Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency.

There is hitherto no literature available for explaining two distinct, but confused, Nrf1 transcription factors, because they shared the same abbreviations from nuclear factor erythroid 2-related factor 1 (also called Nfe2l1) and nuclear respiratory factor (originally designated α-Pal). Thus, we have here identified that Nfe2l1Nrf1 and α-PalNRF1 exert synergistic and antagonistic roles in integrative regulation of the nuclear-to-mitochondrial respiratory and antioxidant transcription profiles. In mouse embryonic fibroblasts (MEFs), knockout of Nfe2l1-/- leads to substantial decreases in expression levels of α-PalNRF1 and Nfe2l2, together with TFAM (mitochondrial transcription factor A) and other target genes. Similar inhibitory results were determined in Nfe2l2-/- MEFs but with an exception that both GSTa1 and Aldh1a1 were distinguishably upregulated in Nfe2l1-/- MEFs. Such synergistic contributions of Nfe2l1 and Nfe2l2 to the positive regulation of α-PalNRF1 and TFAM were validated in Keap1-/- MEFs. However, human α-PalNRF1 expression was unaltered by hNfe2l1α-/- , hNfe2l2-/-ΔTA , or even hNfe2l1α-/-+siNrf2, albeit TFAM was activated by Nfe2l1 but inhibited by Nfe2l2; such an antagonism occurred in HepG2 cells. Conversely, almost all of mouse Nfe2l1, Nfe2l2, and cotarget genes were downexpressed in α-PalNRF1+/- MEFs. On the contrary, upregulation of human Nfe2l1, Nfe2l2, and relevant reporter genes took place after silencing of α-PalNRF1, but their downregulation occurred upon ectopic expression of α-PalNRF1. Furtherly, Pitx2 (pituitary homeobox 2) was also identified as a direct upstream regulator of Nfe2l1 and TFAM, besides α-PalNRF1. Overall, these across-talks amongst Nfe2l1, Nfe2l2, and α-PalNRF1, along with Pitx2, are integrated from the endoplasmic reticulum towards the nuclear-to-mitochondrial communication for targeting TFAM, in order to finely tune the robust balance of distinct cellular oxidative respiratory and antioxidant gene transcription networks, albeit they differ between the mouse and the human. In addition, it is of crucial importance to note that, in view of such mutual interregulation of these transcription factors, much cautions should be severely taken for us to interpret those relevant experimental results obtained from knockout of Nfe2l1, Nfe2l2, α-Pal or Pitx2, or their gain-of-functional mutants.

FMR1 encodes an RNA-binding protein whose absence results in fragile X mental retardation. In most patients, the FMR1 gene is cytosine-methylated and transcriptionally inactive. NRF-1 and Sp1 are known to bind and stimulate the active, but not the methylated/silenced, FMR1 promoter. Prior analysis has implicated a CRE site in regulation of FMR1 in neural cells but the role of this site is controversial. We now show that a phospho-CREB/ATF family member is bound to this site in vivo. We also find that the histone acetyltransferases CBP and p300 are associated with active FMR1 but are lost at the hypoacetylated fragile X allele. Surprisingly, FMR1 is not cAMP-inducible and resides in a newly recognized subclass of CREB-regulated genes. We have also elucidated a role for NRF-2 as a regulator of FMR1 in vivo through a previously unrecognized and highly conserved recognition site in FMR1. NRF-1 and NRF-2 act additively while NRF-2 synergizes with CREB/ATF at FMR1's promoter. These data add FMR1 to the collection of genes controlled by both NRF-1 and NRF-2 and disfavor its membership in the immediate early response group of genes.

