Stochastic Optical Reconstruction Microscopy Imaging of Multiple System Atrophy Inclusions Suggests Stepwise α-Synuclein Aggregation.

BACKGROUND: The architecture and composition of glial (GCI) and neuronal (NCI) α-synuclein inclusions observed in multiple system atrophy (MSA) remain to be precisely defined to better understand the disease. METHODS: Here, we used stochastic optical reconstruction microscopy (STORM) to characterize the nanoscale organization of glial (GCI) and neuronal (NCI) α-synuclein inclusions in cryopreserved brain sections from MSA patients. RESULTS: STORM revealed a dense cross-linked internal structure of α-synuclein in all GCI and NCI. The internal architecture of hyperphosphorylated α-synuclein (p-αSyn) inclusions was similar in glial and neuronal cells, suggesting a common aggregation mechanism. A similar sequence of p-αSyn stepwise intracellular aggregation was defined in oligodendrocytes and neurons, starting from the perinuclear area and growing inside the cells. Consistent with this hypothesis, we found a higher mitochondrial density in GCI and NCI compared to oligodendrocytes and neurons from unaffected donors (P < 0.01), suggesting an active recruitment of the organelles during the aggregation process. CONCLUSIONS: These first STORM images of GCI and NCI suggest stepwise α-synuclein aggregation in MSA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Super-resolution microscopies, technological breakthrough to decipher mitochondrial structure and dynamic.

Mitochondria are complex organelles with an outer membrane enveloping a second inner membrane that creates a vast matrix space partitioned by pockets or cristae that join the peripheral inner membrane with several thin junctions. Several micrometres long, mitochondria are generally close to 300 nm in diameter, with membrane layers separated by a few tens of nanometres. Ultrastructural data from electron microscopy revealed the structure of these mitochondria, while conventional optical microscopy revealed their extraordinary dynamics through fusion, fission, and migration processes but its limited resolution power restricted the possibility to go further. By overcoming the limits of light diffraction, Super-Resolution Microscopy (SRM) now offers the potential to establish the links between the ultrastructure and remodelling of mitochondrial membranes, leading to major advances in our understanding of mitochondria's structure-function. Here we review the contributions of SRM imaging to our understanding of the relationship between mitochondrial structure and function. What are the hopes for these new imaging approaches which are particularly important for mitochondrial pathologies?

Homozygous MFN2 variants causing severe antenatal encephalopathy with clumped mitochondria.

Pathogenic variants in the MFN2 gene are commonly associated with autosomal dominant (CMT2A2A) or recessive (CMT2A2B) Charcot-Marie-Tooth disease, with possible involvement of the CNS. Here, we present a case of severe antenatal encephalopathy with lissencephaly, polymicrogyria and cerebellar atrophy. Whole genome analysis revealed a homozygous deletion c.1717-274_1734 del (NM_014874.4) in the MFN2 gene, leading to exon 16 skipping and in-frame loss of 50 amino acids (p.Gln574_Val624del), removing the proline-rich domain and the transmembrane domain 1 (TM1). MFN2 is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial fusion, shaping large mitochondrial networks within cells. In silico modelling showed that the loss of the TM1 domain resulted in a drastically altered topological insertion of the protein in the mitochondrial outer membrane. Fetus fibroblasts, investigated by fluorescent cell imaging, electron microscopy and time-lapse recording, showed a sharp alteration of the mitochondrial network, with clumped mitochondria and clusters of tethered mitochondria unable to fuse. Multiple deficiencies of respiratory chain complexes with severe impairment of complex I were also evidenced in patient fibroblasts, without involvement of mitochondrial DNA instability. This is the first reported case of a severe developmental defect due to MFN2 deficiency with clumped mitochondria.

Transactive response DNA-binding protein 43 is enriched at the centrosome in human cells.

