*2.2. Mitochondrial Cristae Organization Was Impaired in CTNS*−/− *ciPTEC*

Comparative ultrastructural analysis of mitochondria in ciPTEC, by transmission electron microscopy (TEM), showed the presence of smaller mitochondria in *CTNS*−/− ciPTEC compared to *CTNS*+/+ ciPTEC (0.12 <sup>±</sup> 0.01 <sup>μ</sup>m2 in *CTNS*−/<sup>−</sup> vs. 0.26 <sup>±</sup> 0.01 <sup>μ</sup>m2 in *CTNS*+/+, *p* < 0.0001). Mitochondrial area was completely recovered by MEA treatment (0.32 <sup>±</sup> 0.04 <sup>μ</sup>m2 in *CTNS*−/<sup>−</sup> + MEA vs. 0.12 <sup>±</sup> 0.01 <sup>μ</sup>m2 in *CTNS*−/−, *p* < 0.0001). Moreover, TEM showed a substantial reduction in number of cristae per mitochondrial section in *CTNS*−/− (5.1 ± 0.6 in *CTNS*+/+ vs. 1.6 ± 0.2 in *CTNS*−/−, *p* < 0.0001). This parameter was rescued by MEA by 167.5% (*p* < 0.0001). Because evidences underlined a critical role of cristae junction in mitochondrial function and organization, we measured them. It was found an increase in cristae junction width (39.65 ± 4.83 nm in *CTNS*+/+ vs. 53.21 ± 10.05 nm in *CTNS*−/−, *p* < 0.0001) and cristae lumen width (29.68 ± 3.53 nm in *CTNS*+/+ vs. 37.04 ± 6.13 nm in *CTNS*−/−, *p* < 0.0003) partially rescued by cysteamine (Figure 5).

**Figure 5.** Comparative ultrastructural analysis of mitochondria in ciPTEC. (**a**) Representative images of TEM with magnification 16,000× of untreated and MEA-treated ciPTEC *CTNS*+/+ and *CTNS*−/−; scale bar = 1 μm. As shown in high magnification cropped micrographs and in ad hoc schematic reconstruction, mitochondria kept preserved ultrastructure in ciPTEC *CTNS*+/+ and in MEA-treated ciPTEC *CTNS*+/+ and *CTNS*−/−, whereas ciPTEC *CTNS*−/− showed disruption of mitochondrial cristae and the disarrangement of the internal structures; scale bar = 200 nm. (**b**–**e**) Quantitative analysis was performed with ImageJ v.1.52p in *n* ≥ 5 double-blind acquisitions for each experimental condition, red lines represent median with interquartile range. (**b**) Evaluation of relative mitochondrial size measured as area of *n* ≥ 27 mitochondrial sections. (**c**) Average number of cristae per mitochondrion in each cell (*n* ≥ 27 mitochondrial sections). (**d**) The measure of distance of cristae junction near the inner membrane boundary and (**e**) the measure of cristae lumen, assessed on cristae membranes that outline the lumen boundary, were assessed in *n* ≥ 100 cristae. Non-parametric Mann-Whitney test was applied, \*\*\* *p* < 0.001; \* *p* < 0.05.

#### **3. Discussion**

Nephropathic cystinosis is a rare inherited metabolic disorder, belonging to the group of lysosomal storage diseases (LSD). The disease is the first cause of Fanconi syndrome in children, characterized by loss of electrolytes, glucose, amino acid, low-molecular weight proteins in urine caused by proximal tubule dysfunction [4,22]. The molecular mechanism at the basis of Fanconi syndrome in cystinosis is not completely understood. Several mechanisms have been suggested to contribute to the pathogenesis of cystinosis, including lysosomal overload, endo-lysosomal transport defect, altered chaperone-mediated autophagy, mTOR signaling, transcription factor EB (TFEB) expression [11,13,23–27]. Cysteamine, a cystine-depleting agent, which allows clearance of cystine from lysosomes, represents the only specific treatment for cystinosis. 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 [28–30] and dynamics [31]. In this contest, it should be noted that MEA has been found to improve mitochondrial function in mitochondrial respiratory chain diseases [32]. Mitochondrial dynamics is balanced between rates of fusion and fission that respond to pathophysiologic signals. This finely regulated equilibrium is closely related to the quality control system, which is mainly ascribed to the ubiquitin protease system (UPS) and to the intra-mitochondrial proteolytic systems [33]. In our experimental model, no significant differences were observed on protein levels of phosphorylated Drp1 at Ser-637. The PKA-dependent phosphorylation of Drp1 at Ser-637 is generally recognized to block Drp1 GTPase activity and to suppress mitochondrial fission. However, and in agreement with data previously reported by Yu et al., Drp1pS637 did not contribute substantially to mitochondrial fission regulation in *CTNS*−/− ciPTEC [18]. Several key effector proteins of mitochondrial fusion (MFN1 and MFN2) and fission (Fis1, Mff) are located at the OMM with their domains exposed at the cytosolic side of the membrane. This peculiar topology allows selective removing of fusion or fission components exposed by the UPS, providing fine tuning of this high-level regulatory processes. In mammalian cells, Fis1 accumulates in the mitochondrial outer membrane during the fission process, whereas MFN1 and MFN2 are ubiquitinated and degraded by the proteasome [34]. In this respect, we observed an increase in Fis1, ubiquitinated MFN2 and of the E3 ubiquitin ligase Parkin in *CTNS*−/− ciPTEC, indicating the tendency to mitochondrial fragmentation. The effect of MEA on the reduction of ubiquitilated MFN2 could be ascribed to the modulation of USP30, a mitochondrion-localized deubiquitilase, which counteracts Parkin by deubiquitilating OMM proteins and regulate mitophagy [35]. 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]. The protein expression levels of OPA1 were not significantly changed in mutated cells, compared to control ciPTEC cells (data not shown). However, the activity of OPA1 is also controlled at the post-translational level by proteolytic and acetylation changes [31]. Various stress conditions, including apoptotic stimulation, trigger the complete conversion of L-OPA1 into S-OPA1. The pro-fusion activity of OPA1 depends on the balanced formation of L-OPA1 and S-OPA1 [40]. In this respect, we observed an alteration of OPA1 processing in *CTNS*−/− ciPTEC cells. In particular, cystinotic cells were characterized by a significant increase of S-OPA1, associated with an increase in the protease OMA1 activity, that was not prevented by MEA. In addition to its role as a fusion protein, OPA1 controls the remodeling of mitochondrial cristae. Specifically, OPA1 forms oligomers in the

