Magnetic resonance spectroscopy in MELAS syndrome: correlation with CSF and plasma metabolite levels and change after glutamine supplementation.

PURPOSE: MELAS syndrome is a genetic disorder caused by mitochondrial DNA mutations. We previously described that MELAS patients had increased CSF glutamate and decreased CSF glutamine levels and that oral glutamine supplementation restores these values. Proton magnetic resonance spectroscopy (1H-MRS) allows the in vivo evaluation of brain metabolism. We aimed to compare 1H-MRS of MELAS patients with controls, the 1H-MRS after glutamine supplementation in the MELAS group, and investigate the association between 1H-MRS and CSF lactate, glutamate, and glutamine levels. METHODS: We conducted an observational case-control study and an open-label, single-cohort study with single-voxel MRS (TE 144/35 ms). We assessed the brain metabolism changes in the prefrontal (PFC) and parieto-occipital) cortex (POC) after oral glutamine supplementation in MELAS patients. MR spectra were analyzed with jMRUI software. RESULTS: Nine patients with MELAS syndrome (35.8 ± 3.2 years) and nine sex- and age-matched controls were recruited. Lactate/creatine levels were increased in MELAS patients in both PFC and POC (0.40 ± 0.05 vs. 0, p < 0.001; 0.32 ± 0.03 vs. 0, p < 0.001, respectively). No differences were observed between groups in glutamate and glutamine (Glx/creatine), either in PFC (p = 0.930) or POC (p = 0.310). No differences were observed after glutamine supplementation. A positive correlation was found between CSF lactate and lactate/creatine only in POC (0.85, p = 0.003). CONCLUSION: No significant metabolite changes were observed in the brains of MELAS patients after glutamine supplementation. While we found a positive correlation between lactate levels in CSF and 1H-MRS in MELAS patients, we could not monitor treatment response over short periods with this tool. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04948138; initial release 24/06/2021; first patient enrolled on 1/07/2021. https://clinicaltrials.gov/ct2/show/NCT04948138.

Pathophysiology of Cerebellar Degeneration in Mitochondrial Disorders: Insights from the Harlequin Mouse.

By means of a proteomic approach, we assessed the pathways involved in cerebellar neurodegeneration in a mouse model (Harlequin, Hq) of mitochondrial disorder. A differential proteomic profile study (iTRAQ) was performed in cerebellum homogenates of male Hq and wild-type (WT) mice 8 weeks after the onset of clear symptoms of ataxia in the Hq mice (aged 5.2 ± 0.2 and 5.3 ± 0.1 months for WT and Hq, respectively), followed by a biochemical validation of the most relevant changes. Additional groups of 2-, 3- and 6-month-old WT and Hq mice were analyzed to assess the disease progression on the proteins altered in the proteomic study. The proteomic analysis showed that beyond the expected deregulation of oxidative phosphorylation, the cerebellum of Hq mice showed a marked astroglial activation together with alterations in Ca2+ homeostasis and neurotransmission, with an up- and downregulation of GABAergic and glutamatergic neurotransmission, respectively, and the downregulation of cerebellar "long-term depression", a synaptic plasticity phenomenon that is a major player in the error-driven learning that occurs in the cerebellar cortex. Our study provides novel insights into the mechanisms associated with cerebellar degeneration in the Hq mouse model, including a complex deregulation of neuroinflammation, oxidative phosphorylation and glutamate, GABA and amino acids' metabolism.

High-dose oral glutamine supplementation reduces elevated glutamate levels in cerebrospinal fluid in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome.

