The pathogenic m.8993 T > G mutation in mitochondrial ATP6 gene prevents proton release from the subunit c-ring rotor of ATP synthase.

The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here, we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionary conservation.

The renal effects of droxidopa are maintained in propranolol treated cirrhotic rats.

BACKGROUND & AIMS: Droxidopa improves hemodynamic and renal alterations of cirrhotic rats without changing portal pressure. We aimed to evaluate the effects of a combined treatment with droxidopa and non-selective beta-blockers or statins in order to decrease portal pressure, while maintaining droxidopa beneficial effects. METHODS: Acute studies combining droxidopa with carvedilol, propranolol or atorvastatin in four-week bile-duct ligated (BDL) rats and a chronic study combining propranolol and droxidopa for 5 days in CCl4 -cirrhotic rats were performed. Hemodynamic values were registered and biochemical parameters from blood and urine samples analyzed. RESULTS: Bile-duct ligated rats treated with carvedilol + droxidopa showed no changes in mean arterial pressure (MAP) and portal pressure (PP) compared to vehicles. Atorvastatin + droxidopa combination also failed to reduce PP, but maintained the beneficial increase in MAP and superior mesenteric artery resistance (SMAR) and decrease in blood flow (SMABF) caused by droxidopa. In contrast, the acute administration of propranolol + droxidopa significantly reduced PP maintaining a mild increase in MAP and improving, in an additive way, the decrease in SMABF and increase in SMAR caused by droxidopa. This combination also preserved droxidopa diuretic effect. When chronically administered to CCl4 -cirrhotic rats, propranolol + droxidopa caused a decrease in PP, a significant reduction in SMABF and an increase in SMAR. The combination did not alter liver function and droxidopa diuretic and natriuretic effect, and even improved free water clearance. CONCLUSION: Droxidopa could be effective for the renal alterations of cirrhotic patients on propranolol therapy and the combination of both drugs may balance the adverse effects of each treatment.

Inhibition of neuronal apoptosis and axonal regression ameliorates sympathetic atrophy and hemodynamic alterations in portal hypertensive rats.

BACKGROUND AND AIM: A neuronal pathway participates in the development of portal hypertension: blockade of afferent sensory nerves in portal vein ligated (PVL) rats simultaneously prevents brain cardiovascular regularory nuclei activation, neuromodulator overexpression in superior mesenteric ganglia, sympathetic atrophy of mesenteric innervation and hemodynamic alterations. Here we investigated in PVL rats alterations in neuromodulators and signaling pathways leading to axonal regression or apoptosis in the superior mesenteric ganglia and tested the effects of the stimulation of neuronal proliferation/survival by using a tyrosine kinase receptor A agonist, gambogic amide. RESULTS: The neuronal pathway was confirmed by an increased neuronal afferent activity at the vagal nodose ganglia and the presence of semaphorin3A in sympathetic pre-ganglionic neurons at the intermediolateral nucleus of the spinal cord of PVL rats. Expression of the active form of tyrosine kinase receptor A (phosphorylated), leading to proliferation and survival signaling, showed a significant reduction in PVL comparing to sham rats. In contrast, the apoptotic and axonal retraction pathways were stimulated in PVL, demonstrated by a significant overexpression of semaphorin 3A and its receptor neuropilin1, together with increases of cleaved caspase7, inactive poly(ADP-ribose) polymerase and Rho kinase expression. Finally, the administration of gambogic amide in PVL rats showed an amelioration of hemodynamic alterations and sympathetic atrophy, through the activation of survival pathways together with the inhibition of apoptotic cascades and Rho kinase mediated axonal regression. CONCLUSION: The adrenergic alteration and sympathetic atrophy in mesenteric vessels during portal hypertension is caused by alterations on neuromodulation leading to post-ganglionic sympathetic regression and apoptosis and contributing to splanchnic vasodilation.

Defining the pathogenesis of human mtDNA mutations using a yeast model: the case of T8851C.

