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Glutamate pathway dysfunction in MELAS syndrome is alleviated by ketogenic diet

ABSTRACT: The development of mitochondrial medicine is greatly impaired by the lack of knowledge and identification of efficient therapeutic routes targeting mitochondria. To better understand the pathophysiology of MELAS syndrome, neuronal cybrid cells, carrying different mutant loads of the m.3243A>G mutation, were investigated by a metabolomics and transcriptomics combined approach. Specific signatures, identifying MELAS biochemical biomarkers, disclosed the glutamate pathway as a culprit mechanism, establishing a strong correlation between glutamate concentrations and the m.3243A>G heteroplasmy levels. Transcriptomic analyses further revealed peculiar gene clusters, including glutamate, gamma-aminobutyric acid (GABA) and tricarboxylic acid (TCA) cycle pathways. These results were supported by post-mortem brain tissue analysis of a MELAS patient, confirming the dysregulation of the glutamate metabolic pathway. Ketogenic diet known to reduce glutamate toxicity, induced a significant reduction of glutamate level after 48h of ketone body treatment, improved mitochondrial functions alleviating the accumulation of several intermediate metabolites of the TCA cycle in MELAS cells. Thus, the integrated approach using a multi-OMICs strategy on MELAS cybrid cells, disclosed novel insights in the mitochondrial energy failure, identifying glutamate as a potential biomarker of the disease, while highlighting ketogenic diet, a nutrition based strategy, to treat MELAS patients

ORGANISM(S): Homo sapiens

PROVIDER: GSE165953 | GEO | 2022/08/03

REPOSITORIES: GEO

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Project description:Background: The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the MELAS syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation. Objective: To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome. Methods: Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses. Results and Interpretation: Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM), but also detected the hypoxia-inducible factor 1α (HIF-1α)/HIF-1β complex, nuclear factor Y (NF-Y) and CREB-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction. Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome. Peripheral blood samples were collected from ten A3243G MELAS patients and twenty age- and sex-matched healthy controls (2 controls for each patient). The patient cohort consisted of 4 females and 6 males, mean±s.e.m. age was 44.1±11.9 years (range: 22–63 years), mean±s.e.m. age at disease onset was 27.1±4.9 years (range: 13–55 years), mean±s.e.m. disease duration was 19.0±4.7 years (range: 4–43 years), and disease severity measured by the ‘Newcastle Mitochondrial Disease Adult Scale’ (NMDAS) 8 ranging from 0.0 (no symptoms) to 1.0 (maximum score) was 0.26±0.05 (range: 0.02–0.54).

Project description:The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. We performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic acid receptor alpha (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene-transcription factor mapping-network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. To investigate retrograde signaling pathways induced by the mitochondrial dysfunction caused by the mt3243 mutation, we generated the three types of cybrid cells using a mitochondria-mediated transformation method. Cells lacking mtDNA (rho0) were fused with mtDNAs with the mt3243 mutation isolated from platelets of a diabetic patient with sensorineural hearing loss. We then isolated three types of cybrid cells: W cells had wild-type mtDNA (3243A homoplasmy), H cells had both the mutant and wild-type mtDNA (3243A/G heteroplasmy) with 70% of the mtDNA containing 3243G, and M cells with only mutant mtDNA (3243G homoplasmy). Gene expression profiles of cybrid cells were generated using Illumina HumanHT-12-v3-BeadChip (Illumina, San Diego, CA), which includes 49,896 probes corresponding to 25,202 annotated genes. According to the Illumina protocols, three biological triplicates of each type of cybrid cells were analyzed. Total RNA (500ng) was isolated from cybrid cells using RNeasy Mini Kit (Qiagen, GmbH, Germany). RNA integrity number (RIN) was in the range of RIN = 9.2 and 10 when measured with an Agilent 2100 Bioanalyzer. RNA was reversely transcribed and amplified using IlluminaTotalPrep RNA amplification kit (Ambion, Austin, TX). In vitro transcription was then carried out to prepare cRNA. The cRNAs were hybridized to the array and then labeled with Cy3-streptavidin (Amersham Bioscience, Little Chalfont, UK). The fluorescent signal on the array was measured with a BeadStation 500 System (Illumina, San Diego, CA).

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Glutamate pathway dysfunction in MELAS syndrome is alleviated by ketogenic diet

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