BACKGROUND AND PURPOSE: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous disorder caused by mitochondrial DNA mutations. There are no disease-modifying therapies, and treatment remains mainly supportive. It has been shown previously that patients with MELAS syndrome have significantly increased cerebrospinal fluid (CSF) glutamate and significantly decreased CSF glutamine levels compared to controls. Glutamine has many metabolic fates in neurons and astrocytes, and the glutamate-glutamine cycle couples with many metabolic pathways depending on cellular requirements. The aim was to compare CSF glutamate and glutamine levels before and after dietary glutamine supplementation. It is postulated that high-dose oral glutamine supplementation could reduce the increase in glutamate levels. METHOD: This open-label, single-cohort study determined the safety and changes in glutamate and glutamine levels in CSF after 12 weeks of oral glutamine supplementation. RESULTS: Nine adult patients with MELAS syndrome (66.7% females, mean age 35.8 ± 3.2 years) were included. After glutamine supplementation, CSF glutamate levels were significantly reduced (9.77 ± 1.21 vs. 18.48 ± 1.34 µmol/l, p < 0.001) and CSF glutamine levels were significantly increased (433.66 ± 15.31 vs. 336.31 ± 12.92 µmol/l, p = 0.002). A side effect observed in four of nine patients was a mild sensation of satiety. One patient developed mild and transient elevation of transaminases, and another patient was admitted for an epileptic status without stroke-like episode. DISCUSSION: This study demonstrates that high-dose oral glutamine supplementation significantly reduces CSF glutamate and increases CSF glutamine levels in patients with MELAS syndrome. These findings may have potential therapeutic implications in these patients. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT04948138. Initial release 24 June 2021, first patient enrolled 1 July 2021. https://clinicaltrials.gov/ct2/show/NCT04948138.

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects.

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.

Apoptosis-Inducing Factor Deficiency Induces Tissue-Specific Alterations in Autophagy: Insights from a Preclinical Model of Mitochondrial Disease and Exercise Training Effects.

We analyzed the effects of apoptosis-inducing factor (AIF) deficiency, as well as those of an exercise training intervention on autophagy across tissues (heart, skeletal muscle, cerebellum and brain), that are primarily affected by mitochondrial diseases, using a preclinical model of these conditions, the Harlequin (Hq) mouse. Autophagy markers were analyzed in: (i) 2, 3 and 6 month-old male wild-type (WT) and Hq mice, and (ii) WT and Hq male mice that were allocated to an exercise training or sedentary group. The exercise training started upon onset of the first symptoms of ataxia in Hq mice and lasted for 8 weeks. Higher content of autophagy markers and free amino acids, and lower levels of sarcomeric proteins were found in the skeletal muscle and heart of Hq mice, suggesting increased protein catabolism. Leupeptin-treatment demonstrated normal autophagic flux in the Hq heart and the absence of mitophagy. In the cerebellum and brain, a lower abundance of Beclin 1 and ATG16L was detected, whereas higher levels of the autophagy substrate p62 and LAMP1 levels were observed in the cerebellum. The exercise intervention did not counteract the autophagy alterations found in any of the analyzed tissues. In conclusion, AIF deficiency induces tissue-specific alteration of autophagy in the Hq mouse, with accumulation of autophagy markers and free amino acids in the heart and skeletal muscle, but lower levels of autophagy-related proteins in the cerebellum and brain. Exercise intervention, at least if starting when muscle atrophy and neurological symptoms are already present, is not sufficient to mitigate autophagy perturbations.

Elevated glutamate and decreased glutamine levels in the cerebrospinal fluid of patients with MELAS syndrome.

BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous disorder caused by mitochondrial DNA (mtDNA) mutations in the MT-TL1 gene. The pathophysiology of neurological manifestations is still unclear, but neuronal hyperexcitability and neuron-astrocyte uncoupling have been suggested. Glutamatergic neurotransmission is linked to glucose oxidation and mitochondrial metabolism in astrocytes and neurons. Given the relevance of neuron-astrocyte metabolic coupling and astrocyte function regulating energetic metabolism, we aimed to assess glutamate and glutamine CSF levels in MELAS patients. METHODS: This prospective observational case-control study determined glutamate and glutamine CSF levels in patients with MELAS syndrome and compared them with controls. The plasma and CSF levels of the remaining amino acids and lactate were also determined. RESULTS: Nine adult patients with MELAS syndrome (66.7% females mean age 35.8 ± 3.2 years) and 19 controls (63.2% females mean age 42.7 ± 3.8 years) were included. The CSF glutamate levels were significantly higher in patients with MELAS than in controls (18.48 ± 1.34 vs. 5.31 ± 1.09 µmol/L, p < 0.001). Significantly lower glutamine concentrations in patients with MELAS than controls were shown in CSF (336.31 ± 12.92 vs. 407.06 ± 15.74 µmol/L, p = 0.017). Moreover, the CSF levels of alanine, the branched-chain amino acids (BCAAs) and lactate were significantly higher in patients with MELAS. CONCLUSIONS: Our results suggest the glutamate-glutamine cycle is altered probably due to metabolic imbalance, and as a result, the lactate-alanine and BCAA-glutamate cycles are upregulated. These findings might have therapeutic implications in MELAS syndrome.