DNA breaks activate the DNA damage response and, if left unrepaired, trigger cellular senescence. Telomeres are specialized nucleoprotein structures that protect chromosome ends from persistent DNA damage response activation. Whether protection can be enhanced to counteract the age-dependent decline in telomere integrity is a challenging question. Telomeric repeat-containing RNA (TERRA), which is transcribed from telomeres, emerged as important player in telomere integrity. However, how human telomere transcription is regulated is still largely unknown. We identify nuclear respiratory factor 1 and peroxisome proliferator-activated receptor γ coactivator 1α as regulators of human telomere transcription. In agreement with an upstream regulation of these factors by adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), pharmacological activation of AMPK in cancer cell lines or in normal nonproliferating myotubes up-regulated TERRA, thereby linking metabolism to telomere fitness. Cycling endurance exercise, which is associated with AMPK activation, increased TERRA levels in skeletal muscle biopsies obtained from 10 healthy young volunteers. The data support the idea that exercise may protect against aging.

Tributyltin (TBT), which has been widely used as an antifouling agent in paints, is a common environmental pollutant. Although the toxicity of high-dose TBT has been extensively reported, the effects of low concentrations of TBT are relatively less well studied. We have previously reported that low-concentration TBT decreases α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor subunit 2 (GluR2) expression in cortical neurons and enhances neuronal vulnerability to glutamate. However, the mechanism of this TBT-induced GluR2 decrease remains unknown. Therefore, we examined the effects of TBT on the activity of transcription factors that control GluR2 expression. Exposure of primary cortical neurons to 20 nM TBT for 3 h to 9 days resulted in a decrease in GluR2 mRNA expression. Moreover, TBT inhibited the DNA binding activity of nuclear respiratory factor-1 (NRF-1), a transcription factor that positively regulates the GluR2. This result indicates that TBT inhibits the activity of NRF-1 and subsequently decreases GluR2 expression. In addition, 20 nM TBT decreased the expression of genes such as cytochrome c, cytochrome c oxidase (COX) 4, and COX 6c, which are downstream of NRF-1. Our results suggest that NRF-1 inhibition is an important molecular action of the neurotoxicity induced by low-concentration TBT.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

The mitochondrial respiratory chain is composed of individual complexes that range widely in terms of size and subunit composition. For example, whereas complex II is approximately 260 kDa and is composed of 4 subunits, complex I is almost 1 MDa and contains 45 different subunits. Furthermore, complexes I, III, IV, and V harbor additional complexity, because their subunits are encoded by both nuclear and mitochondrial DNA. Subunits that are encoded by nuclear genes must be imported into mitochondria before undergoing processing, folding, and assembly with other subunits that are synthesized within the organelle. This process requires the coordinated action of assembly factors with the integration of subunits into intermediate assembly complexes. Recent studies have used various techniques to analyze subunit assembly to gain information into the biogenesis of these respiratory chain complexes and to understand how defects in assembly lead to disease. Here we describe methods to monitor the assembly of newly synthesized subunits encoded by mitochondrial DNA from cultured mammalian cells, as well as the import and assembly of individual subunits encoded by nuclear DNA.

Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (psim) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis is a phenotypic hallmark of C2C12 skeletal muscle cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFkappaB (cRel:p50) pathway, NFAT, CREB and C/EBPdelta. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFkappaB (cRel:p50), C/EBPdelta, CREB, and NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional coactivator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes.

microRNAs (miRNAs) are involved in the various processes of DNA damage repair and play crucial roles in regulating response of tumors to radiation therapy. Here, we used nasopharyngeal carcinoma (NPC) radio-resistant cell lines as models and found that the expression of miR-504 was significantly up-regulated. In contrast, the expression of nuclear respiratory factor 1 (NRF1) and other mitochondrial metabolism factors, including mitochondrial transcription factor A (TFAM) and oxidative phosphorylation (OXPHOS) complex III were down-regulated in these cell lines. At the same time, the Seahorse cell mitochondrial stress test results indicated that the mitochondrial respiratory capacity was impaired in NPC radio-resistant cell lines and in a miR-504 over-expressing cell line. We also conducted dual luciferase reporter assays and verified that miR-504 could directly target NRF1. Additionally, miR-504 could down-regulate the expression of TFAM and OXPHOS complexes I, III, and IV and impaired the mitochondrial respiratory function of NPC cells. Furthermore, serum from NPC patients showed that miR-504 was up-regulated during different weeks of radiotherapy and correlated with tumor, lymph nodes and metastasis (TNM) stages and total tumor volume. The radio-therapeutic effect at three months after radiotherapy was evaluated. Results indicated that patients with high expression of miR-504 exhibited a relatively lower therapeutic effect ratio of complete response (CR), but a higher ratio of partial response (PR), compared to patients with low expression of miR-504. Taken together, these results demonstrated that miR-504 affected the radio-resistance of NPC by down-regulating the expression of NRF1 and disturbing mitochondrial respiratory function. Thus, miR-504 might become a promising biomarker of NPC radio-resistance and targeting miR-504 might improve tumor radiation response.

