Human SOD1 Research Articles

In-cell processing enables rapid and in-depth proteome analysis of low-input Caenorhabditis elegans.

Caenorhabditis elegans is a widely used genetic model organism, however, the worm cuticle complicates extraction of intracellular proteins, a prerequisite for typical bottom-up proteomics. Conventional physical disruption procedures are not only time-consuming, but can also cause significant sample loss, making it difficult to perform proteomics with low-input samples. Here, for the first time, we present an on-filter in-cell (OFIC) processing approach, which can digest C. elegans proteins directly in the cells of the organism after methanol fixation. With OFIC processing and single-shot LCMS analysis, we identified over 9,400 proteins from a sample of only 200 worms, the largest C. elegans proteome reported to date that did not require fractionation or enrichment. We systematically evaluated the performance of the OFIC approach by comparing it with conventional lysis-based methods. Our data suggest equivalent and unbiased performance of OFIC processing for C. elegans proteome identification and quantitation. We further evaluated the OFIC approach with even lower input samples, then used this method to determine how the proteome is impacted by loss of superoxide dismutase sod-1, the ortholog of human SOD-1, a gene associated with amyotrophic lateral sclerosis (ALS). Analysis of 8,800 proteins from only 50 worms as the initial input showed that loss of sod-1 affects the abundance of proteins required for stress response, ribosome biogenesis, and metabolism. In conclusion, our streamlined OFIC approach, which can be broadly applied to other systems, minimizes sample loss while offering the simplest workflow reported to date for C. elegans proteomics analysis.

Nuclear Localization of Human SOD1 in Motor Neurons in Mouse Model and Patient Amyotrophic Lateral Sclerosis: Possible Links to Cholinergic Phenotype, NADPH Oxidase, Oxidative Stress, and DNA Damage.

Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1's nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6-14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology.

AAV-NRIP gene therapy ameliorates motor neuron degeneration and muscle atrophy in ALS model mice

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS.MethodsWe used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice.ResultsSOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice.ConclusionsAAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.

Trehalose Protects against Superoxide Dismutase 1 Proteinopathy in an Amyotrophic Lateral Sclerosis Model.

This work aimed to study the effect of trehalose in protecting cells against Sod1 proteinopathy associated with amyotrophic lateral sclerosis (ALS). Humanized yeast cells in which native Sod1 was replaced by wild-type human Sod1 or an ALS mutant (WT-A4V Sod1 heterodimer) were used as the experimental model. Cells were treated with 10% trehalose (p/v) before or after the appearance of hSod1 proteinopathy induced by oxidative stress. In both conditions, trehalose reduced the number of cells with Sod1 inclusions, increased Sod1 activity, and decreased the levels of intracellular oxidation, demonstrating that trehalose avoids Sod1 misfolding and loss of function in response to oxidative stress. The survival rates of ALS Sod1 cells stressed in the presence of trehalose were 60% higher than in their absence. Treatment with trehalose after the appearance of Sod1 inclusions in cells expressing WT Sod1 doubled longevity; after 5 days, non-treated cells did not survive, but 15% of cells treated with sugar were still alive. Altogether, our results emphasize the potential of trehalose as a novel therapy, which might be applied preventively in ALS patients with a family history of the disease or after diagnosis in ALS patients who discover the disease following the first symptoms.

Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Perturb mRNA Splicing through Aberrant Interactions with SRSF2.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that results in the degeneration of neurons in the brain and spinal cord. Although a substantial number of studies have been conducted, much remains to be learned about the cellular mechanisms underlying ALS. In this study, we employed an engineered ascorbate peroxidase (APEX)-based proximity biotinylation, together with affinity pull-down of the ensuing biotinylated peptides, to investigate the proximity proteomes of human SOD1 and its two ALS-linked mutants, G85R and G93A. We were able to identify 25 common biotinylated peptides with preferential enrichment in the proximity proteomes of SOD1G85R and SOD1G93A over wild-type SOD1. Our coimmunoprecipitation followed by Western blot analyses revealed that one of these proteins, SRSF2, binds more strongly with the two SOD1 mutants than its wild-type counterpart. We also observed aberrant splicing of mRNAs in cells with ectopic expression of the two SOD1 mutants relative to cells expressing the wild-type protein. In addition, the aberrations in splicing elicited by the SOD1 variants were markedly attenuated upon knockdown of SRSF2. Collectively, we uncovered that ALS-liked SOD1G85R and SOD1G93A mutants interact more strongly with SRSF2, where the aberrant interactions perturbed mRNA splicing. Thus, our work offered novel mechanistic insights into the contributions of the ALS-linked SOD1 mutants to disease etiology.

