Early nuclear phenotypes and reactive transformation in human iPSC-derived astrocytes from ALS patients with SOD1 mutations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive death of motor neurons (MNs). Glial cells play roles in MN degeneration in ALS. More specifically, astrocytes with mutations in the ALS-associated gene Cu/Zn superoxide dismutase 1 (SOD1) promote MN death. The mechanisms by which SOD1-mutated astrocytes reduce MN survival are incompletely understood. To characterize the impact of SOD1 mutations on astrocyte physiology, we generated astrocytes from human induced pluripotent stem cell (iPSC) derived from ALS patients carrying SOD1 mutations, together with control isogenic iPSCs. We report that astrocytes harboring SOD1(A4V) and SOD1(D90A) mutations exhibit molecular and morphological changes indicative of reactive astrogliosis when compared to isogenic astrocytes. We show further that a number of nuclear phenotypes precede, or coincide with, reactive transformation. These include increased nuclear oxidative stress and DNA damage, and accumulation of the SOD1 protein in the nucleus. These findings reveal early cell-autonomous phenotypes in SOD1-mutated astrocytes that may contribute to the acquisition of a reactive phenotype involved in alterations of astrocyte-MN communication in ALS.

The Ile35 Residue of the ALS-Associated Mutant SOD1 Plays a Crucial Role in the Intracellular Aggregation of the Molecule.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an unknown pathogenesis. It has been reported that mutations in the gene for Cu/Zn superoxide dismutase (SOD1) cause familial ALS. Mutant SOD1 undergoes aggregation and forms amyloid more easily, and SOD1-immunopositive inclusions have been observed in the spinal cords of ALS patients. Because of this, SOD1 aggregation is thought to be related to the pathogenesis of ALS. Some core regions of amyloid have been identified, but the issue of whether these regions form aggregates in living cells remains unclear, and the mechanism responsible for intracellular SOD1 aggregation also remains unclear. The findings reported in this study indicate that the aggregation of the ALS-linked mutant SOD1-EGFP was significantly enhanced when the BioID2 gene was fused to the N-terminus of the mutant SOD1-EGFP plasmid for cellular expression. Expression of a series of BioID2-(C-terminal deletion peptides of SOD1)-EGFP permitted us to identify 1-35 as a minimal N-terminal sequence and Ile35 as an essential amino acid residue that contributes to the intracellular aggregation of SOD1. The findings also showed that an additional substitution of Ile35 with Ser into the ALS mutant SOD1 resulted in the significant suppression of aggregate formation. The fact that no Ile35 mutations have been reported to date in ALS patients indicates that all ALS mutant SOD1s contain Ile35. Taken together, we propose that Ile35 plays a pivotal role in the aggregation of the ALS-linked SOD1 and that this study will contribute to our understanding of the mechanism responsible for SOD1 aggregation.

[Cell membrane-penetrating capacity of hPP10-Cu, Zn-SOD fusion protein and its antioxidant and anti-inflammatory activity].

OBJECTIVE: To investigate the cell membrane-penetrating capacity of human cell-penetrating peptide hPP10 carrying human antioxidant protein Cu-Zn superoxide dismutase (Cu, Zn-SOD) and assess the antioxidant and anti-inflammatory activity of these fusion proteins. METHODS: The fusion protein hPP10-Cu, Zn-SOD was obtained by genetic engineering and identified by Western blotting. The membrane-penetrating ability of the fusion protein was evaluated by immunofluorescence assay, fluorescence colocalization assay and Western blotting, its SOD enzyme activity was detected using a commercial kit, and its effect on cell viability was assessed with MTT assay. In a HEK293 cell model of H2O2-induced oxidative stress, the effect of hPP10-Cu, Zn-SOD on cell apoptosis was analyzed with flow cytometry and RT-qPCR, and its antioxidant effect was assessed using reactive oxygen species (ROS) assay; its anti-inflammatory effect was evaluated in mouse model of TPA-induced ear inflammation by detecting expression of the inflammatory factors using RT-qPCR, Western blotting and immunohistochemistry. RESULTS: The fusion protein hPP10-Cu, Zn-SOD was successfully obtained. Immunofluorescence assay confirmed obvious membrane penetration of this fusion protein in HEK293 cells, localized both in the cell membrane and the cell nuclei after cell entry. hPP10-Cu, Zn-SOD at the concentration of 5 µmol/L exhibited strong antioxidant activity with minimal impact on cell viability at the concentration up to 10 µmol/L. The fusion protein obviously inhibited apoptosis and decreased intracellular ROS level in the oxidative stress cell model and significantly reduced mRNA and protein expression of the inflammatory factors in the mouse model of ear inflammation. CONCLUSION: The fusion protein hPP10-Cu, Zn-SOD capable of penetrating the cell membrane possesses strong antioxidant and anti-inflammatory activities with only minimal cytotoxicity, demonstrating the value of hPP10 as an efficient drug delivery vector and the potential of hPP10-Cu, Zn-SOD in the development of skincare products.

Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent Kd ~ µM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins.

CuZn-superoxide dismutase is differentially modified in localization and expression by three abiotic stresses in miniature rose bushes.

Three abiotic stresses, copper application (CS), mechanical rubbing (MS) and water deprivation (WS) applied on miniature rose bushes specifically activate the expression of the CuZn-Superoxide dismutase (SOD). The Cu/Zn-SOD protein immunodetected in the 4th internode was shown engaged in lignification in phloem, cambium and xylem cells. The SOD occurrence was detailed in the vessel associated cells (VACs), using immunogold labeling observed in transmission electron microscopy. The enzyme was detected in mitochondria, plastids, Golgi vesicles, endoplasmic reticulum and plasma membrane. In addition, in pit-fields without plasmodesmata linking vessel associated cells to vessels, the abiotic stresses increased the transfer apparatus volume. The content in unmethylatedpectins increased in wall ingrowths after CS and MS, but not in WS. In addition to the different localization, the SOD was differentially overexpressed according to the applied stress: an isoform detected at 17 kDa under CuSO4 application, two isoforms respectively detected at 20 and 17 kDa under MS and detected at 17 and 15 kDa under WS. Notably, the only 17 kDa isoform was detected in plasma membrane vesicles from plants submitted to the three stresses. Thus, by increasing the transfer apparatus development, the key role of VACs was emphasized in establishing an adaptative response to abiotic stresses, in miniature rose bushes. Additionally, it has been observed that the differential SOD localization under such stresses sustained the regulatory function of VACs in the transitory sink function of xylem.

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, 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 amyotrophic lateral sclerosis. 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 ß-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 ß-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.

The essential liaison of two copper proteins: the Cu-sensing transcription factor Mac1 and the Cu/Zn superoxide dismutase Sod1 in Saccharomyces cerevisiae.

Although copper is an essential trace element for cell function and viability, its excess can lead to protein oxidation, DNA cleavage, and ultimate cell damage. Cells have established a variety of regulatory mechanisms to ensure copper ion homeostasis. In Saccharomyces cerevisiae, copper sensing and response to copper deficiency are regulated by the transcription factor Mac1. Our group has previously reported that in addition to copper, several chromatin proteins modulate Mac1 functionality. In this study, based on a synthetic growth deficiency phenotype, we showed that the Cu/Zn superoxide dismutase Sod1 plays an important role in Mac1 transcriptional activity, in unchallenged nutrient-rich growth conditions. Sod1 is a multipotent cytoplasmic and mitochondrial enzyme, whose main known function is to detoxify the cell from superoxide ions. It has been previously reported that Sod1 also enters the nucleus and affects the transcription of several genes, some of which are involved in copper homeostasis under Cu-depleted (Wood and Thiele in J Biol Chem 284:404-413, 2009) or only under specific oxidative stress conditions (Dong et al. Mol Cell Biol 33:4041-4050, 2013; Tsang et al. Nar Commun 8:3446, 2014). We have shown that Sod1 physically interacts with Mac1 transcription factor and is important for the transactivation as well as its DNA-binding activities. On the other hand, a constitutively active mutant of Mac1 is not affected functionally by the Sod1 ablation, pointing out that Sod1 contributes to the maintenance of the copper-unchelated state of Mac1. In conclusion, we showed that Sod1-Mac1 interaction is vital for Mac1 functionality, regardless of copper medium deficiency, in unchallenged growth conditions, and we suggest that Sod1 enzymatic activity may modify the redox state of the cysteine-rich motifs in the Mac1 DNA-binding and transactivation domains.

