Childhood Neurodegenerative Lysosomal Storage Disorders

儿童神经退行性溶酶体储存障碍

• 批准号: 10470628
• 负责人: ANIL B MUKHERJEE
• 金额: $ 175.88万
• 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/10470628
• 关键词: Acetylcysteine; Acyltransferase; Adaptor Signaling Protein; Affect; Alzheimer' s Disease; Astrocytes; Autophagocytosis; Autophagosome; Autopsy; Biochemical; Birth; Blood - brain barrier anatomy; Brain; CLN1 gene; CLN10 gene; CLN3 gene; Calcium; Cathepsins; Cause of Death; Cell Nucleus; Cell membrane; Cellular biology; Ceroid; Child; Childhood; Clinical Trials; Cystagon; Cysteamine; Cytoplasm; Defect; Development; Disease; Endoplasmic Reticulum; Enzymes; Fibroblasts; Functional disorder; Genes; Goals; Grant; Homeostasis; Hydrolase; Hydroxylamine; ITPR1 gene; Impairment; Infantile neuronal ceroid lipofuscinosis; Investigation; Laboratories; Laboratory Research; Laboratory Study; Legal patent; Link; Location; Longevity; Lysosomes; Mediating; Membrane; Methods; Microglia; Modeling; Molecular; Molecular Biology; Monomeric GTP-Binding Proteins; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Ceroid-Lipofuscinosis; Neurons; Neurotoxins; Nonsense Mutation; Parkinson Disease; Pathogenesis; Pathogenicity; Pathologic; Patients; Protein Complex Subunit; Proteins; Proton Pump; Protons; Rare Diseases; Recycling; Reporting; Research; Role; Seizures; Spielmeyer-Vogt Disease; Sum; Techniques; Therapeutic; Therapeutic Agents; Toxin; Transgenic Mice; Translational Research; age related; base; bench to bedside; brain tissue; clinical investigation; curative treatments; cytokine; developmental genetics; disease-causing mutation; effective therapy; enzyme activity; in vivo evaluation; insight; juvenile neuronal ceroid lipofuscinosis; lysosomal proteins; mimetics; mouse model; neuron loss; neuropathology; neurotoxic; novel; overexpression; palmitoylation; pre-clinical; preclinical study; prevent; progressive neurodegeneration; small molecule; small molecule therapeutics; thioesterase PPT1 gene product; trafficking; transcription factor; vacuolar H+-ATPase; virtual

Summary Neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, constitute a group of the most prevalent neurodegenerative lysosomal storage disorders (LSDs). As a group, these diseases have no curative treatment. Mutations in at least >14 different genes (called the CLNs) underlie pathogenesis of various forms of NCLs. The infantile NCL (or INCL) is one of the most devastating neurodegenerative LSDs caused by inactivating mutations in the CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme that catalyzes depalmitoylation of proteins (constituents of ceroid) for their recycling or degradation by lysosomal hydrolases. The deficiency of PPT1 prevents the degradation of S-acylated proteins causing the accumulation of ceroid in lysosomes, which leads to INCL. There are several pathological features (e.g. elevated lysosomal pH, accumulation of intracellular autofluorescent material (called GRODs), seizures and shortened life span. These features are common to virtually all NCLs. These findings prompted us to investigate whether there are common pathogenic mechanisms that are shared by all NCLs. We have previously reported that cathepsin D (CD)-deficiency is a common pathogenic link between congenital NCL (CLN10-disease), caused by mutations in the CLN10 gene encoding cathepsin D (CD), and INCL caused by mutations in the CLN1 gene encoding PPT1. Thus, in both INCL (CLN1-disease) and CLN10-disease lysosomal accumulation of ceroid contributes to pathogenesis. During the past year, we uncovered that in the lysosomes of Cln3-/- mice, which mimic juvenile NCL (JNCL), there is lysosomal insufficiency of Ppt1-protein and Ppt1-enzyme activity suggesting that there might be a pathogenic link between INCL (CLN1-disease) and juvenile NCL (CLN3-disease). Defective lysosomal acidification contributes to pathogenesis of virtually all lysosomal storage disorders (LSDs). It is also a contributory factor in the pathogenesis of common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite the critical importance of lysosomal acidification, the mechanism(s) underlying the dysregulation of lysosomal acidification in these diseases until now remained poorly understood. The cellular proton pump, vacuolar-ATPase (v-ATPase), is known to regulate lysosomal pH. A multi-subunit protein complex, v-ATPase is composed of a cytosolic V1-sector and a lysosomal membrane-anchored V0-sector. The V1 subunit breaks down ATP generating energy required for the V0 sector to transport protons from the cytoplasm to the lysosomal lumen to maintain acidic pH. We found that in the brain tissues of Cln1-/- mice, reduced v-ATPase activity correlated with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires