Spinal cord injury (SCI) causes permanent neurological deficits in mammals. Larval Xenopus laevis regenerate after SCI, but post-metamorphic animals lose this capacity, resembling mammalian response. Because mitochondria and metabolism influence cell survival and tissue repair, we examined mitochondrial dynamics and metabolic responses after SCI in non-regenerative X. laevis. Using imaging, ultrastructural analyses, ATP measurements, and transcriptional profiling, we found that, unlike regenerative stages, non-regenerative spinal cords exhibited a delayed response with mitochondrial altered localization, reduced density, increased area, and structural abnormalities, consistent with dysfunction. Despite these defects, ATP levels increased after injury, coinciding with the upregulation of glycolytic enzymes and markers of lipid catabolism and lipid droplet formation. These findings indicate that SCI in non-regenerative X. laevis triggers metabolic reprogramming toward glycolysis and lipid utilization that may compensate for mitochondrial dysfunction. Our results identify mitochondrial and metabolic responses associated with regenerative failure and provide insight into metabolic states linked to repair.

Heart disease is the leading cause of death in the elderly population. Age-related heart failure is frequently associated with energy deficits in cardiomyocytes. These cells rely on their abundant, cristae-rich mitochondria for ATP production. ATP synthase, localized along the cristae rims, is central to this process. It is presumed that its function is tightly bound to its spatial organization, but details remain unclear. Here, we explored the spatiotemporal organization of ATP synthase in senescent human iPSC-derived CM in conjunction with its functions. We found changes in the stoichiometry of F1 and FO subunits in senescent CM. The ratio of FO-SU c to F1-SU β increased. The oligomeric organization of the complex was weakened. Using single-molecule localization and tracking microscopy, we observed an increased enzyme mobility within cristae that displayed increased fenestrations. This coincided with decreased mitochondrial ATP level, increased ATP hydrolysis capacity, and a moderate increase in mitochondrial transition pore opening. Disturbed ATP production was correlated with dysregulated calcium dynamics, characterized by heightened spikes and slower cytosolic clearance. Consequently, senescent cardiomyocytes exhibited irregular autonomous and paced beating patterns. These findings indicate that, in senescent cardiomyocytes, functional decline is closely linked to disrupted ATP metabolism, driven by the aberrant organization, dynamics, and activity of ATP synthase within remodeled cristae.

Abstract Mitochondria undergo fusion and fission. While DRP1 regulates fission, fusion is controlled by OPA1, MFN1, and MFN2. The balance between these processes and the crosstalk between machineries remains poorly understood. MFN2 mutations cause Charcot-Marie-Tooth disease type 2 A (CMT2A), affecting mitochondrial fusion and morphology. However, their role in fission is unclear. Using skin fibroblasts from CMT2A patients (L248H and M376V MFN2 mutations) and wild-type mouse embryonic fibroblasts expressing these variants, we studied how MFN2 mutations impact mitochondrial dynamics beyond fusion. We analyzed mitochondrial morphology and dynamics by live-cell confocal microscopy and tested fusion/fission protein levels, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and oxidative phosphorylation complex subunits. MFN2 mutations impaired mitochondrial fusion and displayed distinct effects on fission and cellular metabolism. L248H-expressing cells showed hyper-elongated mitochondria, impaired fission, and increased OCR, while M376V cells exhibited fragmentation, enhanced fission, and elevated ECAR. These effects correlated with differential Drp1 phosphorylation. Our findings demonstrate that MFN2 mutants differentially influence fission and metabolism, highlighting the need to consider these effects in therapies aimed at modulating mitochondrial dynamics.

Spinal cord injury (SCI) results in severe disruption of communication between the brain and body, causing motor, sensory, and autonomic dysfunctions. While SCI in mammals leads to permanent impairment due to limited regenerative capacity, certain non-mammalian species, such as Xenopus laevis larval stages, exhibit remarkable regenerative abilities. During Xenopus laevis spinal cord regeneration, neural stem precursor cells (NSPCs) surrounding the central canal rapidly proliferate in response to SCI, compensating for cellular loss, restoring canal continuity, and generating new neurons to reestablish lost connections. It has been described that mitochondria and cellular metabolism play essential roles in stem cell proliferation, self-renewal, and differentiation. However, the mitochondrial and cellular metabolic response during spinal cord regeneration remains unexplored. This study uses electron and confocal microscopy to investigate the NSPCs mitochondrial response in Xenopus laevis following SCI. We observed that mitochondria exhibit a rapid and transient response after SCI, characterized by a disruption of the mitochondrial localization, a decrease in mitochondrial number per cell section, and an increase in mitochondrial area and circularity. Furthermore, mitochondria adopted a swollen phenotype, which did not impair mitochondrial function or cellular energy balance. This morphological shift was accompanied by a transient decrease in the mitochondrial membrane potential and a metabolic switch favoring glycolysis. Therefore, these findings demonstrate that a transient metabolic shift toward glycolysis occurs during spinal cord regeneration.

