Early steps in oxidation-induced SOD1 misfolding: implications for non-amyloid protein aggregation in familial ALS

Among the diseases of protein misfolding, amyotrophic lateral sclerosis (ALS) is unusual in that the proteinaceous neuronal inclusions that are the hallmark of the disease have neither the classic fibrillar appearance of amyloid by transmission electron microscopy nor the affinity for the dye Congo red that is a defining feature of amyloid. Mutations in the Cu, Zn superoxide dismutase (SOD1) cause the largest subset of inherited ALS cases. The mechanism by which this highly stable enzyme misfolds to form non-amyloid aggregates is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that SOD1's normal free radical scavenger role puts it at risk of oxidative damage and that it is this damage that triggers the misfolding primed by mutation. Here, we present evidence that hydrogen peroxide treatment, which generates free radical species at the SOD1 active site, causes oxidative damage to active-site histidine residues, leading to major structural changes and non-amyloid aggregation similar to that seen in ALS. Time-resolved measurements of release of bound metal ligands, exposure of hydrophobic surface area, and alterations in the SOD1 proton NMR spectrum have allowed us to model the early structural changes occurring as SOD1 misfolds, prior to aggregation. ALS-causing SOD1 mutations apparently alter this pathway by increasing exposure of buried epitopes in misfolded species populated at endpoint. We have identified a well-populated early misfolding intermediate that could serve as a target for therapies designed to block downstream misfolding and aggregation events and thereby treat SOD1-associated ALS.     

Protein misfolding, aggregation, and degradation in disease

Pathologies associated with protein misfolding have been observed in neurodegenerative diseases such as Alzheimer’s disease, metabolic diseases like phenylketonuria, and diseases affecting structural proteins like collagen or keratin. Misfolding of mutant proteins in these and many other diseases may result in premature degradation, formation of toxic aggregates, or incorporation of toxic conformations into structures. We review common traits of these diverse diseases under the unifying view of protein misfolding. The molecular pathogenesis is discussed in the context of protein quality control systems consisting of molecular chaperones and intracellular proteases that assist the folding and supervise the maintenance of the folded structure. Furthermore, genetic and environmental factors that may modify the severity of these diseases are underscored. The present article represents a partly revised and updated version of chapter 1 published earlier in volume 232 of the series Methods in Molecular Biology (Walker, J. M., ed., Humana Press, Totowa, NJ), Protein Misfolding and Disease: Principles and Protocols (Bross, P. & Gregersen, N., eds.), pp. 3–16 (2003).     

Muscle-specific loss of apoptosis-inducing factor leads to mitochondrial dysfunction, skeletal muscle atrophy, and dilated cardiomyopathy

Cardiac and skeletal muscle critically depend on mitochondrial energy metabolism for their normal function. Recently, we showed that apoptosis-inducing factor (AIF), a mitochondrial protein implicated in programmed cell death, plays a role in mitochondrial respiration. However, the in vivo consequences of AIF-regulated mitochondrial respiration resulting from a loss-of-function mutation in Aif are not known. Here, we report tissue-specific deletion of Aif in the mouse. Mice in which Aif has been inactivated specifically in cardiac and skeletal muscle exhibit impaired activity and protein expression of respiratory chain complex I. Mutant animals develop severe dilated cardiomyopathy, heart failure, and skeletal muscle atrophy accompanied by lactic acidemia consistent with defects in the mitochondrial respiratory chain. Isolated hearts from mutant animals exhibit poor contractile performance in response to a respiratory chain-dependent energy substrate, but not in response to glucose, supporting the notion that impaired heart function in mutant animals results from defective mitochondrial energy metabolism. These data provide genetic proof that the previously defined cell death promoter AIF has a second essential function in mitochondrial respiration and aerobic energy metabolism required for normal heart function and skeletal muscle homeostasis.     

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle

The domestic cat (Felis catus), a carnivore, naturally eats a very low carbohydrate diet. In contrast, the dog (Canis familiaris), a carno-omnivore, has a varied diet. This study was performed to determine the expression of the intestinal brush border membrane sodium/glucose cotransporter, SGLT1, sweet receptor, T1R2/T1R3, and disaccharidases in these species adapted to contrasting diets. The expression (this includes function) of SGLT1, sucrase, maltase and lactase were determined using purified brush border membrane vesicles and by quantitative immunohistochemistry of fixed tissues. The pattern of expression of subunits of the sweet receptor T1R2 and T1R3 was assessed using fluorescent immunohistochemistry. In proximal, middle, and distal small intestine, SGLT1 function in dogs was 1.9- to 2.3-fold higher than in cats (P = 0.037, P = 0.0011, P = 0.027, respectively), and SGLT1 protein abundance followed an identical pattern. Both cats and dogs express T1R3 in a subset of intestinal epithelial cells, and dogs, but not cats, express T1R2. In proximal and middle regions, there were 3.1- and 1.6-fold higher lactase (P = 0.006 and P = 0.019), 4.4- and 2.9-fold higher sucrase (both P < 0.0001), and 4.6- and 3.1-fold higher maltase activity (P = 0.0026 and P = 0.0005), respectively, in the intestine of dogs compared with cats. Dogs have a potential higher capacity to digest and absorb carbohydrates than cats. Cats may suffer from carbohydrate malabsorption following ingestion of high-carbohydrate meals. However, dogs have a digestive ability to cope with diets containing significant levels of carbohydrate.     