More and more mutations are found in the mitochondrial DNA of various patients but ascertaining their pathogenesis is often difficult. Due to the conservation of mitochondrial function from yeast to humans, the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and the amenability of the yeast mitochondrial genome to site-directed mutagenesis, yeast is an excellent model for investigating the consequences of specific human mtDNA mutations. Here we report the construction of a yeast model of a point mutation (T8851C) in the mitochondrially-encoded subunit a/6 of the ATP synthase that has been associated with bilateral striatal lesions, a group of rare human neurological disorders characterized by symmetric degeneration of the corpus striatum. The biochemical consequences of this mutation are unknown. The T8851C yeast displayed a very slow growth phenotype on non-fermentable carbon sources, both at 28°C (the optimal temperature for yeast growth) and at 36°C. Mitochondria from T8851C yeast grown in galactose at 28°C showed a 60% deficit in ATP production. When grown at 36°C the rate of ATP synthesis was below 5% that of the wild-type, indicating that heat renders the mutation much more deleterious. At both growth temperatures, the mutant F(1)F(o) complex was correctly assembled but had only very weak ATPase activity (about 10% that of the control), both in mitochondria and after purification. These findings indicate that a block in the proton-translocating domain of the ATP synthase is the primary cause of the neurological disorder in the patients carrying the T8851C mutation. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Droxidopa, an oral norepinephrine precursor, improves hemodynamic and renal alterations of portal hypertensive rats.

UNLABELLED: We aimed to evaluate the effects of droxidopa (an oral synthetic precursor of norepinephrine) on the hemodynamic and renal alterations of portal hypertensive rats. Sham, portal vein-ligated (PVL), and 4-week biliary duct-ligated (BDL) rats received a single oral dose of droxidopa (25-50 mg/kg) or vehicle and hemodynamic parameters were monitored for 2 hours. Two groups of BDL and cirrhotic rats induced by carbon tetrachloride (CCl(4) ) were treated for 5 days with droxidopa (15 mg/kg, twice daily, orally); hemodynamic parameters and blood and urinary parameters were assessed. The droxidopa effect on the Rho kinase (RhoK) / protein kinase B (AKT) / endothelial nitric oxide synthase (eNOS) pathways was analyzed by western blot in superior mesenteric artery (SMA). The acute administration of droxidopa in PVL and BDL rats caused a significant and maintained increase in arterial pressure and mesenteric arterial resistance, with a significant decrease of mesenteric arterial and portal blood flow, without changing portal pressure and renal blood flow. Two-hour diuresis greatly increased. Carbidopa (DOPA decarboxylase inhibitor) blunted all effects of droxidopa. Chronic droxidopa therapy in BDL rats produced the same beneficial hemodynamic effects observed in the acute study, did not alter liver function parameters, and caused a 50% increase in 24-hour diuresis volume (7.4 ± 0.9 mL/100g in BDL vehicle versus 11.8 ± 2.5 mL/100g in BDL droxidopa; P = 0.01). Droxidopa-treated rats also showed a decreased ratio of p-eNOS/eNOS and p-AKT/AKT and increased activity of RhoK in SMA. The same chronic treatment in CCl(4) rats caused similar hemodynamic effects and produced significant increases in diuresis volume and 24-hour natriuresis (0.08 ± 0.02 mmol/100g in CCl(4) vehicle versus 0.23 ± 0.03 mmol/100g in CCl(4) droxidopa; P = 0.014). CONCLUSION: Droxidopa might be an effective therapeutic agent for hemodynamic and renal alterations of liver cirrhosis and should be tested in cirrhosis patients.

Blockage of the afferent sensitive pathway prevents sympathetic atrophy and hemodynamic alterations in rat portal hypertension.

BACKGROUND AND AIMS: Portal hypertension causes arterial vasodilation and sympathetic atrophy in the splanchnic area. We aimed to demonstrate a relationship between hemodynamic alterations and sympathetic atrophy by investigating a pathway from sensitive afferent signals to mesenteric sympathetic ganglia. METHODS: Experiments were conducted in sham and portal vein ligated (PVL) adult and neonatal rats treated with vehicle or capsaicin. Hemodynamic parameters, and immunohistochemistry, immunofluorescence and Western blot of different tissues were analysed. RESULTS: cFos expression in the brain supraoptic nuclei was used to confirm abrogation of the afferent signal in capsaicin-treated PVL rats (effectively afferent blocked). Neonatal and adult PVL afferent blocked rats showed simultaneous prevention of hemodynamic alterations and sympathetic atrophy (measured by tyrosine hydroxylase expression in nerve structures of splanchnic vasculature). Not effectively afferent blocked rats showed none of these effects, behaving as PVL vehicle. All capsaicin treated animals presented loss of calcitonin gene-related peptide in superior mesenteric artery and ganglia, whereas neuronal nitric oxide synthase remained unaffected. Neuronal markers semaphorin-3A, nerve growth factor, its precursor and p75 neurotrophic receptor, were significantly over-expressed in the PVL sympathetic ganglia compared with sham, but not in effectively afferent blocked rats. Semaphorin-3A staining in mesenteric ganglia co-localized with vesicular acetylcholine transporter, but not with adrenergic, nitrergic and sensory axons, suggesting that semaphorin-3A might originate in preganglionic neurons. CONCLUSION: These results indicate that the nervous system has a central role in the genesis of the circulatory abnormalities of portal hypertension, and support that mesenteric sympathetic atrophy contributes to splanchnic arterial vasodilation.

