Heriditary Neurodegenerative Storage Disorders

遗传性神经退行性存储障碍

• 批准号: 8553856
• 负责人: ANIL B MUKHERJEE
• 金额: $ 127.01万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/8553856
• 关键词: Accounting; Acetylcysteine; Affect; Age; Age-Months; Aminoglycosides; Anesthesia procedures; Animal Disease Models; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Antioxidants; Apoptosis; Birth; Blood - brain barrier anatomy; Body Temperature; Brain; Brown Fat; CD4 Positive T Lymphocytes; Cells; Ceroid; Child; Childhood; Chronic; Cleaved cell; Clinical; Clinical Trials; Complication; Cultured Cells; Cystagon; Cysteamine; Data Analyses; Development; Dietary Supplementation; Disease; Dose; Down-Regulation; Effectiveness; Energy Metabolism; Enzymes; Evaluation; Fibroblasts; Genes; Goals; Infantile neuronal ceroid lipofuscinosis; Inherited; Inpatients; Interleukin-17; Investigation; Investigational Therapies; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Laboratory Study; Lead; Link; Lipids; Lymphocyte; Lysosomes; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Matrix Metalloproteinases; Mediating; Methods; Modeling; Molecular; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Ceroid-Lipofuscinosis; Neurotransmitters; Nonsense Codon; Nonsense Mutation; Palmitoyl Coenzyme A; Pathogenesis; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Physiologic Thermoregulation; Procedures; Production; Proteins; Protocols documentation; Rare Diseases; Reading; Recruitment Activity; Reporting; Research; Resveratrol; Risk; Risk Factors; Spielmeyer-Vogt Disease; Study models; Testing; Therapeutic; Tight Junctions; Time; Toxic effect; Translational Research; Up-Regulation; bench to bedside; clinically relevant; developmental genetics; effective therapy; enzyme activity; enzyme deficiency; improved; in vivo; insight; kinetosome; lymphoblast; lysosomal proteins; mitochondrial dysfunction; mouse model; natural hypothermia; neuropathology; novel; novel therapeutics; peroxisome; polyphenol; receptor; research study; small molecule; therapeutic target; thioester; thioesterase PPT1 gene product; uncoupling protein 1

We have accomplished the following advances during the past year: (A) The availability of Ppt1-knockout (Ppt1-KO) mice, which recapitulate virtually all clinical and pathological features of INCL, provides an opportunity to test the effectiveness of novel therapeutic strategies in vivo. However, such studies will require noninvasive methods that can be used to perform serial evaluations of the same animal receiving an experimental therapy. Thus, the development of noninvasive method(s) of evaluation is urgently needed. We developed a non-invasive method for the evaluation of the progression of neurodegeneration in Ppt1-KO mice starting at 3 months of age by using MRI and MR spectroscopy (MRS) and repeating these tests using the same mice at 4, 5 and 6 months of age. Our findings provide a method to serially examine the brains of mouse models of neurodegenerative diseases (e.g. Ppt1-KO mice) using noninvasive and nonlethal procedures such as MRI and MRS. These methods may be useful in studies to understand the progression of neuropathology in animal models of neurodegenerative diseases as they allow repeated evaluations of the same animal in which experimental therapies are tested.(B) Disruption of the blood-brain barrier (BBB) is a serious complication frequently encountered in neurodegenerative disorders. As stated above, INCL is a devastating childhood neurodegenerative LSD caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. However, it remains unclear whether BBB is disrupted in INCL and if so, what might be the molecular mechanism(s) of this complication. Using Ppt1-KO mice we have delineated that T(H)17 lymphocytes producing IL-17A mediate disruption of the BBB by stimulating production of matrix metalloproteinases (MMPs), which degrade the tight junction proteins essential for maintaining BBB integrity. Importantly, dietary supplementation of resveratrol (RSV), a naturally occurring antioxidant/anti-inflammatory polyphenol, markedly reduced the levels of T(H)17 cells, IL-17A and MMPs, and elevated the levels of tight junction proteins, which improved the BBB integrity in Ppt1-KO mice. Intriguingly, we found that RSV suppressed the differentiation of CD4(+) T lymphocytes to IL-17A-positive T(H)17 cells. Our findings uncover a mechanism by which T(H)17 lymphocytes mediate BBB disruption and suggest that small molecules such