Chaperone-Mediated Autophagy Upregulation Rescues Megalin Expression and Localization in Cystinotic Proximal Tubule Cells

Abstract

Cystinosis is a lysosomal storage disorder caused by defects in CTNS, the gene that encodes the lysosomal cystine transporter cystinosin. Patients with nephropathic cystinosis are characterized by endocrine defects, defective proximal tubule cell (PTC) function, the development of Fanconi syndrome and, eventually, end-stage renal disease. Kidney disease is developed despite the use of cysteamine, a drug that decreases lysosomal cystine overload but fails to correct overload-independent defects. Chaperone-mediated autophagy (CMA), a selective form of autophagy, is defective in cystinotic mouse fibroblasts, and treatment with cysteamine is unable to correct CMA defects in vivo, but whether the vesicular trafficking mechanisms that lead to defective CMA in cystinosis are manifested in human PTCs is not currently known and whether PTC-specific mechanisms are corrected upon CMA upregulation remains to be elucidated. Here, using CRISPR-Cas9 technology, we develop a new human PTC line with defective cystinosin expression (CTNS-KO PTCs). We show that the expression and localization of the CMA receptor, LAMP2A, is defective in CTNS-KO PTCs. The expression of the lipidated form of LC3B, a marker for another form of autophagy (macroautophagy), is decreased in CTNS-KO PTCs indicating decreased autophagosome numbers under basal conditions. However, the autophagic flux is functional, as measured by induction by starvation or by blockage using the v-ATPase inhibitor bafilomycin A, and by degradation of the macroautophagy substrate SQSTM1 under starvation and proteasome-inhibited conditions. Previous studies showed that LAMP2A accumulates in Rab11-positive vesicles in cystinotic cells. Here, we show defective Rab11 expression, localization and trafficking in CTNS-KO PTCs as determined by confocal microscopy, immunoblotting and TIRFM. We also show that both Rab11 expression and trafficking in cystinotic PTCs are rescued by the upregulation of CMA using small-molecule CMA activators. Cystinotic PTCs are characterized by PTC de-differentiation accompanied by loss of the endocytic receptor megalin, and megalin recycling is regulated by Rab11. Here we show that megalin plasma membrane localization is defective in CTNS-KO PTCs and its expression is rescued by treatment with CMA activators. Altogether, our data support that CMA upregulation has the potential to improve PTC function in cystinosis.

Keywords: LAMP2A; Rab 11 GTPase; chaperone-mediated autophagy (CMA); cystinosis; fanconi syndrome; lysosomal storage disorder (LSD); megalin; vesicular trafficking.

Figure 1

Generation of cystinosin knockout human proximal tubule cell line using CRISPR/Cas9. (A) Human CTNS genomic sequence; underlined sequences indicate exon 4, gRNA sequences were marked as red and blue, and PAM sequences are in bold. The allele 1 has 37bp insertion which was marked in pink; allele 2 has 17 bp deletion which was marked as a red dashed line. (B) PCR analysis of CTNS gene expression in WT and CTNS-KO HK-2 cells. (C) Surveyor assay of three potential off-target sites that localize in TMEM2, TMIGD2, and LDB3 genes. (D) Cystine levels in WT and CTNS-KO HK-2 cells were determined by mass spectrometry. The data are represented as mean ± SEM from three independent experiments. ^***p < 0.001.

Figure 2

Mislocalization and decreased expression of LAMP2A in CTNS-KO PTCs. (A,B) Confocal microscopy analysis and quantification of the distribution of endogenous LAMP1 and LAMP2A in WT and CTNS-KO PTCs. Scale bar, 10 μm. mean ± SEM. ^***p < 0.001, Student's t-test. (C) Western blot analysis of LAMP1, LAMP2A, and LC3B expression levels in WT and CTNS-KO PTCs. Left panel, Representative immunoblots. Ponceaus S staining is shown for equal loading. Middle and right panels, Quantification of 3 independent experiments. The individual symbols correspond to independent biological replicates from 3 independent experiments. The bars represent the mean ± SEM. ^*p < 0.05; ^**p < 0.01, Student's t-test. (D) SQSTM1/p62 and LC3B-II levels in WT and CTNS-KO PTCs were analyzed by Western blot under fed and serum starvation conditions, in the absence or presence of 100 nM bafilomycin A (BafA) or 1 μM Clasto-Lactacystin β-lactone (proteasome inhibitor) for 5 h. Left panel, Representative immunoblots. Middle and right panel, Quantification of the expression levels of SQSTM1 (p62) and LC3B-II, respectively. Each value has been normalized to the actin expression level in the same sample. Results are expressed relatively to the wild type fed condition. The individual symbols correspond to independent biological replicates from 3 independent experiments. The bars represent the mean ± SEM. ^*p < 0.05.

