Comparative proteomic profiling reveals mechanisms for early spinal cord vulnerability in CLN1 disease

CLN1 disease is a fatal inherited neurodegenerative lysosomal storage disease of early childhood, caused by mutations in the CLN1 gene, which encodes the enzyme Palmitoyl protein thioesterase-1 (PPT-1). We recently found significant spinal pathology in Ppt1-deficient (Ppt1−/−) mice and human CLN1 disease that contributes to clinical outcome and precedes the onset of brain pathology. Here, we quantified this spinal pathology at 3 and 7 months of age revealing significant and progressive glial activation and vulnerability of spinal interneurons. Tandem mass tagged proteomic analysis of the spinal cord of Ppt1−/−and control mice at these timepoints revealed a significant neuroimmune response and changes in mitochondrial function, cell-signalling pathways and developmental processes. Comparing proteomic changes in the spinal cord and cortex at 3 months revealed many similarly affected processes, except the inflammatory response. These proteomic and pathological data from this largely unexplored region of the CNS may help explain the limited success of previous brain-directed therapies. These data also fundamentally change our understanding of the progressive, site-specific nature of CLN1 disease pathogenesis, and highlight the importance of the neuroimmune response. This should greatly impact our approach to the timing and targeting of future therapeutic trials for this and similar disorders

AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion of Dictyostelium Contractile Vacuoles

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane–coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells