Dataset Information

Post-translational modification analysis of VDAC1 in ALS-SOD1 model cells reveals specific asparagine and glutamine deamination

ABSTRACT: Mitochondria from affected tissues of Amyotrophic Lateral Sclerosis (ALS) patients show morphological and biochemical abnormalities. The impairment of mitochondrial functions causes oxidative damage and accumulation of ROS and represents one of the major features that lead to the selective death of motor neurons in ALS. Our aim was to assess the ability of oxidative stress found in ALS to induce post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific post-translational modifications (PTMs) of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neurons model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS. Selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1 or NSC34wt cells were found. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not fromNSC34control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Rosaria Saletti

LAB HEAD: Rosaria Saletti

PROVIDER: PXD022598 | Pride | 2020-12-10

REPOSITORIES: Pride

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Publications

Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation.

Pittalà Maria Gaetana Giovanna MGG Reina Simona S Cubisino Salvatore Antonio Maria SAM Cucina Annamaria A Formicola Beatrice B Cunsolo Vincenzo V Foti Salvatore S Saletti Rosaria R Messina Angela A

Antioxidants (Basel, Switzerland) 20201202 12

Mitochondria from affected tissues of amyotrophic lateral sclerosis (ALS) patients show morphological and biochemical abnormalities. Mitochondrial dysfunction causes oxidative damage and the accumulation of ROS, and represents one of the major triggers of selective death of motor neurons in ALS. We aimed to assess whether oxidative stress in ALS induces post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants r ...[more]

PMID: 33276691

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Project description:Burgeoning evidence highlights seminal roles for microglia in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). The receptor for advanced glycation end products (RAGE) binds ligands relevant to ALS that accumulate in the diseased spinal cord and RAGE has been previously implicated in the progression of ALS pathology. We generated a novel mouse model to temporally delete Ager from microglia in the murine SOD1G93A model of ALS. Microglia Ager deficient SOD1G93A mice and controls were examined for changes in survival, motor function, gliosis, motor neuron numbers, and transcriptomic analyses of lumbar spinal cord. Furthermore, we examined bulk-RNA-sequencing transcriptomic analyses of human ALS cervical spinal cord. Transcriptomic analysis of human cervical spinal cord reveals a range of AGER expression in ALS patients, which was negatively correlated with age at disease onset and death or tracheostomy. The degree of AGER expression related to differential expression of pathways involved in extracellular matrix, lipid metabolism, and intercellular communication. Microglia display increased RAGE immunoreactivity in the spinal cords of high AGER expressing patients and in the SOD1G93A murine model of ALS vs. respective controls. We demonstrate that microglia Ager deletion at the age of symptomatic onset, day 90, in SOD1G93A mice extends survival in male but not female mice. Critically, many of the pathways identified in human ALS patients that accompanied increased AGER expression were significantly ameliorated by microglia Ager deletion in male SOD1G93A mice. Our results indicate that microglia RAGE disrupts communications with cell types including astrocytes and neurons, intercellular communication pathways that divert microglia from a homeostatic to an inflammatory and tissue-injurious program. In totality, microglia RAGE contributes to the progression of SOD1G93A murine pathology in male mice and may be relevant in human disease.