The familial amyotrophic lateral sclerosis-associated A4V SOD1 mutant is not able to regulate aerobic glycolysis

Under certain stress conditions, astrocytes operate in aerobic glycolysis, a process controlled by pyruvate dehydrogenase (PDH) inhibition through its E1 α subunit (Pda1) phosphorylation. This supplies lactate to neurons, which save glucose to obtain NADPH to, among other roles, counteract reactive oxygen species. A failure in this metabolic cooperation causes severe damage to neurons. In this work, using humanized Saccharomyces cerevisiae cells in which its endogenous Cu/Zn Superoxide Dismutase (SOD1) was replaced by human ortholog, we investigated the role of human SOD1 (hSOD1) in aerobic glycolysis regulation and its implications to amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. Yeast cells ferment glucose even in the presence of oxygen and switch to respiratory metabolism after glucose exhaustion. However, like cells of SOD1-knockout strain, cells expressing A4V mutant of hSOD1 growing on glucose showed a respiratory phenotype, i.e., low glucose and high oxygen consumptions and low intracellular oxidation levels in response to peroxide stress, contrary to cells expressing wild-type (WT) SOD1 (yeast or human). The A4V mutation in hSOD1 is linked to ALS. In contrast to WT SOD1 strains, PDH activity of both sod1Δ and A4V hSOD1 cells did not change in response to a metabolic shift toward oxidative metabolism, which was associated to lower Pda1 phosphorylation levels under growth on glucose. Taken together, our results suggest that A4V mutant cannot regulate aerobic glycolysis via Pda1 phosphorylation the same way WT hSOD1, which might be linked to problems observed in the motor neurons of ALS patients with the SOD1 A4V mutation.

Pan-neuronal expression of human mutant SOD1 in Drosophila impairs survival and motor performance, induces early neuroinflammation and chromosome aberrations

Several mutations in the SOD1 gene encoding for the antioxidant enzyme Superoxide Dismutase 1, are associated with amyotrophic lateral sclerosis, a rare and devastating disease characterized by motor neuron degeneration and patients' death within 2–5 years from diagnosis. Motor neuron loss and related symptomatology manifest mostly in adult life and, to date, there is still a gap of knowledge on the precise cellular and molecular events preceding neurodegeneration.To deepen our awareness of the early phases of the disease, we leveraged two Drosophila melanogaster models pan-neuronally expressing either the mutation A4V or G85R of the human gene SOD1 (hSOD1A4V or hSOD1G85R).We demonstrate that pan-neuronal expression of the hSOD1A4V or hSOD1G85R pathogenic construct impairs survival and motor performance in transgenic flies. Moreover, protein and transcript analysis on fly heads indicates that mutant hSOD1 induction stimulates the glial marker Repo, up-regulates the IMD/Toll immune pathways through antimicrobial peptides and interferes with oxidative metabolism. Finally, cytological analysis of larval brains demonstrates hSOD1-induced chromosome aberrations. Of note, these parameters are found modulated in a timeframe when neurodegeneration is not detected.The novelty of our work is twofold: we have expressed for the first time hSOD1 mutations in all neurons of Drosophila and confirmed some ALS-related pathological phenotypes in these flies, confirming the power of SOD1 mutations in generating ALS-like phenotypes. Moreover, we have related SOD1 pathogenesis to chromosome aberrations and antimicrobial peptides up-regulation. These findings were unexplored in the SOD1-ALS field.

Computational screening of damaging nsSNPs in human SOD1 genes associated with amyotrophic lateral sclerosis identifies destabilising effects of G38R and G42D mutations through in silico evaluation.

Amyotrophic lateral sclerosis (ALS), a complicated neurodegenerative disorder affected by hereditary and environmental variables, is a condition. In this study, the genetic makeup of ALS is investigated, with a focus on the SOD1 gene's single-nucleotide polymorphisms (SNPs) and their ability to affect disease risk. Eleven high-risk missense variations that may impair the functionality of the SOD1 protein were discovered after a thorough examination of SNPs in the SOD1 gene. These mutations were chosen using a variety of prediction approaches, highlighting their importance in the aetiology of ALS. Notably, it was discovered that the stability of the SOD1 wild-type protein structure was compromised by the G38R and G42D SOD1 variants. Additionally, Edaravone, a possible ALS medication, showed a greater affinity for binding mutant SOD1 structures, pointing to potential personalised treatment possibilities. The high-risk SNPs discovered in this investigation seem to have functional effects, especially on the stability of proteins and their interactions with other molecules. This study clarifies the complex genetics of ALS and offers insights into how these genetic variations may affect the effectiveness of therapeutic interventions, particularly in the context of edaravone. In this study advances our knowledge of the genetic mechanisms causing ALS vulnerability and prospective therapeutic strategies. Future studies are necessary to confirm these results and close the gap between individualised clinical applications and improved ALS care.

Oxidized SOD1 accelerates cellular senescence in neural stem cells

BackgroundNeural stem cells (NSCs), especially human NSCs, undergo cellular senescence characterized by an irreversible proliferation arrest and loss of stemness after prolonged culture. While compelling correlative data have been generated to support the oxidative stress theory as one of the primary determinants of cellular senescence of NSCs, a direct cause-and-effect relationship between the accumulation of oxidation-mediated damage and cellular senescence of NSCs has yet to be firmly established. Human SOD1 (hSOD1) is susceptible to oxidation. Once oxidized, it undergoes aberrant misfolding and gains toxic properties associated with age-related neurodegenerative disorders. The present study aims to examine the role of oxidized hSOD1 in the senescence of NSCs.MethodsNSCs prepared from transgenic mice expressing the wild-type hSOD1 gene were maintained in culture through repeated passages. Extracellular vesicles (EVs) were isolated from culture media at each passage. To selectively knock down oxidized SOD1 in NSCs and EVs, we used a peptide-directed chaperone-mediated protein degradation system named CT4 that we developed recently.ResultsIn NSCs expressing the hSOD1 from passage 5, we detected a significant increase of oxidized hSOD1 and an increased expression of biomarkers of cellular senescence, including upregulation of P53 and SA-β-Gal and cytoplasmic translocation of HMGB1. The removal of oxidized SOD1 remarkably increased the proliferation and stemness of the NSCs. Meanwhile, EVs derived from senescent NSCs carrying the wild-type hSOD1 contained high levels of oxidized hSOD1, which could accelerate the senescence of young NSCs and induce the death of cultured neurons. The removal of oxidized hSOD1 from the EVs abolished their senescence-inducing activity. Blocking oxidized SOD1 on EVs with the SOD1 binding domain of the CT4 peptide mitigated its toxicity to neurons.ConclusionOxidized hSOD1 is a causal factor in the cellular senescence of NSCs. The removal of oxidized hSOD1 is a strategy to rejuvenate NSCs and to improve the quality of EVs derived from senescent cells.

Nur77 mitigates endothelial dysfunction through activation of both nitric oxide production and anti-oxidant pathways

