*2.1. CTNS*−/− *ciPTEC Showed Deregulation of Proteins Involved in Mitochondrial Fission*/*Fusion Processes*

Recruitment of the GTPase dynamin-related protein 1 (Drp1) to mitochondria is a key step required for mitochondrial fission and its reversible phosphorylation was implicated in the regulation of this process. The analysis of phosphorylated Drp1 at Ser-637 (Drp1pS637) showed high variability of protein phosphorylation with no significant differences in wild type and CTNS−/− ciPTEC, also 24 h treatment with 100 μM cysteamine (MEA) did not affect Drp1pS637phosphorylation (Figure 1). The western blotting of the pro-fission mitochondrial protein Fis1 revealed the ubiquitinated and not-ubiquitinated forms of protein. *CTNS*−/− ciPTEC showed increased level of Fis1 (2.47 ± 0.27 vs. 1.08 ± 0.08, *p* = 0.0027). Treatment with 100 μM MEA for 24 h further increased total Fis1 protein level (2.47 ± 0.27 vs. 3.59 ± 0.15, *p* = 0.011) but almost completely reduced the ubiquitinated counterpart by 96.3% (*p* < 0.001) (Figure 1). Third key regulatory protein analyzed was mitochondrial fission factor (Mff), which localizes on OMM and promotes the recruitment of DRP1 to the mitochondrial surface. This protein, shown in its four isoforms, was not modified in *CTNS*−/− ciPTEC and the expression was unchanged after MEA treatment (Figure 1).

**Figure 1.** Analysis of proteins involved in fission process of mitochondrial dynamics in untreated and cysteamine (MEA)-treated *CTNS*−/−compared to *CTNS*+/+. Cell cultures were treated with 100 μM cysteamine (MEA) or DMSO (vehicle) for 24 h as specified in the figure. (**a**) Representative immunoblotting analysis in cellular lysate of conditionally immortalized proximal tubular epithelial cells (ciPTEC) from a healthy subject (*CTNS*+/+) and cystinotic patient (*CTNS*−/−). (**b**–**e**) The histograms (Drp1pS637, panel (**b**), *n* = 8; mitochondrial fission factor (Mff), panel (**c**), *n* = 3; mitochondrial fission 1 protein (Fis1), panel (**d**), *n* = 4; ubiquitinated Fis1 (Ub-Fis1), panel (**e**), *n* = 3) represent the means values ± SEM of the relative expression normalized on actin level. Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. *p*-value less than 0.05 was considered as statistically significant, (Student's *t* test, \*\*\* *p* < 0.001; \*\* *p* < 0.01; \* *p* < 0.05). For further details see under "materials and methods" section.

The inner mitochondrial membrane GTPase OPA1 undergoes constitutive processing leading to the conversion of the un-cleaved long OPA1 (L-OPA1) in cleaved short variants (S-OPA1). Various stress conditions, including apoptotic stimuli, trigger the complete conversion of L-OPA1 into S-OPA1. In this regard, *CTNS*−/− ciPTEC were characterized by a significant increase of short variants (52.4%, *p* < 0.05), but 24 h treatment with 100 μM MEA did not show significant effects (Figure 2). In agreement with higher S-OPA1 levels, we found that the active form of mitochondrial metallo-endopetidase OMA1, which catalyze conversion of OPA1 into short isoforms and triggers mitochondrial fragmentation, was increased by 79.8% in *CTNS*−/− ciPTEC (*p* < 0.001), and not rescued by MEA treatment (Figure 2). OPA1 can oligomerize at the inner mitochondrial membrane to keep the cristae junction tight, therefore cell fresh pellets were treated with the cross-linker bis-maleimidohexane (BMH) 1 mM or with vehicle to test the oligomeric state of OPA1. The OPA1 oligomer, immune-revealed as a high molecular-weight band (≈250 kDa), decreased in *CTNS*−/− cells by 23.5% compared to *CTNS*+/+ cells and was not affected by MEA treatment. The absence of OPA1 oligomerization in cells treated with vehicle (DMSO) confirmed the specificity of cross-linking (Figure 2).