The centrosome, as the main microtubule organizing centre, plays key roles in cell polarity, genome stability and ciliogenesis. The recent identification of ribosomes, RNA-binding proteins and transcripts at the centrosome suggests local protein synthesis. In this context, we hypothesized that TDP-43, a highly conserved RNA binding protein involved in the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, could be enriched at this organelle. Using dedicated high magnification sub-diffraction microscopy on human cells, we discovered a novel localization of TDP-43 at the centrosome during all phases of the cell cycle. These results were confirmed on purified centrosomes by western blot and immunofluorescence microscopy. In addition, the co-localization of TDP-43 and pericentrin suggested a pericentriolar enrichment of the protein, leading us to hypothesize that TDP-43 might interact with local mRNAs and proteins. Supporting this hypothesis, we found four conserved centrosomal mRNAs and 16 centrosomal proteins identified as direct TDP-43 interactors. More strikingly, all the 16 proteins are implicated in the pathophysiology of TDP-43 proteinopathies, suggesting that TDP-43 dysfunction in this organelle contributes to neurodegeneration. This first description of TDP-43 centrosomal enrichment paves the way for a more comprehensive understanding of TDP-43 physiology and pathology.

Case report: Thirty-year progression of an EMPF1 encephalopathy due to defective mitochondrial and peroxisomal fission caused by a novel de novo heterozygous DNM1L variant.

Mutations in DNM1L (DRP1), which encode a key player of mitochondrial and peroxisomal fission, have been reported in patients with the variable phenotypic spectrum, ranging from non-syndromic optic atrophy to lethal infantile encephalopathy. Here, we report a case of an adult female patient presenting with a complex neurological phenotype that associates axonal sensory neuropathy, spasticity, optic atrophy, dysarthria, dysphasia, dystonia, and ataxia, worsening with aging. Whole-exome sequencing revealed a heterozygous de novo variant in the GTPase domain of DNM1L [NM_001278464.1: c.176C>A p.(Thr59Asn)] making her the oldest patient suffering from encephalopathy due to defective mitochondrial and peroxisomal fission-1. In silico analysis suggested a protein destabilization effect of the variant Thr59Asn. Unexpectedly, Western blotting disclosed profound decrease of DNM1L expression, probably related to the degradation of DNM1L complexes. A detailed description of mitochondrial and peroxisomal anomalies in transmission electron and 3D fluorescence microscopy studies confirmed the exceptional phenotype of this patient.

Glutamate-Induced Deregulation of Krebs Cycle in Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-Like Episodes (MELAS) Syndrome Is Alleviated by Ketone Body Exposure.

(1) Background: The development of mitochondrial medicine has been severely impeded by a lack of effective therapies. (2) Methods: To better understand Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-like episodes (MELAS) syndrome, neuronal cybrid cells carrying different mutation loads of the m.3243A > G mitochondrial DNA variant were analysed using a multi-omic approach. (3) Results: Specific metabolomic signatures revealed that the glutamate pathway was significantly increased in MELAS cells with a direct correlation between glutamate concentration and the m.3243A > G heteroplasmy level. Transcriptomic analysis in mutant cells further revealed alterations in specific gene clusters, including those of the glutamate, gamma-aminobutyric acid pathways, and tricarboxylic acid (TCA) cycle. These results were supported by post-mortem brain tissue analysis from a MELAS patient, confirming the glutamate dysregulation. Exposure of MELAS cells to ketone bodies significantly reduced the glutamate level and improved mitochondrial functions, reducing the accumulation of several intermediate metabolites of the TCA cycle and alleviating the NADH-redox imbalance. (4) Conclusions: Thus, a multi-omic integrated approach to MELAS cells revealed glutamate as a promising disease biomarker, while also indicating that a ketogenic diet should be tested in MELAS patients.

Next-Generation Sequencing Identifies Novel PMPCA Variants in Patients with Late-Onset Dominant Optic Atrophy.

Dominant Optic Atrophy (DOA) is one of the most common inherited mitochondrial diseases, leading to blindness. It is caused by the chronic degeneration of the retinal ganglion cells (RGCs) and their axons forming the optic nerve. Until now, DOA has been mainly associated with genes encoding proteins involved in mitochondrial network dynamics. Using next-generation and exome sequencing, we identified for the first time heterozygous PMPCA variants having a causative role in the pathology of late-onset primary DOA in five patients. PMPCA encodes an α subunit of the mitochondrial peptidase (MPP), responsible for the cleavage and maturation of the mitochondrial precursor proteins imported from the cytoplasm into mitochondria. Recently, PMPCA has been identified as the gene responsible for Autosomal Recessive Cerebellar Ataxia type 2 (SCAR2) and another severe recessive mitochondrial disease. In this study, four PMPCA variants were identified, two are frameshifts (c.309delA and c.820delG) classified as pathogenic and two are missenses (c.1363G>A and c.1547G>A) classified with uncertain pathological significance. Functional assays on patients' fibroblasts show a hyperconnection of the mitochondrial network and revealed that frameshift variants reduced α-MPP levels, while not significantly affecting the respiratory machinery. These results suggest that alterations in mitochondrial peptidase function can affect the fusion-fission balance, a key element in maintaining the physiology of retinal ganglion cells, and consequently lead to their progressive degeneration.

