L-type voltage-gated calcium channels are important regulators of neuronal activity and are widely expressed throughout the brain. One of the major L-type voltage-gated calcium channel isoforms in the brain is CaV 1.3. Mice lacking CaV 1.3 are reported to have impairments in fear conditioning and depressive-like behaviors, which have been linked to CaV 1.3 function in the hippocampus and amygdala. Genetic variation in CaV 1.3 has been linked to a variety of psychiatric disorders, including autism and schizophrenia, which are associated with altered motor learning, associative learning and social function. Here, we explored whether CaV 1.3 plays a role in these behaviors. We found that CaV 1.3 knockout mice have deficits in rotarod learning despite normal locomotor function. Deletion of CaV 1.3 is also associated with impaired gait adaptation and associative learning on the Erasmus Ladder. We did not observe any impairments in CaV 1.3 knockout mice on assays of anxiety-like, depression-like or social preference behaviors. Our results suggest an important role for CaV 1.3 in neural circuits involved in motor learning and concur with previous data showing its involvement in associative learning.

As cancer survivorship increases, so does the number of patients that suffer from the late effects of radiation therapy. This includes arthrofibrosis, the development of stiff joints near the field of radiation. Previous reports have concentrated on skin fibrosis around the joint but largely ignored the deeper tissues of the joint. We hypothesized that fat, muscle, and the joint tissues themselves would play a more significant role in joint contracture after radiation than the skin surrounding the joint. To address this hypothesis, we irradiated the right hind flanks of mice with fractionated and unfractionated dose schedules, then monitored the mice for 3 months postradiotherapy. Mice were euthanized and physiological indications of arthrofibrosis including limb contracture and joint resting position were assessed. Stifle (knee) joints demonstrated significant arthrofibrosis, but none was observed in the hock (ankle) joints. During these studies, we were surprised to find that male and female mice showed a significantly different response to radiation injury. Female mice developed more injuries, had significantly worse contracture, and showed a greater difference in the expression of all markers studied. These results suggest that women undergoing radiation therapy might be at significantly greater risk for developing arthrofibrosis and may require specific adjustments to their care.

We tested the hypothesis that supplementation of nicotinamide mononucleotide (NMN), a key NAD(+) intermediate, increases arterial SIRT1 activity and reverses age-associated arterial dysfunction and oxidative stress. Old control mice (OC) had impaired carotid artery endothelium-dependent dilation (EDD) (60 ± 5% vs. 84 ± 2%), a measure of endothelial function, and nitric oxide (NO)-mediated EDD (37 ± 4% vs. 66 ± 6%), compared with young mice (YC). This age-associated impairment in EDD was restored in OC by the superoxide (O2-) scavenger TEMPOL (82 ± 7%). OC also had increased aortic pulse wave velocity (aPWV, 464 ± 31 cm s(-1) vs. 337 ± 3 cm s(-1) ) and elastic modulus (EM, 6407 ± 876 kPa vs. 3119 ± 471 kPa), measures of large elastic artery stiffness, compared with YC. OC had greater aortic O2- production (2.0 ± 0.1 vs. 1.0 ± 0.1 AU), nitrotyrosine abundance (a marker of oxidative stress), and collagen-I, and reduced elastin and vascular SIRT1 activity, measured by the acetylation status of the p65 subunit of NFκB, compared with YC. Supplementation with NMN in old mice restored EDD (86 ± 2%) and NO-mediated EDD (61 ± 5%), reduced aPWV (359 ± 14 cm s(-1) ) and EM (3694 ± 315 kPa), normalized O2- production (0.9 ± 0.1 AU), decreased nitrotyrosine, reversed collagen-I, increased elastin, and restored vascular SIRT1 activity. Acute NMN incubation in isolated aortas increased NAD(+) threefold and manganese superoxide dismutase (MnSOD) by 50%. NMN supplementation may represent a novel therapy to restore SIRT1 activity and reverse age-related arterial dysfunction by decreasing oxidative stress.

Cystic fibrosis-related diabetes (CFRD) is one the most common comorbidities in cystic fibrosis (CF). Pancreatic oxidative stress has been postulated in the pathogenesis of CFRD, but no studies have been done to show an association. The main obstacle is the lack of suitable animal models and no immediate availability of pancreas tissue in humans. In the CF porcine model, we found increased pancreatic total glutathione (GSH), glutathione disulfide (GSSG), 3-nitrotyrosine- and 4-hydroxynonenal-modified proteins, and decreased copper zinc superoxide dismutase (CuZnSOD) activity, all indicative of oxidative stress. CF pig pancreas demonstrated increased DHE oxidation (as a surrogate marker of superoxide) in situ compared to non-CF and this was inhibited by a SOD-mimetic (GC4401). Catalase and glutathione peroxidase activities were not different between CF and non-CF pancreas. Isolated CF pig islets had significantly increased DHE oxidation, peroxide production, reduced insulin secretion in response to high glucose and diminished secretory index compared to non-CF islets. Acute treatment with apocynin or an SOD mimetic failed to restore insulin secretion. These results are consistent with the hypothesis that CF pig pancreas is under significant oxidative stress as a result of increased O2 ●- and peroxides combined with reduced antioxidant defenses against reactive oxygen species (ROS). We speculate that insulin secretory defects in CF may be due to oxidative stress.

