Mitochondrial Dysfunction and Fatigue in Sjögren's Disease.

Objectives: Sjögren's disease (SjD) is a common exocrine disorder typified by chronic inflammation and dryness, but also profound fatigue, suggesting a pathological basis in cellular bioenergetics. In healthy states, damaged or dysfunctional mitochondrial components are broken down and recycled by mitophagy, a specialized form of autophagy. In many autoimmune disorders, however, evidence suggests that dysfunctional mitophagy allows poorly functioning mitochondria to persist and contribute to a cellular milieu with elevated reactive oxygen species. We hypothesized that mitophagic processes are dysregulated in SjD and that dysfunctional mitochondria contribute to overall fatigue. We sought to link fatigue with mitochondrial dysfunction directly in SjD, heretofore unexamined, and further sought to assess the pathogenic extent and implications of dysregulated mitophagy in SjD. Methods: We isolated pan T cells via negative selection from the peripheral blood mononuclear cells of 17 SjD and 8 age-matched healthy subjects, all of whom completed fatigue questionnaires prior to phlebotomy. Isolated T cells were analyzed for mitochondrial oxygen consumption rate (OCR) and glycolysis using Seahorse, and linear correlations with fatigue measures were assessed. A mitophagy transcriptional signature in SjD was identified by reanalysis of whole-blood microarray data from 190 SjD and 32 healthy subjects. Differential expression analyses were performed by case/control and subgroup analyses comparing SjD patients by mitophagy transcriptional cluster against healthy subjects followed by bioinformatic interpretation using gene set enrichment analysis. Results: Basal OCR, ATP-linked respiration, maximal respiration, and reserve capacity were significantly lower in SjD compared to healthy subjects with no observed differences in non-mitochondrial respiration, basal glycolysis, or glycolytic stress. SjD lymphocytic mitochondria show structural alterations compared to healthy subjects. Fatigue scores related to pain/discomfort in SjD correlated with the altered OCR. Results from subgroup analyses by mitophagic SjD clusters revealed highly variable inter-cluster differentially expressed genes (DEGs) and expanded the number of SjD-associated gene targets by tenfold within the same dataset. Conclusion: Mitochondrial dysfunction, associated with fatigue, is a significant problem in SjD and warrants further investigation.

Neuroprotective treatment with the nitrone compound OKN-007 mitigates age-related muscle weakness in aging mice.

Despite the universal impact of sarcopenia on compromised health and quality of life in the elderly, promising pharmaceutical approaches that can effectively mitigate loss of muscle and function during aging have been limited. Our group and others have reported impairments in peripheral motor neurons and loss of muscle innervation as initiating factors in sarcopenia, contributing to mitochondrial dysfunction and elevated oxidative stress in muscle. We recently reported a reduction in α motor neuron loss in aging mice in response to the compound OKN-007, a proposed antioxidant and anti-inflammatory agent. In the current study, we asked whether OKN-007 treatment in wildtype male mice for 8-9 months beginning at 16 months of age can also protect muscle mass and function. At 25 months of age, we observed a reduction in the loss of whole-body lean mass, a reduced loss of innervation at the neuromuscular junction and well-preserved neuromuscular junction morphology in OKN-007 treated mice versus age matched wildtype untreated mice. The loss in muscle force generation in aging mice (~ 25%) is significantly improved with OKN-007 treatment. In contrast, OKN-007 treatment provided no protection in loss of muscle mass in aging mice. Mitochondrial function was improved by OKN-007 treatment, consistent with its potential antioxidative properties. Together, these exciting findings are the first to demonstrate that interventions through neuroprotection can be an effective therapy to counter aging-related muscle dysfunction.

