Mutations associated with Charcot-Marie-Tooth disease cause SIMPLE protein mislocalization and degradation by the proteasome and aggresome-autophagy pathways.

Mutations in SIMPLE cause an autosomal dominant, demyelinating form of peripheral neuropathy termed Charcot-Marie-Tooth disease type 1C (CMT1C), but the pathogenic mechanisms of these mutations remain unknown. Here, we report that SIMPLE is an early endosomal membrane protein that is highly expressed in the peripheral nerves and Schwann cells. Our analysis has identified a transmembrane domain (TMD) embedded within the cysteine-rich (C-rich) region that anchors SIMPLE to the membrane, and suggests that SIMPLE is a post-translationally inserted, C-tail-anchored membrane protein. We found that CMT1C-linked pathogenic mutations are clustered within or around the TMD of SIMPLE and that these mutations cause mislocalization of SIMPLE from the early endosome membrane to the cytosol. The CMT1C-associated SIMPLE mutant proteins are unstable and prone to aggregation, and they are selectively degraded by both the proteasome and aggresome-autophagy pathways. Our findings suggest that SIMPLE mutations cause CMT1C peripheral neuropathy by a combination of loss-of-function and toxic gain-of-function mechanisms, and highlight the importance of both the proteasome and autophagy pathways in the clearance of CMT1C-associated mutant SIMPLE proteins.