BackgroundNur77 belongs to the member of orphan nuclear receptor 4A family that plays critical roles in maintaining vascular homeostasis. This study aims to determine whether Nur77 plays a role in attenuating vascular dysfunction, and if so, to determine the molecular mechanisms involved. MethodsBoth Nur77 knockout (Nur77 KO) and Nur77 endothelial specific transgenic mice (Nur77-Tg) were employed to examine the functional significance of Nur77 in vascular endothelium in vivo. Endothelium-dependent vasodilatation to acetylcholine (Ach) and reactive oxygen species (ROS) production was determined under inflammatory and high glucose conditions. Expression of genes was determined by real-time PCR and western blot analysis. ResultsIn response to tumor necrosis factor alpha (TNF-α) treatment and diabetes, the endothelium-dependent vasodilatation to Ach was significantly impaired in aorta from Nur77 KO as compared with those from the wild-type (WT) mice. Endothelial specific overexpression of Nur77 markedly prevented both TNF-α- and high glucose-induced endothelial dysfunction. Compared with WT mice, after TNF-α and high glucose treatment, ROS production in aorta was significantly increased in Nur77 KO mice, but it was inhibited in Nur77-Tg mice, as determined by dihydroethidium (DHE) staining. Furthermore, we demonstrated that Nur77 overexpression substantially increased the expression of several key enzymes involved in nitric oxide (NO) production and ROS scavenging, including endothelial nitric oxide synthase (eNOS), guanosine triphosphate cyclohydrolase 1 (GCH-1), glutathione peroxidase-1 (GPx-1), and superoxide dismutases (SODs). Mechanistically, we found that Nur77 increased GCH1 mRNA stability by inhibiting the expression of microRNA-133a, while Nur77 upregulated SOD1 expression through directly binding to the human SOD1 promoter in vascular endothelial cells. ConclusionOur results suggest that Nur77 plays an essential role in attenuating endothelial dysfunction through activating NO production and anti-oxidant pathways in vascular endothelium. Targeted activation of Nur77 may provide a novel therapeutic approach for the treatment of cardiovascular diseases associated with endothelial dysfunction.

Function and mechanism of the human SOD2 gene in mice cerebral ischemia/ reperfusion injury.

To investigate the neuroprotective effects of the SOD2 gene in cerebral ischemia reperfusion injury function and the underlying mechanisms in a mice model of middle cerebral artery ischemia reperfusion. SOD2 transgenic mice were engineered using transcription activator-like effector nucleases, and the genotype was identified using PCR after every three generations. Transgenic and C57BL/6J wild type mice were simultaneously subjected to the middle cerebral artery occlusion model. SOD2 expression in the brain, heart, kidney, and skeletal muscle of transgenic mice was significantly higher than that in the wild type. Following ischemia reperfusion, the infarct volume of wild type mice decreased after treatment with fenofibrate compared to the CMC group. Infarction volume in SOD2 transgenic mice after CMC and fenofibrate treatment was significantly reduced. The recovery of cerebral blood flow in wild type mice treated with fenofibrate was significantly enhanced compared with that in the CMC group. The expression of SOD2 in transgenic mice was significantly higher than that in wild type mice, the neuroprotective role of fenofibrate depends on an increase in SOD2 expression.

Staphylococcus Aureus and Pseudomonas Aeruginosa Biofilms in Ear-Set Associated Infection

Background and objectives: Ear infections are a serious public health issue in developing countries. Gram-positive and gram-negative Bacteria are both capable of developing biofilms on medical equipment and earphones; however, Staphylococcus aureus, Staphylococcus epidermidis, E. coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecalis are the most prevalent types and may involve in ear infections. Since there was no such study in our region about ear set association with biofilm formation in patients with ear infections, this study was accomplished. The aim of the study: Therefore, this study was suggested in order to evaluate the relationship between the continuous use of Ear set with biofilm formation and ear infections among handlers. Patients, Materials and Methods: This study was carried out in Kirkuk City (The urban region only) from the 15th of January 2023 to the 23rd of May 2023, done on a total number of (168) participants. The total number of the patients who were Ear set users enrolled for the current study was (120), divided into two groups including (60 patients with biofilm positive (including 27 male and 33 female patients respectively) and 60 patients with biofilm negative (including 23 male and 37 female patients separately), with age, ranged from 20 to 60 years old, who were attended at Kirkuk Teaching Hospital. The control group (non-Ear set users) of the presented study was a total number of (48) including (18) males and (30) females. The serological tests included: the estimation of Human GR, SOD, and MDA antioxidants levels through using of the ELISA technique for all the participants, and the biochemical tests included measuring Vitamin D3 levels by using the iChromaTM technique, while the microbial tests were done through using of VITEK 2 compact system. Biofilm formation was detected by using the Congo red agar method. All the steps of methods are conducted based on the manufacturer's instructions. The bacterial growth among the patients group was (22.5%) of Staphylococcus aureus and (14.16%) of Pseudomonas aeruginosa, while the control group revealed (8.33 %) of Staphylococcus aureus and (4.16%) of Pseudomonas aeruginosa. The age range (20-30) years showed the highest rates among the infected patients, with male patients being more vulnerable to the infections. The mean levels of vitamin D3, human GR, SOD, and MDA decreased significantly in patients in matching to control groups. Conclusions: As a conclusion, Staphylococcus aureus and Pseudomonas aeruginosa are more prevalent agents involved in ear infections. Male patients are more susceptible patients to contracting ear infections. Vitamin D3, Human GR, SOD, and MDA antioxidants levels decline in patients with ear infections.