S-palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Unexpectedly, we discovered that in Ppt1-deficient Cln1-/- mice, V0a1 is misrouted to the plasma membrane instead of its normal location on lysosomal membrane. Notably, treatment of the Cln1-/- mice with a thioesterase (Ppt1)-mimetic, non-toxic small molecule, N-tert (Butyl) hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal an unanticipated role of Cln1/Ppt1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase activity may dysregulate lysosomal acidification in other LSDs including various forms of the NCLs and common neurodegenerative diseases. It is increasingly evident that without understanding the precise molecular mechanism(s) of the NCLs, the development of mechanism-based effective therapies is difficult. Despite the discovery that CLN1 mutations cause lysosomal PPT1-deficiency underlies INCL, the precise molecular mechanism(s) of pathogenesis has remained elusive for more than two decades. Thus, our research efforts have been focused on understanding the mechanism(s) of pathogenesis underlying CLN1-disease, CLN-3 disease and CLN-10 disease. We found that autophagy is dysregulated in Cln1 -/- mice, which mimic INCL and in postmortem brain tissues as well as cultured fibroblasts from INCL patients. Moreover, Rab7, a small GTPase, critical for autophagosome-lysosome fusion, requires S-palmitoylation for trafficking to the late endosomal/lysosomal membrane where it interacts with Rab-interacting lysosomal protein (RILP), essential for autophagosome-lysosome fusion. Intriguingly, PPT1-deficiency in Cln1 -/- mice, dysregulated Rab7-RILP interaction and prevents autophagosome-lysosome fusion and impaired degradative functions of the autolysosome leading to INCL pathogenesis. Importantly, treatment of Cln1 -/- mice with a brain-penetrant, PPT1-mimetic, small molecule, N-tert (butyl)hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal a previously unrecognized role of CLN1/PPT1 in autophagy and suggest that small molecules functionally mimicking PPT1 may have therapeutic implications. In virtually all neurodegenerative disorders, neuronal death is followed by proliferation and activation of astrocytes and microglia (hereafter called astroglia). These activated astroglia secrete cytokines that are neurotoxic causing death of viable neurons, which leads to progressive neurodegeneration. Using two different mouse models of INCL, we found that astroglia activation occurs in an age-dependent manner. It has recently been reported that cytokines secreted by the activated microglia stimulates the differentiation and activation of a special type of astrocytes called Astrocyte A1. These astrocytes secrete as yet uncharacterized, extremely potent neurotoxins, which leads to further neuronal death and progressive neurodegeneration. Our ongoing research is attempting to isolate homogeneous cultures of Astrocyte A1 and characterize the neurotoxins. We hope to study how these neurotoxins mediate neuronal death in Cln1-/- mice and screen small molecules to find those that neutralize these toxins. Such compounds may have neuroprotective activities with potential for use as a therapeutic agent for INCL and perhaps other neurodegenerative diseases. A US Patent (US 20140148513 A1), entitled "Small molecule therapeutic compounds targeting thioesterase deficiency disorders and methods of using the same" has been granted. Currently, preclinical studies are being conducted to obtain FDA approval for initiating a clinical trial in INCL patients. To further explore the mechanism underlying INCL pathogenesis we studied lysosomal calcium (Ca++) homeostasis to determine whether dysregulation of lysosomal Ca++ homeostasis contributes to neuropathology in this disease. We found that in Cln1-/- mice, which mimic INCL, low level of IP3R1, which mediates Ca++-transport from the endoplasmic reticulum (ER) to the lysosome, dysregulates lysosomal Ca++ homeostasis. Intriguingly, the transcription factor NFATC4, which promotes IP3R1-expression, requires S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8 as the enzymes that catalyze S-palmitoylation of NFATC4. Remarkably, in Cln1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly suppressed S-palmitoylated NFATC4 level in the nucleus, which suppressed IP3R1-expression, thereby, dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++-dependent lysosomal enzyme activities were suppressed impairing autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Cln1-/- mouse fibroblasts ameliorated this defect. Our results revealed a previously unrecognized role of Cln1 in regulating lysosomal Ca++-homeostasis and suggested that this defect contributes to INCL pathogenesis.