A yeast-based assay identifies drugs active against human mitochondrial disorders.

Due to the lack of relevant animal models, development of effective treatments for human mitochondrial diseases has been limited. Here we establish a rapid, yeast-based assay to screen for drugs active against human inherited mitochondrial diseases affecting ATP synthase, in particular NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome. This method is based on the conservation of mitochondrial function from yeast to human, on the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and on the amenability of the yeast mitochondrial genome to site-directed mutagenesis. Our method identifies chlorhexidine by screening a chemical library and oleate through a candidate approach. We show that these molecules rescue a number of phenotypes resulting from mutations affecting ATP synthase in yeast. These compounds are also active on human cybrid cells derived from NARP patients. These results validate our method as an effective high-throughput screening approach to identify drugs active in the treatment of human ATP synthase disorders and suggest that this type of method could be applied to other mitochondrial diseases.

Physiopathology of splanchnic vasodilation in portal hypertension.

In liver cirrhosis, the circulatory hemodynamic alterations of portal hypertension significantly contribute to many of the clinical manifestations of the disease. In the physiopathology of this vascular alteration, mesenteric splanchnic vasodilation plays an essential role by initiating the hemodynamic process. Numerous studies performed in cirrhotic patients and animal models have shown that this splanchnic vasodilation is the result of an important increase in local and systemic vasodilators and the presence of a splanchnic vascular hyporesponsiveness to vasoconstrictors. Among the molecules and factors known to be potentially involved in this arterial vasodilation, nitric oxide seems to have a crucial role in the physiopathology of this vascular alteration. However, none of the wide variety of mediators can be described as solely responsible, since this phenomenon is multifactorial in origin. Moreover, angiogenesis and vascular remodeling processes also seem to play a role. Finally, the sympathetic nervous system is thought to be involved in the pathogenesis of the hyperdynamic circulation associated with portal hypertension, although the nature and extent of its role is not completely understood. In this review, we discuss the different mechanisms known to contribute to this complex phenomenon.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase.

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled alpha-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.

Atrophy of mesenteric sympathetic innervation may contribute to splanchnic vasodilation in rat portal hypertension.

BACKGROUND AND AIMS: Portal hypertension is associated with downregulation of mRNA and proteins involved in adrenergic transmission in the superior mesenteric artery (SMA) in portal vein-ligated (PVL) and cirrhotic rats. We aimed to investigate whether SMA adrenergic dysfunction was accompanied by sympathetic nerve structural changes and whether it was extensive to resistance mesenteric arteries. We also attempted to localize the origin of mRNA of specific adrenergic genes. METHODS AND RESULTS: In situ hybridization showed tyrosine hydroxylase (Th) mRNA expression in neuronal bodies of superior mesenteric ganglia and inside axonal fibres surrounding proximal SMA sections. Comparison of SMA by Th immunohistochemistry, both in PVL and bile duct-ligated (BDL) rats, demonstrated a significant decrease in the number of nervous structures (69% PVL; 62% BDL), total nervous area (70% PVL; 52% BDL) and Th-stained nervous area (89% PVL; 64% BDL) compared with sham rats. A strong correlation was detected between the Th-stained nervous area and the haemodynamic parameters, mainly with SMA resistance (r=0.9, P<0.001 for PVL and r=0.75, P=0.018 for BDL). Western blot analysis of Th, dopamine beta-hydroxylase and synaptosome-associated protein of 25 kDa indicated a significant inhibition in protein expression (35-58%) in mesenteric resistance arteries from both portal hypertension models compared with sham. By contrast, nervous structure analysis and protein expression in renal arteries showed no differences between sham and PVL rats. CONCLUSION: Portal hypertension is associated with sympathetic nerve atrophy/regression in the mesenteric arterial vasculature that could contribute to the splanchnic vasodilation associated with portal hypertension.

A yeast model of the neurogenic ataxia retinitis pigmentosa (NARP) T8993G mutation in the mitochondrial ATP synthase-6 gene.

NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F(1) coupled to F(0)-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.