Mitochondria harbor their own DNA (mtDNA), which codifies essential proteins of the oxidative phosphorylation (OXPHOS) system and locally feeds them to their surrounding inner mitochondrial membrane (IMM), according to the 'sphere of influence' theory. mtDNA is compacted into nucleoids, which are tethered to the IMM and distributed throughout the mitochondrial network. Some nucleoid subpopulations present distinct intramitochondrial positioning during fission and their correct positioning is associated with mtDNA segregation and selective degradation. This opinion article focuses on different mechanisms that could control nucleoid positioning through intramitochondrial trafficking, either by cristae reshaping or by intercompartment-driven mechanisms involving the mitochondrial membranes and extramitochondrial elements. Understanding nucleoid trafficking promises insights into mitochondrial dysfunction in pathologies with mtDNA distribution and segregation issues.

Aging is closely associated with cardiovascular diseases, the leading cause of mortality worldwide. Mitochondrial dysfunction is a hallmark of cardiovascular aging. Most of the heart's ATP is produced at the cristae, specialized subcompartments where oxidative phosphorylation (OXPHOS) takes place. In this study, we used multiple-scale electron microscopy approaches to evaluate age-related mitochondrial and ultrastructural alterations of cristae in human and mouse hearts. We found that aged patients' hearts displayed reduced cristae density as seen by transmission electron microscopy (TEM), even before any significant decline in the expression of cristae-shaping proteins. Similarly, a multiscale approach that included TEM and serial block-face scanning electron microscopy (SBF-SEM) showed that in aged mice's hearts, cristae undergo ultrastructural remodeling processes, resulting in a decrease in cristae density and width. Electron tomography suggests an apparent decline in cristae connectivity and an increase in fenestration size. These changes were linked to Opa1 downregulation, accompanied by reduced maximal OXPHOS respiration, but unrelated to alterations in the abundance of OXPHOS core subunits and ATP synthase assembly. Altogether, this indicates that alterations in cristae structure alone are sufficient to impair oxidative metabolism, which highlights its potential as an early signal of cardiac aging, even before noticeable changes in mitochondrial morphology occur.

Optic atrophy protein 1 (OPA1) mediates inner mitochondrial membrane (IMM) fusion and cristae organization. Mutations in OPA1 cause autosomal dominant optic atrophy (ADOA), a leading cause of blindness. Cells from ADOA patients show impaired mitochondrial fusion, cristae structure, bioenergetic function, and mitochondrial DNA (mtDNA) integrity. The mtDNA encodes electron transport chain subunits and is packaged into nucleoids spread within the mitochondrial population. Nucleoids interact with the IMM, and their distribution is tightly linked to mitochondrial fusion and cristae shaping. Yet, little is known about the physio-pathological relevance of nucleoid distribution. We studied the effect of OPA1 and ADOA-associated mutants on nucleoid distribution using high-resolution confocal microscopy. We applied a novel model incorporating the mitochondrial context, separating nucleoid distribution into the array in the mitochondrial population and intramitochondrial longitudinal distribution. Opa1-null cells showed decreased mtDNA levels and nucleoid abundance. Also, loss of Opa1 led to an altered distribution of nucleoids in the mitochondrial population, loss of cristae periodicity, and altered nucleoids to cristae proximity partly rescued by OPA1 isoform 1. Overexpression of WT OPA1 or ADOA-causing mutants c.870+5 G > A or c.2713 C > T in WT cells, showed perturbed nucleoid array in the mitochondria population associated with cristae disorganization, which was partly reproduced in Skeletal muscle-derived fibroblasts from ADOA patients harboring the same mutants. Opa1-null and cells overexpressing ADOA mutants accumulated mitochondria without nucleoids. Interestingly, intramitochondrial nucleoid distribution was only altered in Opa1-null cells. Altogether, our results highlight the relevance of OPA1 in nucleoid distribution in the mitochondrial landscape and at a single-organelle level and shed light on new components of ADOA etiology.

