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 神经元蜡样脂褐质沉积症 (NCL)，通常称为巴顿病，是一组最常见的神经退行性溶酶体贮积症 (LSD)。作为一个群体，这些疾病没有治愈方法。至少超过 14 个不同基因（称为 CLN）的突变是各种形式 NCL 发病机制的基础。婴儿 NCL（或 INCL）是最具破坏性的神经退行性 LSD 之一，由 CLN1 基因失活突变引起。 CLN1 编码棕榈酰蛋白硫酯酶-1 (PPT1)，这是一种溶酶体酶，可催化蛋白质（蜡质成分）的去棕榈酰化，以通过溶酶体水解酶进行回收或降解。 PPT1 的缺乏阻止了 S-酰化蛋白的降解，导致蜡质在溶酶体中积聚，从而导致 INCL。有几种病理特征（例如溶酶体 pH 值升高、细胞内自发荧光物质（称为 GROD）积累、癫痫发作和寿命缩短。这些特征几乎是所有 NCL 所共有的。这些发现促使我们研究是否存在共同的致病机制我们之前曾报道过，组织蛋白酶 D (CD) 缺乏是先天性 NCL（CLN10 疾病）、由编码组织蛋白酶 D (CD) 的 CLN10 基因突变引起，而 INCL 由编码 PPT1 的 CLN1 基因突变引起，因此，在 INCL（CLN1 疾病）和 CLN10 疾病中，蜡样物质的溶酶体积累都有助于发病机制。去年，我们在 Cln3-/- 小鼠的溶酶体中发现了这一点，它模仿了幼年 NCL (JNCL)，Ppt1 蛋白和 Ppt1 酶活性的溶酶体不足表明 INCL（CLN1 疾病）和幼年 NCL（CLN3 疾病）之间可能存在致病联系。 溶酶体酸化缺陷导致几乎所有溶酶体贮积症 (LSD) 的发病。它也是阿尔茨海默病和帕金森病等常见神经退行性疾病发病机制的一个促成因素。尽管溶酶体酸化至关重要，但迄今为止，人们对这些疾病中溶酶体酸化失调的机制仍知之甚少。已知细胞质子泵、液泡 ATP 酶 (v-ATP 酶) 可以调节溶酶体 pH 值。 v-ATPase 是一种多亚基蛋白复合物，由胞质 V1 区和溶酶体膜锚定的 V0 区组成。 V1 亚基分解 ATP，产生 V0 部分所需的能量，将质子从细胞质运输到溶酶体腔，以维持酸性 pH 值。我们发现，在 Cln1-/- 小鼠的脑组织中，v-ATP 酶活性降低与溶酶体 pH 值升高相关。此外，V0 部分的 v-ATP 酶亚基 a1 (V0a1) 需要 S-棕榈酰化才能分别与接头蛋白 2 (AP-2) 和 AP-3 相互作用，从而运输到溶酶体膜。出乎意料的是，我们发现在 Ppt1 缺陷的 Cln1-/- 小鼠中，V0a1 被错误地传送到质膜，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、无毒小分子 N-叔（丁基）羟胺 (NtBuHA) 治疗 Cln1-/- 小鼠，改善了这一缺陷。我们的研究结果揭示了 Cln1/Ppt1 在调节溶酶体靶向 V0a1 方面的意想不到的作用，并表明对 v-ATP 酶活性产生不利影响的各种因素可能会导致其他 LSD 中的溶酶体酸化失调，包括各种形式的 NCL 和常见的神经退行性疾病。越来越明显的是，如果不了解 NCL 的精确分子机制，就很难开发基于机制的有效疗法。 尽管发现 CLN1 突变导致溶酶体 PPT1 缺陷是 INCL 的基础，但二十多年来发病机制的精确分子机制仍然难以捉摸。因此，我们的研究工作重点是了解 CLN1 疾病、CLN-3 疾病和 CLN-10 疾病的发病机制。我们发现，在模仿 INCL 的 Cln1 -/- 小鼠、死后脑组织以及 INCL 患者培养的成纤维细胞中，自噬失调。此外，Rab7 是一种小 GTP 酶，对自噬体-溶酶体融合至关重要，需要 S-棕榈酰化才能运输到晚期内体/溶酶体膜，在此处与 Rab 相互作用溶酶体蛋白 (RILP) 相互作用，而 Rab 对于自噬体-溶酶体融合至关重要。有趣的是，Cln1 -/- 小鼠中 PPT1 缺陷，Rab7-RILP 相互作用失调，阻止自噬体-溶酶体融合，并损害自噬体的降解功能，导致 INCL 发病机制。重要的是，用脑渗透性PPT1模拟小分子N-叔丁基羟胺(NtBuHA)治疗Cln1 -/- 小鼠，改善了这一缺陷。我们的研究结果揭示了 CLN1/PPT1 在自噬中先前未被认识的作用，并表明功能上模仿 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，神经元死亡后都会发生星形胶质细胞和小胶质细胞（以下称为星形胶质细胞）的增殖和激活。这些激活的星形胶质细胞分泌具有神经毒性的细胞因子，导致存活神经元死亡，从而导致进行性神经变性。使用两种不同的 INCL 小鼠模型，我们发现星形胶质细胞的激活以年龄依赖性方式发生。最近有报道称，活化的小胶质细胞分泌的细胞因子刺激一种特殊类型的星形胶质细胞（称为星形胶质细胞A1）的分化和活化。这些星形胶质细胞分泌尚未表征的极强神经毒素，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 的同质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-/- 小鼠的神经元死亡，并筛选小分子以找到中和这些毒素的分子。此类化合物可能具有神经保护活性，有可能用作 INCL 以及其他神经退行性疾病的治疗剂。 一项题为“针对硫酯酶缺乏症的小分子治疗化合物及其使用方法”的美国专利（US 20140148513 A1）已获得授权。 目前，正在进行临床前研究，以获得 FDA 批准在 INCL 患者中启动临床试验。 为了进一步探讨 INCL 发病机制，我们研究了溶酶体钙 (Ca++) 稳态，以确定溶酶体 Ca++ 稳态失调是否会导致该疾病的神经病理学。我们发现，在模仿 INCL 的 Cln1-/- 小鼠中，低水平的 IP3R1（介导 Ca++ 从内质网 (ER) 到溶酶体的转运）会失调溶酶体 Ca++ 稳态。有趣的是，促进 IP3R1 表达的转录因子 NFATC4 需要 S-棕榈酰化才能从细胞质运输到细胞核。我们鉴定了两种棕榈酰酰基转移酶 ZDHHC4 和 ZDHHC8 作为催化 NFATC4 S-棕榈酰化的酶。值得注意的是，在 Cln1-/- 小鼠中，ZDHHC4 和 ZDHHC8 水平的降低显着抑制了细胞核中 S-棕榈酰化 NFATC4 的水平，从而抑制了 IP3R1 的表达，从而导致溶酶体 Ca++ 稳态失调。因此，Ca++ 依赖性溶酶体酶活性受到抑制，损害自噬，从而导致未消化物质在溶酶体中储存。重要的是，Cln1-/- 小鼠成纤维细胞中 IP3R1 的过度表达改善了这一缺陷。我们的结果揭示了 Cln1 在调节溶酶体 Ca++ 稳态中的先前未被认识的作用，并表明这种缺陷有助于 INCL 发病机制。