Gene Therapy in Amyotrophic Lateral Sclerosis.

Since the discovery of Cu/Zn superoxide dismutase (SOD1) gene mutation, in 1993, as the first genetic abnormality in amyotrophic lateral sclerosis (ALS), over 50 genes have been identified as either cause or modifier in ALS and ALS/frontotemporal dementia (FTD) spectrum disease. Mutations in C9orf72, SOD1, TAR DNA binding protein 43 (TARDBP), and fused in sarcoma (FUS) genes are the four most common ones. During the last three decades, tremendous effort has been made worldwide to reveal biological pathways underlying the pathogenesis of these gene mutations in ALS/FTD. Accordingly, targeting etiologic genes (i.e., gene therapies) to suppress their toxic effects have been investigated widely. It includes four major strategies: (i) removal or inhibition of abnormal transcribed RNA using microRNA or antisense oligonucleotides (ASOs), (ii) degradation of abnormal mRNA using RNA interference (RNAi), (iii) decrease or inhibition of mutant proteins (e.g., using antibodies against misfolded proteins), and (iv) DNA genome editing with methods such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas). The promising results of these studies have led to the application of some of these strategies into ALS clinical trials, especially for C9orf72 and SOD1. In this paper, we will overview advances in gene therapy in ALS/FTD, focusing on C9orf72, SOD1, TARDBP, and FUS genes.

Senolytic treatment reduces cell senescence and necroptosis in Sod1 knockout mice that is associated with reduced inflammation and hepatocellular carcinoma.

The goal of this study was to test the role cellular senescence plays in the increased inflammation, chronic liver disease, and hepatocellular carcinoma seen in mice null for Cu/Zn-Superoxide dismutase (Sod1KO). To inhibit senescence, wildtype (WT) and Sod1KO mice were given the senolytics, dasatinib, and quercetin (D + Q) at 6 months of age when the Sod1KO mice begin exhibiting signs of accelerated aging. Seven months of D + Q treatment reduced the expression of p16 in the livers of Sod1KO mice to WT levels and the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1ß, CXCL-1, and GDF-15). D + Q treatment also reduced markers of inflammation in livers of the Sod1KO mice, for example, cytokines, chemokines, macrophage levels, and Kupffer cell clusters. D + Q treatment had no effect on various markers of liver fibrosis in the Sod1KO mice but reduced the expression of genes involved in liver cancer and dramatically reduced the incidence of hepatocellular carcinoma. Surprisingly, D + Q also reduced markers of necroptosis (phosphorylated and oligomerized MLKL) in the Sod1KO mice to WT levels. We also found that inhibiting necroptosis in the Sod1KO mice with necrostatin-1s reduced the markers of cellular senescence (p16, p21, and p53). Our study suggests that an interaction occurs between cellular senescence and necroptosis in the liver of Sod1KO mice. We propose that these two cell fates interact through a positive feedback loop resulting in a cycle amplifying both cellular senescence and necroptosis leading to inflammaging and age-associated pathology in the Sod1KO mice.

Histological endpoints and oxidative stress transcriptional responses in the Mediterranean mussel Mytilus galloprovincialis exposed to realistic doses of salicylic acid.

Despite the availability of analytic data, little is known about the toxicity of salicylic acid (SA) on aquatic non-target organisms. The present study aimed at evaluating the impact of SA through a short-term exposure of the Mediterranean mussel Mytilus galloprovincialis to five environmentally relevant concentrations of SA. A set of suitable biomarkers was applied at selected time-points on mussel digestive glands, including histological observations and expression of oxidative stress related genes. The obtained results showed a conspicuous hemocytic infiltration among mussel digestive tubules, as confirmed also by a flow cytometric approach that revealed an increase of halinocytes and granulocytes. Interestingly, a significant dose and time dependent decrease in the expression levels of oxidative stress related genes was found in mussels exposed to SA except for the glutathione S-transferase gene that was significantly up-regulated in a time-dependent manner confirming its important role against oxidant species and in the metabolism of pharmaceuticals.

