1. Mitochondrial Genome and mtDNA Mutations
Mitochondria contain their own genome, which encodes 13 proteins, 22 tRNAs, and 2 rRNAs. The remaining proteins involved in mitochondrial structure and function are encoded by the nuclear DNA, translated in the cytosol and translocated into mitochondria 
. Although mtDNA only encodes ±1% of the mitochondrial proteins, these elements are critical components of the MRC, essential for mitochondrial function 
. Nuclear and mitochondrial intergenomic communication is essential to build the MRC complexes that enables cell bioenergetics and promotes healthy organ function 
If mtDNA mutates, these mutations can be present in all mtDNA copies in the cell (homoplasmy) or only in a fraction (heteroplasmy). Almost all humans carry low levels of heteroplasmic mtDNA point mutations, which could be pathogenic 
. Both the subcellular distribution of mutated mtDNA and the intrinsic pathogenicity of the mutation itself may also be important in determining whether a mtDNA defect is expressed phenotypically 
. mtDNA mutations are also related to the aging process or age-associated diseases 
The distribution of a mutation in different tissues may change with time depending on the mitotic activity of the tissue 
. In tissues with ongoing cell division, there is a chance that a defect in mtDNA may be selected in or out. This may explain the predilection for mitochondrial diseases to manifest clinically in postmitotic tissues such as the central nervous system and skeletal muscle. This mechanism may also explain why some mitochondrial phenotypes change with time 
Pathogenic mtDNA mutations must be present in 60–90% of mitochondria in a cell to trigger MRC dysfunction 
. Computational models estimate that only mtDNA mutations generated in early life could expand to reach the threshold and cause MRC dysfunction in aging human adults 
. One of the reasons is because mtDNA is organized into mitochondrial nucleoids 
and coupled with mitochondrial transcription factor A (TFAM), to make mtDNA less accessible to mutations than naked DNA. In fact, lower amount of TFAM is found in hypothyroid myopathy (endocrine myopathy, Table 2), triggering reduced mtDNA copy number and mitochondrial dysfunction 
Other NMDs associated to mtDNA mutations are metabolic myopathies, ALS, CMT2, mitochondrial myopathies, and Friedreich’s ataxia (Table 2, Table 3 and Table 4).
Apart from mtDNA point mutations, single large deletion in mtDNA at high levels cause multisystem diseases in children and NMDs associated with chronic progressive external ophthalmoplegia in adults and proximal myopathy 
. Other NMDs with mtDNA deletions are sporadic inclusion body myositis (sIBM) (reported in 67% of sIBM patients) 
and giant axonal neuropathy (GAN). These deletions are generally spontaneously acquired 
, probably as accidents during mtDNA replication in the oocyte, in the early embryo or later on, along lifetime. Some multiple large mtDNA deletions are inherited, as they are caused by mutations in nuclear genes involved in mtDNA replication, nucleotide synthesis or transport 
. Spontaneous mtDNA deletions appear to accumulate with time, and may increase the likelihood that tissue function will be impaired with time 
Above all, mtDNA variants (mutations and deletions) have a broad range of impact, from the ones that are causal of monogenic disorders to those that are a risky allele for complex diseases, in which clinical penetrance also depend on environmental factors. For example, de novo mtDNA monogenic mutations m.3243A > G and m.8344A > G trigger MELAS and MERRF, respectively, and can arise spontaneously, because of replication errors. Another high impact variant is a large 4,977-bp deletion of mtDNA, the most common cause of Kearns–Sayre syndrome. These mutations usually occur de novo within two or three human generations and, for deletions, tend to affect only one generation 
. In all cases, the specific mutation, the level of heteroplasmy, the threshold effect that determines the level of phenotypic penetrance and which tissues are affected, directly determine the inheritance of the disease and the onset and progression of these disorders.
