Studies of Hereditary Neurological Disease: Disease Mechanisms

遗传性神经系统疾病的研究：疾病机制

• 批准号: 8557057
• 负责人: Kenneth Fischbeck
• 金额: $ 148.71万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557057
• 关键词: Acute; Address; Alleles; Amino Acyl-tRNA Synthetases; Androgen Receptor; Animal Model; Attenuated; Binding Proteins; Bioavailable; Blinded; Body Weight decreased; Caenorhabditis elegans; Cell Culture Techniques; Charcot-Marie-Tooth Disease; Clinical; Clinical Trials; Cultured Cells; Denervation; Disease; Disease Progression; Dose; Genes; Genetic; Genetic Complementation Test; Glutamine; Glycine-tRNA Ligase; Goals; Heterozygote; Histone Deacetylase Inhibitor; Histone deacetylase inhibition; Histones; Homozygote; Human; IGF1 gene; In Vitro; Inherited; Injection of therapeutic agent; Insulin-Like Growth Factor I; Intervention; Intraperitoneal Injections; Investigation; Kennedy Syndrome; Link; Mediating; Mind; Missense Mutation; Modeling; Motor; Motor Neuron Disease; Mus; Muscle; Muscle Proteins; Muscular Atrophy; Mutant Strains Mice; Mutation; Myogenin; Myopathy; Neurologic; Neuromuscular Diseases; Neuromuscular Junction; Neuropathy; Onset of illness; Orthologous Gene; Pathology; Pathway interactions; Patients; Performance; Peripheral; Peripheral Nervous System Diseases; Phenotype; Phosphorylation; Point Mutation; Proteasome Inhibition; Protein Deficiency; Protein Isoforms; Proteins; Randomized; Receptor Aggregation; Recombinant IGF-I; Recombinants; Research; Ring Finger Domain; Spinal Cord; Spinal Muscular Atrophy; System; Testing; Therapeutic Intervention; Tissues; Toxic effect; Transgenes; Transgenic Mice; Transgenic Organisms; Ubiquitin; behavior test; dosage; effective therapy; gain of function; glycine-tRNA; hereditary neuropathy; human HDAC4 protein; improved; knock-down; loss of function; mouse model; multicatalytic endopeptidase complex; mutant; nervous system disorder; neuromuscular function; new therapeutic target; overexpression; polyglutamine; programs; protein degradation; spinal and bulbar muscular atrophy; therapeutic target; ubiquitin-protein ligase; Acute; Address; Alleles; Amino Acyl-tRNA Synthetases; Androgen Receptor; Animal Model; Attenuated; Binding Proteins; Bioavailable; Blinded; Body Weight decreased; Caenorhabditis elegans; Cell Culture Techniques; Charcot-Marie-Tooth Disease; Clinical; Clinical Trials; Cultured Cells; Denervation; Disease; Disease Progression; Dose; Genes; Genetic; Genetic Complementation Test; Glutamine; Glycine-tRNA Ligase; Goals; Heterozygote; Histone Deacetylase Inhibitor; Histone deacetylase inhibition; Histones; Homozygote; Human; IGF1 gene; In Vitro; Inherited; Injection of therapeutic agent; Insulin-Like Growth Factor I; Intervention; Intraperitoneal Injections; Investigation; Kennedy Syndrome; Link; Mediating; Mind; Missense Mutation; Modeling; Motor; Motor Neuron Disease; Mus; Muscle; Muscle Proteins; Muscular Atrophy; Mutant Strains Mice; Mutation; Myogenin; Myopathy; Neurologic; Neuromuscular Diseases; Neuromuscular Junction; Neuropathy; Onset of illness; Orthologous Gene; Pathology; Pathway interactions; Patients; Performance; Peripheral; Peripheral Nervous System Diseases; Phenotype; Phosphorylation; Point Mutation; Proteasome Inhibition; Protein Deficiency; Protein Isoforms; Proteins; Randomized; Receptor Aggregation; Recombinant IGF-I; Recombinants; Research; Ring Finger Domain; Spinal Cord; Spinal Muscular Atrophy; System; Testing; Therapeutic Intervention; Tissues; Toxic effect; Transgenes; Transgenic Mice; Transgenic Organisms; Ubiquitin; behavior test; dosage; effective therapy; gain of function; glycine-tRNA; hereditary neuropathy; human HDAC4 protein; improved; knock-down; loss of function; mouse model; multicatalytic endopeptidase complex; mutant; nervous system disorder; neuromuscular function; new therapeutic target; overexpression; polyglutamine; programs; protein degradation; spinal and bulbar muscular atrophy; therapeutic target; ubiquitin-protein ligase