A combined clinical and computational approach to understand the SOD1A4T-mediated pathogenesis of rapidly progressive familial amyotrophic lateral sclerosis.

Here, we aim to provide a comprehensive clinical and biomolecular description of familial amyotrophic lateral sclerosis (fALS) in a 25-year-old female patient with respect to the SOD1A4T genotype. The clinical diagnosis of the disease was based on family history, neurological examination, electroneurophysiological studies, and revised El Escorial criteria. The heterozygous presence of the A4T mutation in the proband was confirmed by PCR coupled with Sanger sequencing of exon 1 of the SOD1 gene. The mutation was introduced in silico into the three-dimensional structure of the native protein. After energy minimization and quality assessment, non-covalent interactions around threonine-4 and changes in protein stability were calculated computationally. The patient differed widely in age at onset, initial neurological symptoms and findings, and survival time from her kindred, in which several members are affected. SOD1A4T-linked fALS in this case had bulbar involvement at onset, a combination of lower and upper motor neuron signs and showed rapid progression. Unlike alanine-4, threonine-4 failed to engage in hydrophobic interactions with the vicinal non-polar amino acids. The overall fold of the modeled SOD1A4T mutant remained intact, but unfolding free energy estimations disclosed a decrease in the protein's stability. We report a phenotypically distinct patient with fALS due to the SOD1A4T mutation and further expand the largest pedigree ever published for SOD1A4T-linked fALS. Genotype‒phenotype correlation in fALS is complex, and it demands detailed clinical investigation and advanced scientific research. Awareness of the broadened phenotypic spectrum might potentially enhance the diagnosis and genetic counseling of fALS.

High-throughput quantitative analysis of axonal transport in cultured neurons from SOD1H46R ALS mice by using a microfluidic device.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons. We have previously shown that autophagosome-like vesicular structures are progressively accumulated in the spinal axons of an ALS mouse model, overexpressing human Cu/Zn superoxide dismutase (SOD1) mutant, prior to the onset of motor symptoms. This suggests that axonal transport perturbation can be an early sign of neuronal dysfunction. However, the exact causal relationship between axonal transport deficits and neurodegeneration is not fully understood. To clarify whether axonal transport of organelles even in neurons at early developmental stages was affected by overexpression of mutant SOD1, we conducted a microfluidic device-based high-throughput quantitative analysis of the axonal transport of acidic vesicles and mitochondria in primary cultured cortical neurons established from SOD1H46R transgenic mice. Compared to wild-type (WT), a significantly increased number of motile acidic vesicles, i.e., autophagosomes and/or late-endosomes, was observed in the axons of SOD1H46R neurons. By contrast, mitochondria moving along the axons were significantly decreased in SOD1H46R compared to WT. Since such phenotypes, where the axonal transport of these organelles is differently affected by mutant SOD1 expression, emerge before axonal degeneration, axonal transport deficits could dysregulate axon homeostasis, thereby ultimately accelerating neurodegeneration.

SOD1 Mutation Spectrum and Natural History of ALS Patients in a 15-Year Cohort in Southeastern China.