概括 神经元蛋白脂蛋白酶（NCLS）（通常称为棕褐色疾病）构成了最普遍的神经退行性溶酶体储存障碍（LSD）。作为一个小组，这些疾病没有治愈性治疗。至少14个不同基因（称为CLN）的突变是各种形式的NCL的发病机理。婴儿NCL（或含）是由CLN1基因失活突变引起的最具破坏性的神经退行性LSD之一。 CLN1编码棕榈酰蛋白硫代酶-1（PPT1），一种溶酶体酶，可催化蛋白质（ceroid的成分）的depalmitylations通过溶酶体水解酶进行回收或降解。 PPT1的缺乏阻止了S酰基化蛋白的降解，从而导致CEROID在溶酶体中的积累，从而导致含有。 There are several pathological features (e.g. elevated lysosomal pH, accumulation of intracellular autofluorescent material (called GRODs), seizures and shortened life span. These features are common to virtually all NCLs. These findings prompted us to investigate whether there are common pathogenic mechanisms that are shared by all NCLs. We have previously reported that cathepsin D (CD)-deficiency is a common pathogenic link between congenital NCL（CLN10-疾病）是由编码组织蛋白酶D（CD）的CLN10基因引起的，以及由CLN1基因编码PPT1引起的，因此在含有（CLN1-酶酶）和Cln10-Dis-Dis-Dis-Dis-Dis-Dis-disese酶溶解度上均造成了植物的生理 - 我们在过去的情况下 - 哪个模仿少年NCL（JNCL），PPT1-蛋白质和PPT1-酶活性的溶酶体不足表明，含有（CLN1-疾病）和幼年NCL（CLN3-疾病）之间可能存在致病性联系。 缺陷的溶酶体酸化有助于几乎所有溶酶体储存障碍（LSD）的发病机理。它也是常见神经退行性疾病（如阿尔茨海默氏症和帕金森氏症）的发病机理的一个因素。尽管溶酶体酸化的重要性至关重要，但这些疾病中溶酶体酸化失调的基础机制迄今仍未理解。已知细胞质子泵（V-ATPase）调节溶酶体pH值。 V-ATPase的多支化蛋白复合物由胞质V1扇区和溶酶体膜锚定V0扇区组成。 V1亚基分解了V0扇形将质子从细胞质传输到溶酶体管腔以维持酸性pH的能量所需的ATP。我们发现，在CLN1 - / - 小鼠的脑组织中，V-ATPase活性降低与溶酶体pH值升高有关。此外，V0扇区的V-ATPase亚基A1（V0A1）需要S-膜酰烯酰化，以分别与衔接蛋白-2（AP-2）和AP-3相互作用，以便运输到溶酶体膜上。出乎意料的是，我们发现在PPT1缺陷型CLN1 - / - 小鼠中，V0A1被误入到质膜上，而不是其在溶酶体膜上的正常位置。值得注意的是，用硫酯酶（PPT1）模拟，无毒的小分子，N-TERT（丁基）羟胺（NTBUHA）治疗CLN1 - / - 小鼠，改善了这一缺陷。我们的发现揭示了CLN1/PPT1在调节V0A1的溶酶体靶向中的意外作用，并表明对V-ATPase活性的不同因素可能会导致其他LSD中的溶酶体酸化，包括各种形式的NCLS和常见神经性疾病。