Comparative Study between Ear Set Use in Relation with Bacterial Biofilms Causing Ear-Infections

Ear infections were recently considered as a serious public health issue in many countries; where Gram-positive and Gram-negative bacteria are the most associated organisms. Besides, the ability of many species of those microorganisms to grow biofilms on medical equipments and different head set raised the question about the relationship between widespread use of ear set and complicated ear infections. Therefore, this study was suggested in order to evaluate the relationship between the continuous use of ear set with biofilm formation and ear infections among handlers. Patients, Materials and Methods: This study was carried out in Kirkuk City during January to May 2023 on a total number of (168) participants from both sexes and age range between 20-60 years. Participants were divided into two groups according to ear set use; Group A included (n=120) headphone users with (50 male and 70 female), who attended Kirkuk Teaching Hospital. On the other hand, Group B included non-headphone users of (n=48) participants with (18 male and 30 female) as the control group. Ear swab samples were collected aseptically under the supervision of Otolaryngologist Consultant from both groups and were immediately cultivated on selective media for microbiological and biochemical diagnostic tests. In accordance, the biofilm formation ability by the isolated bacteria was distinguished using the Congo red agar method. In addition, this study involved certain serological test including: the estimation of Human GR, SOD, and MDA antioxidants levels through using of the ELISA technique for all the participants, and the biochemical tests included measuring Vitamin D3 levels by using the ichromaTM technique to through the light on other factors which might be associated with ear infections. Diagnostic tests revealed the isolation of (28.33%) Pseudomonas aeruginosa, and (45%) of Staphylococcus aureus biofilm positive isolates from ear swabs of Group A participants. While, ear swabs collected from Group B had displayed the isolation of (4.16%) of Pseudomonas aeruginosa, and (8.33 %) of Staphylococcus aureus isolates respectively. Besides, the age range of (20-30) years old showed the highest rates among the infected patients, with male patients being more vulnerable to the infections than their female spouse. Moreover, Vitamin D3, human GR, SOD, and MDA antioxidant markers showed a significant decrease among participants of Group A in comparison to Group B Vitamin D3 and antioxidant marker levels.Staphylococcus aureus and Pseudomonas aeruginosa were the most prevalent agents involved in ear infections. Male patients were more susceptible to contracting ear infections. Vitamin D3, Human GR, SOD, and MDA antioxidant Marker levels declined in patients with ear infections especially the users of headphones.

