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1.
Biol Open ; 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34528068

RESUMO

Branaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects; we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMN▵7), and in juvenile Wistar Hanover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

2.
Lancet Neurol ; 20(9): 739-752, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34418401

RESUMO

BACKGROUND: Plasma tau phosphorylated at threonine 217 (p-tau217) and plasma tau phosphorylated at threonine 181 (p-tau181) are associated with Alzheimer's disease tau pathology. We compared the diagnostic value of both biomarkers in cognitively unimpaired participants and patients with a clinical diagnosis of mild cognitive impairment, Alzheimer's disease syndromes, or frontotemporal lobar degeneration (FTLD) syndromes. METHODS: In this retrospective multicohort diagnostic performance study, we analysed plasma samples, obtained from patients aged 18-99 years old who had been diagnosed with Alzheimer's disease syndromes (Alzheimer's disease dementia, logopenic variant primary progressive aphasia, or posterior cortical atrophy), FTLD syndromes (corticobasal syndrome, progressive supranuclear palsy, behavioural variant frontotemporal dementia, non-fluent variant primary progressive aphasia, or semantic variant primary progressive aphasia), or mild cognitive impairment; the participants were from the University of California San Francisco (UCSF) Memory and Aging Center, San Francisco, CA, USA, and the Advancing Research and Treatment for Frontotemporal Lobar Degeneration Consortium (ARTFL; 17 sites in the USA and two in Canada). Participants from both cohorts were carefully characterised, including assessments of CSF p-tau181, amyloid-PET or tau-PET (or both), and clinical and cognitive evaluations. Plasma p-tau181 and p-tau217 were measured using electrochemiluminescence-based assays, which differed only in the biotinylated antibody epitope specificity. Receiver operating characteristic analyses were used to determine diagnostic accuracy of both plasma markers using clinical diagnosis, neuropathological findings, and amyloid-PET and tau-PET measures as gold standards. Difference between two area under the curve (AUC) analyses were tested with the Delong test. FINDINGS: Data were collected from 593 participants (443 from UCSF and 150 from ARTFL, mean age 64 years [SD 13], 294 [50%] women) between July 1 and Nov 30, 2020. Plasma p-tau217 and p-tau181 were correlated (r=0·90, p<0·0001). Both p-tau217 and p-tau181 concentrations were increased in people with Alzheimer's disease syndromes (n=75, mean age 65 years [SD 10]) relative to cognitively unimpaired controls (n=118, mean age 61 years [SD 18]; AUC=0·98 [95% CI 0·95-1·00] for p-tau217, AUC=0·97 [0·94-0·99] for p-tau181; pdiff=0·31) and in pathology-confirmed Alzheimer's disease (n=15, mean age 73 years [SD 12]) versus pathologically confirmed FTLD (n=68, mean age 67 years [SD 8]; AUC=0·96 [0·92-1·00] for p-tau217, AUC=0·91 [0·82-1·00] for p-tau181; pdiff=0·22). P-tau217 outperformed p-tau181 in differentiating patients with Alzheimer's disease syndromes (n=75) from those with FTLD syndromes (n=274, mean age 67 years [SD 9]; AUC=0·93 [0·91-0·96] for p-tau217, AUC=0·91 [0·88-0·94] for p-tau181; pdiff=0·01). P-tau217 was a stronger indicator of amyloid-PET positivity (n=146, AUC=0·91 [0·88-0·94]) than was p-tau181 (n=214, AUC=0·89 [0·86-0·93]; pdiff=0·049). Tau-PET binding in the temporal cortex was more strongly associated with p-tau217 than p-tau181 (r=0·80 vs r=0·72; pdiff<0·0001, n=230). INTERPRETATION: Both p-tau217 and p-tau181 had excellent diagnostic performance for differentiating patients with Alzheimer's disease syndromes from other neurodegenerative disorders. There was some evidence in favour of p-tau217 compared with p-tau181 for differential diagnosis of Alzheimer's disease syndromes versus FTLD syndromes, as an indication of amyloid-PET-positivity, and for stronger correlations with tau-PET signal. Pending replication in independent, diverse, and older cohorts, plasma p-tau217 and p-tau181 could be useful screening tools to identify individuals with underlying amyloid and Alzheimer's disease tau pathology. FUNDING: US National Institutes of Health, State of California Department of Health Services, Rainwater Charitable Foundation, Michael J Fox foundation, Association for Frontotemporal Degeneration, Alzheimer's Association.


