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.

该研究计划的目的是调查遗传性神经系统疾病的机制，最终目的是为这些疾病开发有效的治疗方法。最近，研究集中在两种特定的神经肌肉疾病：由于蛋白SMN缺乏引起的常染色体隐性脊髓性肌萎缩症（SMA），由于雄激素受体中的聚谷氨酰胺扩增引起的X连锁脊髓和延髓肌萎缩症（SBMA）。过去一年的具体研究成果包括： (1)鉴定负责SMN蛋白降解的E3连接酶，mind bomb 1（Mib 1）。SMN通过泛素蛋白酶体系统（UPS）被泛素化和降解。我们以前已经表明，蛋白酶体抑制改善运动功能，并减少脊髓性肌萎缩症小鼠的脊髓，肌肉和神经肌肉接头病理，这表明UPS是这种疾病的潜在治疗靶点。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，但该途径中的特定靶标可能更有效且毒性更低。在这项研究中，我们表明，E3泛素连接酶，Mib 1，与SMN相互作用，并泛素化，促进其降解。敲低Mib 1水平会增加培养细胞中的SMN蛋白水平。此外，敲低Mib 1直系同源物改善了SMN缺陷的秀丽隐杆线虫的神经肌肉功能。这些发现表明Mib 1泛素化并催化SMN的降解，因此代表了脊髓性肌萎缩症的新治疗靶点。 (2)SMA肌肉中组蛋白脱乙酰酶抑制作用的表征。在肌肉萎缩期间，E3连接酶atrogenes、atrogin-1和肌肉环指1（MuRF 1）通过泛素蛋白酶体系统介导肌肉蛋白质分解。Atrogene的表达可由多种上游调节因子诱导。在急性失神经支配期间，它们被肌细胞生成素激活，而肌细胞生成素又被组蛋白脱乙酰酶4和5调节。我们发现，在SMA模型小鼠和SMA患者肌肉中，与肌细胞生成素和组蛋白脱乙酰酶-4（HDAC 4）表达增加相关的萎缩基因被诱导。急性去神经支配和SMA疾病进展期间的这种激活通过组蛋白去乙酰化酶抑制剂治疗受到抑制;然而，当atrogen诱导独立于肌细胞生成素发生时，这种治疗没有效果。这些结果表明，肌细胞生成素依赖性萎缩基因诱导是服从组蛋白去乙酰化酶抑制剂的药理学干预，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)SBMA动物模型中IGF-1作用的表征。我们最近的研究表明，IGF-1通过激活Akt在体外降低突变雄激素受体的毒性，脊髓和延髓肌肉萎缩症的转基因小鼠，也过表达IGF-1的非循环肌肉亚型有一个不太严重的表型。在此，我们试图在表达突变型雄激素受体的转基因小鼠模型中建立每日腹膜内注射mecasermin rinfabate（重组人IGF-1和IGF-1结合蛋白3）的疗效，该突变型雄激素受体具有扩展的97谷氨酰胺通道。本研究以对照、随机、设盲的方式进行，并且为了反映临床环境，在疾病表现发作后开始注射。治疗导致Akt磷酸化增加和肌肉中突变雄激素受体聚集减少。与溶剂处理的对照组相比，IGF-1处理的转基因小鼠表现出运动能力改善、体重减轻减轻和生存率提高。我们的研究结果表明，外周组织可以有针对性地改善脊髓和延髓肌萎缩表型，并表明IGF-1值得进一步研究，在临床试验中作为一种潜在的治疗这种疾病。 (4)甘氨酸tRNA还原酶（加尔斯）突变引起遗传性神经病的机制表征。腓骨肌萎缩症2D型（CMT 2D）是由甘氨酰-tRNA合成酶基因（加尔斯）错义突变引起的显性遗传性周围神经病变。除了加尔斯，其他三种tRNA合成酶基因的突变也会导致类似的神经病变，尽管其潜在机制尚未完全了解。为了解决这个问题，我们产生了普遍过表达野生型加尔斯的转基因小鼠，并将它们与两种CMT 2D显性小鼠模型杂交，以区分功能丧失和功能获得机制。野生型加尔斯的过度表达并不能改善杂合型加尔斯突变小鼠的神经病变表型，这是通过组织学、功能和行为测试确定的。转基因加尔斯能够作为纯合子或在与加尔斯无效等位基因的互补试验中挽救病理性点突变，证明了转基因的功能性并揭示了点突变的隐性功能丧失组分。错义突变作为转基因挽救的纯合子或复合杂合子具有比杂合子更严重的神经病变，表明即使在野生型转基因存在下，致病等位基因的剂量增加也会导致更严重的神经学表型。我们的结论是，虽然错义突变的加尔斯可能会导致一些功能丧失，在小鼠中观察到的显性神经病变表型是由剂量依赖性增益的功能，不减轻过度表达的功能野生型蛋白。