越来越明显的是，如果不了解NCL的精确分子机制，则很难开发基于机制的有效疗法。 尽管发现CLN1突变引起溶酶体PPT1缺乏依赖性的基础，但发病机理的精确分子机制一直难以捉摸二十年来。因此，我们的研究工作集中在理解CLN1-疾病，CLN-3疾病和CLN-10疾病的发病机理机理上。我们发现自噬在CLN1 - / - 小鼠中失调，这些小鼠模仿含量和尸体后脑组织以及来自含患者的培养的成纤维细胞。此外，RAB7是一种小型GTPase，对自噬体散糖体融合至关重要，需要S-膜酰二酰化来运输到晚期内体/溶酶体膜，在该膜与Rab互动溶酶体蛋白（RILP）相互作用，对于自动型体-溶质体 - 溶剂体 - 溶剂体 - 溶酶体 - 溶剂体 - 溶酶体 - 溶酶体 - 大体体 - 溶剂体融合至关重要。有趣的是，CLN1 - / - 小鼠中的PPT1缺陷，RAB7-RILP相互作用失调，并防止自噬体 - 散糖体融合和自溶性的降解功能受损，导致包含发病机理。重要的是，用脑培养剂，PPT1模拟，小分子，N-TERT（丁基）羟胺（NTBUHA）治疗CLN1 - / - 小鼠，改善了此缺陷。我们的发现揭示了Cln1/ppt1在自噬中的先前未知的作用，并表明在功能上模仿PPT1的小分子可能具有治疗意义。 在几乎所有神经退行性疾病中，神经元死亡之后是星形胶质细胞和小胶质细胞的激活（以下称为星形胶质细胞）。这些活化的星形胶质细胞分泌神经毒性的细胞因子，导致可行神经元死亡，从而导致逐渐发展的神经变性。使用两种不同的含有小鼠模型，我们发现星形胶质细胞激活以年龄依赖性方式发生。最近据报道，活化的小胶质细胞分泌的细胞因子刺激了一种称为星形胶质细胞A1的特殊类型的星形胶质细胞的分化和激活。这些星形胶质细胞分泌尚未表征，极有效的神经毒素，从而导致进一步的神经元死亡和进行性神经变性。我们正在进行的研究试图分离星形胶质细胞A1的均匀培养物并表征神经毒素。我们希望研究这些神经毒素如何介导CLN1 - / - 小鼠和筛查小分子中的神经元死亡，以找到中和这些毒素的分子。这样的化合物可能具有具有可用用作含有和其他神经退行性疾病的治疗剂的神经保护活性。 美国专利（US 20140148513 A1），名为“针对硫酯酶缺乏障碍的小分子治疗化合物和使用相同的方法”。 目前，正在进行临床前研究以获得FDA批准，以启动包括患者的临床试验。 为了进一步探索包含发病机理的机制，我们研究了溶酶体钙（CA ++）稳态，以确定溶酶体CA ++稳态的失调是否有助于这种疾病的神经病理学。我们发现，在cln1 - / - 小鼠中，模仿含有低水平的IP3R1的小鼠，它介导了Ca ++ - 从内质网（ER）转移到溶酶体，使溶酶体CA ++稳态失调。有趣的是，促进IP3R1表达的转录因子NFATC4需要s-甲米酰化以从细胞质到核的运输。我们确定了两个棕榈酰酰基转移酶，ZDHHC4和ZDHHC8是催化NFATC4的S-甲酰化的酶。值得注意的是，在Cln1 - / - 小鼠中，ZDHHC4和ZDHHC8水平降低，在核中显着抑制了S-甲酰化的NFATC4水平，从而抑制了IP3R1表达，从而抑制了溶酶体CA ++稳态。因此，Ca ++ - 依赖性的溶酶体酶活性被抑制受损自噬，从而导致溶酶体储存未消化的货物。重要的是，CLN1 - / - 小鼠成纤维细胞中的IP3R1 Over表达改善了此缺陷。我们的结果表明，CLN1在调节溶酶体CA ++稳态中的先前未认识到的作用，并提出这种缺陷有助于包含发病机理。