Macroautophagy and protein aggregate formation in lysosomal disease

Abstract

Lysosomal diseases comprise a group of nearly 60 distinct disorders characterized by dysfunction of the lysosomal system. While most of these disorders present with systemic involvement, nearly two-thirds are associated with severe CNS impairment. To date, lysosomal disease pathogenesis remains largely undefined. In several lysosomal disease models we evaluated a key component of the lysosomal system, the degradative mechanism known as macroautophagy. Macroautophagy is essential for neuronal survival, as impairment of this mechanism is widely associated with neurodegeneration. Our initial studies established protein aggregate accumulation throughout the CNS of several diseases including the Cln2-/- mouse model of late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 is caused by loss of the lysosomal protease tripeptidyl peptidase I and results in lysosomal storage accumulation. We found aggregates in Cln2-/- mice contain the macroautophagy adapter proteins p62 (p62/Sqstm1) and NBR1 (neighbor of BRCA1 gene 1). Both p62 and NBR1 are preferentially degraded by macroautophagy, and their accumulation is implicated in macroautophagy dysfunction. Interestingly, we identified that the appearance of aggregates in Cln2-/- mice did not temporally correlate with changes in macroautophagy, suggesting that macroautophagy impairment may not be causal for aggregate formation. Unexpectedly, we found evidence that p62 co-localizes with the primary lysosomal storage protein subunit c of mitochondrial ATP synthase (SCMAS) in cytosolic extra-lysosomal aggregates in vivo. This led us to hypothesize that protein aggregate formation in Cln2-/- mice results from lysosomal membrane permeability (LMP). Specifically, we predicted that LMP results in the release of lysosomal contents into the cytosol, where it is subsequently sequestered by the adapter proteins p62 and NBR1. Further in vivo studies confirmed evidence for LMP, and in vitro experiments in primary neuronal cultures established that p62 specifically responds to LMP. We conclude that protein aggregate accumulation is a prominent neuropathological feature of several lysosomal diseases and that the formation of aggregates in Cln2-/- mice is stimulated following LMP. Furthermore, we describe a novel role for the protein p62 in responding to LMP. These findings likely have implications for other lysosomal and neurodegenerative diseases characterized by p62 accumulation.