Molecular characterization of lipofuscin pathology in neuronal ceroid lipofuscinosis

神经元蜡状脂褐质沉积症脂褐质病理学的分子特征

基本信息

批准号：
10604677
负责人：
Sofia Massaro Tieze
金额：
$ 4.77万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-16 至 2024-12-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10604677
关键词：
Affect Age Aging Biochemical Biological Assay Blindness Brain CRISPR/Cas technology Carbohydrates Cathepsins Characteristics Chemicals Chemistry Child Childhood Clinical Complement Cycloheximide Data Data Set Disease Disease Progression Dissection Electron Microscopy Enzymes Etiology Exhibits Family Gel Genetic Goals Human Hydrolase Hydroxylamine Immunoprecipitation Impaired cognition Impairment In Situ In Vitro Ions Kinetics Knockout Mice Label Link Lipids Lipofuscin Maps Mass Spectrum Analysis Membrane Membrane Proteins Mentorship Metals Modification Molecular Monitor Motor Mus Nerve Degeneration Neurodegenerative Disorders Neuronal Ceroid-Lipofuscinosis Neurons Other Genetics Palmitates Pathogenesis Pathology Patients Phospholipids Preparation Protein Sortings Proteins Proteome Proteomics Recombinants Research Resolution Retina Retinal Degeneration Role Seizures Slice Spectrometry, Mass, Secondary Ion Symptoms Synapses Techniques Testing Time Training Up-Regulation Western Blotting age related aged base causal variant common symptom enzyme deficiency experimental study galactosylgalactosylglucosylceramidase immunocytochemistry infancy live cell imaging loss of function mutation lysosomal proteins lysosome membrane mass spectrometric imaging mouse model neuron loss neuropathology normal aging novel palmitoylation pathological aging protein transport therapeutic target therapy development thioesterase PPT1 gene product trafficking tripeptidyl aminopeptidase

项目摘要

PROJECT SUMMARY/ABSTRACT Neuronal ceroid lipofuscinosis type 1 (CLN1) is a devastating neurodegenerative disease with infantile onset. CLN1 is caused by loss-of-function mutations in palmitoyl protein thioesterase 1 (PPT1), a critical depalmitoylating enzyme in the brain. CLN1 and other genetic forms of this disorder (NCLs) are characterized by common clinical symptoms and neuropathological hallmarks. Symptoms include seizures, vision loss, and progressive motor and cognitive decline, while the brains of NCL patients exhibit cortical neuron loss, retinal degeneration, and the accumulation of lipofuscin, an autofluorescent lysosomal storage material with largely undescribed composition. The understanding of the molecular basis of CLN1, and hence the development of treatments, has been hindered by the limited repertoire of known PPT1 substrates. We recently identified more than 100 novel PPT1 substrates and documented a small subset of lysosomal proteins that are both highly upregulated and palmitoylated at early-stage disease in a CLN1 mouse model. Strikingly, these proteins include causal genes and genetic modifiers of NCLs and other lysosomal storage disorders. Further, these salient NCL proteins remain upregulated as CLN1 progresses, and are found, along with validated PPT1 substrates, to accumulate in proteomic datasets of human age-related lipofuscin. These data strongly suggest that CLN1, other forms of NCL, and pathological aging share common molecular mechanisms. However, it remains unclear why this subset of lysosomal storage disorder proteins accumulates in CLN1 and during aging and how they contribute to lipofuscin pathology. The proposed research takes advantage of a key opportunity to dissect these putative etiological relationships. In Aim 1, I will determine if NCL proteins are PPT1 substrates with in vitro depalmitoylation assays and uncover if CLN1 is accompanied by deficits in the lysosomal functions of NCL proteins with hydrolase activity panels. This aim will thus expand the known PPT1 substrate repertoire and detail critical mechanistic links between NCLs. The degradation, trafficking, and lysosomal compartmentalization of NCL proteins will then be tested in Aim 2 using cycloheximide chases, live-cell imaging, and immunocytochemistry. Regardless of whether NCL proteins are PPT1 substrates, this approach will provide a mechanistic basis for their upregulation. Finally, in Aim 3, the composition of CLN1 lipofuscin, including the constituency of NCL proteins and palmitate lipids, will be assessed with electron microscopy and state-of-the art mass spectrometry techniques. Together, the realization of these studies will reveal novel roles of PPT1 in neuronal function and convergent molecular mechanisms of NCL and aging pathogenesis, which may provide therapeutic targets. Mentorship from a diverse panel of experts in my proposed techniques will facilitate my training as an independent cellular and molecular neurobiologist.

项目概要/摘要 1 型神经元蜡样质脂褐素沉着症 (CLN1) 是一种以婴儿期发病的破坏性神经退行性疾病。 CLN1 是由棕榈酰蛋白硫酯酶 1 (PPT1) 的功能丧失突变引起的，PPT1 是一种关键的大脑中的去棕榈酰酶。 CLN1 和该疾病的其他遗传形式 (NCL) 的特征通过常见的临床症状和神经病理学特征。症状包括癫痫发作、视力丧失和进行性运动和认知能力下降，而 NCL 患者的大脑表现出皮质神经元丧失、视网膜变性和脂褐质的积累，脂褐素是一种自发荧光的溶酶体储存材料，主要具有未描述的组成。了解 CLN1 的分子基础，从而开发已知 PPT1 底物的功能有限，阻碍了治疗的发展。我们最近发现了更多超过 100 种新颖的 PPT1 底物，并记录了一小部分溶酶体蛋白，它们都高度在 CLN1 小鼠模型的早期疾病中上调和棕榈酰化。引人注目的是，这些蛋白质包括 NCL 和其他溶酶体贮积症的致病基因和遗传修饰因子。此外，这些突出的 NCL 随着 CLN1 的进展，蛋白质保持上调，并与经过验证的 PPT1 底物一起被发现，在人类年龄相关脂褐质的蛋白质组数据集中积累。这些数据强烈表明 CLN1，其他形式的 NCL 和病理性衰老具有共同的分子机制。但目前尚不清楚为什么溶酶体贮积症蛋白的这个子集在 CLN1 中和衰老过程中积聚以及它们如何有助于脂褐质病理学。拟议的研究利用了一个关键的机会来剖析这些假定的病因学关系。在目标 1 中，我将通过体外去棕榈酰化测定确定 NCL 蛋白是否是 PPT1 底物并揭示如果 CLN1 伴有具有水解酶活性的 NCL 蛋白的溶酶体功能缺陷。因此，这一目标将扩展已知的 PPT1 底物库，并详细说明之间的关键机制联系。 NCL。然后，将测试 NCL 蛋白的降解、运输和溶酶体区室化。目标 2 使用放线菌酮追踪、活细胞成像和免疫细胞化学。不管是否NCL 蛋白质是PPT1底物，这种方法将为它们的上调提供机制基础。最后，在目标 3，CLN1 脂褐质的成分，包括 NCL 蛋白和棕榈酸酯脂质的成分，将通过电子显微镜和最先进的质谱技术进行评估。在一起，这些研究的实现将揭示 PPT1 在神经元功能和聚合分子中的新作用 NCL 和衰老发病机制，可能提供治疗靶点。来自多元化的指导我提出的技术专家小组将促进我作为独立细胞和分子的培训神经生物学家。