Preferent Diaphragmatic Involvement in TK2 Deficiency: An Autopsy Case Study.

Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular ß-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.

Plasma LDH: A specific biomarker for lung affectation in COVID-19?

OBJECTIVES: We aimed to determine whether the plasma profile of lactate dehydrogenase (LDH) isoenzymes is altered in patients with COVID-19, and whether this is attributable to a specific release of LDH-3, the main LDH isoenzyme expressed in lungs. DESIGN: We collected fresh plasma aliquots from 17 patients (LDH range, 281-822 U/L) and seven controls (LDH â€‹< â€‹230 U/L). In-gel relative activity of the different LDH isoenzymes was determined by electrophoresis and densitometric analysis. RESULTS: Despite the expected higher total LDH activity levels in patients (p â€‹< â€‹0.001), the in-gel relative activities of LDH isoenzymes did not differ between patients and controls (all p â€‹> â€‹0.05). We found no correlation between total plasma LDH activity and the in-gel relative activities of the different LDH isoenzymes, including LDH-3. Likewise, there was no correlation between LDH-3 and various routine haematological and serum parameters that have been previously reported to be altered in COVID-19 (such as lymphocyte count, albumin, alanine and aspartate aminotransferase, creatinine, C-reactive protein, or ferritin). CONCLUSIONS: Our findings suggest that elevation of plasma LDH activity in patients with COVID-19 is not associated to a specific release of LDH-3 into the bloodstream, and do not support the use of LDH as a specific biomarker for lung affectation in patients with COVID-19.

Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities.

Lethal neonatal encephalopathies are heterogeneous congenital disorders that can be caused by mitochondrial dysfunction. Biallelic large deletions in the contiguous ATAD3B and ATAD3A genes, encoding mitochondrial inner membrane ATPases of unknown function, as well as compound heterozygous nonsense and missense mutations in the ATAD3A gene have been recently associated with fatal neonatal cerebellar hypoplasia. In this work, whole exome sequencing (WES) identified the novel homozygous variant c.1217 T > G in ATAD3A, predicting a p.(Leu406Arg) substitution, in four siblings from a consanguineous family presenting with fatal neonatal cerebellar hypoplasia, seizures, axial hypotonia, hypertrophic cardiomyopathy, hepatomegaly, congenital cataract, and dysmorphic facies. Biochemical phenotypes of the patients included hyperlactatemia and hypocholesterolemia. Healthy siblings and parents were heterozygous for this variant, which is predicted to introduce a polar chain within the catalytic domain of ATAD3A that shortens its beta-sheet structure, presumably affecting protein stability. Accordingly, patient's fibroblasts with the homozygous variant displayed a specific reduction in ATAD3A protein levels associated with profound ultrastructural alterations of mitochondrial cristae and morphology. Our findings exclude the causative role of ATAD3B on this severe phenotype, expand the phenotypical spectrum of ATAD3A pathogenic variants and emphasize the vital role of ATAD3A in mitochondrial biogenesis.

The Mitochondrial Isoform of FASTK Modulates Nonopsonic Phagocytosis of Bacteria by Macrophages via Regulation of Respiratory Complex I.

Phagocytosis is a pivotal process by which innate immune cells eliminate bacteria. In this study, we explore novel regulatory mechanisms of phagocytosis driven by the mitochondria. Fas-activated serine/threonine kinase (FASTK) is an RNA-binding protein with two isoforms, one localized to the mitochondria (mitoFASTK) and the other isoform to cytosol and nucleus. The mitoFASTK isoform has been reported to be necessary for the biogenesis of the mitochondrial ND6 mRNA, which encodes an essential subunit of mitochondrial respiratory complex I (CI, NADH:ubiquinone oxidoreductase). This study investigates the role and the mechanisms of action of FASTK in phagocytosis. Macrophages from FASTK─/─ mice exhibited a marked increase in nonopsonic phagocytosis of bacteria. As expected, CI activity was specifically reduced by almost 50% in those cells. To explore if decreased CI activity could underlie the phagocytic phenotype, we tested the effect of CI inhibition on phagocytosis. Indeed, treatment with CI inhibitor rotenone or short hairpin RNAs against two CI subunits (NDUFS3 and NDUFS4) resulted in a marked increase in nonopsonic phagocytosis of bacteria. Importantly, re-expression of mitoFASTK in FASTK-depleted macrophages was sufficient to rescue the phagocytic phenotype. In addition, we also report that the decrease in CI activity in FASTK─/─ macrophages is associated with an increase in phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) and that its inhibition using Compound C reverted the phagocytosis phenotype. Taken together, our results clearly demonstrate for the first time, to our knowledge, that mitoFASTK plays a negative regulatory role on nonopsonic phagocytosis of bacteria in macrophages through its action on CI activity.

