**1. Introduction**

Multiple sclerosis (MS) is a complex neurodegenerative disease that involves immune and central nervous system (CNS) [1,2]. MS is expressed in di fferent clinical forms including primary progressive (PP), secondary progressive (SP), progressive relapsing (RP) and relapsing-remitting (RR), which is the most prevalent form [3]. The pathogenesis of MS involves the loss of blood–brain barrier integrity with the consequent invasion of lymphocytes into the CNS resulting in tissue damage [4].

Despite the knowledge of genetics, cell biology and immunology, obtained in the last years, the ultimate etiology or specific elements that trigger MS remain unknown. The etiopathogenesis and pathophysiology of MS involves di fferent factors, among others mitochondrial dysfunction and oxidative stress (OS) play a key role and have a further modulatory e ffect on many aspects of the disease. OS plays an important role in activation of immune cells, especially T cells [5–7], and recently

it has been reported that peripheral blood mononuclear cells (PBMCs) of MS patients show impaired redox status associated with mitochondrial alterations [5]. A number of mechanisms participate in the maintenance of the immune homeostasis avoiding the development of autoimmune diseases. The apoptosis is an important anti-autoimmune process that deletes potentially pathogenic autoreactive lymphocytes, limiting the immune response-dependent tissue damage [8,9]. It has been shown that deletion of autoreactive lymphocytes by apoptosis is defective in patients with MS, thereby permitting these cells to perpetuate a continuous cycle of inflammation within the CNS [10,11]. In particular, the impairment of mitochondria-mediated apoptosis in Cd4+ T lymphocytes [12], as well as, a reduction of mitochondrial respiration are reported in MS patients [13]. Mitochondria have a main role in both cell death and life and they are a major source of reactive oxygen species (ROS) production. At the same time, mitochondria are responsive to OS and are critical in modulating apoptosis in response themselves to a variety of stress signals.

Several mitochondria parameters such as mitochondrial respiratory chain activity, ROS production, dynamics (fusion and fission), and mitochondria cristae architecture are involved in mitochondria-mediated apoptosis [14–16]. Among mitochondrial proteins involved in apoptosis mechanism, optic atrophy 1 (OPA1) is a mitochondrial dynamin like GTPase that has attracted grea<sup>t</sup> attention for its role in the regulating mitochondrial fusion and fission, the stability of the mitochondrial respiratory chain complexes, pro-apoptotic cytochrome c release and the maintenance of mitochondrial cristae architecture [17]. Very interesting, it is reported that mutations in OPA1 gene, resulting in autosomal dominant optic atrophy (ADOA), are associated with multiple sclerosis-like disorder in patients [18].

OPA1 undergoes constitutive processing leading to the conversion of the un-cleaved long OPA1 (L-OPA1) in a cleaved short OPA1 (S-OPA1) forms. Various stress conditions, including apoptotic stimulation are associated with the conversion of L-OPA1 into S-OPA1. The processing and activity of OPA1 is regulated by mitochondrial proteases, such as OMA1, cellular energetic condition [19], post-translational modification, such as acetylation status [20,21], and oxidative stress [20,22]. OMA1-mediated processing of OPA1 is a cellular stress response, in fact, although OMA1 is constitutively active, it display strongly enhanced activity in response to OS [23]. Furthermore, OPA1 stability is controlled by prohibitin 2 (PHB2) [24] a chaperon like protein, localizes in nucleus, plasma membrane, and mitochondria. Evidences indicate that mitochondrial PHB2 is over expressed under conditions of oxidative stress [25]. Interesting, PHB2 has been found up-regulated in lymphocytes of MS patients [26]. OPA1 processing is also modulated by its acetylation status mediated by SIRT3 enzyme, a mitochondrial deacetylase that also plays an important role in apoptosis [20]. In this work we have analyzed the protein level, and proteolytic processing of OPA1 and its stress-associated regulators, OMA1, SIRT3, and PHB2 in PBMCs of MS patients.

### **2. Experimental Section**
