SOD1 Gene Silencing Promotes Apoptosis and Suppresses Proliferation of Heat-Stressed Bovine Granulosa Cells via Induction of Oxidative Stress

Heat stress (HS) compromises dairy cattle reproduction by altering the follicular dynamics, oocyte maturation, and normal physiological function of ovarian granulosa cells (GCs), eventually resulting in oxidative damage and cell apoptosis. To protect the cells from oxidative damage, the Superoxide dismutase-1 (SOD1) degraded the hydrogen peroxide (H2O2) to oxygen (O2) and water. The objective of the current study was to investigate the impact of SOD1 silencing on intracellular ROS accumulation, cell viability, MMP, hormone synthesis (P4, E2), cell proliferation, and apoptosis in GCs under HS. The mechanistic role of SOD1 regulation in the heat-stressed GCs was explored. SOD1 gene was successfully silenced in GCs and confirmed at both transcriptional and translational levels. We found that silencing of SOD1 using siRNA under HS aggravated intracellular accumulation of reactive oxygen species, apoptosis, disrupted the mitochondrial membrane potential (MMP), altered transition of the cell cycle, and impaired synthesis of progesterone (P4) and estrogen (E2) in GCs. The associative apoptotic, steroidogenic, and cell cycle genes (BAX, Caspase-3, STAR, Cyp11A1, HSP70, PCNA, and CyclinB1) were used to confirm the results. These results identify a novel role of SOD1 in the modulation of bovine ovarian GC apoptosis, which provides a target for improving the fertility of heat-stressed dairy cows in summer.

Genomic Sequencing of sod1D Yeast That Escape Spore Death

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that impacts nerve cells and the spinal cord, which in some cases are linked to mutations in the Superoxide Dismutase 1 (SOD1) gene. Sod1 is an antioxidant within cells that converts reactive oxygen from superoxide into water using a copper and zinc ion to deactivate the oxygen. When the SOD1 gene is deleted, yeast cells are still able to undergo meiotic divisions and generate four spores, but the spores that are produced are inviable. However, we see that randomly, sod1∆ spores can grow on rich media. This leads us to hypothesize that somewhere in the genome, there is a suppressor mutation that allows these cells to grow. We tested this hypothesis by preparing samples for whole genome sequencing. By comparing the genomic sequences from our suppressor mutants to wild-type controls, we’re able to identify a single point mutation within a gene called NCA2, which codes for a protein that regulates expression of Fo-F1 ATP synthase subunits 6 and 8. Given this result, we are now working to try and understand the relationship between the sod1∆ spore death phenotype and the modulation of ATP synthase activity. In summary, the results from our work have the potential to further help us understand what role Sod1 plays in yeast meiosis and may be able to give us a deeper understanding for ALS cases that are linked to Sod1.

Missense Mutations of Codon 116 in the SOD1 Gene Cause Rapid Progressive Familial ALS and Predict Short Viability With PMA Phenotype

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease, characterized by a great variety of both clinical presentations and genetic causes. Previous studies had identified two different missense mutations in SOD1 (p.R116C and p.R116G) causing familial ALS. In this study, we report a novel heterozygous missense mutation in the SOD1 gene (p.R116S) in a family with inherited ALS manifested as fast-deteriorating pure lower motor neuron symptoms. The patient displayed similar clinical picture and prognostic value to previous reported cases with different R116 substitution mutations. Modeling of all R116 substitutions in the resolved SOD1 protein structure revealed a shared mechanism with destroyed hydrogen bonds between R116 and other two residues, which might lead to protein unfolding and oligomer formation, ultimately conferring neurotoxicity.

Allele and genotype frequencies of the SOD1 gene polymorphism associated with canine degenerative myelopathy in Belgian Malinois dogs in Greece

Background and Aim: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. Materials and Methods: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. Results: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy–Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. Conclusion: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.

Protective effects of baicalin on caerulein-induced AR42J pancreatic acinar cells by attenuating oxidative stress through miR-136-5p downregulation

