**3. Results**

Fifteen HC and fifteen MS subjects (see Table 1) 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). The PBMC cellular lysate was used to investigate on the OPA1 protein level and processing by Western blotting analysis using a specific antibody (Figure 1).

**Figure 1.** Optic atrophy 1 (OPA1) protein level and processing in peripheral blood mononuclear cells (PBMCs) from HC and MS patients. (**A**) Representative images of western blotting analysis. The PBMC proteins from HC and MS were loaded on 7.5% SDS-PAGE. After separation, the proteins were transferred on nitrocellulose membranes and immunoblotted with the antibody against OPA1. Protein loading was assessed with β-actin antibody. (**B**) The total protein level of OPA1 (L+S forms) was evaluated by densitometric analysis. The arbitrary densitometric units (ADU) of OPA1 were normalized to ADU of β-actin and the mean of HC set to 100%. The histograms represent the percentage values MS patients with respect to HC. The values are means ± SEM of different samples. (**C**) The histograms represent the percentage of ADU of long (L) and short (S), forms of OPA1 in each lane. The values are means ± SEM of samples. (**D**) Left panel, representative image of electrophoretic mobility of immune-revealed bands of OPA1 in PBMCs from HC and MS. Right panel, the images of the western blotting were analyzed by Image Lab Touch 2.4 software (BioRAD) for determination of electrophoretic profile of each lane and calculation of relative front (Rf) of L-OPA1 and S-OPA1 bands (see Table S1). Rf indicates the relative movement of the band from the top of the gel. "d" represents the difference between Rf of S-OPA1 and Rf of L-OPA1 in each lane. (**E**) The table reports the means values of "d" ± SEM of different samples. (*p* < 0.001, Student's *t*-test).

The antibody against OPA1 protein, immuno-revealed in both HC and MS groups a long form (L) and short forms (S) of OPA1 (Figure 1A). Densitometric analysis of immuno-revealed bands of OPA1 (L+S) showed the same level of total OPA1protein in MS group with respect to HC (Figure 1B). No difference was observed in percentage of L and S form of OPA1 between HC and SM samples

(Figure 1C). Interestingly, the image analysis of western blotting revealed two bands of S-OPA1 in HC and one band of S-OPA1with a di fferent electrophoretic mobility with respect to HC samples in MS (Figure 1A,D). Analysis by Image Lab Touch 2.4 software confirmed the presence of two S-OPA1 bands in HC and one S-OPA1 band in MS as shown by curve peaks (Figure 1D). Moreover, the determination of Rf of L-OPA1 and S-OPA1 bands revealed a significant decrease in Rf of S-OPA1 in MS sample with respect to HC (see Table S1). No di fferences were observed in Rf of L-OPA1 between HC and MS samples (Table S1). Calculation of di fference between Rf of S-OPA1 band and L-OPA1 band in each lane ("d"), revealed a significant decrease of "d" in MS samples with respect to HC (Figure 1D,E). This suggested that processing of OPA1, in MS samples, generated a S-OPA1 form at a higher molecular weight with respect to HC.

Processing of OPA1 is regulated by di fferent proteins and cellular conditions such as oxidative stress. Measurement of H2O2 level, detected by the redox-sensitive fluorescent probe DCFDA, showed increased ROS production in the PBMCs of MS patients compared to HC (Figure 2).

**Figure 2.** H2O2 production in PBMC of HC and MS patients. H2O2 level was detected spectrofluorimetrically by dichlorodihydrofluorescin (DCFDA) probe. The mean of HC was set to 100% and the mean value of MS expressed as percentage of intensity of fluorescence respect to HC. The histograms represent the means of values ± SEM. (\*\* *p* < 0.01; Student's *t*-test).

This result prompted to investigate on stress regulated protein OMA1, PHB2 and SIRT3 that are involved in OPA1 processing and stabilization [20,22,25]. Activation of OMA1 protease is accompanied by its autocatalytic degradation that results in the complete turnover of protein [22]. Western blotting analysis of OMA1 did not show the activation of this protease in PBMCs from MS samples as revealed by the increased ratio between inactive and active forms (Figure 3A,B). We next examined PHB2 level by western blot analysis with specific antibody. An increased PHB2 protein level was observed in MS patients (Figure 3C,D) compared to HC samples, while no di fference was observed for SIRT3 protein level (Figure 3C,D).

**Figure 3.** OMA1, SIRT3, and PHB2 in PBMCs of HC and MS patients. (**A**,**C**) Representative images of Western blotting analysis. Proteins from PBMC from HC and MS were loaded on 7.5% SDS-PAGE. After separation, the proteins were transferred on nitrocellulose membranes and immunoblotted with the antibodies against OMA1, SIRT3, and PHB2. Protein loading was assessed with β-actin antibody. The immunoblotting against β-actin in panel C is the same shown in the Figure 1A, belonging to the same experiment series (**B**,**D**). (**B**) The histograms represent the means of ratio values ± SEM between the ADU of inactive and active forms of OMA1 in HC and MS subjects. (**D**,**E**) The ADU of PHB2 and SIRT3 were normalized to ADU of β-actin. The histograms represent the means of values of ADU ± SEM of samples. (\* *p* < 0.05, \*\* *p* < 0.01; Student's *t*-test).

To explore whether the alterations in the analyzed molecular parameters can cross correlate with each other, a correlation analysis was performed for HC and MS groups. The results in HC indicated a remarkable positive correlation of SIRT3 changes with changes of L- and S-OPA1 balance. This correlation was lost in MS group. In addition, in MS group, a significant positive correlation was observed between H2O2 level and PHB2 (Figure 4B), and negative correlations between H2O2 and Land S-OPA1 balance (Figure 4C) and between PHB2 and L- and S-OPA1 balance (Figure 4D).

**Figure 4.** Correlation plots. Empty squares indicates HC group, full squares indicates MS group (**A**)Scatter plot and linear regression of data of relationship between SIRT3 protein level, expressed in ADU, and L-OPA1/S-OPA1 balance in HC group (correlation coefficient, r = 0.596; *P*, 0.021). (**B**) Scatter plot and linear regression of data of relationship between H2O2, expressed in intensity of fluorescence (IF), and PHB2 protein level, expressed in ADU in MS group (correlation coefficient, r = −0.691; *P*, 0.004). (**C**) Scatter plot and linear regression of data of relationship between H2O2 and L-OPA1/S-OPA1 balance in MS group (correlation coefficient r = −0.585, *P*,0.021). (**D**) Scatter plot and linear regression of data of relationship between PHB2 protein expression and cleaved long OPA1 (L-OPA1)/cleaved short OPA1 (S-OPA1) balance in MS group (correlation coefficient, r = −0.652; *P* = 0.008). Degree of freedom 13.
