RESUMO

BACKGROUND: We recently reported an efficient formulation of siRNA targeting TNF-alpha, that was able to restore immunological balance in a mouse arthritis model following intravenous injection. METHOD: Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect. RESULTS: We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate. CONCLUSIONS: These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.


Assuntos
DNA/química , Portadores de Fármacos/química , Vetores Genéticos , Lipídeos/química , Lipossomos/química , RNA Interferente Pequeno/química , Transfecção/métodos , Animais , Fenômenos Químicos , DNA/genética , Estabilidade de Medicamentos , Camundongos , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas
17.
J Neurosci Res ; 86(15): 3285-94, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18646207

RESUMO

Sly disease (MPS VII) is an autosomal-recessive lysosomal storage disorder resulting from beta-glucuronidase deficiency, which is characterized by a severe neurological impairment. MPS VII mice accumulate undegraded glycosaminoglycans and mimic the human neurodegenerative disorder, thus appearing to be an excellent tool to delineate disease pathogenesis. The relationship between abnormal glycosaminoglycan storage and neurodysfunction is not yet well understood, but inflammatory components can be involved, as in several neurological lysosomal disorders. Inflammatory biomarkers are thus good candidates to evaluate the neurodegeneration state of the disease. By using quantitative polymerase chain reaction, we have compared the expression of selected genes of normal and MPS VII cerebral tissues, focusing on inflammation and apoptosis-related genes. The gene expression was evaluated in various brain regions throughout the lifetime of the animals. We have identified a specific expression profile for 27 genes, which was strongly marked in the central nervous system posterior region. Finally, new Sly disease markers were characterized that reflect neurological deterioration state, and that can be used in preclinical follow-up studies.


Assuntos
Biomarcadores/análise , Expressão Gênica , Mucopolissacaridose VII/genética , Degeneração Neural/genética , Animais , Apoptose/fisiologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Imuno-Histoquímica , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Mucopolissacaridose VII/imunologia , Mucopolissacaridose VII/patologia , Degeneração Neural/imunologia , Degeneração Neural/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
BMC Mol Biol ; 9: 62, 2008 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-18611280

RESUMO

BACKGROUND: Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models. RESULTS: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0-48 h post-trauma (PT)) of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of beta-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), beta-microtubulin and S100beta were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less: by using geNorm VBA applet, we obtained the following sequence: cDNA(Oligreen); GAPDH > 18S rRNA > S100beta > beta-microtubulin > beta-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen) > S100beta > 18S rRNA > beta-actin > beta-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen) > 18S rRNA; GAPDH > S100beta > beta-microtubulin > beta-actin. CONCLUSION: This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that beta-actin and beta-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted. Pros and cons of the three methods of normalization factors selection are discussed. A generic time- and cost-effective procedure for normalization factor validation is proposed.


Assuntos
Lesões Encefálicas/genética , Perfilação da Expressão Gênica/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Actinas/genética , Animais , DNA Complementar/análise , DNA Complementar/normas , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Camundongos , Modelos Animais , Fatores de Crescimento Neural/genética , RNA Ribossômico 18S/genética , Padrões de Referência , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/genética , Tubulina (Proteína)/genética , Estudos de Validação como Assunto
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