Principles of protein folding, misfolding and aggregation

This review summarises our current understanding of the underlying and universal mechanism by which newly synthesised proteins achieve their biologically functional states. Protein molecules, however, all have a finite tendency either to misfold, or to fail to maintain their correctly folded states, under some circumstances. This article describes some of the consequences of such behaviour, particularly in the context of the aggregation events that are frequently associated with aberrant folding. It focuses in particular on the emerging links between protein aggregation and the increasingly prevalent forms of debilitating disease with which it is now known to be associated.     

Denervation-induced mitochondrial dysfunction and autophagy in skeletal muscle of apoptosis-deficient animals

Skeletal muscle undergoes remarkable adaptations in response to chronic decreases in contractile activity, such as a loss of muscle mass, decreases in both mitochondrial content and function, as well as the activation of apoptosis. Although these adaptations are well known, questions remain regarding the signaling pathways that mediated these changes. Autophagy is an organelle turnover pathway that could contribute to these adaptations. The purpose of this study was to determine whether denervation-induced muscle disuse would result in the activation of autophagy gene expression in both wild-type (WT) and Bax/Bak double knockout (DKO) animals, which display an attenuated apoptotic response. Denervation caused a reduction in muscle mass for WT and DKO animals; however, there was a 40% attenuation in muscle atrophy in DKO animals. Mitochondrial state 3 respiration was significantly reduced, and reactive oxygen species production was increased by two- to threefold in both WT and DKO animals. Apoptotic markers, including cytosolic AIF and DNA fragmentation, were elevated in WT, but not in DKO animals following denervation. Autophagy proteins including LC3II, ULK1, ATG7, p62, and Beclin1 were increased similarly following denervation for both WT and DKO. Interestingly, denervation markedly increased the localization of LC3II to subsarcolemmal mitochondria, and this was more pronounced in the DKO animals. Thus denervation-induced muscle disuse activates both apoptotic and autophagic signaling pathways in muscle, and autophagic protein expression does not exhibit a compensatory increase in the presence of attenuated apoptosis. However, the absence of Bax and Bak may represent a potential signal to trigger mitophagy in muscle.     

Impaired protein quality control system underlies mitochondrial dysfunction in skeletal muscle of streptozotocin-induced diabetic rats

Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality.     

Satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice

To determine whether muscle satellite cells and utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region as an indicator of myofiber degeneration in the masseter, gastrocnemius, soleus, and diaphragm muscles of mdx mice. Furthermore, we analyzed the expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (the makers of muscle satellite cells), and utrophin mRNA. We also investigated the immunolocalization of m-cadherin and utrophin proteins in the muscles of normal C57BL/10J (B10) and mdx mice. The expression level of pax7 mRNA and the percentage of m-cadherin-positive cells among the total number of cell nuclei in the muscle tissues in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there was no significant correlation between muscle damage and expression level for pax7 mRNA (R = –0.140), nor was there a correlation between muscle damage and the percentage of satellite cells among the total number of cell nuclei (R = –0.411) in the mdx mice. The expression level of utrophin mRNA and the intensity of immunostaining for utrophin in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there also was not a significant correlation between muscle damage and expression level of utrophin mRNA (R = 0.231) in the mdx mice, although upregulated utrophin was incorporated into the sarcolemma. These results suggest that satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice. dystrophy; pax7; m-cadherin; dystrophin-related proteins     

Reductions in RIP140 are not required for exercise- and AICAR-mediated increases in skeletal muscle mitochondrial content

