SOD1 Knockout Mice Research Articles

Vitamin D3 exacerbates steatosis while calcipotriol inhibits inflammation in non-alcoholic fatty liver disease in Sod1 knockout mice: a comparative study of two forms of vitamin D.

The role of vitamin D (VD) in non-alcoholic fatty liver disease (NAFLD) remains controversial, possibly due to the differential effects of various forms of VD. In our study, Sod1 gene knockout (SKO) mice were utilized as lean NAFLD models, which were administered 15 000 IU VD3 per kg diet, or intraperitoneally injected with the active VD analog calcipotriol for 12 weeks. We found that VD3 exacerbated hepatic steatosis in SKO mice, with an increase in the levels of Cd36, Fatp2, Dgat2, and CEBPA. However, calcipotriol exerted no significant effect on hepatic steatosis. Calcipotriol inhibited the expression of Il-1a, Il-1b, Il-6, Adgre1, and TNF, with a reduction of NFκB phosphorylation in SKO mice. No effect was observed by either VD3 or calcipotriol on hepatocyte injury and hepatic fibrosis. Co-immunofluorescence stains of CD68, a liver macrophage marker, and VDR showed that calcipotriol reduced CD68 positive cells, and increased the colocalization of VDR with CD68. However, VD3 elevated hepatocyte VDR expression, with no substantial effect on the colocalization of VDR with CD68. Finally, we found that VD3 increased the levels of serum 25(OH)D3 and 24,25(OH)2D3, whereas calcipotriol decreased both. Both VD3 and calcipotriol did not disturb serum calcium and phosphate levels. In summary, our study found that VD3 accentuated hepatic steatosis, while calcipotriol diminished inflammation levels in SKO mice, and the difference might stem from their distinct cellular selectivity in activating VDR. This study provides a reference for the application of VD in the treatment of lean NAFLD.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Endogenous SOD2 (Superoxide Dismutase) Regulates Platelet-Dependent Thrombin Generation and Thrombosis During Aging.

Reactive oxygen species (ROS) contribute to platelet hyperactivation during aging. Several oxidative pathways and antioxidant enzymes have been implicated; however, their mechanistic contributions during aging remain elusive. We hypothesized that mitochondria are an important source of platelet ROS and that mitochondrial SOD2 (superoxide dismutase) protects against mitochondrial ROS-driven platelet activation and thrombosis during aging. We studied littermates of platelet-specific SOD2-knockout (SOD2fl/flPf4Cre, pSOD2-KO) and control (SOD2fl/fl) mice at young (4-5 months) or old (18-20 months) ages. We examined agonist-induced platelet activation, platelet-dependent thrombin generation potential, and susceptibility to in vivo thrombosis. Platelet αIIbβ3 activation, aggregation, and adhesion were increased to similar extents in aged mice of both genotypes compared with young mice. In contrast, the age-dependent increases in mitochondrial and total cellular ROS, calcium elevation, and phosphatidylserine exposure were augmented in platelets from pSOD2-KO mice compared with control mice. Aged pSOD2-KO mice showed increased platelet-dependent thrombin generation compared with aged control mice. In vivo, aged pSOD2-KO mice exhibited enhanced susceptibility to carotid artery and pulmonary thrombosis compared to aged control mice. Adoptive transfer of platelets from aged pSOD2-KO but not aged control mice increased thrombotic susceptibility in aged host mice, suggesting a prothrombotic effect of platelet pSOD2 deficiency. Treatment with avasopasem manganese (GC4419), a SOD mimetic, decreased platelet mitochondrial pro-oxidants, cellular ROS levels, and inhibited procoagulant platelet formation and arterial thrombosis in aged mice. Platelet mitochondrial ROS contributes to age-related thrombosis and endogenous SOD2 protects from platelet-dependent thrombin generation and thrombosis during aging.