Alzheimer's disease affects tens of millions of people worldwide and its prevalence continues to rise. It is caused by a combination of a subject's heredity, environment, lifestyle, and medical condition. The most significant genetic risk factor for late onset Alzheimer's disease is a variant of the apolipoprotein E gene, APOE4. Here we show that the single nucleotide polymorphism rs429358 that defines APOE4 is located in a short sequence motif repeated several times within exon 4 of apolipoprotein E, reminiscent of the structure of transcriptional enhancers. A JASPAR database search predicts that the T to C transition in rs429358 generates a binding motif for nuclear respiratory factor NRF1. This site appears to be part of a binding site cluster for this transcription factor on exon 4 of APOE. This de novo NRF1 binding site has therefore the potential to affect the expression of multiple genes in its genomic vicinity. Our in silico analysis, suggesting a novel function for APOE4 at the DNA level, offers a potential mechanism for the observed tissue specific neurodegeneration and the role of environmental factors in Alzheimer's disease etiology.

Acute respiratory distress syndrome (ARDS) is characterized by an excessive acute inflammatory response in lung parenchyma, which ultimately leads to refractory hypoxemia. One of the earliest abnormalities seen in lung injury is the elevated levels of inflammatory cytokines, among them, the soluble tumor necrosis factor (TNF-α) has a key role, which exerts cytotoxicity in epithelial and endothelial cells thus exacerbates edema. The bacterial lipopolysaccharide (LPS) was used both in vitro (RAW 264.7, THP-1, MLE-12, A549, and BEAS-2B) and in vivo (C57BL/6 mice), as it activates a plethora of overlapping inflammatory signaling pathways involved in ARDS. Nimbolide is a chemical constituent of Azadirachta indica, which contains multiple biological properties, while its role in ARDS is elusive. Herein, we have investigated the protective effects of nimbolide in abrogating the complications associated with ARDS. We showed that nimbolide markedly suppressed the nitrosative-oxidative stress, inflammatory cytokines, and chemokines expression by suppressing iNOS, myeloperoxidase, and nitrotyrosine expression. Moreover, nimbolide mitigated the migration of neutrophils and mast cells whilst normalizing the LPS-induced hypothermia. Also, nimbolide modulated the expression of epigenetic regulators with multiple HDAC inhibitory activity by suppressing the nuclear translocation of NF-κB and HDAC-3. We extended our studies using molecular docking studies, which demonstrated a strong interaction between nimbolide and TNF-α. Additionally, we showed that treatment with nimbolide increased GSH, Nrf-2, SOD-1, and HO-1 protein expression; concomitantly abrogated the LPS-triggered TNF-α, p38 MAPK, mTOR, and GSK-3β protein expression. Collectively, these results indicate that TNF-α-regulated NF-κB and HDAC-3 crosstalk was ameliorated by nimbolide with promising anti-nitrosative, antioxidant, and anti-inflammatory properties in LPS-induced ARDS.