Neuronal deletion of MnSOD in mice leads to demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

Neuronal oxidative stress has been implicated in aging and neurodegenerative disease. Here we investigated the impact of elevated oxidative stress induced in mouse spinal cord by deletion of Mn-Superoxide dismutase (MnSOD) using a neuron specific Cre recombinase in Sod2 floxed mice (i-mn-Sod2 KO). Sod2 deletion in spinal cord neurons was associated with mitochondrial alterations and peroxide generation. Phenotypically, i-mn-Sod2 KO mice experienced hindlimb paralysis and clasping behavior associated with extensive demyelination and reduced nerve conduction velocity, axonal degeneration, enhanced blood brain barrier permeability, elevated inflammatory cytokines, microglia activation, infiltration of neutrophils and necroptosis in spinal cord. In contrast, spinal cord motor neuron number, innervation of neuromuscular junctions, muscle mass, and contractile function were not altered. Overall, our findings show that loss of MnSOD in spinal cord promotes a phenotype of demyelination, inflammation and progressive paralysis that mimics phenotypes associated with progressive multiple sclerosis.

Inhibition of ATR Reverses a Mitochondrial Respiratory Insufficiency.

Diseases that affect the mitochondrial electron transport chain (ETC) often manifest as threshold effect disorders, meaning patients only become symptomatic once a certain level of ETC dysfunction is reached. Cells can invoke mechanisms to circumvent reaching their critical ETC threshold, but it is an ongoing challenge to identify such processes. In the nematode Caenorhabditis elegans, severe reduction of mitochondrial ETC activity shortens life, but mild reduction actually extends it, providing an opportunity to identify threshold circumvention mechanisms. Here, we show that removal of ATL-1, but not ATM-1, worm orthologs of ATR and ATM, respectively, key nuclear DNA damage checkpoint proteins in human cells, unexpectedly lessens the severity of ETC dysfunction. Multiple genetic and biochemical tests show no evidence for increased mutation or DNA breakage in animals exposed to ETC disruption. Reduced ETC function instead alters nucleotide ratios within both the ribo- and deoxyribo-nucleotide pools, and causes stalling of RNA polymerase, which is also known to activate ATR. Unexpectedly, atl-1 mutants confronted with mitochondrial ETC disruption maintain normal levels of oxygen consumption, and have an increased abundance of translating ribosomes. This suggests checkpoint signaling by ATL-1 normally dampens cytoplasmic translation. Taken together, our data suggest a model whereby ETC insufficiency in C. elegans results in nucleotide imbalances leading to the stalling of RNA polymerase, activation of ATL-1, dampening of global translation, and magnification of ETC dysfunction. The loss of ATL-1 effectively reverses the severity of ETC disruption so that animals become phenotypically closer to wild type.

Age Related Changes in Muscle Mass and Force Generation in the Triple Transgenic (3xTgAD) Mouse Model of Alzheimer's Disease.

Emerging evidence suggests that patients with Alzheimer's disease (AD) may show accelerated sarcopenia phenotypes. To investigate whether pathological changes associated with neuronal death and cognitive dysfunction also occur in peripheral motor neurons and muscle as a function of age, we used the triple transgenic mouse model of AD (3xTgAD mice) that carries transgenes for mutant forms of APP, Tau, and presenilin proteins that are associated with AD pathology. We measured changes in motor neurons and skeletal muscle function and metabolism in young (2 to 4 month) female control and 3xTgAD mice and in older (18-20 month) control and 3xTgAD female mice. In older 3xTgAD mice, we observed a number of sarcopenia-related phenotypes, including significantly fragmented and denervated neuromuscular junctions (NMJs) associated with a 17% reduction in sciatic nerve induced vs. direct muscle stimulation induced contractile force production, and a 30% decrease in gastrocnemius muscle mass. On the contrary, none of these outcomes were found in young 3xTgAD mice. We also measured an accumulation of amyloid-ß (Aß) in both skeletal muscle and neuronal tissue in old 3xTgAD mice that may potentially contribute to muscle atrophy and NMJ disruption in the older 3xTgAD mice. Furthermore, the TGF-ß mediated atrophy signaling pathway is activated in old 3xTgAD mice and is a potential contributing factor in the muscle atrophy that occurs in this group. Perhaps surprisingly, mitochondrial oxygen consumption and reactive oxygen species (ROS) production are not elevated in skeletal muscle from old 3xTgAD mice. Together, these results provide new insights into the effect of AD pathological mechanisms on peripheral changes in skeletal muscle.