Non-enzymatic role of SOD1 in intestinal stem cell growth

Superoxide dismutase 1 (SOD1) modulates intestinal barrier integrity and intestinal homeostasis as an antioxidant enzyme. Intestinal homeostasis is maintained by the intestinal stem cells (ISCs). However, whether and how SOD1 regulates ISCs is unknown. In this study, we established intestinal organoids from tamoxifen–inducible intestinal epithelial cell–specific Sod1 knockout (Sod1f/f; Vil-creERT2) mice. We found that loss of Sod1 in organoids suppressed the proliferation and survival of cells and Lgr5 gene expression. SOD1 is known for nearly half a century for its canonical role as an antioxidant enzyme. We identified its enzyme-independent function in ISC: inhibition of SOD1 enzymatic activity had no impact on organoid growth, and enzymatically inactive Sod1 mutants could completely rescue the growth defects of Sod1 deficient organoids, suggesting that SOD1-mediated ISC growth is independent of its enzymatic activity. Moreover, Sod1 deficiency did not affect the ROS levels of the organoid, but induced the elevated WNT signaling and excessive Paneth cell differentiation, which mediates the occurrence of growth defects in Sod1 deficient organoids. In vivo, epithelial Sod1 loss induced a higher incidence of apoptosis in the stem cell regions and increased Paneth cell numbers, accompanied by enhanced expression of EGFR ligand Epiregulin (EREG) in the stromal tissue, which may compensate for Sod1 loss and maintain intestinal structure in vivo. Totally, our results show a novel enzyme-independent function of SOD1 in ISC growth under homeostasis.

ROS-Mediated Enamel Formation Disturbance Characterized by Alternative Cervical Loop Cell Proliferation and Downregulation of RhoA/ROCK in Ameloblasts.

Reactive oxygen stress (ROS) is generally accepted as a signal transducer for coordinating the growth and differentiation of tissues and organs in the oral and maxillofacial region. Although ROS has been confirmed to affect the development of enamel, it is not yet known that the specific mechanism of ROS accumulation induced enamel defects. Given the lack of knowledge of the role of ROS in enamel, the aim of the study is to determine how oxidative stress affects cervical cells and ameloblast cells. Using SOD1 knockout mice, we identified a relationship between ROS fluctuations and abnormal enamel structure with HE staining, micro-CT, and scanning electron microscope. Increased ROS induced by H2O2, certified by the DCFH probe, has resulted in a dual effect on the proliferation and differentiation of cervical cells, indicating a higher tendency to proliferate at low ROS concentrations. Ameloblasts transfected with SOD1 siRNA showed a significant reduction of RhoA and ROCK. This study investigates for the first time that SOD1-mediated ROS accumulation disrupted normal enamel structure through alternative cervical loop cell proliferation and downregulation of RhoA and ROCK in ameloblasts, demonstrating the convoluted role of ROS in monitoring the progress of enamel defects.

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‐1β, 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.

Erythropoietin Gene Therapy Delays Retinal Degeneration Resulting from Oxidative Stress in the Retinal Pigment Epithelium.

Erythropoietin (EPO) plays an important role in erythropoiesis by its action in blocking apoptosis of progenitor cells and protects both photoreceptors and retinal ganglion cells from induced or inherited degeneration. A modified form of EPO, EPO-R76E has attenuated erythropoietic activity but is effective in inhibiting apoptosis, oxidative stress, and inflammation in several models of retinal degeneration. In this study, we used recombinant Adeno Associated Virus (AAV) to provide long-term sustained delivery of EPO-R76E and demonstrated its effects in a mouse model of dry-AMD in which retinal degeneration is induced by oxidative stress in the retinal pigment epithelial (RPE) cells. Experimental vector AAV-EPO-R76E and control vector AAV-GFP were packaged into serotype-1 (AAV1) to enable RPE selective expression. RPE oxidative stress-mediated retinal degeneration was induced by exon specific deletion of the protective enzyme MnSOD (encoded by Sod2) by cre/lox mechanism. Experimental mice received subretinal injection of AAV-EPO-R76E in the right eye and AAV-GFP in the left eye. Western blotting of RPE/choroid protein samples from AAV-EPO-R76E injected eyes showed RPE specific EPO expression. Retinal function was monitored by electroretinography (ERG). EPO-R76E over-expression in RPE delayed the retinal degeneration as measured by light microscopy in RPE specific Sod2 knockout mice. Delivery of EPO-R76E vector can be used as a tool to prevent retinal degeneration induced by RPE oxidative stress, which is implicated as a potential cause of Age-Related Macular Degeneration.