Cancer cells (relative to normal cells) demonstrate alterations in oxidative metabolism characterized by increased steady-state levels of reactive oxygen species (i.e., hydrogen peroxide, H(2)O(2)) that may be compensated for by increased glucose metabolism, but the therapeutic significance of these observations is unknown. In this study, inhibitors of glucose (i.e., 2-deoxy-d-glucose, 2DG) and hydroperoxide (i.e., l-buthionine-S,R-sulfoximine, BSO) metabolism were utilized in combination with a chemotherapeutic agent, paclitaxel (PTX), thought to induce oxidative stress, to treat breast cancer cells. 2DG + PTX was more toxic than either agent alone in T47D and MDA-MB231 human breast cancer cells, but not in normal human fibroblasts or normal human mammary epithelial cells. Increases in parameters indicative of oxidative stress, including steady-state levels of H(2)O(2), total glutathione, and glutathione disulfide, accompanied the enhanced toxicity of 2DG + PTX in cancer cells. Antioxidants, including N-acetylcysteine and polyethylene glycol-conjugated catalase and superoxide dismutase, inhibited the toxicity of 2DG + PTX and suppressed parameters indicative of oxidative stress in cancer cells, whereas inhibition of glutathione synthesis using BSO further sensitized breast cancer cells to 2DG + PTX. These results show that combining inhibitors of glucose (2DG) and hydroperoxide (BSO) metabolism with PTX selectively (relative to normal cells) enhances breast cancer cell killing via H(2)O(2)-induced metabolic oxidative stress, and suggest that this biochemical rationale may be effectively utilized to treat breast cancers.

The ageing kidney exhibits slowly developing chronic kidney disease (CKD) and is associated with nitric oxide (NO) deficiency and increased oxidative stress. The impact of exercise on the ageing kidney is not well understood. Here, we determined whether 12 weeks of treadmill exercise can influence age-dependent CKD in old (22-24 months) Fisher 344 (F344) male rats by comparing sedentary (SED) and exercise (EX) trained rats; young (3 months) rats were also studied. In addition to renal structure and function, we assessed protein levels of various isoforms of the NO synthases (NOS) and superoxide dismutase (SOD) enzymes as well as markers of oxidative stress, in kidney cortex and medulla. Renal function as determined by plasma creatinine, proteinuria, and glomerular structural injury worsened with age and was unaffected by exercise. Ageing also increased the protein abundance of neuronal NOSβ and p22phox while decreasing extracellular (EC) and copper/zinc (CuZn) SOD, in kidney cortex and medulla. H(2)O(2) content and nitrotyrosine abundance also increased in the kidney with age. None of these age-related changes were altered with exercise. However, exercise did increase renal cortical endothelial (e)NOS and EC SOD in young rats. Data indicate that exercise-induced increases in eNOS and EC SOD seen in young rats are lost with age. We conclude that chronic exercise is ineffective in reversing age-dependent CKD in the male F344 rat.

Alpha-1 antitrypsin deficiency is the leading cause of childhood liver failure and one of the most common lethal genetic diseases. The disease-causing mutant A1AT-Z fails to fold correctly and accumulates in the endoplasmic reticulum (ER) of the liver, resulting in hepatic fibrosis and hepatocellular carcinoma in a subset of patients. Furthermore, A1AT-Z sequestration in hepatocytes leads to a reduction in A1AT secretion into the serum, causing panacinar emphysema in adults. The purpose of this work was to elucidate the details by which A1AT-Z is degraded in hepatic cell lines. We identified the ubiquitin ligase FBG1, which has been previously shown to degrade proteins by both the ubiquitin proteasome pathway and autophagy, as being key to A1AT-Z degradation. Using chemical and genetic approaches we show that FBG1 degrades A1AT-Z through both the ubiquitin proteasome system and autophagy. Overexpression of FBG1 decreases the half-life of A1AT-Z and knocking down FBG1 in a hepatic cell line, and in mice results in an increase in ATAT. Finally, we show that FBG1 degrades A1AT-Z through a Beclin1-dependent arm of autophagy. In our model, FBG1 acts as a safety ubiquitin ligase, whose function is to re-ubiquitinate ER proteins that have previously undergone de-ubiquitination to ensure they are degraded.

Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13 C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Age-related arterial endothelial dysfunction, a key antecedent of the development of cardiovascular disease (CVD), is largely caused by a reduction in nitric oxide (NO) bioavailability as a consequence of oxidative stress. Mitochondria are a major source and target of vascular oxidative stress when dysregulated. Mitochondrial dysregulation is associated with primary ageing, but its role in age-related endothelial dysfunction is unknown. Our aim was to determine the efficacy of a mitochondria-targeted antioxidant, MitoQ, in ameliorating vascular endothelial dysfunction in old mice. Ex vivo carotid artery endothelium-dependent dilation (EDD) to increasing doses of acetylcholine was impaired by ∼30% in old (∼27 months) compared with young (∼8 months) mice as a result of reduced NO bioavailability (P < 0.05). Acute (ex vivo) and chronic (4 weeks in drinking water) administration of MitoQ completely restored EDD in older mice by improving NO bioavailability. There were no effects of age or MitoQ on endothelium-independent dilation to sodium nitroprusside. The improvements in endothelial function with MitoQ supplementation were associated with the normalization of age-related increases in total and mitochondria-derived arterial superoxide production and oxidative stress (nitrotyrosine abundance), as well as with increases in markers of vascular mitochondrial health, including antioxidant status. MitoQ also reversed the age-related increase in endothelial susceptibility to acute mitochondrial damage (rotenone-induced impairment in EDD). Our results suggest that mitochondria-derived oxidative stress is an important mechanism underlying the development of endothelial dysfunction in primary ageing. Mitochondria-targeted antioxidants such as MitoQ represent a promising novel strategy for the preservation of vascular endothelial function with advancing age and the prevention of age-related CVD.

The gene CACNA1C, which encodes the pore forming subunit of the L-type calcium channel CaV1.2, is associated with increased risk for neuropsychiatric disorders including schizophrenia, autism spectrum disorder, major depression, and bipolar disorder. Previous rodent work identified that loss or reduction of CaV1.2 results in cognitive, affective, and motor deficits. Most previous work has either included non-neuronal cell populations (haploinsufficient and Nestin-Cre) or investigated a discrete neuronal cell population (e.g. CaMKII-Cre, Drd1-Cre), but few studies have examined the effects of more broad neuron-specific deletion of CaV1.2. Additionally, most of these studies did not evaluate for sex-specific effects or used only male animals. Here, we sought to clarify whether there are sex-specific behavioral consequences of neuron-specific deletion of CaV1.2 (neuronal CaV1.2 cKO) using Syn1-Cre-mediated conditional deletion. We found that neuronal CaV1.2 cKO mice have normal baseline locomotor function but female cKO mice display impaired motor performance learning. Male neuronal CaV1.2 cKO display impaired startle response with intact pre-pulse inhibition. Male neuronal CaV1.2 cKO mice did not display normal social preference, whereas female neuronal CaV1.2 cKO mice did. Neuronal CaV1.2 cKO mice displayed impaired associative learning in both sexes, as well as normal anxiety-like behavior and hedonic capacity. We conclude that deletion of neuronal CaV1.2 alters motor performance, acoustic startle reflex, and social behaviors in a sex-specific manner, while associative learning deficits generalize across sexes. Our data provide evidence for both sex-specific and sex-independent phenotypes related to neuronal expression of CaV1.2.

In genetically heterogeneous mice produced by the CByB6F1 x C3D2F1 cross, the "non-feminizing" estrogen, 17-α-estradiol (17aE2), extended median male lifespan by 19% (p < 0.0001, log-rank test) and 11% (p = 0.007) when fed at 14.4 ppm starting at 16 and 20 months, respectively. 90th percentile lifespans were extended 7% (p = 0.004, Wang-Allison test) and 5% (p = 0.17). Body weights were reduced about 20% after starting the 17aE2 diets. Four other interventions were tested in males and females: nicotinamide riboside, candesartan cilexetil, geranylgeranylacetone, and MIF098. Despite some data suggesting that nicotinamide riboside would be effective, neither it nor the other three increased lifespans significantly at the doses tested. The 17aE2 results confirm and extend our original reports, with very similar results when started at 16 months compared with mice started at 10 months of age in a prior study. The consistently large lifespan benefit in males, even when treatment is started late in life, may provide information on sex-specific aspects of aging.