Project description:Familial amyotrophic lateral sclerosis (ALS) represents about 10% of ALS cases. In about 20% of familial ALS patients, a mutation in superoxide dismutase-1 (SOD1) can be found. The ubiquitous SOD1 protein converts superoxide radical anions to oxygen and hydrogen peroxide. Patients with familial ALS caused by mutations in SOD1 can show comorbidity with frontotemporal dementia and develop cognitive impairment, including apathy, inattention, verbal deficits, and hypersexuality. At the cellular level, pathological signs of ALS may include tau immunoreactive astrocytic and neuronal inclusions, suggesting that cognitive dysfunction in ALS may also reflect abnormal protein metabolism of the microtubule associated protein (MAP), tau. To identify cell-specific expression changes, we performed laser capture microdissection (LCM) to isolate anterior horn motor neurons and surrounding cells. To determine common pathways for the development of ALS due to dysfunction of SOD1 or TAU, we performed whole transcriptome analysis in SOD1 and TAU mouse models. Because ALS is a neurodegenerative disease that specifically affects motor neurons, these cells were the main investigative target. Global transcriptomes of glial cells surrounding the motor neurons were also assessed, because these cells have been implicated as triggers of neurodegeneration. Mouse experiments were performed at the presymptomatic stage, prior to the onset of cell loss, in order to reduce false positive signals due to tissue reactive changes. Lumbar anterior horn anterior horn motor neurons and surrounding cells (glia) were isolated from presymptomatic TAU-P301L and SOD1-G93A transgenic mice using laser capture microdissection (LCM; PixCellÂ® IIe LCM System, Arcturus, Molecular Devices, CA). On average, 500 motor neuron bodies or surrounding glial cells from 20 tissue sections per animal were collected. RNA isolation from LCM collected cells was performed using the Qiagen RNeasy Micro Kit. Only RNA with RNA Integrity Numbers (RIN) above 7.5 were taken for further analysis (Agilent Bioanalyzer 2100). A two-round T7-based amplification and labeling protocol (Agilent Low RNA Input Linear Amplification Kit PLUS) was used to generate high quality labeled cRNA. Agilent Whole Mouse Genome Microarray comparisons of motor neurons and surrounding glia were performed between transgenic (SOD1G93A and TAUP301L) and corresponding nontransgenic (control) littermate animals, producing four independent comparison groups: SOD1G93A motor neurons versus control motor neurons (SOD1mn), SOD1G93A motor neuron surrounding glia versus control glia (SOD1gl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Raw microarray data were acquired using the Agilent DNA Microarray scanner and processed with the accompanying Agilent Feature Extraction 10.5 Image Analysis software using default settings. Normalized signal intensities were used to identify gene expression changes in SOD1G93A and TAUP301L motor neurons and surrounding glial cells, generating four partially overlapping sets of data. For the identification of differential expression, the genes were required to pass two conservative criteria: a ratio beyond the 99.5% confidence interval observed in homotypic comparisons, which corresponded to an approximately 1.5-fold expression change, and a paired t-test (P<0.01) computed using 100 permutations of the data for each gene. Correction for multiple comparisons was performed using the adjusted Bonferroni test. The analysis was performed in the TM4: Microarray Software Suite. These techniques identified 251 transcripts representing 186 known genes for which expression was altered in at least one of the four comparisons. Four-condition experiment, SOD1G93A motor neurons versus control motor neurons (SODmn), SOD1G93A motor neuron surrounding glia versus control glia (SODgl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Biological replicates: 4 SOD1G93A motor neurons (SODmn), 4 non SOD littermate motor neurons (nonSODmn), 4 SOD1G93A motor neuron surrounding glia (SODgl), 4 non SOD littermate motor neuron surrounding glia (nonSODgl), 4 TAUP301L motor neurons (TAUmn), 4 non TAU littermate motor neurons (nonTAUmn), 4 TAUP301L motor neuron surrounding glia (TAUgl), 4 non TAU littermate motor neuron surrounding glia (nonTAUgl). Four independent comparison groups were generated: SOD1G93A motor neurons versus control nonSOD motor neurons, SOD1G93A motor neuron surrounding glia versus control nonSOD glia, TAUP301L motor neurons versus control nonTAU motor neurons and TAUP301L glia versus control nonTAU glia. Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Each replicate was repeated twice with dye flip to correct for unequal dye incorporation rates. Therefore, eight microarray hybridizations were performed for each biological comparison, for a total of 32 microarray slides and generating four groups of differentially expressed genes in SOD1G93A motor neurons (SODmn), SOD1G93A motor neuron surrounding glial cells (SODgl), TAUP301L motor neurons (TAUmn), and TAUP301L surrounding glial cells (TAUgl).

Dataset Information

Post-translational modification analysis of VDAC1 in ALS-SOD1 model cells reveals specific asparagine and glutamine deamination

Publications

Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation.

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