Background: Mutations in superoxide dismutase 1 gene (SOD1) are the most frequent high penetrant genetic cause for amyotrophic lateral sclerosis (ALS) in the Chinese population. A detailed natural history of SOD1-mutated ALS patients will provide key information for ongoing genetic clinical trials. Methods: We screened for SOD1 mutations using whole exome sequencing (WES) in Chinese ALS cases from 2017 to 2021. Functional studies were then performed to confirm the pathogenicity of novel variants. In addition, we enrolled previously reported SOD1 mutations in our centers from 2007 to 2017. The SOD1 mutation spectrum, age at onset (AAO), diagnostic delay, and survival duration were analyzed. Results: We found two novel SOD1 variants (p.G17H and p.E134*) that exerted both gain-of-function and loss-of-function effects in vitro. Combined with our previous SOD1-mutated patients, 32 probands with 21 SOD1 mutations were included with the four most frequently occurring mutations of p.V48A, p.H47R, p.C112Y, and p.G148D. SOD1 mutations account for 58.9% of familial ALS (FALS) cases. The mean (SD) AAO was 46 ± 11.4 years with a significant difference between patients carrying mutations in exon 1 [n = 5, 34.6 (12.4) years] and exon 2 [n = 8, 51.4 (8.2) years] (p = 0.038). The mean of the diagnostic delay of FALS patients is significantly earlier than the sporadic ALS (SALS) patients [9.5 (4.8) vs. 20.3 (9.3) years, p = 0.0026]. In addition, male patients survived longer than female patients (40 vs. 16 months, p = 0.05). Conclusion: Our results expanded the spectrum of SOD1 mutations, highlighted the mutation distribution, and summarized the natural history of SOD1-mutated patients in southeastern China. Male patients were found to have better survival, and FALS patients received an earlier diagnosis. Our findings assist in providing a detailed clinical picture, which is important for ongoing genetic clinical trials.

Prognostic value of oxidative stress in patients with acute myocardial infarction complicated by cardiogenic shock: A prospective cohort study.

INTRODUCTION: Cardiogenic shock is a frequent complication of acute myocardial infarction. Similar to ischemia/reperfusion injury, excessive production of reactive oxygen species can be expected in those who experience cardiogenic shock. The aims of this study were to describe the extent and time course of oxidative stress and evaluate the prognostic value of oxidative stress markers in patients who experienced ST-segment elevation myocardial infarction (STEMI) complicated by cardiogenic shock. METHODS: Plasma/serum levels of selected biomarkers of oxidative stress (oxidised guanine species (OGS), malondialdehyde, and glutathione peroxidase 3) and markers, which simultaneously reflect severe cellular damage (ferric ion reducing antioxidant power (FRAP), Cu/Zn-superoxide dismutase (SOD), and glutathione) were measured seven times per week in a prospective cohort of 82 patients with STEMI complicated by cardiogenic shock. RESULTS: We found elevated OGS levels in patients who died during three months, which persisted significantly increased the next 12 h compared to surviving patients. A similar time course pattern also exhibited concentrations of FRAP and SOD. The other markers did not change significantly and did not show differences between surviving and non-surviving patients during the monitored period. In addition, a strong relationship between OGS, FRAP, and SOD levels (on admission and 12 h after admission) and 3-month mortality was found. CONCLUSION: Levels of OGS, FRAP, and SOD within 12 h after hospital admission were revealed as early predictors of the adverse development of STEMI complicated by cardiogenic shock.

Comparative evaluation of the immunodominant proteins of Brucella abortus for the diagnosis of cattle brucellosis.

BACKGROUND AND AIM: The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis. MATERIALS AND METHODS: Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done. RESULTS: All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden's index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85). CONCLUSION: BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.

Self-Assembled pH-Sensitive Polymeric Nanoparticles for the Inflammation-Targeted Delivery of Cu/Zn-Superoxide Dismutase.

The use of superoxide dismutase (SOD) is currently limited by its short half-life, rapid plasma clearance rate, and instability. We synthesized a small library of biofriendly amphiphilic polymers that comprise methoxy poly(ethylene glycol)-poly(cyclohexane-1,4-diyl acetone dimethyleneketal) (mPEG-PCADK) and mPEG-poly((cyclohexane86.7%, 1,5-pentanediol13.3%)-1,4-diyl acetone dimethylene ketal) (PK3) for the targeted delivery of SOD. The novel polymers could self-assemble into micellar nanoparticles with favorable hydrolysis kinetics, biocompatibility, long circulation time, and inflammation-targeting effects. These materials generated a better pH-response curve and exhibited better hydrolytic kinetic behavior than PCADK and PK3. The polymers showed good biocompatibility with protein drugs and did not induce an acidic microenvironment during degradation in contrast to materials such as PEG-block-poly(lactic-co-glycolic acid) (PLGA) and PLGA. The SOD that contained reverse micelles based on mPEG2000-PCADK exhibited good circulation and inflammation-targeting properties. Pharmacodynamic results indicated exceptional antioxidant and anti-inflammatory activities in a rat adjuvant-induced arthritis model and a rat peritonitis model. These results suggest that these copolymers are ideal protein carriers for targeting inflammation treatment.

