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1.
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
2.
Nat Chem Biol ; 14(12): 1068-1069, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30323218

Assuntos
Splicing de RNA , Éxons
3.
FASEB J ; 27(5): 1820-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23325320

RESUMO

Most neurodegenerative diseases are linked to aberrant accumulation of aggregation-prone proteins. Among them, Huntington's disease (HD) is caused by an expanded polyglutamine repeat stretch in the N terminus of the mutant huntingtin protein (mHTT), which gets cleaved and aggregates in the brain. Recently established human induced pluripotent stem cell-derived HD neurons exhibit some disease-relevant phenotypes and provide tools for HD research. However, they have limitations such as genetic heterogeneity and an absence of mHTT aggregates and lack a robust neurodegeneration phenotype. In addition, the relationship between the phenotype and mHTT levels has not been elucidated. Herein, we present a human embryonic stem cell (hESC)-derived HD neuronal model expressing HTTexon1 fragments, which addresses the deficiencies enumerated above. The wild-type and HD lines are derived from an isogenic background and exhibit insoluble mHTT aggregates and neurodegeneration. We also demonstrate a quantitative relationship between neurodegeneration and soluble monomeric (but not oligomeric or aggregated) mHTT levels. Reduction of ~10% of mHTT is sufficient to prevent toxicity, whereas ~90% reduction of wild-type HTT is safe and well-tolerated in these cells. A known HD toxicity modifier (Rhes) showed expected rescue of neurodegeneration. Therefore, the hESC-derived neuronal models complement existing induced pluripotent stem cell-derived neuronal models and provide valuable tools for HD research.


Assuntos
Células-Tronco Embrionárias/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/genética , Linhagem Celular , Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Modelos Biológicos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Transfecção
6.
Nat Commun ; 10(1): 137, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635584

RESUMO

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Splicing de RNA/efeitos dos fármacos , Proteína bcl-X/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Sinergismo Farmacológico , Compostos de Epóxi/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Macrolídeos/farmacologia , Melanoma/genética , Camundongos , Camundongos Nus , Interferência de RNA , Splicing de RNA/genética , RNA Interferente Pequeno/genética , Spliceossomos/efeitos dos fármacos , Spliceossomos/genética , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Cell Rep ; 23(1): 282-296.e4, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29617667

RESUMO

Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA), and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like), or hotspot mutation profile (oncogene-like). Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis.


Assuntos
Taxa de Mutação , Neoplasias/genética , Fatores de Processamento de RNA/genética , Linhagem Celular Tumoral , Genes Supressores de Tumor , Humanos , Mutação com Perda de Função , Neoplasias/classificação , Oncogenes , Splicing de RNA/genética
8.
Cell Syst ; 7(1): 28-40.e4, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-29936182

RESUMO

Discriminating transcriptional changes that drive disease pathogenesis from nonpathogenic and compensatory responses is a daunting challenge. This is particularly true for neurodegenerative diseases, which affect the expression of thousands of genes in different brain regions at different disease stages. Here we integrate functional testing and network approaches to analyze previously reported transcriptional alterations in the brains of Huntington disease (HD) patients. We selected 312 genes whose expression is dysregulated both in HD patients and in HD mice and then replicated and/or antagonized each alteration in a Drosophila HD model. High-throughput behavioral testing in this model and controls revealed that transcriptional changes in synaptic biology and calcium signaling are compensatory, whereas alterations involving the actin cytoskeleton and inflammation drive disease. Knockdown of disease-driving genes in HD patient-derived cells lowered mutant Huntingtin levels and activated macroautophagy, suggesting a mechanism for mitigating pathogenesis. Our multilayered approach can thus untangle the wealth of information generated by transcriptomics and identify early therapeutic intervention points.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Doença de Huntington/genética , Animais , Encéfalo/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Doença de Huntington/fisiopatologia , Células-Tronco Pluripotentes Induzidas , Masculino , Transcriptoma/genética
9.
Cancer Cell ; 34(2): 225-241.e8, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30107174

RESUMO

Mutations affecting RNA splicing factors are the most common genetic alterations in myelodysplastic syndrome (MDS) patients and occur in a mutually exclusive manner. The basis for the mutual exclusivity of these mutations and how they contribute to MDS is not well understood. Here we report that although different spliceosome gene mutations impart distinct effects on splicing, they are negatively selected for when co-expressed due to aberrant splicing and downregulation of regulators of hematopoietic stem cell survival and quiescence. In addition to this synthetic lethal interaction, mutations in the splicing factors SF3B1 and SRSF2 share convergent effects on aberrant splicing of mRNAs that promote nuclear factor κB signaling. These data identify shared consequences of splicing-factor mutations and the basis for their mutual exclusivity.