Restoration of Sarcoplasmic Reticulum Ca2+ ATPase (SERCA) Activity Prevents Age-Related Muscle Atrophy and Weakness in Mice.

Sarcopenia has a significant negative impact on healthspan in the elderly and effective pharmacologic interventions remain elusive. We have previously demonstrated that sarcopenia is associated with reduced activity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump. We asked whether restoring SERCA activity using pharmacologic activation in aging mice could mitigate the sarcopenia phenotype. We treated 16-month male C57BL/6J mice with vehicle or CDN1163, an allosteric SERCA activator, for 10 months. At 26 months, maximal SERCA activity was reduced 41% in gastrocnemius muscle in vehicle-treated mice but maintained in old CDN1163 treated mice. Reductions in gastrocnemius mass (9%) and in vitro specific force generation in extensor digitorum longus muscle (11%) in 26 versus 16-month-old wild-type mice were also reversed by CDN1163. CDN1163 administered by intra-peritoneal injection also prevented the increase in mitochondrial ROS production in gastrocnemius muscles of aged mice. Transcriptomic analysis revealed that these effects are at least in part mediated by enhanced cellular energetics by activation of PGC1-α, UCP1, HSF1, and APMK and increased regenerative capacity by suppression of MEF2C and p38 MAPK signaling. Together, these exciting findings are the first to support that pharmacological targeting of SERCA can be an effective therapy to counter age-related muscle dysfunction.

Neuron-specific deletion of CuZnSOD leads to an advanced sarcopenic phenotype in older mice.

Age-associated loss of muscle mass and function (sarcopenia) has a profound effect on the quality of life in the elderly. Our previous studies show that CuZnSOD deletion in mice (Sod1-/- mice) recapitulates sarcopenia phenotypes, including elevated oxidative stress and accelerated muscle atrophy, weakness, and disruption of neuromuscular junctions (NMJs). To determine whether deletion of Sod1 initiated in neurons in adult mice is sufficient to induce muscle atrophy, we treated young (2- to 4-month-old) Sod1flox/SlickHCre mice with tamoxifen to generate i-mn-Sod1KO mice. CuZnSOD protein was 40-50% lower in neuronal tissue in i-mn-Sod1KO mice. Motor neuron number in ventral spinal cord was reduced 28% at 10 months and more than 50% in 18- to 22-month-old i-mn-Sod1KO mice. By 24 months, 22% of NMJs in i-mn-Sod1KO mice displayed a complete lack of innervation and deficits in specific force that are partially reversed by direct muscle stimulation, supporting the loss of NMJ structure and function. Muscle mass was significantly reduced by 16 months of age and further decreased at 24 months of age. Overall, our findings show that neuronal-specific deletion of CuZnSOD is sufficient to cause motor neuron loss in young mice, but that NMJ disruption, muscle atrophy, and weakness are not evident until past middle age. These results suggest that loss of innervation is critical but may not be sufficient until the muscle reaches a threshold beyond which it cannot compensate for neuronal loss or rescue additional fibers past the maximum size of the motor unit.

Molecular changes associated with spinal cord aging.