Noninvasive Prenatal Testing: Comparison of Two Mappers and Influence in the Diagnostic Yield.

OBJECTIVE: The aim of this study was to determine if the use of different mappers for NIPT may vary the results considerably. METHODS: Peripheral blood was collected from 217 pregnant women, 58 pathological (34 pregnancies with trisomy 21, 18 with trisomy 18, and 6 with trisomy 13) and 159 euploid. MPS was performed following a manufacturer's modified protocol of semiconductor sequencing. Obtained reads were mapped with two different software programs: TMAP and HPG-Aligner, comparing the results. RESULTS: Using TMAP, 57 pathological samples were correctly detected (sensitivity 98.28%, specificity 93.08%): 33 samples as trisomy 21 (sensitivity 97.06%, specificity 99.45%), 16 as trisomy 18 (sensibility 88.89%, specificity 93.97%), and 6 as trisomy 13 (sensibility 100%, specificity 100%). 11 false positives, 1 false negative, and 2 samples incorrectly identified were obtained. Using HPG-Aligner, all the 58 pathological samples were correctly identified (sensibility 100%, specificity 96.86%): 34 as trisomy 21 (sensibility 100%, specificity 98.91%), 18 as trisomy 18 (sensibility 100%, specificity 98.99%), and 6 as trisomy 13 (sensibility 100%, specificity 99.53%). 5 false positives were obtained. CONCLUSION: Different mappers use slightly different algorithms, so the use of one mapper or another with the same batch file can provide different results.

Health Benefits of an Innovative Exercise Program for Mitochondrial Disorders.

PURPOSE: We determined the effects of an innovative 8-wk exercise intervention (aerobic, resistance, and inspiratory muscle training) for patients with mitochondrial disease. METHODS: Several end points were assessed in 12 patients (19-59 yr, 4 women) at pretraining, posttraining, and after 4-wk detraining: aerobic power, muscle strength/power and maximal inspiratory pressure (main end points), ability to perform activities of daily living, body composition, quality of life, and blood myokines (secondary end points). RESULTS: The program was safe, with patients' adherence being 94% ± 5%. A significant time effect was found for virtually all main end points (P ≤ 0.004), indicating a training improvement. Similar findings (P ≤ 0.003) were found for activities of daily living tests, total/trunk/leg lean mass, total fat mass, femoral fracture risk, and general health perception. No differences were found for blood myokines, except for an acute exertional increase in interleukin 8 at posttraining/detraining (P = 0.002) and in fatty acid binding protein 3 at detraining (P = 0.002). CONCLUSIONS: An intervention including novel exercises for mitochondrial disease patients (e.g., inspiratory muscle training) produced benefits in numerous indicators of physical capacity and induced a previously unreported shift toward a healthier body composition phenotype.

A Case of IL-7R Deficiency Caused by a Novel Synonymous Mutation and Implications for Mutation Screening in SCID Diagnosis.

Reported synonymous substitutions are generally non-pathogenic, and rare pathogenic synonymous variants may be disregarded unless there is a high index of suspicion. In a case of IL7 receptor deficiency severe combined immunodeficiency (SCID), the relevance of a non-reported synonymous variant was only suspected through the use of additional in silico computational tools, which focused on the impact of mutations on gene splicing. The pathogenic nature of the variant was confirmed using experimental validation of the effect on mRNA splicing and IL7 pathway function. This case reinforces the need to use additional experimental methods to establish the functional impact of specific mutations, in particular for cases such as SCID where prompt diagnosis can greatly impact on diagnosis, treatment, and survival.

COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation.