High fat suppresses SOD1 activity by reducing copper chaperone for SOD1 associated with neurodegeneration and memory decline.

High fat consumption leads to reactive oxygen species (ROS) which is associated with age-progressive neurological disorders. Cu/Zn superoxide dismutase (SOD1) is a critical enzyme against ROS. However, the relationship between SOD1 and the high-fat-induced ROS and neurodegeneration is poorly known. Here we showed that, upon treatment with a saturated fatty acid palmitic acid (PA), the SOD1 activity was decreased in mouse neuronal HT-22 cell line accompanied by elevation of ROS, but not in mouse microglial BV-2 cell line. We further showed that PA decreased the levels of copper chaperone for SOD1 (CCS) in HT-22 cells, which promoted the nuclear import of SOD1 and decreased its activity. We demonstrated that the reduction of CCS is involved in the PA-induced decrease of SOD1 activity and elevation of ROS. In addition, compared with the adult mice fed with a standard diet, the high-fat-diet adult mice presented an increase of plasma free fatty acids, reduction of hippocampal SOD1 activity and CCS, mitochondrial degeneration and long-term memory decline. Taken together, our findings suggest that the high-fat-induced lower CCS level is essential for SOD1 suppression which may be associated with neurodegeneration and cognitive decline.

Role of necroptosis in chronic hepatic inflammation and fibrosis in a mouse model of increased oxidative stress.

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase (Sod1-/- or Sod1KO mice) have increased oxidative stress, show accelerated aging and develop spontaneous hepatocellular carcinoma (HCC) with age. Similar to humans, HCC development in Sod1KO mice progresses from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) with fibrosis, which eventually progresses to HCC. Oxidative stress plays a role in NAFLD to NASH progression, and liver inflammation is the main mechanism that drives the disease progression from NASH to fibrosis. Because necroptosis is a major source of inflammation, we tested the hypothesis that increased necroptosis in the liver plays a role in increased inflammation and fibrosis in Sod1KO mice. Phosphorylation of MLKL (P-MLKL), a well-accepted marker of necroptosis, and expression of MLKL protein were significantly increased in the livers of Sod1KO mice compared to wild type (WT) mice indicating increased necroptosis. Similarly, phosphorylation of RIPK3 and RIPK3 protein levels were also significantly increased. Markers of pro-inflammatory M1 macrophages, NLRP3 inflammasome, and transcript levels of pro-inflammatory cytokines and chemokines, e.g., TNFα, IL-6, IL-1ß, and Ccl2 that are associated with human NASH, were significantly increased. Expression of antioxidant enzymes and heat shock proteins, and markers of fibrosis and oncogenic transcription factor STAT3 were also upregulated and autophagy was downregulated in the livers of Sod1KO mice. Short term treatment of Sod1KO mice with necrostatin-1s (Nec-1s), a necroptosis inhibitor, reversed these conditions. Our data show for the first time that necroptosis-mediated inflammation contributes to fibrosis in a mouse model of increased oxidative stress and accelerated aging, that also exhibits progressive HCC development.

Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic.

Cu/Zn superoxide dismutase (SOD1) is a frontline antioxidant enzyme catalysing superoxide breakdown and is important for most forms of eukaryotic life. The evolution of aerobic respiration by mitochondria increased cellular production of superoxide, resulting in an increased reliance upon SOD1. Consistent with the importance of SOD1 for cellular health, many human diseases of the central nervous system involve perturbations in SOD1 biology. But far from providing a simple demonstration of how disease arises from SOD1 loss-of-function, attempts to elucidate pathways by which atypical SOD1 biology leads to neurodegeneration have revealed unexpectedly complex molecular characteristics delineating healthy, functional SOD1 protein from that which likely contributes to central nervous system disease. This review summarises current understanding of SOD1 biology from SOD1 genetics through to protein function and stability.