The purpose of this research program is to investigate the mechanisms of hereditary neurological diseases, with the ultimate intent of developing effective treatments for these disorders. Recently, the research has focused on two specific neuromuscular diseases: autosomal recessive spinal muscular atrophy (SMA) due to deficiency of the protein SMN, X-linked spinal and bulbar muscular atrophy (SBMA) due to polyglutamine expansion in the androgen receptor. Specific research accomplishments in the past year include the following: (1) Identification of an E3 ligase, mind bomb 1 (Mib1), responsible for the degradation of SMN protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy mice, suggesting that the UPS is a potential therapeutic target for this disease. While inhibiting the proteasome provides proof of concept that the UPS can be targeted to increase SMN protein levels, specific targets in this pathway may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, Mib1, interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. In addition, knocking down the Mib1 ortholog improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for spinal muscular atrophy. (2) Characterization of the effects of histone deacetylase inhibition in SMA muscle. During muscle atrophy, the E3 ligase atrogenes, atrogin-1 and muscle ring finger 1 (MuRF1), mediate muscle protein breakdown through the ubiquitin proteasome system. Atrogene expression can be induced by various upstream regulators. During acute denervation, they are activated by myogenin, which is in turn regulated by histone deacetylases 4 and 5. We showed that atrogenes are induced in SMA model mice and in SMA patient muscle in association with increased myogenin and histone deacetylase-4 (HDAC4) expression. This activation during both acute denervation and SMA disease progression is suppressed by treatment with a histone deacetylase inhibitor; however, this treatment has no effect when atrogene induction occurs independently of myogenin. These results indicate that myogenin-dependent atrogene induction is amenable to pharmacological intervention with histone deacetylase inhibitors and help to explain the beneficial effects of these agents on SMA and other denervating diseases. (3) Characterization of the effects of IGF-1 in an animal model of SBMA. Our recent studies have demonstrated that IGF-1 reduces the mutant androgen receptor toxicity through activation of Akt in vitro, and spinal and bulbar muscular atrophy transgenic mice that also overexpress a non-circulating muscle isoform of IGF-1 have a less severe phenotype. Here we sought to establish the efficacy of daily intraperitoneal injections of mecasermin rinfabate, recombinant human IGF-1 and IGF-1 binding protein 3, in a transgenic mouse model expressing the mutant androgen receptor with an expanded 97 glutamine tract. The study was done in a controlled, randomized, blinded fashion, and in order to reflect the clinical settings the injections were started after the onset of disease manifestations. The treatment resulted in increased Akt phosphorylation and reduced mutant androgen receptor aggregation in muscle. In comparison to vehicle-treated controls, IGF-1 treated transgenic mice showed improved motor performance, attenuated weight loss, and increased survival. Our results suggest that peripheral tissue can be targeted to improve the spinal and bulbar muscular atrophy phenotype and indicate that IGF-1 warrants further investigation in clinical trials as a potential treatment for this disease. (4) Characterization of the mechanism of hereditary neuropathy due to mutation in glycine tRNA synthtase (GARS). Charcot-Marie-Tooth disease type 2D (CMT2D) is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS). In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein.