Selective removal of misfolded SOD1 delays disease onset in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. There is no cure currently. The discovery that mutations in the gene SOD1 are a cause of ALS marks a breakthrough in the search for effective treatments for ALS. SOD1 is an antioxidant that is highly expressed in motor neurons. Human SOD1 is prone to aberrant modifications. Familial ALS-linked SOD1 variants are particularly susceptible to aberrant modifications. Once modified, SOD1 undergoes conformational changes and becomes misfolded. This study aims to determine the effect of selective removal of misfolded SOD1 on the pathogenesis of ALS. Based on the chaperone-mediated protein degradation pathway, we designed a fusion peptide named CT4 and tested its efficiency in knocking down intracellularly misfolded SOD1 and its efficacy in modifying the pathogenesis of ALS. Expression of the plasmid carrying the CT4 sequence in human HEK cells resulted in robust removal of misfolded SOD1 induced by serum deprivation. Co-transfection of the CT4 and the G93A-hSOD1 plasmids at various ratios demonstrated a dose-dependent knockdown efficiency on G93A-hSOD1, which could be further increased when misfolding of SOD1 was enhanced by serum deprivation. Application of the full-length CT4 peptide to primary cultures of neurons expressing the G93A variant of human SOD1 revealed a time course of the degradation of misfolded SOD1; misfolded SOD1 started to decrease by 2 h after the application of CT4 and disappeared by 7 h. Intravenous administration of the CT4 peptide at 10mg/kg to the G93A-hSOD1 reduced human SOD1 in spinal cord tissue by 68% in 24 h and 54% in 48 h in presymptomatic ALS mice. Intraperitoneal administration of the CT4 peptide starting from 60days of age significantly delayed the onset of ALS and prolonged the lifespan of the G93A-hSOD1 mice. The CT4 peptide directs the degradation of misfolded SOD1 in high efficiency and specificity. Selective removal of misfolded SOD1 significantly delays the onset of ALS, demonstrating that misfolded SOD1 is the toxic form of SOD1 that causes motor neuron death. The study proves that selective removal of misfolded SOD1 is a promising treatment for ALS.

Structure-based discovery of an antipsychotic drug as a modulator of human SOD1: a potential therapeutic target in ALS

Structure-based discovery of an antipsychotic drug as a modulator of human SOD1: a potential therapeutic target in ALS

The ε-Isozyme of Protein Kinase C (PKCε) Is Impaired in ALS Motor Cortex and Its Pulse Activation by Bryostatin-1 Produces Long Term Survival in Degenerating SOD1-G93A Motor Neuron-like Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, characterized by a progressive depletion of upper and lower motor neurons (MNs) in the brain and spinal cord. The aberrant regulation of several PKC-mediated signal transduction pathways in ALS has been characterized so far, describing either impaired expression or altered activity of single PKC isozymes (α, β, ζ and δ). Here, we detailed the distribution and cellular localization of the ε-isozyme of protein kinase C (PKCε) in human postmortem motor cortex specimens and reported a significant decrease in both PKCε mRNA (PRKCE) and protein immunoreactivity in a subset of sporadic ALS patients. We furthermore investigated the steady-state levels of both pan and phosphorylated PKCε in doxycycline-activated NSC-34 cell lines carrying the human wild-type (WT) or mutant G93A SOD1 and the biological long-term effect of its transient agonism by Bryostatin-1. The G93A-SOD1 cells showed a significant reduction of the phosphoPKCε/panPKCε ratio compared to the WT. Moreover, a brief pulse activation of PKCε by Bryostatin-1 produced long-term survival in activated G93A-SOD1 degenerating cells in two different cell death paradigms (serum starvation and chemokines-induced toxicity). Altogether, the data support the implication of PKCε in ALS pathophysiology and suggests its pharmacological modulation as a potential neuroprotective strategy, at least in a subgroup of sporadic ALS patients.

Intrinsic structural vulnerability in the hydrophobic core induces species-specific aggregation of canine SOD1 with degenerative myelopathy–linked E40K mutation

Canine degenerative myelopathy (DM), a fatal neurodegenerative disease in dogs, shares clinical and genetic features with amyotrophic lateral sclerosis (ALS), a human motor neuron disease. Mutations in the SOD1 gene encoding Cu/Zn superoxide dismutase (SOD1) cause canine DM and a subset of inherited human ALS. The most frequent DM causative mutation is homozygous E40K mutation which induces the aggregation of canine SOD1 but not of human SOD1. However, the mechanism through which canine E40K mutation induces species-specific aggregation of SOD1 remains unknown. By screening human/canine chimeric SOD1s, we identified that the humanized mutation of the 117th residue (M117L), encoded by exon 4, significantly reduced aggregation propensity of canine SOD1E40K. Conversely, introducing a mutation of leucine 117 to methionine, a residue homologous to canine, promoted E40K-dependent aggregation in human SOD1. M117L mutation improved protein stability and reduced cytotoxicity of canine SOD1E40K. Furthermore, crystal structural analysis of canine SOD1 proteins revealed that M117L increased the packing within the hydrophobic core of the β-barrel structure, contributing to the increased protein stability. Our findings indicate that the structural vulnerability derived intrinsically from Met 117 in the hydrophobic core of the β-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.