Assuntos
Mutação , Neoplasias/genética , Spliceossomos , Animais , Caspase 8/genética , Feminino , Hematopoese , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética
10.
Nat Commun ; 8: 15522, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28541300

RESUMO

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome Bact complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A-SF3B1 forms a central node for binding to these splicing modulators.


Assuntos
Adenosina/química , Processamento Alternativo , Proteínas de Transporte/química , Fosfoproteínas/química , Fatores de Processamento de RNA/química , Proliferação de Células , Sobrevivência Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Compostos de Epóxi/química , Éxons , Álcoois Graxos/química , Células HCT116 , Humanos , Íntrons , Macrolídeos/química , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Piranos/química , Interferência de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Análise de Sequência de RNA , Compostos de Espiro/química , Spliceossomos/metabolismo , Transativadores
11.
Nat Med ; 22(6): 672-8, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27135740

RESUMO

Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition. Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 (Srsf2(P95H))-which commonly occurs in individuals with MDS and AML-in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 (refs. 7,8) resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML.


Assuntos
Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Fatores de Processamento de Serina-Arginina/genética , Spliceossomos/genética , Anemia Aplástica/genética , Animais , Doenças da Medula Óssea/genética , Transtornos da Insuficiência da Medula Óssea , Transplante de Medula Óssea , Catálise , Linhagem Celular Tumoral , Compostos de Epóxi/farmacologia , Citometria de Fluxo , Técnicas de Introdução de Genes , Hemizigoto , Hemoglobinúria Paroxística/genética , Humanos , Macrolídeos/farmacologia , Camundongos , Camundongos Knockout , Mutação , Transplante de Neoplasias , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Elife ; 42015 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-25738228

RESUMO

Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.


Assuntos
Corpo Estriado/metabolismo , Doença de Huntington/metabolismo , Íntrons , Receptores Acoplados a Proteínas G/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Células Cultivadas , Corpo Estriado/citologia , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Feminino , Humanos , Doença de Huntington/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Atividade Motora/genética , Atividade Motora/fisiologia , Mutação , Desempenho Psicomotor/fisiologia , Interferência de RNA , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética
13.
Trends Pharmacol Sci ; 35(2): 53-62, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24388390

RESUMO

Classical targeted drug discovery is based on targeting druggable targets, typically kinases and receptors of which the function can be agonized or antagonized. This strategy meets difficulties in cases such as Huntington's disease (HD) and similar neurodegenerative disorders, where the pathological function of the protein causing the disease is not clear. HD is caused by mutant HTT protein (mHTT) containing an expanded polyglutamine (polyQ) stretch, but the function of mHTT and how mHTT causes HD are unknown, thus preventing efforts to screen for mHTT 'inhibitors'. However, HD is appealing for drug discovery because the genetic mutation is clear, as compared with other major neurodegenerative disorders. Although mHTT is not a conventional 'druggable' target, one approach that appears promising is lowering its level, which might be applicable to other neurodegenerative disorders and proteinopathies linked to aberrant accumulation of proteins. Here we review mHTT lowering strategies that might provide promising avenues for drugging such diseases.


Assuntos
Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Animais , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
14.
Nat Neurosci ; 16(5): 562-70, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23525043

RESUMO

Huntington's disease is caused by expanded CAG repeats in HTT, conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model, and 6 exhibited activity consistent with the in vitro screening results. Among these, negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment, interferon-ß lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus, we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease.


Assuntos
Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteínas Culina/metabolismo , Modelos Animais de Doenças , Drosophila/efeitos dos fármacos , Drosophila/metabolismo , Embrião de Mamíferos , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Proteína Huntingtina , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína NEDD8 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/toxicidade , Neurônios/efeitos dos fármacos , Gravidez , Fatores de Transcrição/genética , Ubiquitinas/metabolismo
16.
Mol Neurodegener ; 5: 58, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21156064

RESUMO

BACKGROUND: The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. RESULTS: Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. CONCLUSION: We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified.