2. Mitochondrial Respiratory Chain (MRC)
The MRC contains five multimeric enzymatic complexes. Complexes I–IV oxidize and transfer their electrons, in the form of hydrogen ions, to the next complex. Once in complex IV, electrons are transferred to molecular oxygen, producing water 
. Complex V generates ATP from adenosine diphosphate (ADP) using the transmembrane electrical potential generated by proton translocation along the MRC 
. This production of ATP from the reduction of oxygen, known as mitochondrial coupling, is what generates the energy needed for cellular function 
. In parallel, MRC generates ROS as a subproduct of oxygen metabolism, especially in case of disease. Mutations in mtDNA can trigger the impairment of MRC, affecting energy production and cellular dysfunction 
, observed in some NMDs and mitochondrial diseases 
. mtDNA mutates more than ten times faster than nuclear DNA, and this high mutation rate in mtDNA is based on the proximity of mtDNA to the inner mitochondrial membrane, where ROS are generated as by-products of MRC chain, coupled with a lack of histones and efficient DNA repair mechanisms. In addition, mtDNA has low number of noncoding regions between genes, leading to random mutations that impact coding sequences of genes responsible of MRC.
In particular, most of the muscular dystrophies (MD) have some MRC complexes with impaired or decreased function (Table 1): complex I in Becker MD (BMD), complexes III and IV in DMD, and complexes I and V in oculopharyngeal (OPMD) and in distal MD (DD). Other NMDs with MRC alterations are myofibrillar myopathies (complexes I and IV) (Table 2), ALS (CIV subunit I) (Table 3), SMA (complexes I and IV) (Table 3), GAN (complexes I and IV) (Table 4), and sIBM (complex IV) 
. Impaired MRC function promotes mitochondrial dysfunction, eventually leading to progressive weakness and muscle wasting.
To understand the extent of the how MRC dysfunction affects the progression of a NMDs or vice versa, here we present the case of hypothyroid myopathy (endocrine myopathy, Table 2) and ALS (motor neuron diseases, Table 3).
In hypothyroid myopathy, there is an abnormal activity of the thyroid gland, leading to reduced hormone levels. Thyroid hormones affect skeletal muscle through T3 receptors, located on the mitochondrial membrane of skeletal muscle fibers 
. This MRC-thyroid hormones connection is mediated by several molecular mechanisms that include both direct and indirect effects on mitochondrial structure, function, and biogenesis. One effect in hypothyroid myopathy is a reduced mitochondrial oxidative metabolism, due to decreased hormonal levels and a reduced amount and activity of complex IV (cytochrome c oxidase negative fibers (or COX-)). In addition, T3 receptors, together with the thyroid hormone receptor, increase the expression of some nuclear-encoded respiratory genes, such as cytochrome c1 and b-F1- ATPase subunit genes 
Regarding ALS, ALS-associated P525L mutant interacts with ATP synthase complex subunit TP5B, disrupting the formation of ATP synthase complex, thus inhibiting the production of ATP 
. Both are examples of the complex and intricated pathways governing NMDs in which mitochondria, and herein explained, MRC, play a role.
4. Mitochondrial ROS and ER Stress
ROS comprises superoxide anion (O2•−
), hydrogen peroxide (H2
), hydroxyl radical (OH•
), peroxyl radical (ROO•
), and nitric oxide (NO•
). Although mitochondrial oxidative metabolism is a major source of ROS in many cell types, outside mitochondria there are other enzymes associated with ROS production, like NADPH oxidase, neural nitric oxide synthase, and monoamine oxidase 
. A balance between oxidative species and antioxidant defense mechanisms is required for cell homeostasis 
. Briefly, the antioxidant response is mediated by enzymes responsible for metabolizing or neutralizing ROS, such as catalase, glutathione peroxidase, or superoxide dismutase (SODs) 
The high proportion of ROS generation in MRC is due to the transfer of electrons in the inner mitochondrial membrane until they encounter oxygen, when electrons are converted in water. Under hypoxia or pathologic conditions this process is not completed, resulting in an increase of superoxide anions 
. For example, mutation in SOD2, an antioxidant enzyme encoded in the mitochondrial genome, triggers the development of CMT, a NMD 
. Mutations in SOD1 gene are found in ALS. SOD1 encodes the Cu-Zn superoxide dismutase, one of three proteins involved in the conversion of free superoxide radicals to molecular oxygen and hydrogen peroxide. Mutations in the SOD1 gene are found in 10–20% of familial ALS cases and 1–5% of sporadic ALS cases globally 
; so far, more than 170 mutations of the SOD1 gene are known in ALS 
Other NMDs in which high ROS levels are not compensated and cells hold excessive oxidative stress are BMD, DMD, FSHD, ALS, SBMA, mitochondrial myopathies, and FA.