Assuntos
Doença de Alzheimer/diagnóstico , Disfunção Cognitiva/diagnóstico , Degeneração Lobar Frontotemporal/diagnóstico , Proteínas tau/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Biomarcadores/sangue , Disfunção Cognitiva/sangue , Disfunção Cognitiva/líquido cefalorraquidiano , Diagnóstico Diferencial , Feminino , Degeneração Lobar Frontotemporal/sangue , Degeneração Lobar Frontotemporal/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/fisiologia , Tomografia por Emissão de Pósitrons , Valor Preditivo dos Testes , Estudos Retrospectivos , Proteínas tau/sangue , Proteínas tau/líquido cefalorraquidiano
3.
Nature ; 596(7871): 291-295, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321659

RESUMO

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Edição de Genes/métodos , Terapia Genética/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Eritropoetina/biossíntese , Eritropoetina/genética , Eritropoetina/metabolismo , Éxons/genética , Feminino , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular Espinal/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Progranulinas/biossíntese , Progranulinas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
4.
Neurology ; 96(18): e2296-e2312, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33827960

RESUMO

OBJECTIVE: We tested the hypothesis that plasma neurofilament light chain (NfL) identifies asymptomatic carriers of familial frontotemporal lobar degeneration (FTLD)-causing mutations at risk of disease progression. METHODS: Baseline plasma NfL concentrations were measured with single-molecule array in original (n = 277) and validation (n = 297) cohorts. C9orf72, GRN, and MAPT mutation carriers and noncarriers from the same families were classified by disease severity (asymptomatic, prodromal, and full phenotype) using the CDR Dementia Staging Instrument plus behavior and language domains from the National Alzheimer's Disease Coordinating Center FTLD module (CDR+NACC-FTLD). Linear mixed-effect models related NfL to clinical variables. RESULTS: In both cohorts, baseline NfL was higher in asymptomatic mutation carriers who showed phenoconversion or disease progression compared to nonprogressors (original: 11.4 ± 7 pg/mL vs 6.7 ± 5 pg/mL, p = 0.002; validation: 14.1 ± 12 pg/mL vs 8.7 ± 6 pg/mL, p = 0.035). Plasma NfL discriminated symptomatic from asymptomatic mutation carriers or those with prodromal disease (original cutoff: 13.6 pg/mL, 87.5% sensitivity, 82.7% specificity; validation cutoff: 19.8 pg/mL, 87.4% sensitivity, 84.3% specificity). Higher baseline NfL correlated with worse longitudinal CDR+NACC-FTLD sum of boxes scores, neuropsychological function, and atrophy, regardless of genotype or disease severity, including asymptomatic mutation carriers. CONCLUSIONS: Plasma NfL identifies asymptomatic carriers of FTLD-causing mutations at short-term risk of disease progression and is a potential tool to select participants for prevention clinical trials. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT02372773 and NCT02365922. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that in carriers of FTLD-causing mutations, elevation of plasma NfL predicts short-term risk of clinical progression.


Assuntos
Progressão da Doença , Degeneração Lobar Frontotemporal/sangue , Degeneração Lobar Frontotemporal/diagnóstico por imagem , Proteínas de Neurofilamentos/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Imageamento por Ressonância Magnética/tendências , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Adulto Jovem
5.
J Med Chem ; 64(8): 4744-4761, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33822618

RESUMO

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from a deletion or loss of function mutation of the survival motor neuron 1 (SMN1) gene. Branaplam (1) elevates levels of full-length SMN protein in vivo by modulating the splicing of the related gene SMN2 to enhance the exon-7 inclusion and increase levels of the SMN. The intramolecular hydrogen bond present in the 2-hydroxyphenyl pyridazine core of 1 enforces a planar conformation of the biaryl system and is critical for the compound activity. Scaffold morphing revealed that the pyridazine could be replaced by a 1,3,4-thiadiazole, which provided additional opportunities for a conformational constraint of the biaryl through intramolecular 1,5-sulfur-oxygen (S···O) or 1,5-sulfur-halogen (S···X) noncovalent interactions. Compound 26, which incorporates a 2-fluorophenyl thiadiazole motif, demonstrated a greater than 50% increase in production of full-length SMN protein in a mouse model of SMA.