as RSV that suppress T(H)17 differentiation are therapeutic targets for neurodegenerative disorders such as INCL. (C) We have previously reported that children with INCL have increased risk of hypothermia during anesthesia. Moreover, we have shown that PPT1-deficiency is associated with disruption of adaptive energy metabolism, downregulation of peroxisome roliferator-activated receptor γ coactivator 1α (PGC-1α), and mitochondrial dysfunction. In a collaborative study with Dr. Quezado and colleagues, we tested a hypothesis PPT1-deficiency in mice impair thermoregulation observed in children with INCL involving the upregulation of PGC-1α and uncoupling protein 1 (UCP-1) in brown adipose tissue. We found that the Ppt1-KO mice, a well-studied model of INCL, have lower basal body temperature as they age and develop hypothermia during cold exposure. This inability to maintain body temperature during cold exposure in Ppt1-KO mice was associated with upregulation of PGC-1α and UCP-1 but with lower levels of sympathetic neurotransmitters in brown adipose tissue. The results of our experiments uncover previously unknown phenotypes associated with PPT1-deficiency and suggest that in patients with this disease, impaired thermoregulation and hypothermia are potential risk factors.(D)Nonsense mutations in a gene generate premature termination codons producing truncated, nonfunctional or deleterious proteins. PPT1 nonsense-mutations account for approximately 31% of INCL patients in the US. Currently, there is no effective treatment for this disease. While aminoglycosides such as gentamycin suppress nonsense mutations, inherent toxicity of aminoglycosides prohibits chronic use inpatients. PTC124 is a non-toxic compound that induces ribosomal read-through of premature termination codons. We sought to determine whether PTC124-treatment of cultured cells from INCL patients carrying nonsense mutations in the PPT1 gene would correct PPT1 enzyme-deficiency with beneficial effects. Our results showed that PTC124-treatment of cultured cells from INCL patients carrying PPT1 nonsense-mutations induced PPT1 enzymatic activity in a dose- and time-dependent manner. This low level of PPT1 enzyme activity induced by PTC124 is virtually identical to that induced by gentamycin-treatment. Even though only a modest increase in PPT1 activity was achieved by PTC124-treatment of INCL cells, this treatment reduced the levels of thioester (constituent of ceroid) load. Our results suggest that PTC124-treatment induces PPT1 enzymatic activity in cultured cells from INCL patients carrying PPT1 nonsense-mutations, and this modest enzymatic activity has demonstrable beneficial effects on these cells. The clinical relevance of these effects may be tested in animal models of INCL carrying nonsense mutations in the PPT1 gene. Our ongoing studies are attempting to generate a mouse model of INCL in which the most common nonsense mutations in the Ppt1 gene is knocked-in (Ppt1-KI mice). We plan to test whether PTC124 can induce PPT1 activity in these mice and ameliorate INCL pathogenesis.(E) PPT1 is a lysosomal enzyme that catalyzes the cleavage of thioester linkage in palmitoylated (S-acylated) proteins and its deficiency impairs degradation of fatty-acylated proteins by lysosomal proteases. Consequently, accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL pathogenesis. Given that thioester linkages are susceptible to nucleophilic attack, drugs that can cleave such linkages may have therapeutic potential for INCL. The results of our laboratory studies showed that two drugs, phosphocysteamine and N-acetylcysteine, cleave thioester linkage in 14C palmitoyl-CoA, a model substrate of PPT1. Moreover, these small molecules also mediate depletion of ceroid from cultured lymphoblasts and fibroblasts from INCL patients. The drugs also inhibit apoptosis in these cells. These laboratory results prompted us to initiate a bench-to-bedside clinical trial to determine whether a combination of Cystagon (cysteamine bitartrate) and Mucomyst (N-acetylcysteine) is beneficial for patients with INCL. To date, we have treated 9 INCL patients and the study results are being analyzed to evaluate whether recruitment of more patients are needed to arrive at a meaningful conclusion of this study. Thus, at present our protocol is not recruiting any patients until our analysis of the data is complete and a determination is made as to whether we need to admit more patients to this protocol.