Human SOD1 is secreted via a conventional secretion pathway in Saccharomyces cerevisiae

Soluble proteins sorted through the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER) from the cytosol. However, a few proteins that lack a signal peptide are still translocated into the ER, such as SOD1. SOD1 is a causative gene of amyotrophic lateral sclerosis (ALS). A relationship has been suggested between the secretion of SOD1 and the pathogenesis of ALS; however, the transport mechanism of SOD1 remains unclear. We herein report that SOD1 was translocated into the ER lumen through the translocon Sec61 and was then secreted extracellularly. The present results indicate the potential of suppressing the secretion of SOD1 as a therapeutic target for ALS.

Nanozyme mediated SOD1 strategy linked to nanochaperone facilitate aggregation inhibition in amyotrophic lateral sclerosis.

Unlike natural enzymes, nanomaterial based ‘nanozyme’ offers higher stability and easy storage to address a steady function even in the extreme conditions of pH, temperature and molecular resistance to digestion from proteases. Nanozyme scavenging surplus reactive oxygen species (ROS) can serve as an effective tool to treat various neurodegenerative diseases. Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of the upper motor neuron and lower motor neuron leading to destruction of motor neurons and extra motor symptoms. Thus, loss/inefficiency of SOD1 enzymatic activity and its aggregation, both are known to be associated with ALS disease pathology. Here, nanoparticle (NP) functionalization strategy is used to develop a histidine (His) based superoxide dismutase-I (SOD1) therapeutic substitute with a dual role of nanozyme and nanochaperone activity. Zinc and Copper are an essential micronutrient of biological milieu in humans and act as an integral part of several enzymes active sites. His-functionalized nanozyme was found significant for scavenging excess ROS and targeting mitochondria with cytoprotective effect in the ALS model in-vitro. The nanochaperone role was characterized by its ability to reduce the aggregation of human apoSOD1WT and apoSOD1G93A mutant. In particular, a critical analysis of Amide I and III Raman band, specified to protein secondary structural elements suggested the decrease of a-helix and the early predominant b-sheets in human SOD1 in the absence of nanochaperone. But a reverse phenomenon was observed in the presence of nanochaperons. The dual role of nanozyme-nanochaperone provides a promising insightful into ALS pathology and may open new avenues for future therapeutics.

Sequence adaptation in various environments across organisms; similar yet different superoxide dismutase.

Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium that attack the host immune system and killed millions of people annually. The body first defense mechanism is through phagocytosis where an outburst of radical oxygen species (ROS) is released and potentially kill the bacterium. M. tuberculosis superoxide dismutase (MtSOD) survives the phagocytosis and suppresses the host immune system by converting reactive oxygen species to H2O2 and O2. SOD proteins are important for cell survival to prevent the buildup of reactive ROS in the cell that may cause damage to DNA, RNA, proteins, and even cell death. MtSOD (oxidized) is homologous to human SOD1 (hSOD1, reduced) which contains both zinc and copper metals along with the disulfide bond. Though, MtSOD lacks the zinc binding site which proposed to play a role in stability, dimerization, and function of SOD. Utilizing Circular Dichroism, an apparent correlation of MtSOD stabilization with the copper and disulfide bond as more stable secondary structure is observed for oxidized apoprotein compared to the reduced apo and oxidized holoprotein. To further investigate the novel role of zinc in SOD (ZnMtSOD), a mutation of zinc binding site consists of T119H, A123H, T192K (modeled from hSOD1) is introduced in the wild type MtSOD. With the low sequence identity of 19.7% but similar in protein structures, the thermodynamic and kinetics results are compared for hSOD1, ZnMtSOD, and wt MtSOD.