To date, the specific causes of multiple sclerosis (MS) remain uncertain, and in its pathogenesis an interaction between environmental and genetic factors has been implicated leading to inflammation, demyelination and neurodegeneration of the central nervous system (CNS). Epidemiological studies conducted in ethnic groups of families, twins, half sibs and conjugate pairs support a genetic component to this process. The risk for monozygotic twins is 300-times, and for first-degree relatives 20-50-times higher than for an individual in the general population of Northern-European origin with a prevalence rate of 0.1. The transmission patterns observed are not compatible with an autosomal dominant, recessive or X-linked inheritance. MS is a complex trait disorder, defined by several genes, each exerting small effect, and in an interaction with the environment. Phenotypic expressions of MS suggest the involvement of complex mechanisms with features of autoimmunity and neurodegeneration. The currently approved disease modifying drugs are mainly targeted towards the inflammatory components, but exert a limited effect on neurodegeneration in MS. The cognitive impairment can be an early feature of the demyelinating disease process, and in a few cases dementia has been documented in the absence of severe neurological signs. There are some correlation between disease subtype and cognitive impairment: in fact, it is well known that cognitive impairment occurs more frequently and is more severe in patients with progressive rather than in relapsing-remitting MS. Impairment of cognitive domains such as memory, mental processing speed attention and executive function can occur from the early stage of the diseases and tend to worsen over time. Moreover, the underlying pathophysiological mechanisms of the cognitive impairment and neuropsychiatric disorders observed in MS are not fully understood. White matter abnormalities alone cannot fully explain the extent of clinical symptoms in MS, including cognitive impairment. Furthermore, several MRI techniques have shown the involvement of gray matter in MS and the association between gray matter damage, physical disability and cognitive impairment. Therefore, biomarkers that reliably capture the different aspects of disease heterogeneity are needed, and might help to better understand MS disease aetiopathogenesis, diagnosis, and prognosis, to predict response outcome to treatments, and to develop new treatments.

In particular, there is increasing effort to develop molecular diagnostic markers that meet requirements like easy accessibility e.g., from blood, high specificity and sensitivity, low costs and applicability by laboratories with standard equipment. Several blood, plasma, or serum MS biomarkers have been proposed to meet these criteria. In order of this the circulating markers are represented, in addition to the classic serum markers, also by the cells and by free circulating nucleic acids (DNA, RNA). In recent years, among the circulating nucleic acids, a possible role of mitochondrial DNA has emerged as a biomarker in the diagnosis of numerous pathologies.