inner mitochondrial membrane that keep the cristae junctions tight. During apoptosis, oligomers are destabilized causing the opening of cristae and release of cytochrome *c* out of the mitochondria. OPA1 oligomers were decreased in cystinotic cells. MEA did not rescue this phenotype. This defect correlates with increased cristae junction width that we observed in our TEM ultrastructural analyses.

In summary, our study shows deregulation of several proteins involved in mitochondrial dynamics in *CTNS*−/− cells. We observed mitochondrial fragmentation in cystinotic cells associated with altered proteolytic processing of OPA1, increased Fis1 and parkin protein levels. The deregulation of parkin could result in increase of ubiquitination of MFN2. The cristae number was decreased while the cristae lumen was increased in cystinotic cells, which parallels the previously reported bioenergetic defects in these cells. The cristae junction width was increased in *CTNS*−/− cells, which is most likely secondary to low OPA1 oligomerization levels. MEA treatment restored mitochondrial size, cristae number, and lumen, but had no effect on cristae junction width, making tubular cells more susceptible to apoptotic stimuli. In this contest, we highlight several cellular mediators of mitochondrial dynamics that could be useful to develop new therapeutic interventions [41].

#### **4. Materials and Methods**

#### *4.1. Cell Culture*

Conditionally immortalized proximal tubular epithelial cells (ciPTEC), from healthy donor and cystinotic patients were obtained from Radboud University Medical Center, Nijmegen, The Netherlands and cultured as described in [42]. Cells were grown in a humidified atmosphere with 5% CO2 at 37 ◦C. Where indicated, the cells were treated with 100 μM MEA or water (vehicle) for 24 h.

#### *4.2. SDS-PAGE and Western Blotting*

Monolayer cell cultures were harvested with 0.05% trypsin, 0.02% EDTA. After trypsinization, cells were centrifuged at 500× *g* and resuspended in RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris/HCl, 0.1% SDS, 1% Triton X-100, pH 7.4), in the presence of a protease inhibitor (0.25 mM PMSF). Cell lysate proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a nitrocellulose membrane and immunoblotted with antibodies against OPA1 (Thermo scientific, Waltham, MA, USA; Pierce Antibodies); Fis1, Mfn2 (Merck Millipore, Burlington, MA, USA); parkin, USP30 (Santa Cruz Biotechnology, Dallas, TX, USA); OMA1 (Abcam, Cambridge, UK); Drp1, Mff (Cell Signaling, Danvers, MA, USA) and actin (Merck, Kenilworth, NJ, USA). After being washed in TTBS, the membranes were incubated for 1 h with anti-mouse or anti-rabbit IgG peroxidase-conjugate antibody. Immunodetection was performed with the enhanced chemiluminescence (ECL) (Thermo scientific, Waltham, MA, USA). VersaDoc imaging system (BioRad, Milan, Italy) was used for densitometric analysis.

### *4.3. Analysis of OPA1 Oligomers*

To investigate on OPA1 oligomerization, cell fresh pellets were treated with the cross-linker bismaleimidohexane (BMH) 1 mM or with vehicle (DMSO) for 30 min at 37 ◦C. After incubation, the samples were centrifuged, resuspended in SDS lysis buffer and then subjected to SDS-PAGE and western blotting analysis with the antibody against OPA1.

#### *4.4. Electron Microscopy*

For routine EM the cells were grown in 12 well plates as a monolayer. At the end of the experiment the cells were fixed with 1% Glutaraldehyde prepared in 0.2 M Hepes buffer. Then the cells were scraped, pelleted, post-fixed in OsO4 and uranyl acetate and embedded in Epon as described previously [43]. From each sample, thin 60 nm sections were cut using Leica EM UC7 (Leica Microsystems, Vienna, Austria). EM images were acquired from thin sections using a FEI Tecnai-12 electron microscope

(FEI, Eindhoven, Netherlands) equipped with a VELETTA CCD digital camera (Soft Imaging Systems GmbH, Munster, Germany).

**Author Contributions:** F.B. and D.D.R. conceptualization; A.S., E.D.L., E.V.P., A.F., R.R., and S.R. investigation; F.B. and D.D.R. writing—original draft preparation; F.B., D.D.R., A.S., R.P., and F.E. writing—review and editing; F.B. and D.D.R. funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Cystinosis Research Foundation, grant number CRFF-2017-001 and by Ricerca Corrente of the Italian Ministry of Health.

**Acknowledgments:** We thank Paolo Lattanzio for technical assistance and Manuela Colucci for statistical analysis contribution.

**Conflicts of Interest:** The authors declare no conflict of interest.