Receptor interacting protein 1 (RIP140) has recently been demonstrated to be a key player in the regulation of skeletal muscle mitochondrial content. We have shown that β-guanadinopropionic acid (β-GPA) feeding reduces RIP140 protein content and mRNA levels concomitant with increases in mitochondrial content (Williams DB, Sutherland LN, Bomhof MR, Basaraba SA, Thrush AB, Dyck DJ, Field CJ, Wright DC. Am J Physiol Endocrinol Metab 296: E1400-E1408, 2009). Since β-GPA feeding reduces high-energy phosphate levels and activates AMPK, alterations reminiscent of exercise, we hypothesized that exercise training would reduce RIP140 protein content. We further postulated that an acute bout of exercise, or interventions known to induce the expression of mitochondrial enzymes or genes involved in mitochondrial biogenesis, would result in decreases in nuclear RIP140 content. Two weeks of daily swim training increased markers of mitochondrial content in rat skeletal muscle independent of reductions in RIP140 protein. Similarly, high-intensity exercise training in humans failed to reduce RIP140 content despite increasing skeletal muscle mitochondrial enzymes. We found that 6 wk of daily 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) injections had no effect on RIP140 protein content in rat skeletal muscle while RIP140 content from LKB1 knockout mice was unaltered despite reductions in mitochondria. An acute bout of exercise, AICAR treatment, and epinephrine injections increased the mRNA levels of PGC-1α, COXIV, and lipin1 independent of decreases in nuclear RIP140 protein. Surprisingly these interventions increased RIP140 mRNA expression. In conclusion our results demonstrate that decreases in RIP140 protein content are not required for exercise and AMPK-dependent increases in skeletal muscle mitochondrial content, nor do acute perturbations alter the cellular localization of RIP140 in parallel with the induction of genes involved in mitochondrial biogenesis.     

Mitochondrial fission contributes to mitochondrial dysfunction and insulin resistance in skeletal muscle

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance and type 2 diabetes. Considering the importance of mitochondrial dynamics in mitochondrial and cellular functions, we hypothesized that obesity and excess energy intake shift the balance of mitochondrial dynamics, further contributing to mitochondrial dysfunction and metabolic deterioration in skeletal muscle. First, we revealed that excess palmitate (PA), but not hyperglycemia, hyperinsulinemia, or elevated tumor necrosis factor alpha, induced mitochondrial fragmentation and increased mitochondrion-associated Drp1 and Fis1 in differentiated C2C12 muscle cells. This fragmentation was associated with increased oxidative stress, mitochondrial depolarization, loss of ATP production, and reduced insulin-stimulated glucose uptake. Both genetic and pharmacological inhibition of Drp1 attenuated PA-induced mitochondrial fragmentation, mitochondrial depolarization, and insulin resistance in C2C12 cells. Furthermore, we found smaller and shorter mitochondria and increased mitochondrial fission machinery in the skeletal muscle of mice with genetic obesity and those with diet-induced obesity. Inhibition of mitochondrial fission improved the muscle insulin signaling and systemic insulin sensitivity of obese mice. Our findings indicated that aberrant mitochondrial fission is causally associated with mitochondrial dysfunction and insulin resistance in skeletal muscle. Thus, disruption of mitochondrial dynamics may underlie the pathogenesis of muscle insulin resistance in obesity and type 2 diabetes.     

Regional skeletal muscle remodeling and mitochondrial dysfunction in right ventricular heart failure

Exercise intolerance is a cardinal symptom of right ventricular heart failure (RV HF) and skeletal muscle adaptations play a role in this limitation. We determined regional remodeling of muscle structure and mitochondrial function in a rat model of RV HF induced by monocrotaline injection (MCT; 60 mg·kg(-1); n = 11). Serial sections of the plantaris were stained for fiber type, succinate dehydrogenase (SDH) activity and capillaries. Mitochondrial function was assessed in permeabilized fibers using respirometry, and isolated complex activity by blue native gel electrophoresis (BN PAGE). All measurements were compared with saline-injected control animals (CON; n = 12). Overall fiber cross-sectional area was smaller in MCT than CON: 1,843 ± 114 vs. 2,322 ± 120 μm(2) (P = 0.009). Capillary-to-fiber ratio was lower in MCT in the oxidative plantaris region (1.65 ± 0.09 vs. 1.93 ± 0.07; P = 0.03), but not in the glycolytic region. SDH activity (P = 0.048) and maximal respiratory rate (P = 0.012) were each ～15% lower in all fibers in MCT. ADP sensitivity was reduced in both skeletal muscle regions in MCT (P = 0.032), but normalized by rotenone. A 20% lower complex I/IV activity in MCT was confirmed by BN PAGE. MCT-treatment was associated with lower mitochondrial volume density (lower SDH activity), quality (lower complex I activity), and fewer capillaries per fiber area in oxidative skeletal muscle. These features are consistent with structural and functional remodeling of the determinants of oxygen supply potential and utilization that may contribute to exercise intolerance and reduced quality of life in patients with RV HF.     