In humans, mtDNA is significantly smaller when compared with nuclear DNA (16.569bp vs. 3.2 billion bp), and it possesses only 37 genes, among which 13 encode proteins belonging to the respiratory electron transport chain. Unlike nuclear DNA, mtDNA is devoid of protective histones and sophisticated DNA repair mechanisms, which makes it vulnerable to genotoxic stimuli including oxidative stress. In fact, high levels of reactive oxygen species (ROS) are generated around mtDNA during oxidative phosphorylation occurring in mitochondria. Such an oxidative environment contributes to a high susceptibility of mtDNA to mutagenesis. In fact, mtDNA possesses roughly a 10- to 200-fold higher rate of mutagenesis than nuclear DNA under a comparable oxidative stress environment. This may be detrimental for those high-energy-demanding and post-mitotic cells including neurons and myocytes, which are mostly sensitive to altered respiratory chain activity and ROS-mediated damage yielded by mtDNA changes. Such a specific vulnerability of mtDNA determines the occurrence of a detectable amount of mitochondrial DNA fragments, which are released into the bloodstream as circulating, cell-free fragments (ccf-mtDNA). These correspond to double-stranded DNA molecules, which are biologically fragmented into both short (lower than 1 Kb) and long (up to 21 kb) segments. The high rate of mtDNA fragmentation is key in generating ccf-mtDNA, though it remains unclear whether mtDNA is released due to a disruption of the plasma membrane or it is actively extruded from the cell. For instance, oxidative stress or other stimuli can damage cell integrity, while producing apoptosis or necrosis, which in turn lead to mtDNA extrusion from the cell or release into the blood. Nonetheless, even in baseline conditions when the plasma membrane is intact, fragments of mutated mtDNA could be compartmentalized within cytosolic organelles and then released extracellularly. This latter mechanism would guarantee the preservation of mitochondrial function by removing dysfunctional mutated DNA fragments. This is supported by recent work from C. elegans neurons, which expel dysfunctional mitochondria when exposed to neurotoxic stress. Nonetheless, the biological role of ccf-mtDNA and its fragments is still controversial and it needs to be fully understood. In fact, DNA fragments may act as toxic molecules, which in turn impair mitochondrial function and cell membrane, and could also act on cell integrity and tissue repair. This is largely bound to the established, yet double faceted, involvement of mtDNA in innate immunity and inflammation. In fact, similar to bacterial DNA, mtDNA possesses non-methylated CpG sites, which once released in either cytosol or extracellular space behave as damage-associated molecular patterns (DAMPs) to activate innate immunity and inflammation. This occurs via specific biochemical cascades involving the binding of mtDNA to Toll-like receptor 9 (TLR9) and subsequent activation of the stimulator of interferon genes (STING) pathway. These are key in generating inflammatory responses including antimicrobial immunity and neuro-immunological disorders. In fact, DAMPs accumulation activates resident macrophages and fosters tissue infiltration by leukocytes. As for most molecules involved in the immune response, the bulk of evidence concerning the measurement of ccf-mtDNA and its role in physiology and disease stems from studies carried out outside the CNS. In fact, ccf-mtDNA has been analysed in various clinical conditions like neoplasia, trauma, infections, stroke and cardiovascular diseases, where it has been tested as diagnostic and predictive biomarker. Only recently, mtDNA started being evaluated in neurological disorders. In line with the higher resistance of mtDNA to nuclease-dependent degradation compared with nDNA, mtDNA persists as ccf-mtDNA within extracellular ﬂuids including the CSF.

CNS disorders featuring a strong inﬂammatory response are characterized by elevated plasma mtDNA level. In fact, elevated CSF ccf-mtDNA occur in relapsing-remitting (RRMS) and PMS. RRMS is characterized by an acute inﬂammatory response, which precedes neurodegeneration. Thus, the increase in ccf-mtDNA observed in RRMS is a direct consequence of increased activation of inﬂammatory cells. These cells release mtDNA in addition to nDNA, into the CSF. A persistent inflammatory reaction may recruit circulating immune cells while triggering a systemic response through the activation of mtDNA-induced inflammatory pathways. In this way, a vicious circle occurs where inflammatory cytokines and ROS may induce further damage to mitochondria and mtDNA. In this scenario, elevated ccf-mtDNA concentration in MS may reflect early, active inflammatory activity, which eventually culminates in mitochondrial damage, neural loss and brain atrophy. In this condition, the measurement of ccf-mtDNA concentration configures as a potential biomarker for acute inflammatory stress. Whether this phenomenon is specific for MS or it rather reflects a generic neuro-inflammation still needs to be investigated. Since mitochondrial damage occurs in active MS lesions, mtDNA in the CSF could reflect its role as a DAMP. Considering mtDNA as a DAMP in MS, may explain the "inside-out theory" which suggests that inflammation is secondary to a primary intrinsic process within neurons or other cells such as oligodendrocytes. This neuro-immune concept consists in the formation of intracellular compounds, which trigger biochemical cascades leading to immunity activation (inflammasome) which once released from the cell recruit in turn a focal immune response. In this scenario, the "inside" mtDNA fragment would be the inflammatory stimulus, which clusters the intracellular cascade leading to a molecular complex, which triggers the immune response. Once such a complex is exposed "out" of the cell, immunity is strongly activated.

Thus, ccf-mtDNA may be a potential biomarker of cell death and non-specific tissue injury, and in the near future, it is supposed to become an innovative diagnostic tool in early stage screening and prognosis of several disorders.