Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport, Alleviates Endoplasmic Reticulum Stress, and Reduces Lysosome Overload in Cystinosis

Abstract

bCystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis. We found that Rab27a was expressed in proximal tubular cells (PTCs) and partially colocalized with the lysosomal marker LAMP-1. The expression of Rab27a but not other small GTPases, including Rab3 and Rab7, was downregulated in kidneys from Ctns ؊/؊ mice and in human PTCs from cystinotic patients. Using total internal reflection fluorescence microscopy, we found that lysosomal transport is impaired in Ctns ؊/؊ cells. Ctns ؊/؊ cells showed significant ER expansion and a marked increase in the unfolded protein response-induced chaperones Grp78 and Grp94. Upregulation of the Rab27a-dependent vesicular trafficking mechanisms rescued the defective lysosomal transport phenotype and reduced ER stress in cystinotic cells. Importantly, reconstitution of lysosomal transport mediated by Rab27a led to decreased lysosomal overload, manifested as reduced cystine cellular content. Our data suggest that upregulation of the Rab27a-dependent lysosomal trafficking and secretory pathways contributes to the correction of some of the cellular defects induced by lysosomal overload in cystinosis, including ER stress.

Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation

“…TIRF Microscopy-The analysis of vesicular trafficking in LysoTracker-labeled fibroblasts treated with Nexinhib20 or DMSO was performed by TIRF microscopy using a ϫ100 1.45 numerical aperture TIRF objective (Nikon) on a Nikon TE2000U microscope custom-modified with a TIRF illumination module as described (34). Images were acquired on a 14-bit cooled charge-coupled device camera (Hamamatsu) controlled through NIS-Elements software.…”

A Mouse Model Suggests Two Mechanisms for Thyroid Alterations in Infantile Cystinosis: Decreased Thyroglobulin Synthesis Due to Endoplasmic Reticulum Stress/Unfolded Protein Response and Impaired Lysosomal Processing

“…As an additional hypothesis, nonmutually exclusive upstream mechanism, cystinosis causes ER stress (37) to which thyrocytes are particularly prone (38) so that ER stress would impair Tg synthesis and its supply to lysosomes. ER stress triggers the complex adaptive unfolded protein response (UPR) (for review see reference 39).…”

Time Course of Pathogenic and Adaptation Mechanisms in Cystinotic Mouse Kidneys

“…15 Among candidate genes promoting lysosomal discharge are Rab27a and related machineries. 38 Amorphous inclusions are then converted into membrane-bound micrometric crystals that coalesce into huge aggregates, deforming the membrane of lysosomes and becoming progressively excluded from endocytic trafficking (i.e., residual bodies). Distorted lysosomes can discharge their WT, n=3 Ctns 2/2 ).…”