Superoxide produced by mitochondrial site IQ inactivates cardiac succinate dehydrogenase and induces hepatic steatosis in Sod2 knockout mice

Superoxide produced by mitochondria has been implicated in numerous physiologies and pathologies. Eleven different mitochondrial sites that can produce superoxide and/or hydrogen peroxide (O2.-/H2O2) have been identified in vitro, but little is known about their contributions in vivo. We introduce novel variants of S1QELs and S3QELs (small molecules that suppress O2.-/H2O2 production specifically from mitochondrial sites IQ and IIIQo, respectively, without compromising bioenergetics), that are suitable for use in vivo. When administered by intraperitoneal injection, they achieve total tissue concentrations exceeding those that are effective in vitro. We use them to study the engagement of sites IQ and IIIQo in mice lacking functional manganese-superoxide dismutase (SOD2). Lack of SOD2 is expected to elevate superoxide levels in the mitochondrial matrix, and leads to severe pathologies and death about 8 days after birth. Compared to littermate wild-type mice, 6-day-old Sod2−/− mice had significantly lower body weight, lower heart succinate dehydrogenase activity, and greater hepatic lipid accumulation. These pathologies were ameliorated by treatment with a SOD/catalase mimetic, EUK189, confirming previous observations. A 3-day treatment with S1QEL352 decreased the inactivation of cardiac succinate dehydrogenase and hepatic steatosis in Sod2−/− mice. S1QEL712, which has a distinct chemical structure, also decreased hepatic steatosis, confirming that O2.- derived specifically from mitochondrial site IQ is a significant driver of hepatic steatosis in Sod2−/− mice. These findings also demonstrate the ability of these new S1QELs to suppress O2.- production in the mitochondrial matrix in vivo. In contrast, suppressing site IIIQo using S3QEL941 did not protect, suggesting that site IIIQo does not contribute significantly to mitochondrial O2.- production in the hearts or livers of Sod2−/− mice. We conclude that the novel S1QELs are effective in vivo, and that site IQ runs in vivo and is a significant driver of pathology in Sod2−/− mice.

Novel SOD1 monoclonal antibodies against the electrostatic loop preferentially detect misfolded SOD1 aggregates

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that leads to motor neuron degeneration and paralysis. Superoxide dismutase (SOD1) mutations are the second most common cause of familial ALS and are responsible for up to 20 % of familial ALS cases. In ALS patients, SOD1 can form toxic misfolded aggregates that deposit in the brain and spinal cord. To better detect SOD1 aggregates and expand the repertoire of conformational SOD1 antibodies, SOD1 monoclonal antibodies were generated by immunizing SOD1 knockout mice with an SOD1 fragment consisting of amino acids 129–146, which make up part of the electrostatic loop. A series of hybridomas secreting antibodies were screened and five different SOD1 monoclonal antibodies (2C10, 2F8, 4B11, 5H5, and 5A10) were found to preferentially detect denatured or aggregated SOD1 by enzyme-linked immunosorbent assay (ELISA), filter trap assay, and immunohistochemical analysis in SOD1 mouse models. The staining with these antibodies was compared to Campbell-Switzer argyrophilic reactivity of pathological inclusions. These new conformational selective SOD1 antibodies will be useful for clinical diagnosis of SOD1 ALS and potentially for passive immunotherapy.

Defective biosynthesis of ascorbic acid in Sod1-deficient mice results in lethal damage to lung tissue

Superoxide dismutase 1 (Sod1) plays pivotal roles in antioxidation via accelerating the conversion of superoxide anion radicals into hydrogen peroxide, thus inhibiting the subsequent radical chain reactions. While Sod1 deficient cells inevitably undergo death in culture conditions, Sod1-knockout (KO) mice show relatively mild phenotypes and live approximately two years. We hypothesized that the presence of abundant levels of ascorbic acid (AsA), which is naturally produced in mice, contributes to the elimination of reactive oxygen species (ROS) in Sod1-KO mice. To verify this hypothesis, we employed mice with a genetic ablation of aldehyde reductase (Akr1a), an enzyme that is involved in the biosynthesis of AsA, and established double knockout (DKO) mice that lack both Sod1 and Akr1a.Supplementation of AsA (1.5 mg/ml in drinking water) was required for the DKO mice to breed, and, upon terminating the AsA supplementation, they died within approximately two weeks regardless of age or gender. We explored the etiology of the death from pathophysiological standpoints in principal organs of the mice. Marked changes were observed in the lungs in the form of macroscopic damage after the AsA withdrawal. Histological and immunological analyses of the lungs indicated oxidative damage of tissue and activated immune responses. Thus, preferential oxidative injury that occurred in pulmonary tissues appeared to be primary cause of the death in the mice. These collective results suggest that the pivotal function of AsA in coping with ROS in vivo, is largely in pulmonary tissues that are exposed to a hyperoxygenic microenvironment.

