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.

项目总结/文摘