Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis

Abstract

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS) or with compound heterozygous loss-of-function mutations (mutCTNS). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis

“…These findings are consistent with data showing that an increase of parkin expression results in mitochondrial fragmentation [36] and is associated with MFN2 ubiquitination [37]. In addition, the increase of FIS1, in cystinotic cell, might be due to Sirt3 protein [38] that was found downregulated in the same cystinotic cell line [8]. OPA1, an inner mitochondrial membrane GTPase protein, has gained attention because it regulates important mitochondrial functions, including the balance between mitochondrial fusion and fission processes, the stability of the mitochondrial respiratory chain complexes, the proapoptotic release of cytochrome c molecules sequestered within the mitochondrial cristae and the maintenance of mitochondrial cristae architecture [39].…”

“…However, cysteamine does not correct the above cited cellular alterations and does not stop the progress of the Fanconi syndrome. Our recent studies have shown in CTNS−/− ciPTEC a higher mitochondrial fragmentation index associated with lower mitochondrial potential and mitochondrial cyclic AMP levels, rescued by 24 h treatment with 100 µM cysteamine or with the cell-permeant analogue of cyclic AMP, 8-Br-cAMP [8]. cAMP, in fact, is one of the major regulators of mitochondrial function [28][29][30] and dynamics [31].…”

“…Renal tubular cells are very rich in mitochondria due to the intense reabsorption and excretion processes that occur in this district. We recently reported, in CTNS−/− cells derived from proximal tubules, mitochondrial fragmentation associated with respiratory chain dysfunction and low mitochondrial 3 ,5 -cyclic adenosine monophosphate (cAMP) levels [8]. Furthermore, enhanced apoptosis [9,10], defect of autophagic flux [11,12], and endo-lysosomal dysfunction [13,14] were observed.…”

Enhanced Intrinsic Skin Aging in Nephropathic Cystinosis Assessed by High-Definition Optical Coherence Tomography

“…Thus, we exclude the possible confounding of steroid treatment in KTx recipients, while demonstrating, in a quantitative manner, the presence of a skin phenotype that points to enhanced intrinsic skin aging in cystinosis (Moloney et al, 2005;Schoepe et al, 2006). As the pathophysiology of cystinosis shares similar features associated with cellular senescence as in intrinsic skin aging, including enhanced apoptosis and impaired autophagic flux resulting in mitochondrial dysfunction and increased generation of reactive oxygen species, it is likely that these mechanisms underlie the skin phenotype (Bellomo et al, 2018;Cherqui and Courtoy, 2017;Jenkins, 2002;Levtchenko et al, 2005;Luciani et al, 2018 Figure 1. Epidermal and papillary dermis thinning is present in nonekidney-transplanted patients with cystinosis in adolescent and young adult age group as assessed by HD-OCT. (a) HD-OCT image acquisition analysis workflow.…”

DYNC1LI2 regulates localization of the chaperone-mediated autophagy-receptor LAMP2A and improves cellular homeostasis in cystinosis

“…Mitochondrial fission is an important mechanism that precedes elimination of damaged mitochondrial by mitophagy 41 and mitochondrial fragmentation is induced by oxidative stress and has been associated with human disease. Mitochondrial dysfunction has long been demonstrated in many LSDs including cystinosis 42 . Here, to establish the impact of DYNC1LI2 reconstitution on mitochondrial fragmentation in cystinotic cells, we measured mitochondria fragmentation in wild-type and Ctns -/cells expressing DYNC1LI2 or DYNC1LI1.…”