SOD1 suppresses pro-inflammatory immune responses by protecting against oxidative stress in colitis

Superoxide dismutase 1 (SOD1) binds copper and zinc ions and is one of three superoxide dismutases responsible for destroying free superoxide radicals in the body. Reactive oxygen species (ROS), including free superoxide radicals, play important roles in colitis. However, the role of SOD1 in oxidative stress under colitis remains unclear. Here, we examined the role of SOD1 in the DSS-induced mouse model of colitis. SOD1 deficiency resulted in severe oxidative stress with body weight loss, epithelial barrier disruption and decreased antioxidant enzyme activities. The levels of neutrophils, monocytes, pro-inflammatory CD11c+ macrophages and CD11b+CD103- dendritic cells (DCs) were increased, while anti-inflammatory CD206+ macrophages and CD11b−CD103+ DCs were decreased, in DSS-treated SOD1-knockout (KO) mice compared to DSS-treated wild-type mice. Furthermore, rescue of SOD activity in SOD1-KO mice by oral gavage of B. amyloliquefaciens SOD (BA SOD) significantly ameliorated enhanced DSS-induced colitis in these mice by suppressing p38-MAPK/NF-κB signaling, which can induce inflammation and apoptosis. Taken together, our results suggest that SOD1-mediated inhibitory responses play a crucial role in limiting the development of DSS-induced colitis, and that BA SOD is a promising candidate for treating colitis.

SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy

Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O2•-) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2Δ mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O2 consumption, particularly during basal conditions and after the addition of FCCP (H+ ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2fl littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy.

The Effects of 3% Diquafosol Sodium Eye Drops on Tear Function and the Ocular Surface of Cu, Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice Treated with Antiglaucoma Eye Medications.

Anti-glaucoma eye drop treatment often induces dry eyes and can lead to poor medication adherence. This study aimed to investigate the effects of 3% diquafosol sodium eye drops on tear function and the ocular surface epithelium in Sod1−/− mice after treatment with anti-glaucoma eye drops. The mice were divided into four groups: group 1, control group; group 2, anti-glaucoma eye drop; group 3, anti-glaucoma eye drops followed by a secretagogue eye drop (3% diquafosol); and group 4, simultaneous anti-glaucoma and secretagogue eye drop. Mice underwent assessments of tear quantity, tear film breakup time, and vital staining score. Mice in groups 3 and 4 showed significantly better tear stability and lower corneal staining scores than mice in group 2 after eye drop instillations (p < 0.05). Mice in group 4 showed significantly better tear stability, lower corneal staining scores, and higher goblet cell densities than those in group 1 after eye drop instillations (p < 0.05). The conjunctival epithelium showed stratification and abundance of Muc5AC-positive goblet cells in group 4, whereas thinning with desquamation was observed with a few goblet cells in group 2. Thus, simultaneous administration of 3% diquafosol sodium eye drops with topical anti-glaucoma drops showed favorable effects on tear stability and the corneal epithelium against the ocular surface toxicity inflicted by the anti-glaucoma eye drops.

SOD1 is essential for oncogene-driven mammary tumor formation but dispensable for normal development and proliferation.

We previously reported that the dismutase SOD1 is overexpressed in breast cancer. However, whether SOD1 plays an active role in tumor formation in vivo has never been demonstrated. Further, as luminal cells of normal breast epithelial cells are enriched in SOD1, whether SOD1 is essential for normal mammary gland development has never been determined. We initiated this study to investigate the role of SOD1 in mammary gland tumorigenesis as well as in normal mammary gland development. We crossed the inducible erbB2 (MMTV-iErbB2) and Wnt (MMTV-Wnt) transgenic mice to the SOD1 heterozygote or knockout mice. Our results show that SOD1 is essential for oncogene-driven proliferation, but not normal proliferation of the mammary gland associated with pregnancy or other normal proliferative tissues such as skin and intestines. We show that activation of the oncogene ErbB2 is associated with increased ROS and that high ROS sub-population of ErbB2 cancer cells show elevated SOD1. In the same cells, decrease in SOD1 is associated with an elevation in both apoptosis as well as oncogene-induced senescence. Based on these results, we suggest that SOD1 carries a housekeeping function that maintains ROS levels below a threshold that supports oncogene-dependent proliferation, while allowing escape from oncogene-induced senescence, independently of the oncogene driving tumor formation. These results identify SOD1 as an ideal target for cancer therapy as SOD1 inhibitors hold the potential to prevent the growth of cancers cells of diverse genotypes, activate multiple modes of cell death therefore making acquired resistance more difficult, while sparing normal tissues.