Age-related muscle weakness and loss of muscle mass (sarcopenia) is a universal problem in the elderly. Our previous studies indicate that alpha motor neurons (α-MNs) play a critical role in this process. The goal of the current study is to uncover changes in the aging spinal cord that contribute to loss of innervation and the downstream degenerative processes that occur in skeletal muscle. The number of α-MNs is decreased in the spinal cord of wildtype mice during aging, beginning in middle age and reaching a 41% loss by 27 months of age. There is evidence for age-related loss of myelin and mild inflammation, including astrocyte and microglia activation and an increase in levels of sICAM-1. We identified changes in metabolites consistent with compromised neuronal viability, such as reduced levels of N-acetyl-aspartate. Cleaved caspase-3 is more abundant in spinal cord from old mice, suggesting that apoptosis contributes to neuronal loss. RNA-seq analysis revealed changes in the expression of a number of genes in spinal cord from old mice, in particular genes encoding extracellular matrix components (ECM) and a 172-fold increase in MMP-12 expression. Furthermore, blood-spinal cord barrier (BSCB) permeability is increased in old mice, which may contribute to alterations in spinal cord homeostasis and exacerbate neuronal distress. Together, these data show for the first time that the spinal cord undergoes significant changes during aging, including progressive α-MNs loss that is associated with low-grade inflammation, apoptosis, changes in ECM, myelination, and vascular permeability.

Disparate Central and Peripheral Effects of Circulating IGF-1 Deficiency on Tissue Mitochondrial Function.

Age-related decline in circulating levels of insulin-like growth factor (IGF)-1 is associated with reduced cognitive function, neuronal aging, and neurodegeneration. Decreased mitochondrial function along with increased reactive oxygen species (ROS) and accumulation of damaged macromolecules are hallmarks of cellular aging. Based on numerous studies indicating pleiotropic effects of IGF-1 during aging, we compared the central and peripheral effects of circulating IGF-1 deficiency on tissue mitochondrial function using an inducible liver IGF-1 knockout (LID). Circulating levels of IGF-1 (~ 75%) were depleted in adult male Igf1f/f mice via AAV-mediated knockdown of hepatic IGF-1 at 5 months of age. Cognitive function was evaluated at 18 months using the radial arm water maze and glucose and insulin tolerance assessed. Mitochondrial function was analyzed in hippocampus, muscle, and visceral fat tissues using high-resolution respirometry O2K as well as redox status and oxidative stress in the cortex. Peripherally, IGF-1 deficiency did not significantly impact muscle mass or mitochondrial function. Aged LID mice were insulin resistant and exhibited ~ 60% less adipose tissue but increased fat mitochondrial respiration (20%). The effects on fat metabolism were attributed to increases in growth hormone. Centrally, IGF-1 deficiency impaired hippocampal-dependent spatial acquisition as well as reversal learning in male mice. Hippocampal mitochondrial OXPHOS coupling efficiency and cortex ATP levels (~ 50%) were decreased and hippocampal oxidative stress (protein carbonylation and F2-isoprostanes) was increased. These data suggest that IGF-1 is critical for regulating mitochondrial function, redox status, and spatial learning in the central nervous system but has limited impact on peripheral (liver and muscle) metabolism with age. Therefore, IGF-1 deficiency with age may increase sensitivity to damage in the brain and propensity for cognitive deficits. Targeting mitochondrial function in the brain may be an avenue for therapy of age-related impairment of cognitive function. Regulation of mitochondrial function and redox status by IGF-1 is essential to maintain brain function and coordinate hippocampal-dependent spatial learning. While a decline in IGF-1 in the periphery may be beneficial to avert cancer progression, diminished central IGF-1 signaling may mediate, in part, age-related cognitive dysfunction and cognitive pathologies potentially by decreasing mitochondrial function.

Mitochondrial oxidative stress impairs contractile function but paradoxically increases muscle mass via fibre branching.