17.
J Neurochem ; 97(6): 1585-99, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16805770

RESUMO

While transplanted neural stem cells (NSCs) have been shown to hold promise for cell replacement in models of a number of neurological disorders, these examples have typically been under conditions where the host cells become dysfunctional due to a cell autonomous etiology, i.e. a 'sick' cell within a relatively supportive environment. It has long been held that cell replacement in a toxic milieu would not likely be possible; donor cells would succumb in much the same way as endogenous cells had. Many metabolic diseases are characterized by this situation, suggesting that they would be poor targets for cell replacement therapies. On the other hand, models of such diseases could prove ideal for testing the capacity for cell replacement under such challenging conditions. In the twitcher (twi ) mouse -- as in patients with Krabbe or globoid cell leukodystrophy (GLD), for which it serves as an authentic model -- loss of galactocerebrosidase (GalC) activity results in the accumulation of psychosine, a toxic glycolipid. Twi mice, like children with GLD, exhibit inexorable neurological deterioration presumably as a result of dysfunctional and ultimately degenerated oligodendrocytes with loss of myelin. It is believed that GLD pathophysiology is related to a psychosine-filled environment that kills not only host oligodendrocytes but theoretically any new cells placed into that milieu. Through the implantation of NSCs into the brains of both neonatal and juvenile/young adult twi mice, we have determined that widespread oligodendrocyte replacement and remyelination is feasible. NSCs appear to be intrinsically resistant to psychosine -- more so in their undifferentiated state than when directed ex vivo to become oligodendrocytes. This resistance can be enhanced by engineering the NSCs to over-express GalC. Some twi mice grafted with such engineered NSCs had thicker white tracts and lived 2-3 times longer than expected. While their brains had detectable levels of GalC, it was probably more significant that their psychosine levels were lower than in twi mice that died at a younger age. This concept of resistance based on differentiation state extended to human NSCs which could similarly survive within the twi brain. Taken together, these results suggest a number of points regarding cellular therapies against degenerative diseases with a prominent cell non-autonomous component: Cell replacement is possible if cells resistant to the toxic environment are employed. Furthermore, an important aspect of successful treatment will likely be not only cell replacement but also cross-correction of host cells to provide them with enzyme activity and hence resistance. While oligodendrocyte replacement alone was not a sufficient treatment for GLD (even when extensive), the replacement of both cells and molecules -- e.g. with NSCs that could both become oligodendrocytes and 'pumps' for GalC -- emerges as a promising basis for a multidisciplinary strategy. Most neurological disease are complex in this way and will likely require multifaceted approaches, perhaps with NSCs serving as the 'glue'.


Assuntos
Galactosilceramidase/biossíntese , Leucodistrofia de Células Globoides/cirurgia , Neurônios/fisiologia , Transplante de Células-Tronco/métodos , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Galactosilceramidase/deficiência , Terapia Genética/métodos , Humanos , Imuno-Histoquímica , Leucodistrofia de Células Globoides/patologia , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Transmissão/métodos , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Neurônios/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/fisiologia , Psicosina/toxicidade , Células-Tronco/efeitos dos fármacos , Transdução Genética/métodos
18.
J Biol Chem ; 279(18): 18614-22, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-14985362

RESUMO

Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.


Assuntos
Mitocôndrias/fisiologia , Doenças Mitocondriais/genética , Estresse Oxidativo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Antioxidantes , Gânglios da Base/ultraestrutura , Respiração Celular , Camundongos , Camundongos Knockout , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/análise , Fosforilação Oxidativa , Doença de Parkinson/etiologia , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio , Ubiquitina-Proteína Ligases/genética , Aumento de Peso
19.
J Biol Chem ; 278(44): 43628-35, 2003 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-12930822

RESUMO

Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin-/- mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin-/- mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin-/- mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin-/- mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and alpha-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin-/- brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice.


Assuntos
Encéfalo/patologia , Dopamina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologia , Alelos , Animais , Comportamento Animal , Western Blotting , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Eletrofisiologia , Mutação em Linhagem Germinativa , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Neurônios/metabolismo , Doença de Parkinson/genética , Receptores Dopaminérgicos/metabolismo , Substância Negra/metabolismo , Fatores de Tempo
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