权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Genetic Metabolic Myopathy - Acid Maltase Deficiency

遗传代谢性肌病 - 酸性麦芽糖酶缺乏症

基本信息

批准号：
9573215
负责人：
Vittorio Sartorelli
金额：
$ 36.52万
依托单位：
NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/9573215
关键词：
Address Affect Amino Acids Antibodies Area Arginine Autophagocytosis Autophagosome Binding Binding Sites Biogenesis Biotechnology Cardiac Cardiomegaly Cell Line Cell model Cells ChIP-seq Characteristics Clinical Data Clinical Research Collaborations Complex Conflict (Psychology)DNA Binding Data Defect Deposition Development Disease Disease model E-Box Elements Environment Environmental air flow Enzymes Evaluation Excision Exocytosis FDA approved FRAP1 gene Functional disorder Gene Targeting Genes Genetic Genotype Glucan 1,4-alpha-Glucosidase Glucose Glycogen Glycogen storage disease type II Heart failure Hepatomegaly Heterogeneity Human Hypertrophic Cardiomyopathy Impairment Infant Infection Inherited Institution Knockout Mice Lead Life Lipofuscin Literature Liver Lysosomes MAP Kinase Gene MAPK3 gene Macroglossia Messenger RNA Metabolic Mitochondria Modeling Motor Mus Muscle Muscle Cells Muscle Fibers Muscle Weakness Muscle hypotonia Muscular Atrophy Mutation Myoblasts Myocardium Nuclear Translocation Nutrient Organ Pathology Pathway interactions Patients Pharmaceutical Preparations Pharmacologic Substance Phenotype Phosphotransferases Process Production Protein Biosynthesis Recombinants Recruitment Activity Regulation Research Residual state Respiratory Insufficiency Respiratory Muscles Role Scientist Signal Pathway Signal Transduction Site Skeletal Muscle Small Interfering RNA Symptoms TSC2 gene Testing Therapeutic Therapeutic Intervention Therapeutic Uses Thoracic Radiography Transcriptional Regulation Transgenic Mice Up-Regulation Variant Wheelchairs Work base dietary supplements effective therapy enzyme activity enzyme replacement therapy feeding insight metabolic myopathies mouse model muscle form negative affect neuromuscular novel novel strategies novel therapeutics overexpression pre-clinical preclinical study programs promoter response scoliosis therapeutic target trafficking transcription factor treatment strategy uptake vacuolar H+-ATPase