BACKGROUND: Excess reactive oxygen species (ROS) and muscle weakness occur in parallel in multiple pathological conditions. However, the causative role of skeletal muscle mitochondrial ROS (mtROS) on neuromuscular junction (NMJ) morphology and function and muscle weakness has not been directly investigated. METHODS: We generated mice lacking skeletal muscle-specific manganese-superoxide dismutase (mSod2KO) to increase mtROS using a cre-Lox approach driven by human skeletal actin. We determined primary functional parameters of skeletal muscle mitochondrial function (respiration, ROS, and calcium retention capacity) using permeabilized muscle fibres and isolated muscle mitochondria. We assessed contractile properties of isolated skeletal muscle using in situ and in vitro preparations and whole lumbrical muscles to elucidate the mechanisms of contractile dysfunction. RESULTS: The mSod2KO mice, contrary to our prediction, exhibit a 10-15% increase in muscle mass associated with an ~50% increase in central nuclei and ~35% increase in branched fibres (P < 0.05). Despite the increase in muscle mass of gastrocnemius and quadriceps, in situ sciatic nerve-stimulated isometric maximum-specific force (N/cm2 ), force per cross-sectional area, is impaired by ~60% and associated with increased NMJ fragmentation and size by ~40% (P < 0.05). Intrinsic alterations of components of the contractile machinery show elevated markers of oxidative stress, for example, lipid peroxidation is increased by ~100%, oxidized glutathione is elevated by ~50%, and oxidative modifications of myofibrillar proteins are increased by ~30% (P < 0.05). We also find an approximate 20% decrease in the intracellular calcium transient that is associated with specific force deficit. Excess superoxide generation from the mitochondrial complexes causes a deficiency of succinate dehydrogenase and reduced complex-II-mediated respiration and adenosine triphosphate generation rates leading to severe exercise intolerance (~10 min vs. ~2 h in wild type, P < 0.05). CONCLUSIONS: Increased skeletal muscle mtROS is sufficient to elicit NMJ disruption and contractile abnormalities, but not muscle atrophy, suggesting new roles for mitochondrial oxidative stress in maintenance of muscle mass through increased fibre branching.

Restoration of SERCA ATPase prevents oxidative stress-related muscle atrophy and weakness.

Molecular targets to reduce muscle weakness and atrophy due to oxidative stress have been elusive. Here we show that activation of Sarcoplasmic Reticulum (SR) Ca2+ ATPase (SERCA) with CDN1163, a novel small molecule allosteric SERCA activator, ameliorates the muscle impairment in the CuZnSOD deficient (Sod1-/-) mouse model of oxidative stress. Sod1-/- mice are characterized by reduced SERCA activity, muscle weakness and atrophy, increased oxidative stress and mitochondrial dysfunction. Seven weeks of CDN1163 treatment completely restored SERCA activity and reversed the 23% reduction in gastrocnemius mass and 22% reduction in specific force in untreated Sod1-/- versus wild type mice. These changes were accompanied by restoration of autophagy protein markers to the levels found in wild-type mice. CDN1163 also reversed the increase in mitochondrial ROS generation and oxidative damage in muscle tissue from Sod1-/- mice. Taken together our findings suggest that the pharmacological restoration of SERCA is a promising therapeutic approach to counter oxidative stress-associated muscle impairment.

Oxidative stress-induced dysregulation of excitation-contraction coupling contributes to muscle weakness.

BACKGROUND: We have previously shown that the deletion of the superoxide scavenger, CuZn superoxide dismutase, in mice (Sod1-/- mice) results in increased oxidative stress and an accelerated loss of skeletal muscle mass and force that mirror the changes seen in old control mice. The goal of this study is to define the effect of oxidative stress and ageing on muscle weakness and the Excitation Contraction (EC) coupling machinery in age-matched adult (8-10 months) wild-type (WT) and Sod1-/- mice in comparison with old (25-28 months) WT mice. METHODS: In vitro contractile assays were used to measure muscle contractile parameters. The activity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump was measured using an NADH-linked enzyme assay. Immunoblotting and immunofluorescence techniques were used to measure protein expression, and real-time reverse transcription PCR was used to measure gene expression. RESULTS: The specific force generated by the extensor digitorum longus muscle was reduced in the Sod1-/- and old WT mice compared with young WT mice along with significant prolongation of time to peak force, increased half relaxation time, and disruption of intracellular calcium handling. The maximal activity of the SERCA calcium uptake pump was significantly reduced in gastrocnemius muscle from both old WT (≈14%) and adult Sod1-/- (≈33%) mice compared with young WT mice along with increased expression of sarcolipin, a known inhibitor of SERCA activity. Protein levels of the voltage sensor and calcium uptake channel proteins dihydropyridine receptor α1 and SERCA2 were significantly elevated (≈45% and ≈57%, respectively), while the ratio of calstabin, a channel stabilizing protein, to ryanodine receptor was significantly reduced (≈21%) in Sod1-/- mice compared with young WT mice. The changes in calcium handling were accompanied by substantially elevated levels of global protein carbonylation and lipid peroxidation. CONCLUSIONS: Our data suggest that the muscle weakness in Sod1-/- and old WT mice is in part driven by reactive oxygen species-mediated EC uncoupling and supports a role for reduced SERCA pump activity in compromised muscle function. The novel quantitative mechanistic data provided here can lead to potential therapeutic interventions of SERCA dysfunction for sarcopenia and muscle diseases.