Effects of weight loss and physical activity on skeletal muscle mitochondrial function in obesity

The current study was undertaken to address responsiveness of skeletal muscle mitochondrial electron transport chain (ETC) activity to weight loss (WL) and exercise in overweight or obese, sedentary volunteers. Fourteen middle-aged participants (7 male/7 female) had assessments of mitochondrial ETC activity and mitochondrial (mt)DNA in vastus lateralis muscle, obtained by percutaneous biopsy, before and after a 16-wk intervention. Mean WL was 9.7 (1.5%) and the mean increase in Vo(2 max) was [means (SD)] 21.7 (3.7)%. Total ETC activity increased significantly, from 0.13 (0.02) to 0.19 (0.03) U/mU creatine kinase (CK; P < 0.001). ETC activity was also assessed in mitochondria isolated into subsarcolemmal (SSM) and intermyofibrillar (IMF-M) fractions. In response to intervention, there was a robust increase of ETC activity in SSM (0.028 (0.007) to 0.046 (0.011) U/mU CK, P < 0.001), and in IMF-M [0.101 (0.015) to 0.148 (0.018) U/mU CK, P < 0.005]. At baseline, the percentage of ETC activity contained in the SSM fraction was low and remained unchanged following intervention [19 (3) vs. 22 (2)%], despite the increase in ETC activity. Also, muscle mtDNA content did not change significantly [1665 (213) vs. 1874 (214) mtDNA/nuclear DNA], denoting functional improvement rather than proliferation of mitochondria as the principal mechanism of enhanced ETC activity. Increases in ETC activity were correlated with energy expenditure during exercise sessions, and ETC activity in SSM correlated with insulin sensitivity after adjustment for Vo(2 max). In summary, skeletal muscle ETC activity is increased by WL and exercise in previously sedentary obese men and women. We conclude that improved skeletal muscle ETC activity following moderate WL and improved aerobic capacity contributes to associated alleviation of insulin resistance.     

SIRT1 attenuates high glucose-induced insulin resistance via reducing mitochondrial dysfunction in skeletal muscle cells

Insulin resistance is often characterized as the most critical factor contributing to the development of type 2 diabetes mellitus (T2DM). Sustained high glucose is an important extracellular environment that induces insulin resistance. Acquired insulin resistance is associated with reduced insulin-stimulated mitochondrial activity as a result of increased mitochondrial dysfunction. Silent information regulator 1 (SIRT1) is one member of the SIRT2 (Sir2)-like family of proteins involved in glucose homeostasis and insulin secretion in mammals. Although SIRT1 has a therapeutic effect on metabolic deterioration in insulin resistance, it is still not clear how SIRT1 is involved in the development of insulin resistance. Here, we demonstrate that pcDNA3.1 vector-mediated overexpression of SIRT1 attenuates insulin resistance in the high glucose-induced insulin-resistant skeleton muscle cells. These beneficial effects were associated with ameliorated mitochondrial dysfunction. Further studies have demonstrated that SIRT1 restores mitochondrial complex I activity leading to decreased oxidative stress and mitochondrial dysfunction. Furthermore, SIRT1 significantly elevated the level of another SIRT which is named SIRT3, and SIRT3 siRNA-suppressed SIRT1-induced mitochondria complex activity increments. Taken together, these results showed that SIRT1 improves insulin sensitivity via the amelioration of mitochondrial dysfunction, and this is achieved through the SIRT1–SIRT3–mitochondrial complex I pathway.     

Estimation of the mitochondrial redox state in human skeletal muscle during exercise

The mitochondrial redox (NAD+/NADH) state can be used as a reflection of oxygen availability within the mitochondrion. Previous studies using isolated muscle preparations suggest that active muscle is not hypoxic during lactate production, whereas experiments with humans come to the opposite conclusion. Six men exercised for 5 min at 75% maximal O2 consumption (VO2max) and then at 100% VO2max to exhaustion. Ammonia, oxoglutarate (alpha-ketoglutarate), and glutamate, as well as lactate, were measured in biopsies (vastus lateralis) taken at the end of each exercise. The three former metabolites were used to determine the mass action ratio of glutamate dehydrogenase and thus were used as an estimate of the mitochondrial redox state. Muscle lactate increased (P less than 0.05) to 14.5 and 24.5 mmol/kg wet wt after 75 and 100% VO2max, respectively. At both exercise intensities, muscle ammonia rose (P less than 0.05), glutamate fell (P less than 0.05) to only 30-35% of rest levels, and oxoglutarate declined (P less than 0.05). Despite the high levels of muscle lactate accumulation, the estimated mitochondrial redox rate rose 300% (P less than 0.05) in both exercise bouts. This response should increase the activity of key oxidative enzymes and promote increased VO2. Furthermore the data do not support the concept that muscle lactate is formed because of tissue hypoxia.