While the exact role of β1 subunit-containing GABA-A receptors (GABARs) in brain function is not well understood, altered expression of the β1 subunit gene (GABRB1) is associated with neurological and neuropsychiatric disorders. In particular, down-regulation of β1 subunit levels is observed in brains of patients with epilepsy, autism, bipolar disorder and schizophrenia. A pathophysiological feature of these disease states is imbalance in energy metabolism and mitochondrial dysfunction. The transcription factor, nuclear respiratory factor 1 (NRF-1), has been shown to be a key mediator of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Using a variety of molecular approaches (including mobility shift, promoter/reporter assays, and overexpression of dominant negative NRF-1), we now report that NRF-1 regulates transcription of GABRB1 and that its core promoter contains a conserved canonical NRF-1 element responsible for sequence specific binding and transcriptional activation. Our identification of GABRB1 as a new target for NRF-1 in neurons suggests that genes coding for inhibitory neurotransmission may be coupled to cellular metabolism. This is especially meaningful as binding of NRF-1 to its element is sensitive to the kind of epigenetic changes that occur in multiple disorders associated with altered brain inhibition.

In this study, we show that porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 1α (nsp1α) facilitates PRRSV escape from innate immune by modulating nuclear to cytoplasmic translocation and distribution ratio of TRAIP to promote virus proliferation. Mechanistically, TRAIP interacts with PRRSV nsp1α via its K205 site, while NSP1α decreases the SUMOylation and K48 ubiquitination independent of the TRAIP interaction K205 site. Modulation of the dual modification of TRAIP by PRRSV nsp1α results in over-enrichment of TRAIP in the cytoplasm. Enrichment of nsp1α-induced cytoplasmic TRAIP in turn leads to excessive K48 ubiquitination and degradation of serine/threonine-protein kinase (TBK1), thereby antagonizing TBK1-IRF3-IFN signaling. This study proposes a novel mechanism by which PRRSV utilizes host proteins to regulate innate immunity. Findings from this study provides novel perspective to advance our understanding in the pathogenesis of PRRSV.

GABPB1, known as nuclear respiratory factor 2 (Nrf2), activates mitochondrial genes that are responsible for oxidative phosphorylation. Earlier studies on GABPB1 reported that two single nucleotide polymorphisms (SNPs) such as rs7181866 and rs8031031, to be associated with increased endurance in athletes. In the present study, a cohort of 302 South Indians, including normoglycemic healthy controls, T2DM with and without obesity were genotyped for the two SNPs by PCR-RFLP method and correlated with serum adipokines. The 'G' allele of rs7181866 was found to be associated with obesity whereas rs8031031 didn't show any significant association with obese individuals. The increased levels of adipokines such as Leptin, IL-6 and TNF-α and decreased adiponectin were found among obese-T2DM, when compared to non-obese T2DM subjects. Further, Factor analysis on metabolic components revealed four factors which accounts for 71.5% for non-obese control and 88.3% for obese T2DM of variance. The bias-corrected and accelerated bootstrap analysis revealed GG genotype to have significant positive and negative correlation with both TNF-α and adiponectin. In conclusion, the G allele of (rs7181866 A/G) was found to be significantly associated with risk for obesity among T2DM subjects.

The reduction in muscle mass and strength with age, sarcopenia, is a prevalent condition among the elderly, linked to skeletal muscle dysfunction and cell apoptosis. We demonstrated that testosterone protects against H2O2-induced apoptosis in C2C12 muscle cells. Here, we analyzed the effect of testosterone on mitochondrial gene expression in C2C12 skeletal muscle cells. We found that testosterone increases mRNA expression of genes encoded by mitochondrial DNA, such as NADPH dehydrogenase subunit 1 (ND1), subunit 4 (ND4), cytochrome b (CytB), cytochrome c oxidase subunit 1 (Cox1) and subunit 2 (Cox2) in C2C12. Additionally, the hormone induced the expression of the nuclear respiratory factors 1 and 2 (Nrf-1 and Nrf-2), the mitochondrial transcription factors A (Tfam) and B2 (TFB2M), and the optic atrophy 1 (OPA1). The simultaneous treatment with testosterone and the androgen receptor antagonist, Flutamide, reduced these effects. H2O2-oxidative stress induced treatment, significantly decreased mitochondrial gene expression. Computational analysis revealed that mitochondrial DNA contains specific sequences, which the androgen receptor could recognize and bind, probably taking place a direct regulation of mitochondrial transcription by the receptor. These findings indicate that androgen plays an important role in the regulation of mitochondrial transcription and biogenesis in skeletal muscle.