Heart disease is the leading cause of death in the elderly population and the heart is a highly energy-consuming tissue. The high energy requirement is reflected in the abundance of mitochondria in cardiomyocytes and the cristae dense architecture of the organelles. The ATP synthase is well known for its involvement in ATP synthesis, but it also plays an important structural role. This is reflected in its spatiotemporal organisation, making single molecule localisation microscopy in living cells a valuable tool to study ATP synthase under different conditions. In the present study, we studied the effects of cellular senescence on the ATP Synthase in cardiomyocytes. We used human induced pluripotent stem cell derived cardiomyocytes as a model system and induced senescence with low doses of doxorubicin. We observed reduced function of the ATP Synthase while membrane potential was increased, indicating a malfunction of the proton sink. These impairments could however not be related to changes in neither expression nor dimerization levels of the complex. Using single-molecule tracking of ATP synthase, we observed stronger confinement of the enzyme in the cristae. This suggests that the altered spatiotemporal organisation of ATP synthase is linked with impaired ATP production in senescent cardiomyocytes. ### Competing Interest Statement The authors have declared no competing interest.

Dysfunction of the aging heart is a major cause of death in the human population. Amongst other tasks, mitochondria are pivotal to supply the working heart with ATP. The mitochondrial inner membrane (IMM) ultrastructure is tailored to meet these demands and to provide nano-compartments for specific tasks. Thus, function and morphology are closely coupled. Senescent cardiomyocytes from the mouse heart display alterations of the inner mitochondrial membrane. To study the relation between inner mitochondrial membrane architecture, dynamics and function is hardly possible in living organisms. Here, we present two cardiomyocyte senescence cell models that allow in cellular studies of mitochondrial performance. We show that doxorubicin treatment transforms human iPSC-derived cardiomyocytes and rat neonatal cardiomyocytes in an aged phenotype. The treated cardiomyocytes display double-strand breaks in the nDNA, have β-galactosidase activity, possess enlarged nuclei, and show p21 upregulation. Most importantly, they also display a compromised inner mitochondrial structure. This prompted us to test whether the dynamics of the inner membrane was also altered. We found that the exchange of IMM components after organelle fusion was faster in doxorubicin-treated cells than in control cells, with no change in mitochondrial fusion dynamics at the meso-scale. Such altered IMM morphology and dynamics may have important implications for local OXPHOS protein organization, exchange of damaged components, and eventually the mitochondrial bioenergetics function of the aged cardiomyocyte.

Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion; yet, their specific roles remain elusive. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity.

Many people around the world suffer from some form of paralysis caused by spinal cord injury (SCI), which has an impact on quality and life expectancy. The spinal cord is part of the central nervous system (CNS), which in mammals is unable to regenerate, and to date, there is a lack of full functional recovery therapies for SCI. These injuries start with a rapid and mechanical insult, followed by a secondary phase leading progressively to greater damage. This secondary phase can be potentially modifiable through targeted therapies. The growing literature, derived from mammalian and regenerative model studies, supports a leading role for mitochondria in every cellular response after SCI: mitochondrial dysfunction is the common event of different triggers leading to cell death, cellular metabolism regulates the immune response, mitochondrial number and localization correlate with axon regenerative capacity, while mitochondrial abundance and substrate utilization regulate neural stem progenitor cells self-renewal and differentiation. Herein, we present a comprehensive review of the cellular responses during the secondary phase of SCI, the mitochondrial contribution to each of them, as well as evidence of mitochondrial involvement in spinal cord regeneration, suggesting that a more in-depth study of mitochondrial function and regulation is needed to identify potential targets for SCI therapeutic intervention.

The life quality of the elderly is compromised by various cardiovascular diseases. Mitochondria are the principal source of energy in the heart, and their functional impairment is key in the pathophysiology of the senescent heart. Mitochondrial bioenergetics is determined by dynamic reshaping including, fusion, fission, and inner mitochondrial membrane folding into cristae, the domain where oxidative phosphorylation occurs. Disorders in cristae organization are associated with organelle dysfunction. In this study, we analyzed the mitochondrial ultrastructure of adult and senescent mice hearts. C57/BL6 mice aged 6 and 17-19 months old, adults and senescent, respectively, were sacrificed by cervical dislocation. The left ventricle was divided into i) apex, fixed in 2.5% glutaraldehyde, for transmission electron microscopy; ii) lateral wall for biochemical analysis. Longitudinal sections of the apex were observed in the TALOS electron microscope, Thermo Scientific. The mRNA levels of mitochondrial dynamics-related genes were tested by RT-qPCR: Mfn1, Mfn2 and OPA1 (fusion), Drp1 (fission), and Mic60 and Mic19 (MICOS, mitochondrial cristae organizing system). The mRNA levels of Mfn1, OPA1, Drp1, Mic60, and Mic19 did not show significant changes in the different evaluated ages, but Mfn2 mRNA showed higher levels, in the elder samples. Mitochondrial area and roundness are significantly increased the senescent heart, consistent with Mfn2 mRNA elevated levels. Ultrastructure studies indicate that the density of mitochondrial cristae and the cristae packing are decreased in senescent mice compared to 6 month-old animals. Interestingly, the cristae width of senescent hearts are narrower than that of adult animals: 6.66 ± 0.18 and 7.62 ± 0.23 nm, respectively (100/27/2; cristae/mitochondria/animals), suggesting an adaptation in the metabolic capacity of the mitochondria of senescent cardiomyocytes. The hearts of senescent animals show augmented area and mitochondrial cristae ultrastructural changes, possibly explaining the functional bioenergetics adaptations of the adult heart during aging.