Loss of mitochondrial protease ClpP protects mice from diet-induced obesity and insulin resistance.

Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPRmt on metabolism, ClpP knockout (ClpP-/-) mice were analyzed. ClpP-/- mice fed ad libitum have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole-body energy expenditure and markers of mitochondrial biogenesis are selectively up-regulated in the white adipose tissue (WAT) of ClpP-/- mice. When challenged with a metabolic stress such as high-fat diet, despite similar caloric intake, ClpP-/- mice are protected from diet-induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPRmt initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial.

A fish oil diet induces mitochondrial uncoupling and mitochondrial unfolded protein response in epididymal white adipose tissue of mice.

White adipose tissue (WAT) mitochondrial dysfunction is linked to the pathogenesis of obesity driven insulin resistance. Dietary conditions that alter fat mass are known to affect white adipocyte mitochondrial function, however, the impact of high calorie diets on white adipocyte mitochondria is not fully understood. The aim of this study is to assess the effect of a diet rich in saturated or polyunsaturated fat on mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that maintains mitochondrial homeostasis, in epididymal WAT (eWAT). Mice were fed a low fat diet (LFD), saturated fat diet (SFD) or fish oil (unsaturated fat diet, UFD) and assessed changes in eWAT mitochondria. Compared to mice fed a LFD, SFD-fed mice have reduced mitochondrial biogenesis markers, mitochondrial fatty acid oxidation enzymes and TCA cycle enzymes, suggesting an impaired mitochondrial function that could contribute to increased fat mass. In contrast, isocaloric UFD-fed mice have increased expression of mitochondrial uncoupling protein 1 (UCP1) and peroxisomal fatty acid oxidation enzymes suggesting that elevated mitochondrial uncoupling and peroxisomal fatty acid oxidation could contribute to the reduction in fat mass. Interestingly, expression of UPRmt-associated proteins caseinolytic peptidase (ClpP) and heat shock protein 60 (Hsp60) are induced by UFD, whereas SFD reduced the expression of ClpP. Based on our data, we propose that induction of UPRmt helps to preserve a functional mitochondria and efficient utilization of fat by UFD whereas a dampened UPRmt response might impair mitochondrial function and promote fat accumulation by SFD. Thus, our findings suggest a potential role of UPRmt in mediating the beneficial effects of fish oil.

A new mouse model of frailty: the Cu/Zn superoxide dismutase knockout mouse.