Baicalin, the main active component of Scutellaria baicalensis, has antioxidant and anti-apoptotic effects and is used to treat acute pancreatitis; however, its specific mechanism is unclear. This study aims to determine the protective effect and underlying mechanism of baicalin on AR42J pancreatic acinar cell injury. AR42J acinar cells (caerulein, 10 nmol/L) were induced in vitro to establish a cell model for acute pancreatitis. Cell relative survival was measured by thiazolyl blue tetrazolium bromide, and cell apoptosis and death were examined by flow cytometry. The expression levels of superoxide dismutase1 (SOD1), Bax, survivin, Bcl-2, caspase-3, and caspase-7 proteins were analyzed by Western blot, and those of SOD1 mRNA and miR-136-5p were determined by RT-PCR. The activities of GSH, SOD1, ROS, and MDA were also investigated. Compared with those of the caerulein group, the relative survival rate and activity of AR42J pancreatic acinar cells with different baicalin concentrations were significantly increased ( p < 0.05), and the supernatant amylase level was markedly decreased ( p < 0.05). In addition, the ROS and MDA activities and mir-136-5p expression were significantly decreased, and the GSH activities and SOD1 gene and protein expression levels were markedly increased ( p < 0.05). These results suggest that baicalin reduced the caerulein-induced death of AR42J acinar cells and alleviated the caerulein-induced injury in pancreatic acinar cells by inhibiting oxidative stress. The mechanism may be related to the decreased expression of Mir-136-5p and the increased expression of SOD1 gene and protein.

Dysregulations of Expression of Genes of the Ubiquitin/SUMO Pathways in an In Vitro Model of Amyotrophic Lateral Sclerosis Combining Oxidative Stress and SOD1 Gene Mutation

Protein aggregates in affected motor neurons are a hallmark of amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to their formation remain incompletely understood. Oxidative stress associated with age, the major risk factor in ALS, contributes to this neurodegeneration in ALS. We show that several genes coding for enzymes of the ubiquitin and small ubiquitin-related modifier (SUMO) pathways exhibit altered expression in motor neuronal cells exposed to oxidative stress, such as the CCNF gene mutated in ALS patients. Eleven of these genes were further studied in conditions combining oxidative stress and the expression of an ALS related mutant of the superoxide dismutase 1 (SOD1) gene. We observed a combined effect of these two environmental and genetic factors on the expression of genes, such as Uhrf2, Rbx1, Kdm2b, Ube2d2, Xaf1, and Senp1. Overall, we identified dysregulations in the expression of enzymes of the ubiquitin and SUMO pathways that may be of interest to better understand the pathophysiology of ALS and to protect motor neurons from oxidative stress and genetic alterations.

Changes in the Activity and Concentration of Superoxide Dismutase Isoenzymes (Cu/Zn SOD, MnSOD) in the Blood of Healthy Subjects and Patients with Acute Pancreatitis

This study was aimed at evaluating the changes in the concentration and activity of all superoxide dismutase isoenzymes (SOD1, SOD2, SOD3) in the blood of patients with acute pancreatitis (AP) and healthy subjects, taking into account the extracellular (plasma) and intracellular (erythrocyte lysate) compartment. The relationships between the activity/concentration of SODs, metal concentration and the markers of inflammation were evaluated. To assess the pro/antioxidative imbalance, the malonyldialdehyde (MDA) concentration and the value of total antioxidant capacity (TAC) were measured. The impact of single-nucleotide polymorphism (SNP) in the SOD1 gene (rs2070424) on the activity/concentration of SOD1 as the main isoenzyme of the SOD family was also analyzed in this study. The SOD2 activity in erythrocytes was increased compared to plasma: 10-fold in the AP patient group and 5-fold in healthy subjects. The plasma of AP patients showed an increased SOD1 concentration and decreased SOD2 and SOD3 concentrations compared to healthy subjects. The Cu/Zn SOD (SOD1 + SOD3) concentration in plasma of AP patients was elevated compared to healthy subjects, but changes in plasma Cu/Zn SOD (SOD1 + SOD3) activity in the examined groups were not observed. An influence of SNP rs2070424 in the SOD1 gene on the total activity of SOD in AP patients (with AG genotype), accompanied by an increased IL-6 concentration, was observed. In oxidative stress conditions induced by inflammation, the participation of individual forms of plasma SOD isoenzymes in total antioxidative activity of SOD changed. A significant increase in the intracellular SOD1 concentration in plasma of AP patients proves the important role of this isoenzyme in the neutralization of oxidative stress induced by impaired Cu and Zn homeostasis. The presence of increased concentration of SOD2 in erythrocytes of healthy subjects and AP patients confirms the important function of this isoenzyme in the antioxidative defense.

Clinical and Molecular Landscape of ALS Patients with SOD1 Mutations: Novel Pathogenic Variants and Novel Phenotypes. A Single ALS Center Study

Mutations in the copper zinc superoxide dismutase 1 (SOD1) gene are the second most frequent cause of familial amyotrophic lateral sclerosis (ALS). Nearly 200 mutations of this gene have been described so far. We report all SOD1 pathogenic variants identified in patients followed in the single ALS center of Lyon, France, between 2010 and 2020. Twelve patients from 11 unrelated families are described, including two families with the not yet described H81Y and D126N mutations. Splice site mutations were detected in two families. We discuss implications concerning genetic screening of SOD1 gene in familial and sporadic ALS.