Cancer/Testis Antigen 55 is required for cancer cell proliferation and mitochondrial DNA maintenance.

Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.

The Long Non-Coding RNA SAMMSON Is a Regulator of Chemosensitivity and Metabolic Orientation in MCF-7 Doxorubicin-Resistant Breast Cancer Cells.

Despite improvements in therapeutic strategies for treating breast cancers, tumor relapse and chemoresistance remain major issues in patient outcomes. Indeed, cancer cells display a metabolic plasticity allowing a quick adaptation to the tumoral microenvironment and to cellular stresses induced by chemotherapy. Recently, long non-coding RNA molecules (lncRNAs) have emerged as important regulators of cellular metabolic orientation. In the present study, we addressed the role of the long non-coding RNA molecule (lncRNA) SAMMSON on the metabolic reprogramming and chemoresistance of MCF-7 breast cancer cells resistant to doxorubicin (MCF-7dox). Our results showed an overexpression of SAMMSON in MCF-7dox compared to doxorubicin-sensitive cells (MCF-7). Silencing of SAMMSON expression by siRNA in MCF-7dox cells resulted in a metabolic rewiring with improvement of oxidative metabolism, decreased mitochondrial ROS production, increased mitochondrial replication, transcription and translation and an attenuation of chemoresistance. These results highlight the role of SAMMSON in the metabolic adaptations leading to the development of chemoresistance in breast cancer cells. Thus, targeting SAMMSON expression levels represents a promising therapeutic route to circumvent doxorubicin resistance in breast cancers.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

LPS-enriched small extracellular vesicles from metabolic syndrome patients trigger endothelial dysfunction by activation of TLR4.

BACKGROUND: Metabolic syndrome (MetS) is characterized by a cluster of interconnected risk factors -hyperglycemia, dyslipidemia, hypertension and obesity- leading to an increased risk of cardiovascular events. Small extracellular vesicles (sEVs) can be considered as new biomarkers of different pathologies, and they are involved in intercellular communication. Here, we hypothesize that sEVs are implicated in MetS-associated endothelial dysfunction. METHODS: Circulating sEVs of non-MetS (nMetS) subjects and MetS patients were isolated from plasma and characterized. Thereafter, sEV effects on endothelial function were analyzed by measuring nitric oxide (NO) and reactive oxygen species (ROS) production, and mitochondrial dynamic proteins on human endothelial aortic cells (HAoECs). RESULTS: Circulating levels of sEVs positively correlated with anthropometric and biochemical parameters including visceral obesity, glycaemia, insulinemia, and dyslipidemia. Treatment of HAoECs with sEVs from MetS patients decreased NO production through the inhibition of the endothelial NO-synthase activity. Injection of MetS-sEVs into mice impaired endothelium-dependent relaxation induced by acetylcholine. Furthermore, MetS-sEVs increased DHE and MitoSox-associated fluorescence in HAoECs, reflecting enhanced cytosolic and mitochondrial ROS production which was not associated with mitochondrial biogenesis or dynamic changes. MetS patients displayed elevated circulating levels of LPS in plasma, and, at least in part, it was associated to circulating sEVs. Pharmacological inhibition and down-regulation of TLR4, as well as sEV-carried LPS neutralization, results in a substantial decrease of ROS production induced by MetS-sEVs. CONCLUSION: These results evidence sEVs from MetS patients as potential new biomarkers for this syndrome, and TLR4 pathway activation by sEVs provides a link between the endothelial dysfunction and metabolic disturbances described in MetS.

Mutations in the m-AAA proteases AFG3L2 and SPG7 are causing isolated dominant optic atrophy.