Assuntos
Desenho de Fármacos , Splicing de RNA , Tiadiazóis/química , Animais , Meia-Vida , Halogênios/química , Humanos , Masculino , Camundongos , Conformação Molecular , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Oxigênio/química , Piridazinas/química , Splicing de RNA/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Enxofre/química , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Tiadiazóis/metabolismo , Tiadiazóis/farmacologia
6.
Neurology ; 96(5): e671-e683, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33199433

RESUMO

OBJECTIVE: To test the hypothesis that plasma total tau (t-tau) and neurofilament light chain (NfL) concentrations may have a differential role in the study of frontotemporal lobar degeneration syndromes (FTLD-S) and clinically diagnosed Alzheimer disease syndromes (AD-S), we determined their diagnostic and prognostic value in FTLD-S and AD-S and their sensitivity to pathologic diagnoses. METHODS: We measured plasma t-tau and NfL with the Simoa platform in 265 participants: 167 FTLD-S, 43 AD-S, and 55 healthy controls (HC), including 82 pathology-proven cases (50 FTLD-tau, 18 FTLD-TDP, 2 FTLD-FUS, and 12 AD) and 98 participants with amyloid PET. We compared cross-sectional and longitudinal biomarker concentrations between groups, their correlation with clinical measures of disease severity, progression, and survival, and cortical thickness. RESULTS: Plasma NfL, but not plasma t-tau, discriminated FTLD-S from HC and AD-S from HC. Both plasma NfL and t-tau were poor discriminators between FLTD-S and AD-S. In pathology-confirmed cases, plasma NfL was higher in FTLD than AD and in FTLD-TDP compared to FTLD-tau, after accounting for age and disease severity. Plasma NfL, but not plasma t-tau, predicted clinical decline and survival and correlated with regional cortical thickness in both FTLD-S and AD-S. The combination of plasma NfL with plasma t-tau did not outperform plasma NfL alone. CONCLUSION: Plasma NfL is superior to plasma t-tau for the diagnosis and prediction of clinical progression of FTLD-S and AD-S. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that plasma NfL has superior diagnostic and prognostic performance vs plasma t-tau in FTLD and AD.


Assuntos
Doença de Alzheimer/sangue , Degeneração Lobar Frontotemporal/sangue , Proteínas de Neurofilamentos/sangue , Proteínas tau/sangue , Adulto , Idoso , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Degeneração Lobar Frontotemporal/diagnóstico , Degeneração Lobar Frontotemporal/diagnóstico por imagem , Degeneração Lobar Frontotemporal/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Tomografia por Emissão de Pósitrons , Proteína FUS de Ligação a RNA/metabolismo , Sensibilidade e Especificidade , Taxa de Sobrevida , Proteínas tau/metabolismo
7.
J Med Chem ; 61(24): 11021-11036, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30407821

RESUMO

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.


Assuntos
Encéfalo/efeitos dos fármacos , Canal de Potássio ERG1/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Piridazinas/química , Administração Oral , Animais , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1/antagonistas & inibidores , Humanos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/genética , Piridazinas/farmacologia , Relação Quantitativa Estrutura-Atividade , Splicing de RNA , Ratos Sprague-Dawley , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética
8.
Science ; 358(6370): 1617-1622, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29192133

RESUMO

The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.


Assuntos
Cromatina/metabolismo , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Ativação Transcricional , Fatores de Elongação da Transcrição/síntese química , Fatores de Elongação da Transcrição/metabolismo , Inativação Gênica , Humanos , RNA Polimerase II/metabolismo , Transcrição Genética
11.
Nat Chem Biol ; 11(7): 511-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26030728

RESUMO

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.


Assuntos
Processamento Alternativo , Atrofia Muscular Espinal/tratamento farmacológico , RNA de Cadeia Dupla/agonistas , Ribonucleoproteína Nuclear Pequena U1/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/mortalidade , Atrofia Muscular Espinal/patologia , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteólise , Precursores de RNA/agonistas , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismo , Análise de Sobrevida , Proteína 2 de Sobrevivência do Neurônio Motor/química , Proteína 2 de Sobrevivência do Neurônio Motor/genética
12.
Proc Natl Acad Sci U S A ; 110(26): E2371-80, 2013 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23757500

RESUMO

The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA.


