Abstract: Microglial cells and perivascular macrophages are the only resident immune cells of the brain parenchyma and act as innate immune sentinels in the central nervous system (CNS). Microglial cells are vital for the maintenance of CNS homeostasis thanks to their strict interaction with neurons. When the homeostasis of the microenvironment is disrupted, microglia can alter their phenotype acquiring pro- or anti-inflammatory function to defend the brain. On the other hand, the excessive activation of proinflammatory microglia in response to primary neurodegeneration, axonal degeneration, and additional peripheral activation processes linked to systemic inflammation can trigger or maintain chronic inflammation. Therefore, under such conditions, the proinflammatory phenotype of microglia could be harmful and associated with the pathogenesis of neurological disease characterized by inflammation, such as neurodegenerative diseases, demyelinating diseases, CNS trauma, and epilepsy. Despite the numerous studies on that field, the primary stimuli that provoke and maintain such inflammation, as well as the biological pathways and mechanisms that cause detrimental actions of microglia are still a subject of debate. Microglia can sense cellular damage and stress by recognizing the damage-associated molecular patterns (DAMPs) through the pattern recognition receptors (PRRs). Several lines of evidence, obtained from studies in humans and animal models, suggest that DAMPs could play a relevant role in the pathogenesis of several neurodegenerative diseases (Gong et al., 2020). The category of DAMPs includes several molecules, some of them can be released from damaged mitochondria (the so-called mitochondrial DAMPs, mtDAMPs), such as N-formyl peptides, cardiolipin, the mitochondrial transcription factor A (TFAM), succinate, adenosine triphosphate, and mitochondrial DNA (mtDNA). Damaged cells accumulate dysfunctional mitochondria that trigger processes such as cell senescence, apoptosis, or necrosis. In all of these cases, mtDAMPs can be released in the extracellular space and could be recognized through different pattern recognition receptors by innate immune cells recruited to remove cellular debris of dying cells. Recently, increasing attention has been paid to mtDNA, as DAMP able to strongly stimulate cells through Toll-like receptor (TLR) 9 contributing to inflammation even in the absence of infection (sterile inflammation) (Riley et al., 2020). After an extensive cell injury, several mitochondrial products, including mtDNA, can enter the bloodstream or cerebrospinal fluid (CSF), where they are recognized by the innate immune system and evoke a local or systemic response. The cell-free mtDNA is stable and resistant to nuclease digestion, more than genomic DNA, and could be detected in blood or CSF. Even in healthy people, mtDNA is present at relatively high levels in the blood and easily measurable. Over the past few years, there has been a growing interest in mtDNA as a potential biomarker as its levels are increased in several physio-pathological conditions characterized by chronic inflammation (Cossarizza et al., 2011; Pinti et al., 2014; Nasi et al., 2016), including neurodegenerative diseases such as multiple sclerosis (MS) (Nasi et al., 2020a). Interestingly, mtDNA levels were found higher also in CSF from people with MS but not in people affected by Parkinson’s disease or Alzheimer’s disease (Gambardella et al., 2019). Parkinson’s disease and Alzheimer’s disease are characterized by a loss of neuronal mitochondria (where probably the low levels of mtDNA in the CSF come from) followed by neuronal death, while MS is characterized by a strong inflammatory response in which mtDNA could be released into the CSF. Thus, MS represents a valuable model of neuro-inflammation, in which mtDAMPs could have a prominent role. On the other hand, the neuro-inflammation itself is strictly associated with mitochondrial dysfunction that could trigger a vicious circle: dysfunctional mitochondria can induce inflammation and inflammation induces mitochondrial dysfunction followed by the further release of mtDAMPs. However, the triggers by which mtDAMPs are released are still unknown, as well as the precise role of mtDNA and mtDAMPs in patients with MS has poorly been investigated.