OBJECTIVE: To improve the genetic diagnosis of dominant optic atrophy (DOA), the most frequently inherited optic nerve disease, and infer genotype-phenotype correlations. METHODS: Exonic sequences of 22 genes were screened by new-generation sequencing in patients with DOA who were investigated for ophthalmology, neurology, and brain MRI. RESULTS: We identified 7 and 8 new heterozygous pathogenic variants in SPG7 and AFG3L2. Both genes encode for mitochondrial matricial AAA (m-AAA) proteases, initially involved in recessive hereditary spastic paraplegia type 7 (HSP7) and dominant spinocerebellar ataxia 28 (SCA28), respectively. Notably, variants in AFG3L2 that result in DOA are located in different domains to those reported in SCA28, which likely explains the lack of clinical overlap between these 2 phenotypic manifestations. In comparison, the SPG7 variants identified in DOA are interspersed among those responsible for HSP7 in which optic neuropathy has previously been reported. CONCLUSIONS: Our results position SPG7 and AFG3L2 as candidate genes to be screened in DOA and indicate that regulation of mitochondrial protein homeostasis and maturation by m-AAA proteases are crucial for the maintenance of optic nerve physiology.

Oxidative stress contributes differentially to the pathophysiology of Charcot-Marie-Tooth disease type 2K.

Charcot-Marie-Tooth (CMT) disease is a common inherited peripheral neuropathy. The CMT2K axonal form is associated with GDAP1 dominant mutations, which according to the affected domain cause a gradient of severity. Indeed, the p.C240Y mutation, located within GDAP1 glutathione S-transferase (GST) domain and associated to a mitochondrial complex I defect, is related to a faster disease progression, compared to other mutations, such as the p.R120W located outside the GST domain. Here, we analysed the pathophysiology of six CMT2K fibroblast cell lines, carrying either the p.C240Y or p.R120W mutations. We show that complex I deficiency leads to a redox potential alteration and a significant reduction of sirtuin 1 (SIRT1) expression, a major deacetylase sensitive to the cellular redox state, and NRF1 the downstream target of SIRT1. In addition, we disclosed that the p.C240Y mutation is associated with a greater mitochondrial oxidative stress than the p.R120W mutation. Moreover, complex I activity is further restored in CMT2K mutant cell lines exposed to resveratrol. Together, these results suggest that the reduction of oxidative stress may constitute a promising therapeutic strategy for CMT2K.

Lipidomics Reveals Triacylglycerol Accumulation Due to Impaired Fatty Acid Flux in Opa1-Disrupted Fibroblasts.

OPA1 is a dynamin GTPase implicated in mitochondrial membrane fusion. Despite its involvement in lipid remodeling, the function of OPA1 has never been analyzed by whole-cell lipidomics. We used a nontargeted, reversed-phase lipidomics approach, validated for cell cultures, to investigate OPA1-inactivated mouse embryonic fibroblasts ( Opa1 -/- MEFs). This led to the identification of a wide range of 14 different lipid subclasses comprising 212 accurately detected lipids. Multivariate and univariate statistical analyses were then carried out to assess the differences between the Opa1 -/- and Opa1 +/+ genotypes. Of the 212 lipids identified, 69 were found to discriminate between Opa1 -/- MEFs and Opa1 +/+ MEFs. Among these lipids, 34 were triglycerides, all of which were at higher levels in Opa1 -/- MEFs with fold changes ranging from 3.60 to 17.93. Cell imaging with labeled fatty acids revealed a sharp alteration of the fatty acid flux with a reduced mitochondrial uptake. The other 35 discriminating lipids included phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamine, and sphingomyelins, mainly involved in membrane remodeling, and ceramides, gangliosides, and phosphatidylinositols, mainly involved in apoptotic cell signaling. Our results show that the inactivation of OPA1 severely affects the mitochondrial uptake of fatty acids and lipids through membrane remodeling and apoptotic cell signaling.

Warburg-like effect is a hallmark of complex I assembly defects.

Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.

The Metabolomic Bioenergetic Signature of Opa1-Disrupted Mouse Embryonic Fibroblasts Highlights Aspartate Deficiency.