项目摘要

The major limitation of the current therapy is the difficulty of the enzyme to reach skeletal muscle and its preferential uptake by the liver. We have shown that on top of that, the trafficking and delivery of the replacement enzyme to the lysosomes in skeletal muscle are negatively affected by the presence of massive autophagic buildup and large lipofuscin deposits within the areas of autophagic accumulation. A critical step in the autophagic pathway, which is profoundly impaired in Pompe muscle, is the fusion between autophagosomes and lysosomes where the contents of autophagosomes is digested and recycled. Importantly, both pathologies, autophagic buildup and lipofuscin inclusions, are not amenable to ERT. An entirely novel approach has recently been proposed for treatment of lysosomal storage disorders, which relies on the ability of the DNA-binding transcription factors EB (TFEB) and E3 (TFE3) to stimulate autophagosomal-lysosomal fusion and induce lysosomal exocytosis (expulsion of the lysosomal content outside the cell) leading to cellular clearance. We have demonstrated that overexpression of TFEB or TFE3 in Pompe muscle cells reduced the size of LAMP-positive lysosomes, decreased the amount of accumulated glycogen, and alleviated autophagic buildup. These data established TFEB/TFE3 as valid therapeutic targets in Pompe disease. This approach circumvents the inefficient enzyme delivery to skeletal muscle and restores autophagic flux. In addition, we have identified a compound that induces nuclear translocation (activation) of endogenous TFE3 and demonstrated that two kinases, mTORC1and MAPK (ERK1/2), are involved in the regulation of TFEB and TFE3 in skeletal muscle. Both defective autophagy and accelerated production of lipofuscin pointed to the mitochondrial abnormalities in Pompe skeletal muscle. The autophagic pathway is responsible for the removal of worn-out and damaged mitochondria, a process known as mitophagy. Indeed, we have found multiple mitochondrial defects in mouse and human models of Pompe disease. ChIP-seq analysis of C2C12 cells with specific anti-TFE3 antibody showed the similarity to the previously established TFEB-binding sites in other cells. In addition, TFE3 binding in muscle cells was strongly associated with mitochondrial genes; inspection of several mitochondria-related genes revealed the presence of E-box sequences in their promoters. These data suggested that upregulation of TFE3 may have an additional benefit by stimulating mitochondrial biogenesis in Pompe muscle cells. To further explore the connection between TFE3 and mitochondrial genes and to gain insight into the functional significance of the TFE3 binding in muscle cells, we performed mRNA-seq of non-transfected, Ad-TFE3-transfected, and TFE3 siRNA-transfected C2C12 myoblasts. These data were overlapped with the Chip-seq data (comparing the sets of genes with TFE3 peaks within 1 kb from the TSS); the overlap was represented by 169 genes associated with TFE3 overexpression, and by 211 genes associated with TFE3 silencing. Using MitoCarta 2.0, as a reference, we have identified a reliable list of TFE3-induced mitochondria-related genes in myoblasts, and selected genes were verified by western analysis. Thus, we have identified mitochondrial genes as new TFE3-target genes in myoblasts. Overexpression of Ad-TFE3 in C2C12 cells and in immortalized KO myotubes significantly enhanced mitochondrial mass as evidenced by the increased level of the mitochondrial marker, COXIV. Our second major project included analysis of the mTORC1 signaling pathway in Pompe skeletal muscle. The evaluation of the mTORC1 status is particularly relevant to Pompe disease, because it is a muscle wasting disorder, and mTORC1 is directly involved in the control of muscle mass. The signaling pathways responsible for the loss of muscle mass in Pompe disease are largely unknown, and the limited data in the literature on the subject are conflicting. Understanding the mechanism of the disturbed mTOR signaling in Pompe muscle cells opens the possibility for much needed novel treatment strategies. We anticipated that lysosomal enlargement and the acidification defect (as we have shown previously in Pompe muscle cells) would affect the interaction of the components of a complex machinery involved in the recruitment of mTORC1 to the lysosome (activation) and its release from the lysosome (inactivation). Therefore, we systematically analyzed mTOR pathway by looking at the mTOR downstream targets and the upstream inputs in Pompe muscle cells. We conducted an extended analysis of mTOR pathway in Pompe muscle cells by evaluating mTOR activity, localization, regulation in response to nutrients, and its role in the control of protein synthesis and autophagy. This study is the first systematic analysis of mTORC1 signaling in Pompe muscle cells. Based on the extensive experimental data, we proposed a model of mTOR dysregulation in Pompe disease. Most importantly, we have identified sites of therapeutic intervention and used targeted approaches to reinstate mTOR activity in Pompe muscle cells: 1) manipulation of v-ATPase activity by addressing the lysosomal acidification defect to force proper mTORC1 localization; and 2) manipulation of TSC2 to relieve its inhibitory effect on mTOR. Recent data demonstrated that arginine can activate mTORC1 by suppressing lysosomal localization of the TSC2 complex. Considering the site of arginine action, this amino acid seems ideally suited for correction of the defect in KO cells. Most exciting, we could reinstate mTOR activity in KO cells and in KO mice by providing excess of L-arginine. The attractiveness of this approach is obvious in that this amino acid can be taken as a natural dietary supplement. This safe and effective treatment strategy may have broad relevance for a large group of metabolic, neuromuscular, and lysosomal storage disorders. In addition, we have established a collaboration with Amicus Therapeutics, a biotechnology company, which is developing novel enzyme replacement therapy for Pompe disease. We have tested the new replacement enzyme in our mouse model of the disease, and we have found that the new compound works far better than the currently available drug.