When overproduction of ROS cannot be compensated by antioxidant defenses or mitophagy, ER receives ROS signals through MAMs, which may eventually activate cell stress responses to compensate and overcome this adverse situation 
ER is the main organelle in charge of protein biosynthesis and folding and one of the main cell sensors to stress insults. Alterations of the ER redox status can negatively affect protein folding and result in ER stress. ER stress occurs when the rate at which new protein entering the ER exceeds its folding capacity, which can lead to the activation of three transmembrane proteins: inositol-requiring enzyme 1α (IRE1α), PKR-like ER kinase (PERK), and the activation transcription factor 6 (ATF6), which altogether trigger a UPR 
. The UPR is comprised of a series of transcriptional, translational, and post-translational processes that can lower the rate of protein synthesis and increase the protein folding capacity of the ER machinery. This results in an increase of misfolded protein elimination, and escalating the size of the ER compartment thereby reducing the ER stress 
. Usually, transient ER stress can be overcome by UPR, but if the damaging stimulus persists, inflammatory response genes are activated 
ER-mitochondria crosstalk is involved in several pathways including cell proliferation, apoptosis, autophagy, lipid metabolism, Ca2+
signaling, UPR, inflammation, and bioenergetics. Alterations in this ER-mitochondria crosstalk are associated with multiple diseases, like motor neuron diseases, myotonic dystrophy, OPMD, endocrine myopathies, myofibrillar myopathies, SBMA, CMT, and mitochondrial myopathies. In ALS, over expression of both wild-type and mutated TDP-43 leads to a decrease in ER-mitochondria physical and functional coupling, affecting Ca2+
dysregulation is considered to be a primary cause of motor neuron death in ALS 
is responsible for the link between metabolic impairment and MAMs interaction. This is seen in cancer cells, which are able to remodel their intracellular Ca2+
signaling, enhancing their survival and proliferation. Modulation of ER-mitochondrial Ca2+
crosstalk favors resistance to apoptosis 
In addition to triggering ER stress, if ROS damage overwhelms these repair mechanisms, MMP may decrease, thus potentially leading to the increase in the permeability of mitochondrial membranes. Higher permeability in mitochondrial membrane can activate the mitochondrial permeability transition pore (MPTP) and thus, the release of DAMPs to the cytosol (mtDNA), ceramides, formaldehyde) 
. MPTP and ROS can also promote the activation of mitochondrial fission and mitophagy to eliminate damaged mitochondria (Figure 2). In mitochondrial fission, MAMs signal the ER tubules to encircle mitochondria and tag the sites for mitochondrial division 
. Accordingly to mitochondrial implication in NMDs, dysregulation of MPTP opening and defective autophagy have been associated with muscular dystrophies, like collagen VI myopathies (CMD), BMD, DMD, LGMD, and DD 
and, remarkably, most of NMDs show some level of deregulation in most of these pathways.
