Defining the Role of CLN6 in Lysosomal Function and Neurodegeneration

The student will investigate neuronal ceroid lipofuscinosis type 6 (CLN6 disease) by generating a CLN6 knockout cell model and deriving induced pluripotent stem cells (iPSCs) from patient fibroblasts with homozygous CLN6 mutations. Although CLN6 is an endoplasmic reticulum (ER) transmembrane protein, its deficiency leads to lysosomal storage disease, underscoring a critical ER-lysosome functional connection. The research will focus on elucidating the downstream effects of CLN6 loss on both ER and lysosomal functions. Utilizing our existing CLN6 knockout mouse model, the student will further explore disease mechanisms in vivo. Additionally, the student will acquire skills in immunoprecipitation of organelles, such as the ER and lysosomes, followed by downstream multi-omics analyses to investigate molecular alterations resulting from CLN6 deficiency.

The student will be assigned specific tasks from the outlined deliverables, with the selection and sequencing of these tasks contingent upon the project's progression and current needs within the laboratory. This adaptive approach ensures that the student's efforts align with the project's dynamic requirements, fostering both effective learning and meaningful contributions to the research objectives. Desired Deliverables: 1. Generation of CLN6 Knockout Cell Models: o Develop and validate CLN6 knockout cell lines to serve as in vitro models for studying neuronal ceroid lipofuscinosis type 6 (CLN6 disease). 2. Derivation of Patient-Specific iPSCs: o Establish induced pluripotent stem cells (iPSCs) from fibroblasts of patients with homozygous CLN6 mutations, ensuring their pluripotency and genomic integrity. 3. Comprehensive In vitro and In vivo Multi-Omics Analysis: o Conduct integrated multi-omics analyses—including proteomics and transcriptomics—on CLN6-deficient cell models, differentiated neurons, and brain tissues from CLN6 knockout mouse models to identify molecular alterations and affected pathways. 4. Investigation of Pathogenic CLN6 Mutations: o Examine the impact of various pathogenic CLN6 mutations on protein function and subcellular localization, providing insights into genotype-phenotype correlations. 5. Application of TurboID Proximity Labeling: o Utilize TurboID-based proximity labeling to map protein-protein interactions and organelle-specific proteomes associated with CLN6, enhancing the understanding of its role in cellular pathways. These deliverables aim to advance the understanding of CLN6 disease mechanisms and contribute to the development of potential therapeutic strategies.