The expression of MFN2, an outer mitochondrial membrane GTPase involved in fusion processes, was not changed in *CTNS*−/− ciPTEC with respect to control cells (Figure 3). However, the higher molecular weight band, corresponding to ubiquitinated MFN2, indicated an increase of ubiquitination in *CTNS*−/− ciPTEC by 70.8% (*p* < 0.001). Treatment with MEA showed 37.8% reduction of ubiquitination but the effect was not statistically significant (Figure 3).

**Figure 2.** Processing and oligomerization of optic atrophy 1 (OPA1) fusion protein in untreated and MEA-treated *CTNS*−/− compared to *CTNS*+/+. (**a**) Representative immunoblotting analysis of ciPTEC obtained from *CTNS*+/+ and *CTNS*−/−. Where indicated, the cells were treated with MEA or DMSO (vehicle) for 24 h. The histograms of OPA1 (**b**) represent the percentage of relative expression of L and S forms of OPA1 in each lane (*n* = 3). The histograms of OMA1 (**c**) represent the means values ± SEM of the relative expression normalized on actin level (*n* = 3). (**d**) The fresh collected cells were treated with the cross-linker 1,6-bismaleimidohexane (BMH) 1 mM or with vehicle (DMSO) for 30 min at 37 ◦C, then centrifuged and resuspended in sodium dodecyl sulfate (SDS) lysis buffer for western blotting analysis with the antibody against OPA1. (**e**) The histograms represent the means values ± SEM of the relative expression of OPA1 oligomers (*n* = 3). Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student's *t* test, \*\*\* *p* < 0.001; \* *p* < 0.05. For further details see under "materials and methods" section.

Numerous mitochondrial outer membrane proteins are modified with K48- and K63-linked ubiquitin chains, including the mitochondrial fusion factors MFN1 and MFN2 and fission factors Fis1 and Drp1, triggering a cascade of events that result in mitophagy. According with previous results, we found in *CTNS*−/− ciPTEC a significant increase in protein levels of the E3 ubiquitin ligase parkin (2.92 ± 0.22 in *CTNS*−/− vs. 1.0 ± 0.04 in *CTNS*+/+, *p* = 0.001). MEA treatment did not change parkin expression (Figure 4A). Ubiquitin carboxyl-terminal hydrolase 30 (USP30) mediates the removal of the ubiquitin chains added by parkin to ubiquitilated forms of mitofusins, such as MFN2, therefore we analyzed the expression of this deubiquitinating enzyme tethered to the OMM and showed in *CTNS*−/− ciPTEC 62.9% decreasing compared to wild type cells (*p* < 0.001). Treatment with MEA rescued USP30 expression in *CTNS*−/− ciPTEC by 87.9% (*p* = 0.037) (Figure 4B).

**Figure 3.** Expression and ubiquitination of mitofusin 2 (MFN2) in untreated and MEA-treated *CTNS*+/+ and *CTNS*−/−. (**a**) Representative immunoblotting analysis of untreated and MEA-treated ciPTEC *CTNS*+/+ and *CTNS*−/−. (**b**) The histogram of MFN2, *n* = 3, and (**c**) the histogram of ubiquitinated MFN2 Ub-MFN2, *n* = 3, represent the mean values ± SEM of the relative expression normalized on actin level. Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student's *t* test, \*\*\* *p* < 0.001.

**Figure 4.** Parkin and ubiquitin carboxyl-terminal hydrolase 30 (USP30) proteins levels and MEA effect in *CTNS*+/+ and *CTNS*−/−. (**a**) Immunoblotting analysis of untreated and MEA-treated ciPTEC *CTNS*+/+ and *CTNS*−/−. (**b**) The histogram represents the means values ± SEM of the relative expression of Parkin normalized on actin level (*n* = 3). (**c**) The histogram represents the means values ± SEM of the relative expression of USP30 normalized on actin level (*n* = 3). Densitometric analysis was performed by Versa-Doc imaging system BioRad, using Quantity One software. Student's *t* test, \*\*\* *p* < 0.001; \* *p* < 0.05.