Distal denervation in the SOD1 knockout mouse correlates with loss of mitochondria at the motor nerve terminal

Impairment of mitochondrial transport has long been implicated in the pathogenesis of neuropathy and neurodegeneration. However, the role of mitochondria in stabilizing motor nerve terminals at neuromuscular junction (NMJ) remains unclear. We previously demonstrated that mice lacking the antioxidant enzyme, superoxide dismutase-1 (Sod1−/−), develop progressive NMJ denervation. This was rescued by expression of SOD1 exclusively in the mitochondrial intermembrane space (MitoSOD1/Sod1−/−), suggesting that oxidative stress within mitochondria drives denervation in these animals. However, we also observed reduced mitochondrial density in Sod1−/− motor axons in vitro. To investigate the relationship between mitochondrial density and NMJ innervation in vivo, we crossed Sod1−/− mice with the fluorescent reporter strains Thy1-YFP and Thy1-mitoCFP. We identified an age-dependent loss of mitochondria at motor nerve terminals in Sod1−/− mice, that closely correlated with NMJ denervation, and was rescued by MitoSOD1 expression. To test whether augmenting mitochondrial transport rescues Sod1−/− axons, we generated transgenic mice overexpressing the mitochondrial cargo adaptor, Miro1. This led to a partial rescue of mitochondrial density at motor nerve terminals by 12 months of age, but was insufficient to prevent denervation. These findings suggest that loss of mitochondria in the distal motor axon may contribute to denervation in Sod1−/− mice, perhaps via loss of key mitochondrial functions such as calcium buffering and/or energy production.

Mitochondrial oxidative stress in the retinal pigment epithelium (RPE) led to metabolic dysfunction in both the RPE and retinal photoreceptors

Age-related macular degeneration (AMD) is the leading cause of vision loss in the western world. Recent evidence suggests that RPE and photoreceptors have an interconnected metabolism and that mitochondrial damage in RPE is a trigger for degeneration in both RPE and photoreceptors in AMD. To test this hypothesis, this study was designed to induce mitochondrial damage in RPE in mice to determine whether this is sufficient to cause RPE and photoreceptor damage characteristic of AMD. In this study, we conditionally deleted the gene encoding the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD encoded by Sod2) in the retinal pigment epithelium (RPE) of albino BALB/cJ mice. VMD2-Cre;Sod2flox/flox BALB/cJ mice were housed in either 12-h dark, 12-h 200 lux white lighting (normal light), or 12-h dark, 12-h <10 lux red lighting (dim light). Electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT) were performed to assess retinal function and morphology. Immunofluorescence was used to examine protein expression; quantitative RT-PCR was used to measure gene expression. Sod2 knockout (KO) mice had reduced RPE function with age and increased oxidative stress compared to wild type (WT) controls as expected by the cell-specific deletion of Sod2. This was associated with alterations in RPE morphology and the structure and function of RPE mitochondria. In addition, data show a compensatory increase in RPE glycolytic metabolism. The metabolic shift in RPE correlated with severe disruption of photoreceptor mitochondria including a reduction in TOMM20 expression, mitochondrial fragmentation, and reduced COXIII/β-actin levels. These findings demonstrate that mitochondrial oxidative stress can lead to RPE dysfunction and metabolic reprogramming of RPE. Secondary to these changes, photoreceptors also undergo metabolic stress with increased mitochondrial damage. These data are consistent with the hypothesis of a linked metabolism between RPE and photoreceptors and suggest a mechanism of retinal degeneration in dry AMD.

