Childhood Neurodegenerative Lysosomal Storage Disorders

儿童神经退行性溶酶体储存障碍

基本信息

批准号：
10898488
负责人：
ANIL B MUKHERJEE
金额：
$ 191.09万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
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/10898488
关键词：
Ablation Acetylcysteine Acyltransferase Adaptor Signaling Protein Affect Alzheimer&apos s Disease Astrocytes Autophagocytosis Autophagosome Autopsy Biochemical Biological Birth Brain CLN1 gene CLN10 gene CLN3 gene Calcium Cathepsins Cause of Death Cell Nucleus Cell membrane Cells Ceroid Child Childhood Clinical Trials Cystagon Cysteamine Cytoplasm Defect Development Disease Endoplasmic Reticulum Enzymes Fibroblasts Functional disorder Genes Goals Homeostasis Human Hydrolase Hydroxylamine ITPR1 gene Impairment Infantile neuronal ceroid lipofuscinosis Inflammation Inflammatory Investigation Knockout Mice Laboratories Laboratory Research Laboratory Study Link Location Longevity Lysosomal Storage Diseases Lysosomes Mediating Membrane MicroRNAs Microglia Modeling Molecular Monomeric GTP-Binding Proteins Mus Mutation Nerve Degeneration Neurodegenerative Disorders Neuronal Ceroid-Lipofuscinosis Neurons Neurotoxins Nonsense Mutation Parkinson Disease Pathogenesis Pathogenicity Pathologic Patients Proliferating Protein Complex Subunit Proteins Proton Pump Protons Rare Diseases Recycling Reporting Research Role Seizures Spielmeyer-Vogt Disease Techniques Therapeutic Transgenic Mice Translational Research age related bench to bedside blood-brain barrier crossing brain tissue clinical investigation curative treatments cytokine developmental genetics effective therapy enzyme activity functional mimics in vivo evaluation infancy insight juvenile neuronal ceroid lipofuscinosis late endosome lysosomal proteins mimetics mouse model neuroinflammation neuron loss neuropathology neuroprotection neurotoxic novel palmitoylation pre-clinical preclinical study prevent progressive neurodegeneration small molecule thioesterase PPT1 gene product trafficking transcription factor vacuolar H+-ATPase virtual

项目摘要

Summary Neuronal ceroid lipofuscinoses (NCLs), as a group are commonly called Batten disease. These are the most common neurodegenerative lysosomal storage disorders (LSDs) that mostly affect children. As a group, these diseases have no curative treatment. Mutations in at least 13 different genes (called the CLNs) underlie various forms of NCLs. The infantile NCL (or INCL), also called infantile Batten disease,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 S-palmitoylated (S-acylated) 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 causing 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. We are attempting to isolate homogeneous cultures of A1 Astrocytea and characterize the neurotoxins. The Cln1-/- mice, which mimic human INCL, develop progressive neuroinflammation contributing to neurodegeneration. However, the underlying mechanism of neuroinflammation in INCL and in Cln1-/- mice has remained elusive. Previously, it has been reported that microRNA-155 (miR-155) regulates inflammation and miR profiling in Cln1-/- mouse brain showed that the level of miR-155 was upregulated. Thus, we sought to determine whether ablation of miR-155 in Cln1-/- mice may suppress neuroinflammation in these mice. Towards this goal, we generated Cln1-/-/miR-155-/- double-knockout mice and evaluated the inflammatory signatures in the brain. We found that the brains of double-KO mice manifest progressive neuroinflammatory changes virtually identical to those found in Cln1-/- mice. We conclude that ablation of miR-155 in Cln1-/- mice does not alter the neuroinflammatory trajectory in INCL mouse model. To 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 impaired autophagy,causing neuronal deat

概括神经元蜡质脂褐质沉积症（NCL）通常被称为巴顿病。这些是最常见的神经退行性溶酶体贮积症 (LSD)，主要影响儿童。作为一个群体，这些疾病没有治愈方法。至少 13 个不同基因（称为 CLN）的突变是各种形式的 NCL 的基础。婴儿 NCL（或 INCL），也称为婴儿 Batten 病，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 S-棕榈酰化（S-酰化）蛋白（蜡质成分）的去棕榈酰化，以便通过溶酶体水解酶对其进行回收或降解。 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。我们正在尝试分离 A1 星形胶质细胞的同质培养物并表征神经毒素。 Cln1-/- 小鼠模仿人类 INCL，会出现进行性神经炎症，导致神经变性。然而，INCL 和 Cln1-/- 小鼠神经炎症的潜在机制仍不清楚。此前有报道称，microRNA-155 (miR-155) 调节炎症，Cln1-/- 小鼠大脑中的 miR 分析显示 miR-155 水平上调。因此，我们试图确定 Cln1-/- 小鼠中 miR-155 的消除是否可以抑制这些小鼠的神经炎症。为了实现这一目标，我们培育了 Cln1-/-/miR-155-/- 双敲除小鼠，并评估了大脑中的炎症特征。我们发现双敲除小鼠的大脑表现出进行性神经炎症变化，与 Cln1-/- 小鼠的大脑几乎相同。我们得出的结论是，Cln1-/- 小鼠中 miR-155 的消除不会改变 INCL 小鼠模型中的神经炎症轨迹。为了探索 INCL 发病机制的潜在机制，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，自噬受损，导致神经元死亡

项目成果

期刊论文数量（28）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Suppression of agrin-22 production and synaptic dysfunction in Cln1 (-/-) mice.

DOI：
10.1002/acn3.261
发表时间：
2015-12
期刊：
Annals of clinical and translational neurology
影响因子：
5.3
作者：
Peng S;Xu J;Pelkey KA;Chandra G;Zhang Z;Bagh MB;Yuan X;Wu LG;McBain CJ;Mukherjee AB
通讯作者：
Mukherjee AB

Neuroprotection and lifespan extension in Ppt1(-/-) mice by NtBuHA: therapeutic implications for INCL.

DOI：
10.1038/nn.3526
发表时间：
2013-11
期刊：
NATURE NEUROSCIENCE
影响因子：
25
作者：
Sarkar, Chinmoy;Chandra, Goutam;Peng, Shiyong;Zhang, Zhongjian;Liu, Aiyi;Mukherjee, Anil B.
通讯作者：
Mukherjee, Anil B.

Children with infantile neuronal ceroid lipofuscinosis have an increased risk of hypothermia and bradycardia during anesthesia.

DOI：
10.1213/ane.0b013e3181aa6e95
发表时间：
2009-08
期刊：
Anesthesia and analgesia
影响因子：
5.7
作者：
Miao N;Levin SW;Baker EH;Caruso RC;Zhang Z;Gropman A;Koziol D;Wesley R;Mukherjee AB;Quezado ZM
通讯作者：
Quezado ZM

Subdural fluid collections in patients with infantile neuronal ceroid lipofuscinosis.

DOI：
10.1001/archneurol.2009.263
发表时间：
2009-12
期刊：
ARCHIVES OF NEUROLOGY
影响因子：
0
作者：
Levin, Sondra W.;Baker, Eva H.;Gropman, Andrea;Quezado, Zenaide;Miao, Ning;Zhang, Zhongjian;Jollands, Alice;Di Capua, Matteo;Caruso, Rafael;Mukherjee, Anil B.
通讯作者：
Mukherjee, Anil B.

Evaluation of disease progression in INCL by MR spectroscopy.