Figure 3

Rab11 is down-regulated and mislocalized in CTNS-KO PTCs. (A) Confocal microscopy analysis of the distribution of endogenous Rab11 and LAMP2A in WT and CTNS-KO PTCs. Scale bar, 10 μm. (B) WT and CTNS-KO PTCs were treated with DMSO or 20 μM QX77 for 48 h, and Rab11 expression levels were analyzed by Western blot. Quantitative analysis of Rab11 expression levels. The individual symbols correspond to independent biological replicates from 4 independent experiments. The expression level of Rab11 was normalized to actin in each sample. The bars represent the mean ± SEM. ^**p < 0.01, and ^***p < 0.001, Student's t-test.

Figure 4

Rab11 trafficking is impaired in CTNS-KO PTCs and is enhanced by CMA activation. (A) Representative images of GFP-Rab11 in WT, CTNS-KO PTCs, and CTNS-KO PTCs treated with QX77. Scale bar, 10 μm. (B,C) Quantitative analysis of the trafficking of GFP-Rab11 in WT, CTNS-KO PTCs, and CTNS-KO PTCs treated with 20 μM QX77 for 72 h. (B) Quantitative analysis of the numbers of vesicles with decreased motility (speed < 0.1 μm/s) in wild type, CTNS-KO PTCs and CTNS-KO PTCs treated with QX77. The individual symbols correspond to independent biological replicates from 3 independent experiments. The bars indicate the mean ± SEM. ^*p < 0.05. (C) Histograms represent the speeds of GFP-Rab11-containing organelles. The speeds for the independent vesicles were binned in 0.05 μm/s increments and plotted as a percentage of total vesicles for a given cell. Results are represented as mean ± SEM from at least 22 cells from 3 independent experiments. The statistically significant differences between the groups are indicated in the figure. Student's t-test.

Figure 5

Decreased membrane localization and expression of megalin in CTNS-KO PTCs is rescued by CMA activation. (A) Confocal microscopy analysis of the distribution of endogenous megalin. WT, CTNS-KO PTCs, and CTNS-KO PTCs treated with 20 μM QX77 or vehicle for 72 h were fixed and endogenous megalin was detected by immunofluorescence (green) and nuclei stained with DAPI (blue). The arrows indicate plasma membrane localization of megalin. Scale bar, 10 μm. (B) Megalin expression was quantified by analysis of the mean fluorescence intensity of megalin in each cell using ImageJ. Each symbol represents a cell. The data are from one experiment, and are representative of three independent experiments with the same result. (C) Megalin expression was analyzed as in B. The individual symbols correspond to independent biological replicates from 3 independent experiments. The bars indicate the mean ± SEM. The data are normalized to the values observed in wild type cells. Significant differences between CTNS-KO untreated and QX77-treated CTNS-KO PTCs were calculated using the Student's t-test. ^*p < 0.05.

Figure 6

Schematic representation of the effect of chaperone-mediated autophagy (CMA) enhancers on cystinotic proximal tubule cells (PTCs). Left panel, Cystinotic PTCs are characterized by decreased localization of the CMA receptor LAMP2A at the lysosomal membrane, reduced Rab11 expression, impaired Rab11 trafficking and decreased plasma membrane localization and expression of the apical receptor megalin, a phenotype that is associated with the development of Fanconi syndrome in cystinosis. Right panel, upon treatment with CMA enhancers, cystinotic PTCs show increased LAMP2A at the lysosomal membrane, enhanced Rab11 trafficking and increased apical distribution of megalin along with increased megalin expression, suggesting that CMA upregulation has direct positive implications for PTC function.