该研究计划的目的是研究遗传神经疾病的机制，最终是为这些疾病开发有效治疗的目的。最近，这项研究集中在两种特定的神经肌肉疾病上：由于蛋白质SMN，X连锁脊柱和鳞茎肌肉萎缩（SBMA）导致的常染色体隐性脊柱肌肉萎缩（SMA），这是由于雄激素受体中的多氯丁胺膨胀而引起的。过去一年的特定研究成就包括以下内容： （1）识别E3连接酶，Mind Bomb 1（MIB1），负责SMN蛋白的降解。 SMN通过泛素蛋白酶体系统（UPS）泛素化并降解。我们先前已经表明，蛋白酶体抑制可改善运动功能，并减少脊髓肌肉萎缩小鼠的脊髓，肌肉和神经肌肉连接病理学，这表明UPS是该疾病的潜在治疗靶标。虽然抑制蛋白酶体提供了可以将UPS靶向提高SMN蛋白水平的概念证明，但该途径中的特定靶标可能更有效，毒性较小。在这项研究中，我们表明E3泛素连接酶MIB1与SMN相互作用并泛素化并促进其降解。击倒MIB1水平会增加培养细胞中的SMN蛋白水平。此外，击倒MIB1直系同源物可以改善秀丽隐杆线虫缺乏SMN的神经肌肉功能。这些发现表明，MIB1泛素化并催化SMN的降解，因此代表了脊柱肌肉萎缩的新型治疗靶标。 （2）表征组蛋白脱乙酰基酶在SMA肌肉中的作用。在肌肉萎缩期间，E3连接酶垂体，Atrogin-1和肌肉环1（MURF1），通过泛素蛋白酶体系统介导肌肉蛋白分解。各种上游调节剂可以诱导阳性蛋白表达。在急性神经支配期间，它们被肌蛋白激活，而肌蛋白又受组蛋白脱乙酰基酶4和5的调节。我们表明，SMA模型小鼠和SMA患者肌肉中诱导了肌激素，与肌蛋白蛋白和组蛋白脱乙酰基酶-4（HDAC4）的表达相关。用组蛋白脱乙酰基酶抑制剂治疗急性神经疾病和SMA疾病进展过程中的这种激活均被抑制。但是，当抗蛋白独立于肌蛋白时，这种治疗方法无效。这些结果表明，组蛋白脱乙酰基酶抑制剂的药理学干预措施，依赖肌蛋白依赖蛋白诱导，并有助于解释这些药物对SMA和其他退化疾病的有益作用。 （3）表征IGF-1在SBMA动物模型中的作用。我们最近的研究表明，IGF-1通过在体外激活AKT以及脊柱和鳞茎肌肉萎缩性转基因小鼠降低了突变的雄激素受体毒性，该小鼠也过表达IGF-1的非循环肌肉同工型的IGF-1同工型的严重表型较低。在这里，我们试图建立每日腹膜内注射麦芽剂仁菜蛋白，重组人IGF-1和IGF-1结合蛋白3，在表达突变体雄激素受体的转基因小鼠模型中，并具有膨胀的97谷氨酰胺株。这项研究是以受控的，随机的，盲目的方式进行的，为了反映临床环境，疾病表现开始后开始注射。治疗导致肌肉中Akt磷酸化增加并降低突变体雄激素受体聚集。与媒介物处理的对照相比，IGF-1处理的转基因小鼠的运动性能提高，体重减轻减弱和生存率提高。我们的结果表明，可以针对外围组织来改善脊柱和鳞茎肌肉萎缩表型，并表明IGF-1值得在临床试验中进一步研究，以此作为该疾病的潜在治疗方法。 （4）表征由于甘氨酸TRNA合成酶突变（GARS）突变引起的遗传神经病机理。 Charcot-Marie-Tooth疾病型2D（CMT2D）是由糖基-TRNA合成酶基因（GARS）中的错义突变引起的主要遗传性周围神经病。除GARS外，尽管尚未完全了解潜在的机制，但其他三个TRNA合成酶基因中的突变引起了相似的神经病。为了解决这个问题，我们生成了过分表达野生型GARS的转基因小鼠，并将它们越过了CMT2D的两种主要小鼠模型，以区分功能丧失和功能获取机制。野生型GARS的过表达不能改善杂合子GARS突变小鼠中的神经病表型，这取决于组织学，功能和行为测试。转基因GARS能够作为纯合子或与GARS NULL等位基因进行互补测试来挽救病理点突变，从而证明了转基因的功能，并揭示了点突变的缺陷丧失功能分量。与杂合子相比，传染性突变作为转基因反应的纯合子或化合物杂合子的神经性更严重，这表明即使存在野生型转移元素，也会增加引起疾病的等位基因剂量的剂量更严重。我们得出的结论是，尽管GARS的错义突变可能会导致功能的损失，但在小鼠中观察到的主要神经病表型是由功能的剂量依赖性获得引起的，而功能的剂量依赖性增加，而不是由于功能性野生型蛋白的过表达而无法减轻。