An SOD1 deficiency aggravates proteasome inhibitor bortezomib-induced testicular damage in mice

Proteasomes play a key role in maintaining cellular homeostasis by the proteolytic removal of proteins, including ubiquitinated proteins and/or oxidatively-damaged proteins. The proteasome inhibitor bortezomib (BTZ) has been reported to exert testicular toxicity in mice. In the current study, we treated SOD1-knockout (KO) mice with BTZ and investigated the issue of whether oxidative stress is involved in the development of testicular toxicity. The BTZ treatment significantly increased superoxide production and cell death in the testes of SOD1-KO mice compared to wild-type (WT) mice. We also found that high levels of both ubiquitinated proteins and p62 accumulated and underwent aggregation in the seminiferous tubules of BTZ-injected SOD1-KO mice. Furthermore, the proteolytic activities of proteasomes were significantly decreased in the testes of BTZ-injected SOD1-KO mice compared to their WT counterparts. These results suggest that a combination of oxidative stress caused by an SOD1 deficiency and proteasome inhibition by BTZ accelerates the impairment of proteasomes, which results in severe testicular damage in SOD1-KO mice.

Systematic Injection of Low-Dose LPS Transiently Improves the Retina Function and Structure of aMouse Model of Geographic Atrophy.

Geographic atrophy (GA), the advanced form of AMD, has been linked to oxidative stress within the RPE and with low-grade inflammation. The RPE-specific Sod2 knockout mouse model of GA develops increase oxidative stress and slow retinal degeneration. Mice of the SOD2floxed::VMD2Cre+ genotype were injected subcutaneously with either saline or 3mg/kg of lipopolysaccharide (LPS) at 8weeks of age. Mice were evaluated by electroretinography (ERG) and spectral domain optical coherence tomography. Inflammatory cells within the retina were studied by CD45 immunofluorescence. Systemic low-dose LPS transiently, but significantly, improved both function and structure of RPE-specific Sod2 KO mice retina when compared to saline-injected mice. There was no difference in CD45 positive cells between saline and LPS treatment. Low-grade activation of the immune system leads to a preconditioning effect that transiently protects the retina of a mouse model of geographic atrophy.

Unveiling systemic organ disorders associated with impaired lipid catabolism in fasted SOD1-deficient mice

Oxidative stress triggers the formation of lipid droplets in the liver by stimulating lipogenesis and simultaneously suppresses lipoprotein secretion under hypernutritional conditions. Herein we report on the observation of systemic organ failure that is associated with lipid droplet accumulation in fasting, SOD1-knockout (KO) mice. Upon a three-day fasting period, the KO mice were observed to be vulnerable, could not be rescued by refeeding and had largely died, while wild-type mice were totally recovered. Visceral fat was rapidly consumed during fasting, which resulted in energy shortage and increased fatality in the KO mice. Lipid droplets had accumulated and continued to remain in KO mouse organs that routinely catalyze fatty acids via β-oxidation, even though the levels of free fatty acids and β-hydroxybutyrate, a ketone body, in blood plasma were less in KO mice compared to WT mice during the fasting period. The fasting-triggered organ failure in the KO mice was effectively mitigated by feeding a high calorie-diet for 2 weeks prior to fasting, even though the mice had an excessive accumulation of lipid droplets in the liver. These collective data suggest that the lipid-catabolizing system is the sensitive target of oxidative stress triggered by fasting conditions in the KO mice.

Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice.

Mitochondrial DNA (mtDNA) mutations become more prevalent with age and are postulated to contribute to the ageing process. Point mutations of mtDNA have been suggested to originate from two main sources, i.e. replicative errors and oxidative damage, but the contribution of each of these processes is much discussed. To elucidate the origin of mtDNA mutations, we measured point mutation load in mice with deficient mitochondrial base-excision repair (BER) caused by knockout alleles preventing mitochondrial import of the DNA repair glycosylases OGG1 and MUTYH (Ogg1 dMTS, Mutyh dMTS). Surprisingly, we detected no increase in the mtDNA mutation load in old Ogg1 dMTS mice. As DNA repair is especially important in the germ line, we bred the BER deficient mice for five consecutive generations but found no increase in the mtDNA mutation load in these maternal lineages. To increase reactive oxygen species (ROS) levels and oxidative damage, we bred the Ogg1 dMTS mice with tissue specific Sod2 knockout mice. Although increased superoxide levels caused a plethora of changes in mitochondrial function, we did not detect any changes in the mutation load of mtDNA or mtRNA. Our results show that the importance of oxidative damage as a contributor of mtDNA mutations should be re-evaluated.