Frailty is a geriatric syndrome that is an important public health problem for the older adults living in the USA. Although several methods have been developed to measure frailty in humans, we have very little understanding of its etiology. Because the molecular basis of frailty is poorly understood, mouse models would be of great value in determining which pathways contribute to the development of frailty. More importantly, mouse models would be critical in testing potential therapies to treat and possibly prevent frailty. In this article, we present data showing that Sod1KO mice, which lack the antioxidant enzyme, Cu/Zn superoxide dismutase, are an excellent model of frailty, and we compare the Sod1KO mice to the only other mouse model of frailty, mice with the deletion of the IL-10 gene. Sod1KO mice exhibit four characteristics that have been used to define human frailty: weight loss, weakness, low physical activity, and exhaustion. In addition, Sod1KO mice show increased inflammation and sarcopenia, which are strongly associated with human frailty. The Sod1KO mice also show alterations in pathways that have been proposed to play a role in the etiology of frailty: oxidative stress, mitochondrial dysfunction, and cell senescence. Using Sod1KO mice, we show that dietary restriction can delay/prevent characteristics of frailty in mice.

Muscle fiber type diversification during exercise and regeneration.

The plasticity of skeletal muscle can be traced down to extensive metabolic, structural and molecular remodeling at the single fiber level. Skeletal muscle is comprised of different fiber types that are the basis of muscle plasticity in response to various functional demands. Resistance and endurance exercises are two external stimuli that differ in their duration and intensity of contraction and elicit markedly different responses in muscles adaptation. Further, eccentric contractions that are associated with exercise-induced injuries, elicit varied muscle adaptation and regenerative responses. Most adaptive changes are fiber type-specific and are highly influenced by diverse structural, metabolic and functional characteristics of individual fiber types. Regulation of signaling pathways by reactive oxygen species (ROS) and oxidative stress also plays an important role in muscle fiber adaptation during exercise. This review focuses on cellular and molecular responses that regulate the adaptation of skeletal muscle to exercise and exercise-related injuries.

Down-regulation of the mitochondrial matrix peptidase ClpP in muscle cells causes mitochondrial dysfunction and decreases cell proliferation.

The caseinolytic peptidase P (ClpP) is the endopeptidase component of the mitochondrial matrix ATP-dependent ClpXP protease. ClpP degrades unfolded proteins to maintain mitochondrial protein homeostasis and is involved in the initiation of the mitochondrial unfolded protein response (UPR(mt)). Outside of an integral role in the UPR(mt), the cellular function of ClpP is not well characterized in mammalian cells. To investigate the role of ClpP in mitochondrial function, we generated C2C12 muscle cells that are deficient in ClpP using siRNA or stable knockdown using lentiviral transduction. Reduction of ClpP levels by ~70% in C2C12 muscle cells resulted in a number of mitochondrial alterations including reduced mitochondrial respiration and reduced oxygen consumption rate in response to electron transport chain (ETC) complex I and II substrates. The reduction in ClpP altered mitochondrial morphology, changed the expression level of mitochondrial fission protein Drp1 and blunted UPR(mt) induction. In addition, ClpP deficient cells showed increased generation of reactive oxygen species (ROS) and decreased membrane potential. At the cellular level, reduction of ClpP impaired myoblast differentiation, cell proliferation and elevated phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) suggesting an inhibition of translation. Our study is the first to define the effects of ClpP deficiency on mitochondrial function in muscle cells in vitro. In addition, we have uncovered novel effects of ClpP on mitochondrial morphology, cell proliferation and protein translation pathways in muscle cells.

A dominant-negative F-box deleted mutant of E3 ubiquitin ligase, ß-TrCP1/FWD1, markedly reduces myeloma cell growth and survival in mice.

