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

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.

这项研究计划的目的是探讨遗传性神经疾病的机制，最终目的是开发这些疾病的有效治疗方法。最近的研究集中在两种特定的神经肌肉疾病上：由于SMN蛋白缺乏引起的常染色体隐性遗传性脊髓性肌萎缩症(SMA)和由于雄激素受体多聚谷氨酰胺扩张引起的X连锁脊髓和延髓性肌萎缩(SBMA)。过去一年的具体研究成果包括： (1)鉴定与SMN蛋白降解有关的E3连接酶--心灵炸弹1(Mib1)。SMN通过泛素蛋白酶体系统(UPS)被泛素化和降解。我们先前已经证明，蛋白酶体抑制可以改善脊髓性肌萎缩症小鼠的运动功能，并减轻脊髓、肌肉和神经肌肉接头的病理改变，这表明UPS是治疗这种疾病的潜在靶点。虽然抑制蛋白酶体提供了UPS可以靶向增加SMN蛋白水平的概念证据，但这一途径中的特定靶点可能更有效，毒性更低。在这项研究中，我们发现E3泛素连接酶Mib1与SMN相互作用并使其泛素化，并促进其降解。下调Mib1水平会增加培养细胞中SMN蛋白的水平。此外，敲除Mib1同源基因可以改善缺乏SMN的秀丽线虫的神经肌肉功能。这些发现表明，Mib1泛素化并催化SMN的降解，从而成为治疗脊髓性肌萎缩症的新靶点。 (2)对SMA肌中组蛋白脱乙酰酶抑制作用的研究。在肌肉萎缩过程中，E3连接酶萎缩基因-1和肌肉环指1(MuRF1)通过泛素蛋白酶体系统介导肌肉蛋白质的分解。Atrogene的表达可被多种上游调控因子诱导。在急性去神经支配过程中，它们被肌生成素激活，而肌生成素又受组蛋白脱乙酰基酶4和5的调控。我们发现，在SMA模型小鼠和SMA患者肌肉中，随着肌生成素和组蛋白脱乙酰基酶-4(HDAC4)表达的增加，可以诱导出萎缩基因。在急性失神经和SMA疾病进展期间，这种激活都可以通过组蛋白脱乙酰酶抑制剂来抑制；然而，当不依赖肌生成素发生萎缩基因诱导时，这种治疗没有效果。这些结果表明，用组蛋白脱乙酰酶抑制剂进行药物干预可以诱导肌生成素依赖的阿托品，并有助于解释这些药物对SMA和其他去神经疾病的有益作用。 (3)胰岛素样生长因子-1在SBMA动物模型中的作用。我们最近的研究表明，IGF-1在体外通过激活Akt来降低突变型雄激素受体的毒性，并且过表达非循环肌肉异构体IGF-1的脊髓和延髓肌萎缩症转基因小鼠的表型不那么严重。在这里，我们试图确定每日腹腔注射甲卡色明、重组人IGF-1和IGF-1结合蛋白3，在表达突变雄激素受体的转基因小鼠模型中具有扩大的97谷氨酰胺区。这项研究是以对照、随机、盲法进行的，为了反映临床环境，注射是在疾病症状出现后开始的。治疗导致Akt磷酸化增加，肌肉中突变的雄激素受体聚集减少。与赋形剂处理的对照组相比，经IGF-1处理的转基因小鼠表现出更好的运动能力，减轻体重减轻，并提高存活率。我们的结果表明，外周组织可以靶向改善脊髓和延髓肌萎缩的表型，并表明IGF-1作为一种潜在的治疗方法，值得在临床试验中进行进一步的研究。 (4)甘氨酸tRNA合成酶(GARS)突变所致遗传性神经病的机制研究。2D型Charcot-Marie-Tooth病(CMT2D)是一种由甘氨酰-tRNA合成酶基因(GARS)错义突变引起的遗传性周围神经病。除了GARS，另外三个tRNA合成酶基因的突变也会导致类似的神经疾病，尽管潜在的机制还不完全清楚。为了解决这个问题，我们产生了普遍过度表达野生型GARS的转基因小鼠，并将它们与两种主要的CMT2D小鼠模型进行杂交，以区分功能丧失和功能获得的机制。组织学、功能和行为测试表明，野生型GARS的过度表达并不能改善杂合子GARS突变小鼠的神经病变表型。转基因GARS能够将病理性点突变挽救为纯合子或在与GARS零等位基因的互补试验中拯救，证明了转基因的功能，并揭示了点突变的隐性功能丧失成分。错义突变作为转基因挽救的纯合子或复合杂合子比杂合子有更严重的神经病变，这表明即使存在野生型转基因，致病等位基因剂量的增加也会导致更严重的神经表型。我们的结论是，尽管GARS错义突变可能导致一些功能丧失，但在小鼠中观察到的主要神经病变表型是由剂量依赖的功能获得引起的，而功能野生型蛋白的过度表达并不能缓解这种情况。