目前疗法的主要限制是酶难以到达骨骼肌并且优先被肝脏吸收。我们已经表明，除此之外，替代酶向骨骼肌溶酶体的运输和递送受到自噬积累区域内大量自噬堆积和大量脂褐素沉积的负面影响。在庞贝肌中严重受损的自噬途径中的一个关键步骤是自噬体和溶酶体之间的融合，其中自噬体的内容物被消化和回收。重要的是，自噬堆积和脂褐素包涵体这两种病理学都不适合 ERT。最近提出了一种全新的方法来治疗溶酶体贮积症，该方法依赖于 DNA 结合转录因子 EB (TFEB) 和 E3 (TFE3) 刺激自噬体-溶酶体融合并诱导溶酶体胞吐作用（将溶酶体内容物排出细胞外）从而导致细胞清除的能力。我们已经证明，庞贝氏肌细胞中 TFEB 或 TFE3 的过度表达可减小 LAMP 阳性溶酶体的大小，减少积累的糖原量，并减轻自噬的积累。这些数据确立了 TFEB/TFE3 作为庞贝病的有效治疗靶点。这种方法避免了酶向骨骼肌的低效传递并恢复自噬通量。此外，我们还鉴定了一种诱导内源性 TFE3 核易位（激活）的化合物，并证明两种激酶 mTORC1 和 MAPK (ERK1/2) 参与骨骼肌中 TFEB 和 TFE3 的调节。自噬缺陷和脂褐素产生加速都表明庞贝氏骨骼肌中存在线粒体异常。自噬途径负责清除磨损和受损的线粒体，这一过程称为线粒体自噬。事实上，我们在庞贝病小鼠和人类模型中发现了多种线粒体缺陷。使用特异性抗 TFE3 抗体对 C2C12 细胞进行 ChIP-seq 分析，结果显示与之前在其他细胞中建立的 TFEB 结合位点相似。此外，TFE3 在肌肉细胞中的结合与线粒体基因密切相关。对几个线粒体相关基因的检查揭示了它们的启动子中存在 E-box 序列。这些数据表明，TFE3 的上调可能会通过刺激庞贝氏肌细胞中的线粒体生物发生而产生额外的好处。为了进一步探索 TFE3 和线粒体基因之间的联系，并深入了解 TFE3 结合在肌肉细胞中的功能意义，我们对未转染、Ad-TFE3 转染和 TFE3 siRNA 转染的 C2C12 成肌细胞进行了 mRNA 测序。这些数据与 Chip-seq 数据重叠（比较距离 TSS 1 kb 范围内具有 TFE3 峰的基因组）；重叠由 169 个与 TFE3 过度表达相关的基因和 211 个与 TFE3 沉默相关的基因代表。使用 MitoCarta 2.0 作为参考，我们确定了成肌细胞中 TFE3 诱导的线粒体相关基因的可靠列表，并通过蛋白质分析验证了所选基因。因此，我们已将线粒体基因鉴定为成肌细胞中新的 TFE3 靶基因。 Ad-TFE3 在 C2C12 细胞和永生化 KO 肌管中的过表达显着增加了线粒体质量，线粒体标记物 COXIV 水平增加证明了这一点。我们的第二个主要项目包括庞贝骨骼肌中 mTORC1 信号通路的分析。 mTORC1状态的评估与庞贝病特别相关，因为它是一种肌肉消耗性疾病，而mTORC1直接参与肌肉质量的控制。导致庞贝病肌肉质量损失的信号通路在很大程度上是未知的，并且有关该主题的文献中的有限数据是相互矛盾的。了解庞贝氏肌细胞中 mTOR 信号传导紊乱的机制为急需的新型治疗策略提供了可能性。我们预计溶酶体增大和酸化缺陷（正如我们之前在庞贝肌细胞中所显示的那样）将影响复杂机制的组件之间的相互作用，该复杂机制涉及将 mTORC1 招募到溶酶体（激活）及其从溶酶体释放（失活）。因此，我们通过观察庞贝肌细胞中的 mTOR 下游靶标和上游输入来系统分析 mTOR 通路。我们通过评估 mTOR 活性、定位、对营养物质的反应调节及其在控制蛋白质合成和自噬中的作用，对庞贝肌细胞中的 mTOR 通路进行了扩展分析。这项研究是对庞贝肌细胞中 mTORC1 信号传导的首次系统分析。基于大量的实验数据，我们提出了庞贝病中 mTOR 失调的模型。最重要的是，我们已经确定了治疗干预的位点，并使用有针对性的方法来恢复庞贝肌细胞中的 mTOR 活性：1）通过解决溶酶体酸化缺陷来操纵 v-ATPase 活性，以强制 mTORC1 正确定位； 2）操纵TSC2以减轻其对mTOR的抑制作用。最近的数据表明，精氨酸可以通过抑制 TSC2 复合物的溶酶体定位来激活 mTORC1。考虑到精氨酸的作用位点，这种氨基酸似乎非常适合纠正 KO 细胞的缺陷。最令人兴奋的是，我们可以通过提供过量的 L-精氨酸来恢复 KO 细胞和 KO 小鼠中的 mTOR 活性。这种方法的吸引力是显而易见的，因为这种氨基酸可以作为天然膳食补充剂。这种安全有效的治疗策略可能对一大群代谢、神经肌肉和溶酶体贮积症具有广泛的相关性。此外，我们还与生物技术公司 Amicus Therapeutics 建立了合作，该公司正在开发针对庞贝氏症的新型酶替代疗法。我们在小鼠疾病模型中测试了这种新的替代酶，我们发现这种新化合物的效果比目前可用的药物要好得多。