在过去的一年中，我们已经取得了以下进展：（a）PPT1敲除（PPT1-KO）小鼠的可用性，这些小鼠实际上概括了含有含的所有临床和病理特征，提供了一个机会来测试体内新型治疗策略的有效性。但是，此类研究将需要无创方法，这些方法可用于对接受实验疗法的同一动物进行连续评估。因此，迫切需要迫切需要开发评估的无创方法。我们开发了一种非侵入性方法，用于评估PPT1-KO小鼠神经退行性的进展，从3个月大时才使用MRI和MR光谱学（MRS）（MRS），并在4、5和6个月大时使用相同的小鼠重复这些测试。我们的发现提供了一种方法，可以使用非侵入性和非致死性程序（例如MRI和MRS）串行检查神经退行性疾病（例如PPT1-KO小鼠）的小鼠模型的大脑。这些方法在研究神经退行性疾病模型中的神经病理学的进展中可能很有用，因为它们允许对测试实验疗法的同一动物进行重复评估。（b）血脑屏障（BBB）的破坏是神经变性分解的严重并发症。如上所述，含含棕榈酰蛋白硫代硫代酶-1（PPT1）缺乏症引起的毁灭性的儿童神经退行性LSD。但是，目前尚不清楚BBB是否在含有含义中被破坏，如果是，则可能是这种并发症的分子机制。使用PPT1-KO小鼠，我们描述了产生IL-17A的T（h）17个淋巴细胞通过刺激基质金属蛋白酶（MMP）的产生来介导BBB的破坏，这会降低紧密连接蛋白维持BBB完整性所必需的紧密连接蛋白。重要的是，饮食补充白藜芦醇（RSV）是一种天然存在的抗氧化剂/抗炎多酚，显着降低了T（H）17细胞，IL-17A和MMP的水平，并提高了紧密连接蛋白的水平，从而改善了PPT1-KKO小鼠中BBB完整性的水平。有趣的是，我们发现RSV抑制了CD4（+）T淋巴细胞与IL-17A阳性T（H）17细胞的分化。我们的发现发现了一种机制，t（h）17淋巴细胞介导了BBB的破坏，并表明抑制t（h）17分化的小分子（例如RSV）是神经退行性疾病（如含）的治疗靶标。 （c）我们以前曾报道说，患有包括麻醉期间体温过低风险的儿童。此外，我们已经表明，PPT1缺陷与自适应能量代谢的破坏有关，过氧化物酶体罗脂蛋白激活的受体γ共激活因子1α（PGC-1α）和线粒体功能障碍的下调。在与Quezado博士及其同事的一项合作研究中，我们测试了小鼠的假设PPT1缺乏症会损害涉及PGC-1α上调的儿童，涉及PGC-1α的上调和棕色脂肪组织中的解偶联蛋白1（UCP-1）。我们发现，随着年龄的增长，PPT1-KO小鼠的基础温度较低，并且在冷暴露期间发展体温过低。 PPT1-KO小鼠冷暴露期间的这种无法维持体温与PGC-1α和UCP-1的上调有关，但与棕色脂肪组织中的交感神经递质水平较低有关。我们的实验结果揭示了先前未知的表型与PPT1缺陷性相关的表型，并表明在患有这种疾病的患者中，体温调节受损和体温过低是潜在的危险因素。（d）基因中产生过早终止密码子的无义突变，产生截短，无肢体或有害蛋白质。 PPT1废话占美国的包含患者的31％。目前，这种疾病还没有有效的治疗方法。虽然氨基糖苷（例如庆大霉素）抑制了胡说八道突变，但氨基糖苷的固有毒性禁止长期使用患者。 PTC124是一种无毒的化合物，可诱导过早终止密码子的核糖体读取。我们试图确定PTC124在PPT1基因中携带废话突变的培养细胞的PTC124治疗是否会纠正PPT1酶缺乏症具有有益作用。我们的结果表明，携带PPT1废话的患者的PTC124治疗培养细胞以剂量和时间依赖性的方式诱导PPT1酶活性。 PTC124诱导的这种低水平的PPT1酶活性实际上与庆大霉素治疗引起的PTC124相同。尽管通过PTC124对含含细胞的治疗仅实现了PPT1活性的适度增加，但这种处理降低了硫酯的水平（CEROID）载荷的水平。我们的结果表明，PTC124处理可诱导携带PPT1废话的患者的培养细胞中的PPT1酶活性，而这种适度的酶促活性对这些细胞具有可证明的有益作用。这些作用的临床相关性可以在PPT1基因中携带废话突变的动物模型中进行测试。我们正在进行的研究试图生成一个含有小鼠模型，其中PPT1基因中最常见的胡说八道突变被敲入（PPT1-KI小鼠）。我们计划测试PTC124是否可以在这些小鼠中诱导PPT1活性并改善含有发病机理。（E）PPT1是一种溶酶体酶，是一种催化硫化（S-酰化）蛋白质及其缺陷及其缺乏蛋白蛋白质蛋白质蛋白质蛋白质蛋白质的裂解的硫酯链接的裂解。因此，这些脂质改性蛋白（Ceroid的成分）在溶酶体中的积累会导致包含发病机理。鉴于硫酯连接易受亲核攻击的影响，因此可以裂解这种连接的药物可能具有含有治疗的潜力。我们的实验室研究的结果表明，在14C棕榈酰-COA中，两种药物，磷脂天stemine和n-乙酰半胱氨酸，裂解硫酯链接，是PPT1的模型底物。此外，这些小分子还介导了培养的淋巴细胞和成纤维细胞的CEROID耗竭。这些药物还抑制这些细胞中的凋亡。这些实验室结果促使我们开始进行基准层临床临床试验，以确定囊肿（cysteamine botartrate）和粘霉素（N-乙酰半胱氨酸）的组合是否对含有含有的患者有益。迄今为止，我们已经治疗了9名含患者，并且正在分析研究结果，以评估是否需要更多患者的招募才能得出这项研究的有意义的结论。因此，目前，我们的方案没有招募任何患者，直到我们对数据的分析完成并确定我们是否需要接受更多患者使用该方案。