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.

Initial biopsy of basal cell carcinoma (BCC) may fail to show aggressive histologic subtypes. Additionality, the clinical evaluation of BCC before surgery can miss subclinical extension. Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) are emerging tools that can help in the presurgical evaluation of BCCs.To assess the feasibility of a combined RCM-OCT imaging modality for presurgical evaluation of biopsy-proven BCCs for residual tumor, margin status, and depth.Thirty-eight BCCs in 35 patients referred to a tertiary cancer center for Mohs micrographic surgery (MMS) were imaged with combined RCM-OCT. Images were correlated to MMS frozen sections.Thirty-eight BCCs were analyzed. The mean age of patients was 67.34 years (range, 36-84 years), and 20 patients were female (57.14%). Twenty four BCCs were located on the head (63.16%) , and the mean size was 8.58 mm (range, 3-30 mm). RCM-OCT showed an overall agreement of 91.1% with MMS frozen sections. A sensitivity of 82.6% (95% confidence interval [CI], 69%-92%), specificity of 93.8% (95% CI, 88%-97%), and receiver operating characteristic curve of 0.88 (95% CI, 0.82-0.94) was found. OCT depth was highly correlated with MMS depth (r2 = 0.9).Small sample size and difficulty evaluating certain challenging anatomic sites.Combined RCM-OCT may emerge as a useful tool for presurgical evaluation of BCCs.

Autosomal Dominant Optic Atrophy (ADOA), a disease that causes blindness and other neurological disorders, is linked to OPA1 mutations. OPA1, dependent on its GTPase and GED domains, governs inner mitochondrial membrane (IMM) fusion and cristae organization, which are central to oxidative metabolism. Mitochondrial dynamics and IMM organization have also been implicated in Ca2+ homeostasis and signaling but the specific involvements of OPA1 in Ca2+ dynamics remain to be established. Here we studied the possible outcomes of OPA1 and its ADOA-linked mutations in Ca2+ homeostasis using rescue and overexpression strategies in Opa1-deficient and wild-type murine embryonic fibroblasts (MEFs), respectively and in human ADOA-derived fibroblasts. MEFs lacking Opa1 required less Ca2+ mobilization from the endoplasmic reticulum (ER) to induce a mitochondrial matrix [Ca2+] rise ([Ca2+]mito). This was associated with closer ER-mitochondria contacts and no significant changes in the mitochondrial calcium uniporter complex. Patient cells carrying OPA1 GTPase or GED domain mutations also exhibited altered Ca2+ homeostasis, and the mutations associated with lower OPA1 levels displayed closer ER-mitochondria gaps. Furthermore, in Opa1 −/− MEF background, we found that acute expression of OPA1 GTPase mutants but no GED mutants, partially restored cytosolic [Ca2+] ([Ca2+]cyto) needed for a prompt [Ca2+]mito rise. Finally, OPA1 mutants’ overexpression in WT MEFs disrupted Ca2+ homeostasis, partially recapitulating the observations in ADOA patient cells. Thus, OPA1 modulates functional ER-mitochondria coupling likely through the OPA1 GED domain in Opa1 −/− MEFs. However, the co-existence of WT and mutant forms of OPA1 in patients promotes an imbalance of Ca2+ homeostasis without a domain-specific effect, likely contributing to the overall ADOA progress.

