Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A

Abstract

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

FYCO1 mediates clearance of α‐synuclein aggregates through a Rab7‐dependent mechanism

“…Our finding that RILP over-expression had no beneficial effect on autophago-lysosomal aggregate clearance was unexpected given the important role of RILP for lysosomal degradation of other substrates, including EGF receptor and LDL (Cantalupo et al 2001) and its involvement in chaperone-mediated autophagy (Zhang et al 2017). Yet, RILPdependent EGF receptor degradation was supported by Rab7 mutants associated with Charcot-Marie-Tooth (CMT) neuropathy (Spinosa et al 2008), whereas we previously Fig.…”

“…) and its involvement in chaperone‐mediated autophagy (Zhang et al . ). Yet, RILP‐dependent EGF receptor degradation was supported by Rab7 mutants associated with Charcot–Marie–Tooth (CMT) neuropathy (Spinosa et al .…”

Chaperone-mediated autophagy and endosomal microautophagy: Jointed by a chaperone

“…Upon CMA activation, LAMP2A is actively excluded from these microdomains (60). Recent studies have shown that defective targeting of LAMP2A from Golgi to lysosomes is behind the low efficiency of CMA in the lysosomal storage disease cystinosis (61,62). These findings highlight LAMP2A trafficking as a possible additional mechanism for regulation of CMA activity.…”

Molecular Basis of Cystinosis: Geographic Distribution, Functional Consequences of Mutations in the <b><i>CTNS</i></b> Gene, and Potential for Repair

“…Another role of CTNS, in addition to its potential involvement in mTORC1 signalling, is the altered chaperone mediated autophagy, as shown by two studies from Napolitano et al [37] and Zhang et al [36]. They observed impaired chaperone mediated autophagy (CMA) in cystinotic mouse skin fibroblasts presenting dislocation of LAMP2A (CMA receptor) and accumulation of GAPDH (CMA substrate).…”

“…This was also true for CTNS-LKG, which is an alternative splice variant of CTNS, primarily targeted to the plasma membrane and other lysosomal/endosomal vesicles. Based on these results, it was suggested that CTNS is a necessary cofactor for LAMP2A trafficking [36,37].…”