CHIP protects lysosomes from CLN4 mutant-induced membrane damage

Understanding how cells mitigate lysosomal damage is critical for unraveling pathogenic mechanisms of lysosome-related diseases. Here we generated and characterized iPSC-derived neurons (i3Neuron) bearing Ceroid Lipofuscinosis Neuronal 4 (CLN4)-linked DNAJC5 mutations, which revealed extensive lysosomal abnormality in mutant neurons. In vitro membrane-damaging experiments establish lysosome damages, caused by lysosome-associated CLN4 mutant aggregates, as a critical pathogenic linchpin in CLN4-associated neurodegeneration. Intriguingly, in non-neuronal cells, a ubiquitin-dependent microautophagy mechanism downregulates CLN4 aggregates to counteract CLN4-associated lysotoxicity. Genome-wide CRISPR screens identify the ubiquitin ligase CHIP as a central microautophagy regulator that confers ubiquitin-dependent lysosome protection. Importantly, CHIP's lysosome protection function is transferrable: ectopic CHIP improves lysosomal function in CLN4 i3Neurons and effectively alleviates lipofuscin accumulation and cell death in a Drosophila CLN4 disease model. Our study establishes CHIP-mediated microautophagy as a key organelle guardian that preserves lysosome integrity, offering new insights into therapeutic development for lysosome-related neurodegenerative diseases. Overall design: 293T cells stably expressing either mKeima-tagged human DNAJC5 wild type or J-domain deleted mutant (delJ or DJ) were infected with GeckoV2 libraries. Keima neutral (3% of cells) and Keima acidic (97%) were sorted by a FACS Ariall cell sorter. Genomic DNA was extracted for amplification of sgRNAs by PCB. Amplified sgRNAs were deep-sequenced by an Illumina platform.