Mitochondrial respiratory chain (MRC) complexes I, III, and IV associate into a variety of supramolecular structures known as supercomplexes and respirasomes. While COX7A2L was originally described as a supercomplex-specific factor responsible for the dynamic association of complex IV into these structures to adapt MRC function to metabolic variations, this role has been disputed. Here, we further examine the functional significance of COX7A2L in the structural organization of the mammalian respiratory chain. As in the mouse, human COX7A2L binds primarily to free mitochondrial complex III and, to a minor extent, to complex IV to specifically promote the stabilization of the III2+IV supercomplex without affecting respirasome formation. Furthermore, COX7A2L does not affect the biogenesis, stabilization, and function of the individual oxidative phosphorylation complexes. These data show that independent regulatory mechanisms for the biogenesis and turnover of different MRC supercomplex structures co-exist.

Secondary coenzyme Q10 deficiencies in oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders.

We evaluated the coenzyme Q10 (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n=72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary CoQ deficiency is a common finding in OXPHOS and non-OXPHOS disorders. We hypothesize that cases of CoQ deficiency associated with OXPHOS defects could be an adaptive mechanism to maintain a balanced OXPHOS, although the mechanisms explaining these deficiencies and the pathophysiological role of secondary CoQ deficiency deserves further investigation.

iTRAQ-based analysis of progerin expression reveals mitochondrial dysfunction, reactive oxygen species accumulation and altered proteostasis.

INTRODUCTION: Nuclear accumulation of a mutant form of the nuclear protein Lamin-A, called Progerin (PG) or Lamin AΔ50, occurs in Hutchinson-Gilford Progeria Syndrome (HGPS) or Progeria, an accelerated aging disease. One of the main symptoms of this genetic disorder is a loss of sub-cutaneous fat due to a dramatic lipodystrophy. METHODS: We stably induced the expression of human PG and GFP -Green Fluorescent Protein- as control in 3T3L1 cells using a lentiviral system to study the effect of PG expression in the differentiation capacity of this cell line, one of the most used adipogenic models. Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation. Several of the modulated proteins were validated by immunoblotting and real-time PCR. Mitochondrial function was analyzed by measurement of a) the mitochondrial basal activity, b) the superoxide anion production and c) the individual efficiency of the different complex of the respiratory chain. RESULTS: We found that over-expression PG by lentiviral gene delivery leads to a decrease in the proliferation rate and to defects in adipogenic capacity when compared to the control. Quantitative proteomics analysis showed 181 proteins significantly (p<0.05) modulated in PG-expressing preadipocytes. Mitochondrial function is impaired in PG-expressing cells. Specifically, we have detected an increase in the activity of the complex I and an overproduction of Superoxide anion. Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio. CONCLUSION: PG expression in 3T3L1 cells promotes changes in several Biological Processes, including structure of cytoskeleton, lipid metabolism, calcium regulation, translation, protein folding and energy generation by the mitochondria. Our data strengthen the contribution of ROS accumulation to the premature aging phenotype and establish a link between mitochondrial dysfunction and loss of proteostasis in HGPS.

Exome sequencing identifies a CHKB mutation in Spanish patient with megaconial congenital muscular dystrophy and mtDNA depletion.

BACKGROUND: Choline kinase beta gene (CHKB) mutations have been identified in Megaconial Congenital Muscular Dystrophy (MDCMC) patients, but never in patients with an additional combined deficiency of complexes I, III and IV and mitochondrial DNA (mtDNA) depletion. AIMS: To report mutations in carry genes for MDCMC with respiratory chain defects and mtDNA depletion. METHODS: Whole-exome sequencing (WES) was used to identify the carry genes in a Spanish child with muscle weakness, mild hypotonia at lower limb muscles, mildly elevated creatine kinase (CK), enlarged mitochondria in the periphery of the fibers, combined deficiency of complex I, III and IV and depletion of mtDNA. RESULTS: With WES data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The first filter of WES data with the nuclear-encoded mitochondrial genes (MitoCarta) did not get any candidate. However, the analysis of whole exome uncovered a homozygous nonsense pathogenic mutation in CHKB gene (NM_005198.4:c.810T>A, p.Tyr270*). CONCLUSIONS: Our data confirm the role of CHKB in MDCMC and point to this gene as unique candidate for the combined deficiency of respiratory chain and mtDNA depletion observed in this patient.