OPA1 (Optic Atrophy 1) is a multi-isoform dynamin GTPase involved in the regulation of mitochondrial fusion and organization of the cristae structure of the mitochondrial inner membrane. Pathogenic OPA1 variants lead to a large spectrum of disorders associated with visual impairment due to optic nerve neuropathy. The aim of this study was to investigate the metabolomic consequences of complete OPA1 disruption in Opa1-/- mouse embryonic fibroblasts (MEFs) compared to their Opa1+/+ counterparts. Our non-targeted metabolomics approach revealed significant modifications of the concentration of several mitochondrial substrates, i.e. a decrease of aspartate, glutamate and α-ketoglutaric acid, and an increase of asparagine, glutamine and adenosine-5'-monophosphate, all related to aspartate metabolism. The signature further highlighted the altered metabolism of nucleotides and NAD together with deficient mitochondrial bioenergetics, reflected by the decrease of creatine/creatine phosphate and pantothenic acid, and the increase in pyruvate and glutathione. Interestingly, we recently reported significant variations of five of these molecules, including aspartate and glutamate, in the plasma of individuals carrying pathogenic OPA1 variants. Our findings show that the disruption of OPA1 leads to a remodelling of bioenergetic pathways with the central role being played by aspartate and related metabolites.

The accumulation of assembly intermediates of the mitochondrial complex I matrix arm is reduced by limiting glucose uptake in a neuronal-like model of MELAS syndrome.

Ketogenic diet (KD) which combined carbohydrate restriction and the addition of ketone bodies has emerged as an alternative metabolic intervention used as an anticonvulsant therapy or to treat different types of neurological or mitochondrial disorders including MELAS syndrome. MELAS syndrome is a severe mitochondrial disease mainly due to the m.3243A > G mitochondrial DNA mutation. The broad success of KD is due to multiple beneficial mechanisms with distinct effects of very low carbohydrates and ketones. To evaluate the metabolic part of carbohydrate restriction, transmitochondrial neuronal-like cybrid cells carrying the m.3243A > G mutation, shown to be associated with a severe complex I deficiency was exposed during 3 weeks to glucose restriction. Mitochondrial enzyme defects were combined with an accumulation of complex I (CI) matrix intermediates in the untreated mutant cells, leading to a drastic reduction in CI driven respiration. The severe reduction of CI was also paralleled in post-mortem brain tissue of a MELAS patient carrying high mutant load. Importantly, lowering significantly glucose concentration in cell culture improved CI assembly with a significant reduction of matrix assembly intermediates and respiration capacities were restored in a sequential manner. In addition, OXPHOS protein expression and mitochondrial DNA copy number were significantly increased in mutant cells exposed to glucose restriction. The accumulation of CI matrix intermediates appeared as a hallmark of MELAS pathophysiology highlighting a critical pathophysiological mechanism involving CI disassembly, which can be alleviated by lowering glucose fuelling and the induction of mitochondrial biogenesis, emphasizing the usefulness of metabolic interventions in MELAS syndrome.

Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions.

OBJECTIVE: Sporadic amyotrophic lateral sclerosis (sALS) is a fatal neurodegenerative disorder affecting upper and lower motor neurons. In view of the heterogeneous presentation of the disease, one of the current challenges is to identify diagnostic and prognostic markers in order to diagnose sALS at early stage and to stratify patients in trials. In this study, we sought to identify cytological hallmarks of sALS in patient-derived fibroblasts with the aim of finding new clinical-related markers of the disease. METHODS: Primary fibroblasts were prospectively collected from patients affected with classical, rapid, and slow forms of sALS. TDP-43 localization, cytoskeleton distribution, mitochondrial network architecture, and stress granules formation were analyzed using 3D fluorescence microscopy and new super-resolution imaging. Intracellular reactive oxygen species (ROS) production was assessed using live imaging techniques. RESULTS: Six sALS patients (two classical, two rapid, and two slow) and four age-matched controls were included. No difference in fibroblasts cell growth, morphology, and distribution was noticed. The analysis of TDP-43 did not reveal any mislocalization nor aggregation of the protein. The cytoskeleton was harmoniously distributed among the cells, without any inclusion noticed, and no difference was observed regarding the mitochondrial network architecture. Basal ROS production and response to induced stress were similar among patient and control fibroblasts. CONCLUSIONS: ALS cytological lesions are absent in patient-derived fibroblasts and thus cannot contribute as diagnostic nor prognostic markers of the disease.