Manganese superoxide dismutase, encoded by the Sod2 gene, is a ubiquitously expressed mitochondrial antioxidant enzyme that is essential for mammalian life. Mice born with constitutive genetic knockout of Sod2 do not survive the neonatal stage, which renders the longitudinal study of the biochemical and metabolic effects of Sod2 loss difficult. However, multiple studies have demonstrated that tissue-specific knockout of Sod2 in murine liver yields no observable gross pathology or injury to the mouse. We hypothesized that Sod2 loss may have sub-pathologic effects on liver biology, including the acquisition of reactive oxygen species-mediated mitochondrial DNA mutations. To evaluate this, we established and verified a hepatocyte-specific knockout of Sod2 in C57/B6 mice using Cre-LoxP recombination technology. We utilized deep sequencing to identify possible mutations in Sod2 (-/-) mitochondrial DNA as compared to wt, and both RT-PCR and traditional biochemical assays to evaluate baseline differences in redox-sensitive pathways in Sod2 (-/-) hepatocytes. Surprisingly, no mutations in Sod2 (-/-) mitochondrial DNA were detected despite measurable increases in dihydroethidium staining in situ and concomitant decreases in complex II activity indicative of elevated superoxide in the Sod2 (-/-) hepatocytes. In contrast, numerous compensatory alterations in gene expression were identified that suggest hepatocytes have a remarkable capacity to adapt and overcome the loss of Sod2 through transcriptional means. Taken together, these results suggest that murine hepatocytes have a large reserve capacity to cope with the presence of additional mitochondrial reactive oxygen species.

Mitochondrial dysregulation and associated excessive reactive oxygen species (mtROS) production is a key source of oxidative stress in aging arteries that reduces baseline function and may influence resilience (ability to withstand stress). We hypothesized that voluntary aerobic exercise would increase arterial resilience in old mice. An acute mitochondrial stressor (rotenone) caused greater (further) impairment in peak carotid EDD in old (~27 mo., OC, n=12; -32.5±-10.5%) versus young (~7 mo., YC n=11; -5.4±- 3.7%) control male mice, whereas arteries from young and old exercising (YVR n=10 and OVR n=11, 10-wk voluntary running; -0.8±-2.1% and -8.0±4.9%, respectively) mice were protected. Ex-vivo simulated Western diet (WD, high glucose and palmitate) caused greater impairment in EDD in OC (-28.5±8.6%) versus YC (-16.9±5.2%) and YVR (-15.3±2.3%), whereas OVR (-8.9±3.9%) were more resilient (not different versus YC). Simultaneous ex-vivo treatment with mitochondria-specific antioxidant MitoQ attenuated WD-induced impairments in YC and OC, but not YVR or OVR, suggesting that exercise improved resilience to mtROS-mediated stress. Exercise normalized age-related alterations in aortic mitochondrial protein markers PGC-1α, SIRT-3 and Fis1 and augmented cellular antioxidant and stress response proteins. Our results indicate that arterial aging is accompanied by reduced resilience and mitochondrial health, which are restored by voluntary aerobic exercise.

Severe and recurrent cisplatin-induced acute kidney injury (AKI) as part of standard cancer therapy is a known risk factor for development of chronic kidney disease (CKD). The specific role of superoxide (O2•-)-mediated disruption of mitochondrial oxidative metabolism in CKD after cisplatin treatment is unexplored. Cisplatin is typically administered in weekly or tri-weekly cycles as part of standard cancer therapy. To investigate the role of O2•- in predisposing patients to future renal injury and in CKD, mice were treated with cisplatin and a mitochondrial-specific, superoxide dismutase (SOD) mimetic, GC4419. Renal function, biomarkers of oxidative stress, mitochondrial oxidative metabolism, and kidney injury markers, as well as renal histology, were assessed to evaluate the cellular changes that occur one week and one month (CKD phase) after the cisplatin insult. Cisplatin treatment resulted in persistent upregulation of kidney injury markers, increased steady-state levels of O2•-, increased O2•--mediated renal tubules damage, and upregulation of mitochondrial electron transport chain (ETC) complex I activity both one week and one month following cisplatin treatment. Treatment with a novel, clinically relevant, small-molecule superoxide dismutase (SOD) mimetic, GC4419, restored mitochondrial ETC complex I activity to control levels without affecting complexes II-IV activity, as well as ameliorated cisplatin-induced kidney injury. These data support the hypothesis that increased mitochondrial O2•- following cisplatin administration, as a result of disruptions of mitochondrial metabolism, may be an important contributor to both AKI and CKD progression.

Cisplatin contributes to acute kidney injury (AKI) and chronic kidney disease (CKD) that occurs with greater frequency and severity in older patients. Age-associated cisplatin sensitivity in human fibroblasts involves increased mitochondrial superoxide produced by older donor cells.