**4. Discussion**

The pathogenesis of MS involves autoreactive T lymphocytes that have the capacity to invade the CNS causing demyelization and axonal damage [4]. The deletion of autoreactive lymphocytes is normally mediated by apoptosis, however, an escape from mitochondria-mediated apoptosis has been reported in lymphocytes of MS patients [12]. Mitochondria from MS lymphocytes also show a decrease of mitochondrial respiration [13] associated with a specific decrease of complex I and complex IV activities [28], and, of note, mitochondria have been found to range in shape and size and showed thickened cristae [29]. Mitochondrial dependent apoptosis is also depending on mitochondrial respiration, shape and structure. A growing body of evidence suggests that OPA1 participates through several mechanisms in defining mitochondrial shape and structure of cristae [14] and modulating mitochondrial respiration [17]. This modulates cell susceptibility towards apoptotic stimuli [14]. Augmented level of OPA1, formation of OPA1 oligomers and a correct balance of L and S forms are considered anti-apoptotic factors [17]. In light of this and the data reporting that OPA1 mutations are associated with multiple sclerosis-like symptoms [18], in this work, we analyzed OPA1 and its modulators in PBMCs of MS patients compared to HC. Fifteen healthy controls and fifteen subjects affected by MS were enrolled in the study. The patients group included 12 females and 3 males and 11 RR forms and 4 SP forms of MS (see Table 1). No significant difference was observed between males and females subjects as well as between RR and SP forms.

Our results showed the same level of OPA1 total protein (L+S) in HC and SM samples and no differences were observed in the balance between L and S forms of OPA1 in PBMC of HC and MS patients. Interesting the analysis of the electrophoretic migration of immune-revealed bands of OPA1 in lymphocytes of MS samples showed only one S-form at a higher molecular weight in MS with respect to the two S forms observed in HC, thus suggesting a characteristic processing of OPA1 in MS samples. The functions of OPA1 are regulated, under stress conditions, by OMA1-dependent proteolytic cleavage. As expected PBMCs of MS patients show increased level of ROS that, anyway, not results in the activation of stress-induced OMA1 [22], as shown by the accumulation of inactive form of OMA1, thus suggesting an adaptation to exposure at chronic OS. Of note, it was reported that suppression of OMA1 activity strongly prevented cytochrome *c* release into the cytosol [30] and cells lacking the protease OMA1 showed an increased resistance to external apoptotic stimuli [31,32]. OPA1, beyond the proteolytic control, is also under the control of alternative splicing. Eight di fferent OPA1 isoforms, generated by alternative splicing of four exons near the amino terminus (Figure S1), are characterized for the presence, or absence, of at least three di fferent proteolytic cleavage sites, named S1, S2, and S3 [33]. The S1 site is cleaved by OMA1 while S2 and S3 by Ymel1 protease. The presence of isoform 3, which contains S3 and S1 cleavage sites (see Figure S1), together with the inactivation of OMA1 protease (see also [32]), could explain the shift of electrophoretic mobility observed in MS patients. Although YME1L and OMA1 constitutively control OPA1, the proteolytic processing of OPA1 is more complex, in fact, other proteases can act on OPA1 under particular stress condition or metabolic demands [33,34], thus, the involvement of other mitochondrial proteases cannot be ruled out. In this contest, should be noted that, in MS, several proteases are involved [35,36] and that mitochondrial proteases are often involved in the pathogenesis of neurological diseases [37].

It has been reported that SIRT3 has an important role in a variety ofoxidative stress-mediated cellular responses [38], in the regulation of bioenergetic function and antioxidant defense of mitochondria under OS conditions [39]. OS regulated SIRT3 protein level that, in turn, is involved in mitochondrial apoptosis by modulating OPA1 acetylation/processing [20]. In particular, sustained level of SIRT3 protein favors the apoptosis resistance, while a decrease promotes cell death [20]. We have also investigated on SIRT3 protein level; however, despite the increased ROS production in MS, no di fference has been observed in MS lymphocytes compared to HC samples. Although the SIRT3 protein level was unchanged, this could be interpreted as a loss of response of lymphocytes of MS patients to OS. The data on SIRT3 and OMA1 sugges<sup>t</sup> a deregulation of normally stress response mechanisms of these proteins in PBMCs of MS patients.

Furthermore, OPA1 processing and stability is also controlled by PHB2 protein. In mitochondrial inner membrane, PHB2 protein forms with PHB1 a large membrane-bound complex [40,41]. This complex is required for OPA1 stability [42], indeed the deletion of PHB2 leads to the impaired cellular proliferation, aberrant mitochondrial cristae morphogenesis, and apoptosis [24,42], while an over-expression of PHB2 is reported to protect cells from apoptosis [43]. We show, according with others [26] and as response to OS [25], a strong increase of PHB2 protein level in MS samples, thus representing another element of resistance to apoptosis. It worth mentioning that an elevated autophagic flux has been reported in autoreactive T cells, both in patients and in the mouse model of experimental autoimmune encephalomyelitis [44]. It has been found that PHB2 participates in the mitophagy process (selective autophagy) by functioning as mitochondrial receptor in autophagosome formation [45].

Interestingly, using Pearson's correlation analysis, we found a positive correlation in HC group between SIRT3 changes and changes of L- and S-OPA1 balance. This is in agreemen<sup>t</sup> with the findings showing that the SIRT3-dependent deacetylation of L-OPA1 inhibits its proteolytic processing to form S-OPA1 [20]. This correlation was lost in the MS group, while changes of L- and S-OPA1 balance, PHB2 protein level and H2O2 correlated each other. In facts, under condition of oxidative stress, an increased cleavage of L-OPA1 to produce S-OPA1 has been observed [20] as well as an increase of PHB2 expression [25]. The over expression of PHB2 has been found to protect the cell from oxidative stress-dependent apoptosis [43].