Assuntos
Proteínas de Drosophila/genética , Genes de Insetos , Proteínas de Ligação a RNA/genética , Animais , Animais Geneticamente Modificados , Redes Reguladoras de Genes , Humanos , Bases de Conhecimento , Junção Neuromuscular/genética , Fenótipo , Interferência de RNA , Especificidade da Espécie , Atrofias Musculares Espinais da Infância/genética
13.
PLoS One ; 4(4): e5197, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19381295

RESUMO

BACKGROUND: Lipid metabolism in mammals is orchestrated by a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control the expression of genes required for the uptake and synthesis of cholesterol, fatty acids, and triglycerides. SREBPs are thus essential for insulin-induced lipogenesis and for cellular membrane homeostasis and biogenesis. Although multiple players have been identified that control the expression and activation of SREBPs, gaps remain in our understanding of how SREBPs are coordinated with other physiological pathways. METHODOLOGY: To identify novel regulators of SREBPs, we performed a genome-wide cDNA over-expression screen to identify proteins that might modulate the transcription of a luciferase gene driven from an SREBP-specific promoter. The results were verified through secondary biological assays and expression data were analyzed by a novel application of the Gene Set Enrichment Analysis (GSEA) method. CONCLUSIONS/SIGNIFICANCE: We screened 10,000 different cDNAs and identified a number of genes and pathways that have previously not been implicated in SREBP control and cellular cholesterol homeostasis. These findings further our understanding of lipid biology and should lead to new insights into lipid associated disorders.


Assuntos
Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Transcrição Genética , Sequência de Bases , Linhagem Celular , Colesterol/metabolismo , DNA Complementar , Homeostase , Humanos , Regiões Promotoras Genéticas , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo
14.
J Neurochem ; 102(4): 1151-61, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17488279

RESUMO

Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.


Assuntos
Astrócitos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Imunossupressores/farmacologia , Propilenoglicóis/farmacologia , Receptores de Lisoesfingolipídeo/fisiologia , Esfingosina/análogos & derivados , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Astrócitos/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Cloridrato de Fingolimode , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Fosfatos de Inositol/metabolismo , Técnicas de Cultura de Órgãos , Oxidiazóis/farmacologia , Ratos , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Esfingosina/farmacologia , Tiofenos/farmacologia , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia
15.
Nat Neurosci ; 7(3): 261-8, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14770187

RESUMO

Successful axon regeneration in the mammalian central nervous system (CNS) is at least partially compromised due to the inhibitors associated with myelin and glial scar. However, the intracellular signaling mechanisms underlying these inhibitory activities are largely unknown. Here we provide biochemical and functional evidence that conventional isoforms of protein kinase C (PKC) are key components in the signaling pathways that mediate the inhibitory activities of myelin components and chondroitin sulfate proteoglycans (CSPGs), the major class of inhibitors in the glial scar. Both the myelin inhibitors and CSPGs induce PKC activation. Blocking PKC activity pharmacologically and genetically attenuates the ability of CNS myelin and CSPGs to activate Rho and inhibit neurite outgrowth. Intrathecal infusion of a PKC inhibitor, Gö6976, into the site of dorsal hemisection promotes regeneration of dorsal column axons across and beyond the lesion site in adult rats. Thus, perturbing PKC activity could represent a therapeutic approach to stimulating axon regeneration after brain and spinal cord injuries.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cones de Crescimento/metabolismo , Proteínas da Mielina/metabolismo , Regeneração Nervosa/fisiologia , Proteína Quinase C/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/lesões , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/ultraestrutura , Proteínas da Mielina/farmacologia , Glicoproteína Associada a Mielina/metabolismo , Glicoproteína Associada a Mielina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Proteínas Nogo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Proteínas Repressoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Resultado do Tratamento
16.
Nature ; 420(6911): 74-8, 2002 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-12422217

RESUMO

In inhibiting neurite outgrowth, several myelin components, including the extracellular domain of Nogo-A (Nogo-66), oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG), exert their effects through the same Nogo receptor (NgR). The glycosyl phosphatidylinositol (GPI)-anchored nature of NgR indicates the requirement for additional transmembrane protein(s) to transduce the inhibitory signals into the interior of responding neurons. Here, we demonstrate that p75, a transmembrane protein known to be a receptor for the neurotrophin family of growth factors, specifically interacts with NgR. p75 is required for NgR-mediated signalling, as neurons from p75 knockout mice are no longer responsive to myelin and to each of the known NgR ligands. Blocking the p75-NgR interaction also reduces the activities of these inhibitors. Moreover, a truncated p75 protein lacking the intracellular domain, when overexpressed in primary neurons, attenuates the same set of inhibitory activities, suggesting that p75 is a signal transducer of the NgR-p75 receptor complex. Thus, interfering with p75 and its downstream signalling pathways may allow lesioned axons to overcome most of the inhibitory activities associated with central nervous system myelin.