5. Mitochondrially Induced Inflammatory Response
Inflammatory responses recognize pathogen-associated molecular patterns (PAMPs), derived from infection, through a variety of receptors. However, because of the bacterial origin of mitochondria, their proteins are structurally similar to those in bacteria and enable their recognition by the same receptors of the immune system, reinforcing the notion of mitochondria as hubs of immunity 
. Thus, mitochondrial DAMPs can also trigger an inflammatory response 
. DAMPs include proteins and peptides, such as N-formyl peptides and TFAM, as well as lipids, and metabolites such as cardiolipin, succinate and ATP, and mtDNA 
5.1. Implication of mtDNA in Inflammation
In particular, mtDNA is recognized by multiple innate immune receptors: cytosolic cyclic GMP-AMP synthase (cGAS), endosomal Toll-like receptor 9 (TLR9), and NOD, LRR, and Pyrin domain-containing protein 3 (NLRP3) [140
]. Here we briefly remark the main effects of these innate immune responses.
Cytosolic mtDNA enhances cytosolic antiviral signaling and expression of interferon-stimulated genes (IRF3/IRF7), which results in the activation of cGAS DNA sensor and STING-IRF3-dependent signaling [141
]. These pathways activate transcription factors NF- kB and IRF3 through the kinases IKK and TBK1, respectively. MtDNA stress triggered by TFAM deficiency has been reported to release mtDNA to the cytosol [101
TLR9 is a nucleic acid sensor that binds to unmethylated CpG DNA, like mtDNA. In basal conditions, TLR9 is located in the ER, but when it is stimulated, TLR9 translocates to the membrane of endosomes or lysosomes, where it binds to ligands and triggers cell inflammation [140
TLR9 interaction with mtDNA occurs in the endolysosomal compartment and activates the myeloid differentiation primary response protein 88 (MyD88), which induces a number of kinases and transcriptional factors, namely mitogen-activated protein kinases (MAPK) [101
]. MAPK leads to nuclear factor-kB (NF-B) and IRF7 activation to enhance pro-inflammatory and interferon type I (IFN-1) responses, respectively [89
]. NF-kB signaling increases the expression of other pro-inflammatory cytokines such as tumor necrosis factor-a (TNF-a), interleukin (IL)-6, IL-1b [128
]. Furthermore, TLR9 senses incomplete digestion in lysosomes of damaged mitochondria, because of impaired mitophagy, without the mtDNA release to cytosol.
The activation of the inflammasome NLRP3 involves two sequential signals. The inflammasome priming and assembly signal is induced by NF-B, which acts downstream of TLRs and other immune receptors [142
]. NF-kB activates the expression of inflammasome components and inactive forms of the cytokines, including pro-IL-1b. When active, NLRP3 inflammasome activates caspase-1, which processes pro-IL-1 and pro-IL-18 [100
]. DAMPs directly activate NLRP3 inflammasome as
well. Mitochondria are suggested to regulate the activity of the NLRP3 inflammasome complex, by: activating mitophagy (reduces inflammation by clearing mitochondrial-bound NLRP3 complexes) and releasing mitochondrial ROS (amplifies inflammasome immunogenic signal) [140
In normal conditions, NLRP3 protein is located on the ER. Under oxidative stress and in response to inflammation, NLRP3 can be translocated to MAMs. MAMs transmit danger signals through physical interaction, promoting coordinated responses to oxidative stress, such as triggering mitophagy to remove damaged mitochondria [131
Altogether, mitochondria-induced inflammation usually is initiated by mitochondrial ROS, which can cause the activation of MAPKs by inhibiting MAPK phosphatase. Activated MAPK may aid in the production of IL-6 and TNF. Mitochondrial ROS accumulation can also activate NLRP3 inflammasome, which promotes the maturation of IL-1 and IL-18. mtDNA accumulation in the cytosol can interact with TLR-9 (in lysosomes) to induce inflammatory responses (e.g., in autosomal
dominant optic atrophy [101
]. This pathologic feature has been described in sIBM and in LMNA-related NMDs (CMD, Emery-Dreifuss MD, LGMD, CMT, etc.)) [144
]. Further, cytosolic mtDNA can activate cGAS-STING to induce IFN-1 or the NLRP3 inflammasome which can induce the maturation/secretion of pro-inflammatory cytokines [101
]. Thus, mtDNA can activate major innate immune responses by acting as a DAMP from the mitochondria. Overall, mtDNA is released from mitochondria to the cytosol, where it is recognized by immune receptors, which trigger a signaling cascade that leads to the production of cytokines or transcription of inflammatory genes in the nucleus (Figure 2) [143
]. This close association between mitochondria and inflammation triggered from affected cells, explains why NMDs are usually associated with inflammatory effects.
