*Article* **Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis**

**Domenico De Rasmo 1,\*,**†**, Anna Signorile 2,**†**, Ester De Leo 3, Elena V. Polishchuk 4, Anna Ferretta 1, Roberto Raso 3, Silvia Russo 2, Roman Polishchuk 4, Francesco Emma <sup>5</sup> and Francesco Bellomo 3,\***


Received: 29 November 2019; Accepted: 24 December 2019; Published: 26 December 2019

**Abstract:** Nephropathic cystinosis is a rare lysosomal storage disorder caused by mutations in *CTNS* gene leading to Fanconi syndrome. Independent studies reported defective clearance of damaged mitochondria and mitochondrial fragmentation in cystinosis. Proteins involved in the mitochondrial dynamics and the mitochondrial ultrastructure were analyzed in *CTNS*−/− cells treated with cysteamine, the only drug currently used in the therapy for cystinosis but ineffective to treat Fanconi syndrome. *CTNS*−/− cells showed an overexpression of parkin associated with deregulation of ubiquitination of mitofusin 2 and fission 1 proteins, an altered proteolytic processing of optic atrophy 1 (OPA1), and a decreased OPA1 oligomerization. According to molecular findings, the analysis of electron microscopy images showed a decrease of mitochondrial cristae number and an increase of cristae lumen and cristae junction width. Cysteamine treatment restored the fission 1 ubiquitination, the mitochondrial size, number and lumen of cristae, but had no effect on cristae junction width, making *CTNS*−/− tubular cells more susceptible to apoptotic stimuli.

**Keywords:** Fanconi syndrome; nephropathic cystinosis; mitochondrial dynamics; cysteamine; mitochondrial fusion; mitochondrial fission; mitochondrial cristae

### **1. Introduction**

Nephropathic cystinosis (MIM 219800) is a rare inherited metabolic disease characterized by an impaired transport of the amino acid cystine out of lysosomes due to reduced or absent function of the specific carrier cystinosin, which is encoded by *CTNS* gene [1–3]. Kidneys are affected at the initial stage of the disease, leading to early onset Fanconi syndrome, which is characterized by polyuria, glycosuria, phosphaturia, aminoaciduria, and urinary loss of electrolytes and low-molecular-weight proteins [4]. The cystine-depleting agent, cysteamine (MEA), significantly delays symptoms [5,6], but does not treat Fanconi syndrome and is ineffective to prevent the progression of kidney disease. Fanconi syndrome has also been reported in children with specific mitochondrial syndromes [7]. 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.

Communication between mitochondria and the endo-lysosomal system is complex. Increasing evidences show close relationship between these two cellular compartments [15,16]. Mitochondria constantly undergo fission and fusion processes to adapt to environmental changes in a continuous and balanced way, in order to maintain morphology and regulate cellular ATP levels. Mitochondrial fission regulates the production of new mitochondria and the segregation of damaged mitochondria. In this process, receptor proteins such as mitochondrial fission factor (Mff), mitochondrial fission 1 protein (Fis1), mitochondrial dynamics protein of 49 kDa (MiD49), and mitochondrial dynamics protein of 51 kDa (MiD51) recruit the large GTPase dynamin-related protein 1 (Drp1) from the cytosol to the outer mitochondrial membrane (OMM), which forms a multimeric complex with mitochondrial membrane adaptors [17,18]. Mitochondrial fusion is mediated by three key regulatory fusion proteins: the dynamin-related GTPases mitofusin 1 (MFN1) and mitofusin 2 (MFN2), responsible for the fusion of OMM and the dynamin-related GTPases optic atrophy 1 (OPA1), which mediates fusion of the inner mitochondrial membrane (IMM) and contributes to the maintenance of mitochondrial potential, to control respiratory chain activity and apoptosis [19,20]. When mitochondrial dynamics are impaired, dysfunctional mitochondria are selectively eliminated through mitophagy, which is initiated by ubiquitin-dependent or ubiquitin-independent signals [21]. The lysosomal alterations in cystinosis lead to defective autophagic clearance of damaged mitochondria [12], therefore the purpose of our study was to investigate mitochondrial dynamics in *CTNS*−/− conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion in the intent of identifying new therapeutic targets and biomarkers for treatment follow-up.

#### **2. Results**