Recent studies highlighted how exoscopes may be employed to approach the lateral skull base. The use of exoscope-assisted procedures to repair middle cranial fossa (MCF) defects has not been fully explored. The surgical microscope in the same circumstances has been associated with relevant limitations, such as its physical obstruction, among others. The aim of this study was to present a proof of concept of exoscope-assisted surgery for MCF defects.A detailed step-by-step MCF approach was performed on 2 alcohol-preserved, latex-injected cadaveric specimens under exoscopic magnification. An illustrative clinical case of encephalocele secondary to a spontaneous tegmen tympani defect repaired via an exoscope-assisted MCF approach was presented.The most common sites of MCF defects, the tegmen tympani and the arcuate eminence, were successfully exposed under exoscopic magnification. Dissection was easily performed; no damage to the dura mater or to vascular or neural structures occurred. In the clinical case, the exoscope-assisted technique demonstrated adequate maneuverability and magnification quality. After localization, the encephalocele was resected, and the MCF defect was repaired. The surgeon's position was comfortable, and operative time was not prolonged.The exoscope allows adequate exposure of the MCF floor with identification and preservation of key anatomical structures. The exoscope represents a valuable alternative to the microscope in reconstruction of MCF defects, offering high-quality magnification and proven maneuverability.

Mitochondrial morphology is the result of the balance between mitochondrial fusion and fission processes. Mitochondrial fission is mediated by the cytosolic GTPase DRP1 which is anchored to the outer mitochondrial membrane (OMM) by MiD49, MiD51, and Mff. Changes in mitochondrial morphology due to alterations in fusion or fission proteins impact cytoplasmic ([Ca2+]cyt) and mitochondrial ([Ca2+]mt) calcium homeostasis. Previous studies from our laboratory showed that cells from a patient bearing a MiD49 deleterious mutation showed elongated mitochondria. However, it is unclear if the ablation of MiD49 alters the [Ca2+]cyt and [Ca2+]mt homeostasis associated. Mouse embryonic fibroblasts from MiD49 Knock out (KO) cells, and MiD49 p.Q92∗ cells were transfected with a cDNA encoding a mitochondrial matrix-targeted fluorescent protein, mt-DsRed, to evaluate mitochondrial morphology. To evaluate [Ca2+]mt, cells were transfected with a cDNA encoding a Ca2+-sensitive mitochondrial matrix-targeted fluorescent protein, mtRCaMP. To evaluate [Ca2+]cyt, cells were loaded with Fluo 4-AM or Fura 2-AM. Live-cell imaging was performed using epifluorescence microscopy. Furthermore, mitochondrial membrane potential (ΔΨmt) was tested by incubating the cells with the ΔΨmt-sensitive fluorescent probe, TMRE. The cells were estimulated with ATP or Histamine. We observed that the two genotypes studied present elongated mitochondria. However, upon 100 nM ATP stimulation, WT and MiD49 KO cells showed no difference in [Ca2+]cyt and [Ca2+]mt transients amplitude and kinetics, consistent with comparable resting ΔΨmt. In addition, Histamine (100 mM)-induced comparable [Ca2+]cyt and [Ca2+]mt transients amplitude in patient-derived MiD49 p.Q92∗ cells, but the patient's cells displayed increased mitochondrial [Ca2+]mt extrusion kinetics than cells from a control individual. Our results suggest that MiD49 protein does not determine [Ca2+]cyt and [Ca2+]mt homeostasis, and the ΔΨmt. Future research is needed to elucidate the role of these Drp1 adaptive proteins on intracellular Ca2+ homeostasis.

Part of the mitochondrial dynamics is the fusion-fission balance. Although, the key proteins that regulate the individual processes are known, the mechanism that maintains the balance between fusion and fusion it is poorly understood. Mutations in mitochondrial fusion protein Mfn2 cause Charcot Marie-Tooth 2A, a motor-neuron degenerative disease. Some, but not all MFN2 mutants alter mitochondrial morphology. Whether Mfn2 mutations alters mitochondrial fission is unknown. In this work, we evaluate the effect of Mfn2 disease-causing mutants in fusion and fission balance.Patient-derived fibroblasts carrying MFN2-L248H or MFN2-M376V, or WT MEF acutely expressing each mutant were co-transfected with mitochondria-targeting mtDsRed and mtPAGFP. Mitochondrial morphology, continuity; and fusion and fission events were evaluated by live-cell confocal microscopy.Patient-derived cells carrying L248H and M376V mutations decreased mitochondrial fusion events. However, Mfn2-L248H induces an hyperelongated phenotype opposite to the fragmentation observed in MFN2-M376V in patients and MEF-WT cells. Consistently, patient cells carrying MFN2-L248H mutant shows diminished fission frequency and augmented lag time. Fusion and fission proteins presented altered levels in Mfn2-mutant carrying cells. Mfn2 mutations, located in different domains of the protein, alter mitochondrial fusion and fission in different ways. Particularly, MFN2-L-248H-bearing cells, inhibit both mitochondrial fusion and fission, causing an elongated phenotype. Our data suggest distinct interaction of MFN2 domains with mitochondrial fission machinery, opening a new possible mechanism contributing to the pathophysiology of CMT2A.