5.2. Implication of Mitochondrial ROS in Inflammation
Regarding mitochondrial ROS, they are sensed by mitochondrial antiviral signaling protein (MAVS), key in viral RNA infections. MAVS can activate pathways that regulate NF-kB and IRF3/IRF7 to induce gene expression [145
]. It also can interact with the outer mitochondrial membrane, where mitochondrial ROS can trigger MAVS oligomerization, promoting IFN-1 production. In addition, MAVS can associate with NLRP3 and triggers its oligomerization, leading to caspase-1 activation [146
NLRP3 is activated by mitochondrial ROS but also by cardiolipin, an inner mitochondrial membrane lipid. Cardiolipin translocates to the outer mitochondrial membrane after mitochondrial membrane depolarization, where it can recruit NLRP3. Cardiolipin-NLRP3 interaction suggests the role of mitochondria as a hub for the activation of innate immunity [143
Similar to MAVS, NLRP3 also responds to mitochondrial ROS and can cause mitochondrial damage that promotes the generation of additional ROS. NLRP3 drives the production of IL-1, IL-18, and pyroptosis. mtDNA can also activate NLRP3, and is also sensed by TLR9, which leads to immune and inflammatory gene expression. Mitochondria are therefore critical for signaling by three major innate immune pathways: RIG-I/MAVS, NLRP3, and TLR9 [143
]. All these pathways rely on mitochondria-inflammatory response and, consequently, are altered in numerous NMDs.
5.3. Feedback Regulation of the Mitochondrial Inflammatory Response
NF-kB has a role as a pro-inflammatory and anti-inflammatory transcription factor in mitochondrial-induced inflammation. NF-kB anti-inflammatory functions prevent premature and excessive NLRP3-inflammasome activation, avoiding chronic inflammatory disease and inflammation caused by cell and tissue stress [147
]. Self-limiting inflammation is also important for maintaining homeostasis, tissue repair, and regeneration, which restores integrity of epithelial barriers and thereby
attenuates PAMP and DAMP availability [100
Autophagy is the mechanism underlying NF-kB-mediated inhibition of NLRP3 inflammasome. After inducing the expression of pro-inflammatory cytokines and NLRP3, NF-kB is able to switch the initial pro-inflammatory to an anti-inflammatory state to avoid excessive inflammation. Thus, NF-kB induces the expression of p62, which eliminates NLRP3 by mitophagy [138
]. Therefore, the “NF-kB-p62/SQSTM1-mitophagy” pathway provides an essential regulatory loop through which NF-kB orchestrates a reparative inflammatory response and prevents excessive collateral damage [100
Dysregulation of the NLRP3-inflammasome is frequently associated with diverse inflammatory, metabolic, and malignant diseases, including gouty arthritis, Alzheimer’s disease, obesity, type II diabetes, and colorectal cancer [148
]. Other NMDs with increased inflammation are ALS (with aberrant activation of NF-kB) [17
], muscular dystrophies (like BMD and DMD), and inflammatory myopathies. Therefore, proper control of NLRP3-inflammasome activity and overall inflammatory cascade are
critical for preventing disease development.
The aging process itself is linked to elevated low-grade inflammation or para-inflammation, characterized by constitutive production of low amounts of IL-1b, TNF, and IL-6. The NLRP3-inflammasome activation and insucient mitophagy link accumulation of damaged mitochondria to the para-inflammatory state. Normal, healthy aging may be compromised and greatly enhanced by accumulation of somatically mutated mitochondria, which are more likely to release mitochondrial ROS and fragmented mtDNA that act as direct NLRP3-inflammasome activators [100
]. This explains why most of NMDs arise hand by hand to aging processes.