Treatment of multiple myeloma with bortezomib can result in severe adverse effects, necessitating the development of targeted inhibitors of the proteasome. We show that stable expression of a dominant-negative F-box deleted (∆F) mutant of the E3 ubiquitin ligase, SCFß-TrCP/FWD1, in murine 5TGM1 myeloma cells dramatically attenuated their skeletal engraftment and survival when inoculated into immunocompetent C57BL/KaLwRij mice. Similar results were obtained in immunodeficient bg-nu-xid mice, suggesting that the observed effects were independent of host recipient immune status. Bone marrow stroma offered no protection for 5TGM1-∆F cells in cocultures treated with tumor necrosis factor (TNF), indicating a cell-autonomous anti-myeloma effect. Levels of p100, IκBα, Mcl-1, ATF4, total and cleaved caspase-3, and phospho-ß-catenin were elevated in 5TGM1-∆F cells whereas cIAP was down-regulated. TNF also activated caspase-3 and downregulated Bcl-2, correlating with the enhanced susceptibility of 5TGM1-∆F cells to apoptosis. Treatment of 5TGM1 tumor-bearing mice with a ß-TrCP1/FWD1 inhibitor, pyrrolidine dithiocarbamate (PDTC), significantly reduced tumor burden in bone. PDTC also increased levels of cleaved Mcl-1 and caspase-3 in U266 human myeloma cells, correlating with our murine data and validating the development of specific ß-TrCP inhibitors as an alternative therapy to nonspecific proteasome inhibitors for myeloma patients.

Neuron specific reduction in CuZnSOD is not sufficient to initiate a full sarcopenia phenotype.

Our previous studies showed that adult (8 month) mice lacking CuZn-superoxide dismutase (CuZnSOD, Sod1KO mice) have neuromuscular changes resulting in dramatic accelerated muscle atrophy and weakness that mimics age-related sarcopenia. We have further shown that loss of CuZnSOD targeted to skeletal muscle alone results in only mild weakness and no muscle atrophy. In this study, we targeted deletion of CuZnSOD specifically to neurons (nSod1KO mice) and determined the effect on muscle mass and weakness. The nSod1KO mice show a significant loss of CuZnSOD activity and protein level in brain and spinal cord but not in muscle tissue. The masses of the gastrocnemius, tibialis anterior and extensor digitorum longus (EDL) muscles were not reduced in nSod1KO compared to wild type mice, even at 20 months of age, although the quadriceps and soleus muscles showed small but statistically significant reductions in mass in the nSod1KO mice. Maximum isometric specific force was reduced by 8-10% in the gastrocnemius and EDL muscle of nSod1KO mice, while soleus was not affected. Muscle mitochondrial ROS generation and oxidative stress measured by levels of reactive oxygen/nitrogen species (RONS) regulatory enzymes, protein nitration and F2-isoprostane levels were not increased in muscle from the nSod1KO mice. Although we did not find evidence of denervation in the nSod1KO mice, neuromuscular junction morphology was altered and the expression of genes associated with denervation acetylcholine receptor subunit alpha (AChRα), the transcription factor, Runx1 and GADD45α) was increased, supporting a role for neuronal loss of CuZnSOD initiating alterations at the neuromuscular junction. These results and our previous studies support the concept that CuZnSOD deficits in either the motor neuron or muscle alone are not sufficient to initiate a full sarcopenic phenotype and that deficits in both tissues are required to recapitulate the loss of muscle observed in Sod1KO mice.

Synthesis and antiprogestational properties of novel 17-fluorinated steroids.

Progesterone receptor (PR) plays a key role in reproductive functions, and compounds that inhibit progesterone action (antiprogestins) have potential use in the treatment of estrogen- and progesterone-dependent diseases, including uterine leiomyomas and breast cancer. In the present study, we chemically synthesized novel 17-fluorinated steroids and evaluated the cytotoxicity profiles of these compounds in T47D breast cancer cells compared to the activity of known antiprogestins, including ZK230 211, RU-486, CDB2914, CDB4124 and ORG33628. We analyzed in vitro receptor-binding assays and PR-transactivation assays to establish the antiprogestational activity of these molecules. The representative antiprogestin EC304 was found to inhibit in vitro tumorigenicity in a dose-dependent fashion in T47D cells by a colony formation assay at 1 and 10nM concentrations. The potent in vivo antiprogestational activity of EC304 was also demonstrated in an antinidation assay for the interruption of early pregnancy in rats. The strong antiprogestational activity and absence of antiglucocorticoid activity in EC compounds may demonstrate their utility in the treatment of leiomyoma, endometriosis and breast cancer.