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

• 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
• 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

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.

项目摘要/摘要 1型神经元粘膜脂肪肌动症（CLN1）是一种毁灭性的神经退行性疾病，伴有婴儿发作。 CLN1是由棕榈酰蛋白硫代酶1（PPT1）中功能丧失突变引起的，这是一个关键 大脑中的deplitoyling酶。 CLN1和该疾病的其他遗传形式（NCLS）的特征是 以常见的临床症状和神经病理学的标志。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，以及脂肪霉素的积累，一种自动荧光溶酶体储存材料，很大程度上 未描述的成分。对CLN1的分子基础的理解，因此 已知的PPT1底物的有限曲目阻碍了处理。我们最近确定了更多 超过100个新型PPT1底物，并记录了一小部分溶酶体蛋白 在CLN1小鼠模型中，在早期疾病上上调和棕榈酰。令人惊讶的是，这些蛋白质包括 NCLS和其他溶酶体储存障碍的因果基因和遗传修饰剂。此外，这些显着的NCL 随着CLN1的进行，蛋白质保持上调，并发现，以及经过验证的PPT1底物 积聚在与人类年龄相关脂肪霉素的蛋白质组学数据集中。这些数据强烈表明Cln1， 其他形式的NCL和病理衰老具有共同的分子机制。但是，还不清楚 为什么溶酶体储存障碍蛋白的这一子集在CLN1和衰老过程中积累 有助于脂肪霉素病理学。拟议的研究利用了一个关键机会来剖析这些 推定的病因关系。 在AIM 1中，我将确定NCL蛋白是否是具有体外去氨木酰化测定和揭开的PPT1底物 如果CLN1伴有NCL蛋白与水解酶活性面板的溶酶体功能的缺陷。 因此，此目标将扩大已知的PPT1基板曲目和细节之间的关键机理联系 NCLS。 NCL蛋白的降解，贩运和溶酶体隔室将进行测试 AIM 2使用环己酰亚胺追逐，活细胞成像和免疫细胞化学。不管NCL是否 蛋白质是PPT1底物，该方法将为其上调提供机械基础。最后，在 AIM 3，CLN1脂肪霉素的组成，包括NCL蛋白和棕榈酸酯脂质的组成，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在神经元功能和收敛分子中的新作用 NCL和衰老发病机理的机制，可能提供治疗靶标。来自多元化的指导 我提出的技术专家小组将有助于我作为独立的细胞和分子的培训 神经生物学家。