Assuntos
Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Cricetinae , Proteínas Ligadas por GPI , Gânglios Espinais/citologia , Humanos , Camundongos , Camundongos Knockout , Proteínas da Mielina/química , Proteínas da Mielina/genética , Glicoproteína Mielina-Oligodendrócito , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Nogo , Receptor Nogo 1 , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/genética , Transdução de Sinais/efeitos dos fármacos
17.
Neuron ; 35(2): 283-90, 2002 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12160746

RESUMO

Myelin inhibitors of axonal regeneration, like Nogo and MAG, block regrowth after injury to the adult CNS. While a GPI-linked receptor for Nogo (NgR) has been identified, MAG's receptor is unknown. We show that MAG inhibits regeneration by interaction with NgR. Binding of and inhibition by MAG are lost if neuronal GPI-linked proteins are cleaved. Binding of MAG to NgR-expressing cells is GPI dependent and sialic acid independent. Conversely, NgR binds to MAG-expressing cells. MAG, but not a truncated MAG that binds neurons but does not inhibit regeneration, precipitates NgR from NgR-expressing cells, DRG, and cerebellar neurons. Importantly, NgR antibody, soluble NgR, or dominant-negative NgR each prevent inhibition of neurite outgrowth by MAG. Also, MAG and Nogo66 compete for binding to NgR. These results suggest redundancy in myelin inhibitors and indicate therapies for CNS injuries.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/lesões , Glicoproteína Associada a Mielina/deficiência , Fibras Nervosas Mielinizadas/metabolismo , Regeneração Nervosa/fisiologia , Neuritos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Animais Recém-Nascidos , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Sistema Nervoso Central/citologia , Cricetinae , Proteínas Ligadas por GPI , Glicosilfosfatidilinositóis/metabolismo , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Substâncias de Crescimento/metabolismo , Imuno-Histoquímica , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/farmacologia , Ácido N-Acetilneuramínico/metabolismo , Fibras Nervosas Mielinizadas/ultraestrutura , Neuritos/ultraestrutura , Receptor Nogo 1 , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/fisiologia , Ratos
18.
Nature ; 417(6892): 941-4, 2002 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-12068310

RESUMO

The inhibitory activity associated with myelin is a major obstacle for successful axon regeneration in the adult mammalian central nervous system (CNS). In addition to myelin-associated glycoprotein (MAG) and Nogo-A, available evidence suggests the existence of additional inhibitors in CNS myelin. We show here that a glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of neurite outgrowth in cultured neurons. Like Nogo-A, OMgp contributes significantly to the inhibitory activity associated with CNS myelin. To further elucidate the mechanisms that mediate this inhibitory activity of OMgp, we screened an expression library and identified the Nogo receptor (NgR) as a high-affinity OMgp-binding protein. Cleavage of NgR and other GPI-linked proteins from the cell surface renders axons of dorsal root ganglia insensitive to OMgp. Introduction of exogenous NgR confers OMgp responsiveness to otherwise insensitive neurons. Thus, OMgp is an important inhibitor of neurite outgrowth that acts through NgR and its associated receptor complex. Interfering with the OMgp/NgR pathway may allow lesioned axons to regenerate after injury in vivo.


Assuntos
Proteínas da Mielina , Glicoproteína Associada a Mielina/metabolismo , Neuritos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Células COS , Bovinos , Divisão Celular , Tamanho Celular , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Embrião de Galinha , Proteínas Ligadas por GPI , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Cones de Crescimento/metabolismo , Humanos , Ligantes , Camundongos , Bainha de Mielina/metabolismo , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Regeneração Nervosa , Neuritos/efeitos dos fármacos , Receptor Nogo 1 , Fosfatidilinositol Diacilglicerol-Liase , Testes de Precipitina , Ligação Proteica , Receptores de Superfície Celular/genética , Retina/citologia , Retina/efeitos dos fármacos , Retina/metabolismo , Fosfolipases Tipo C/metabolismo
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