Not really finished (nrf), a larval-lethal mutation in zebrafish generated by retroviral insertion, causes specific retinal defects. Analysis of mutant retinae reveals an extensive loss of photoreceptors and their precursors around the onset of visual function. These neurons undergo apoptosis during differentiation, affecting all classes of photoreceptors, suggesting an essential function of nrf for the development of all types of photoreceptors. In the mutant, some photoreceptors escape cell death, are functional and, as judged by opsin expression, belong to at least three classes of cones and one class of rods. The protein encoded by nrf is a close homologue of human Nuclear Respiratory Factor 1 and avian Initiation Binding Repressor, transcriptional regulators binding the upstream consensus sequence RCGCRYGCGY. At 24 hours of development, prior to neuronal differentiation, nrf is expressed ubiquitously throughout the developing retina and central nervous system. At 48 hours of development, expression of nrf is detected in the ganglion cell layer, in the neurons of the inner nuclear layer, and in the optic nerve and optic tracts, and, at 72 hours of development, is no longer detectable by in situ hybridization. Mutants contain no detectable nrf mRNA and die within 2 weeks postfertilization as larvae with reduced brain size. On the basis of its similarity with NRF-1 and IBR, nrf is likely involved in transcriptional regulation of multiple target genes, including those that encode mitochondrial proteins, growth factor receptors and other transcription factors. This demonstrates the power of insertional mutagenesis as a means for characterizing novel genes necessary for vertebrate retinal development.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex.

RNA activation (RNAa) is an uncharacterized mechanism of transcriptional activation mediated by small RNAs, such as microRNAs (miRNAs). A critical issue in RNAa research is that it is difficult to distinguish between changes in gene expression caused indirectly by post-transcriptional regulation and direct induction of gene expression by RNAa. Therefore, in this study, we seek to identify a key factor involved in RNAa, using the induction of ZMYND10 by miR-34a as a system to evaluate RNAa. We identify the positive transcription elongation factors CDK9 and DDX21, which form a complex with nuclear AGO and TNRC6A, as important transcriptional activators of RNAa. In addition, we find that inhibition of DDX21 suppresses RNAa by miR-34a and other miRNAs without inhibiting post-transcriptional regulation. Our findings reveal a strong connection between RNAa and release of paused Pol II, facilitating RNAa research by making it possible to separately analyze post-transcriptional regulation and RNAa.

Maternally inherited mitochondrial respiratory disorders are rare, progressive, and multi-systemic diseases that remain intractable, with no effective therapeutic interventions. Patients share a defective oxidative phosphorylation pathway responsible for mitochondrial ATP synthesis, in most cases due to pathogenic mitochondrial variants transmitted from mother to child or to a rare de novo mutation or large-scale deletion of the mitochondrial genome. The clinical diagnosis of these mitochondrial diseases is difficult due to exceptionally high clinical variability, while their genetic diagnosis has improved with the advent of next-generation sequencing. The mechanisms regulating the penetrance of the mitochondrial variants remain unresolved with the patient's nuclear background, epigenomic regulation, heteroplasmy, mitochondrial haplogroups, and environmental factors thought to act as rheostats. The lack of animal models mimicking the phenotypic manifestations of these disorders has hampered efforts toward curative therapies. Patient-derived cellular paradigms provide alternative models for elucidating the pathogenic mechanisms and screening pharmacological small molecules to enhance mitochondrial function. Recent progress has been made in designing promising approaches to curtail the negative impact of dysfunctional mitochondria and alleviate clinical symptoms: 1) boosting mitochondrial biogenesis; 2) shifting heteroplasmy; 3) reprogramming metabolism; and 4) administering hypoxia-based treatment. Here, we discuss their varying efficacies and limitations and provide an outlook on their therapeutic potential and clinical application.