Correlation between genetic polymorphisms and stroke recovery: analysis of the GAIN Americas and GAIN International Studies

BACKGROUND AND PURPOSE: Recovery after stroke occurs on the basis of specific molecular events. Genetic polymorphisms associated with impaired neural repair or plasticity might reduce recovery from stroke and might also account for some of the intersubject variability in stroke recovery. This study hypothesized that the ApoE epsilon4 polymorphism and the val(66) met polymorphism for brain-derived neurotrophic factor (BDNF) are each associated with poorer outcome after stroke. Associations with mitochondrial genotype were also explored. METHODS: Genotypes were determined in 255 stroke patients who also received behavioral evaluations in the Glycine Antagonist In Neuroprotection (GAIN) clinical trials. The primary outcome measure was recovery during the first month post-stroke, as this is the time when neural repair is at a maximum and so when genetic influences might have their largest impact. Two secondary outcome measures at 3 months post-stroke were also examined. RESULTS: Genotype groups were similar acutely post-stroke. Presence of the ApoE epsilon4 polymorphism was associated with significantly poorer recovery over the first month post-stroke (P = 0.023) and with a lower proportion of subjects with minimal or no disability (modified Rankin score 0-1, P = 0.01) at 3 months post-stroke. Indeed, those with this polymorphism were approximately half as likely to achieve minimal or no disability (18.2%) versus those with polymorphism absent (35.5%). Findings were confirmed in multivariate models. Results suggested possible effects from the val(66) met BDNF polymorphism and from the R0 mitochondrial DNA haplotype. CONCLUSIONS: Genetic factors, particularly the ApoE epsilon4 polymorphism, might contribute to variability in outcomes after stroke

Mitochondrial energetics and therapeutics

Mitochondrial dysfunction has been linked to a wide range of degenerative and metabolic diseases, cancer, and aging. All these clinical manifestations arise from the central role of bioenergetics in cell biology. Although genetic therapies are maturing as the rules of bioenergetic genetics are clarified, metabolic therapies have been ineffectual. This failure results from our limited appreciation of the role of bioenergetics as the interface between the environment and the cell. A systems approach, which, ironically, was first successfully applied over 80 years ago with the introduction of the ketogenic diet, is required. Analysis of the many ways that a shift from carbohydrate glycolytic metabolism to fatty acid and ketone oxidative metabolism may modulate metabolism, signal transduction pathways, and the epigenome gives us an appreciation of the ketogenic diet and the potential for bioenergetic therapeutics

Correlation between genetic polymorphisms and stroke recovery: analysis of the GAIN Americas and GAIN International Studies.

Background and purposeRecovery after stroke occurs on the basis of specific molecular events. Genetic polymorphisms associated with impaired neural repair or plasticity might reduce recovery from stroke and might also account for some of the intersubject variability in stroke recovery. This study hypothesized that the ApoE ε4 polymorphism and the val(66) met polymorphism for brain-derived neurotrophic factor (BDNF) are each associated with poorer outcome after stroke. Associations with mitochondrial genotype were also explored.MethodsGenotypes were determined in 255 stroke patients who also received behavioral evaluations in the Glycine Antagonist In Neuroprotection (GAIN) clinical trials. The primary outcome measure was recovery during the first month post-stroke, as this is the time when neural repair is at a maximum and so when genetic influences might have their largest impact. Two secondary outcome measures at 3 months post-stroke were also examined.Results  Genotype groups were similar acutely post-stroke. Presence of the ApoE ε4 polymorphism was associated with significantly poorer recovery over the first month post-stroke (P = 0.023) and with a lower proportion of subjects with minimal or no disability (modified Rankin score 0-1, P = 0.01) at 3 months post-stroke. Indeed, those with this polymorphism were approximately half as likely to achieve minimal or no disability (18.2%) versus those with polymorphism absent (35.5%). Findings were confirmed in multivariate models. Results suggested possible effects from the val(66) met BDNF polymorphism and from the R0 mitochondrial DNA haplotype.Conclusions  Genetic factors, particularly the ApoE ε4 polymorphism, might contribute to variability in outcomes after stroke

Genomic and non-genomic regulation of PGC1 isoforms by estrogen to increase cerebral vascular mitochondrial biogenesis and reactive oxygen species protection

We previously found that estrogen exerts a novel protective effect on mitochondria in brain vasculature. Here we demonstrate in rat cerebral blood vessels that 17beta-estradiol (estrogen), both in vivo and ex vivo, affects key transcriptional coactivators responsible for mitochondrial regulation. Treatment of ovariectomized rats with estrogen in vivo lowered mRNA levels of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1alpha) but increased levels of the other PGC-1 isoforms: PGC-1beta and PGC-1 related coactivator (PRC). In vessels ex vivo, estrogen decreased protein levels of PGC-1alpha via activation of phosphatidylinositol 3-kinase (PI3K). Estrogen treatment also increased phosphorylation of forkhead transcription factor, FoxO1, a known pathway for PGC-1alpha downregulation. In contrast to the decrease in PGC-1alpha, estrogen increased protein levels of nuclear respiratory factor 1, a known PGC target and mediator of mitochondrial biogenesis. The latter effect of estrogen was independent of PI3K, suggesting a separate mechanism consistent with increased expression of PGC-1beta and PRC. We demonstrated increased mitochondrial biogenesis following estrogen treatment in vivo; cerebrovascular levels of mitochondrial transcription factor A and electron transport chain subunits as well as the mitochondrial/nuclear DNA ratio were increased. We examined a downstream target of PGC-1beta, glutamate-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. In vivo estrogen increased protein levels of both GCL subunits and total glutathione levels. Together these data show estrogen differentially regulates PGC-1 isoforms in brain vasculature, underscoring the importance of these coactivators in adapting mitochondria in specific tissues. By upregulating PGC-1beta and/or PRC, estrogen appears to enhance mitochondrial biogenesis, function and reactive oxygen species protection

Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants

The mitochondrial DNA (mtDNA) encompasses two classes of functionally important sequence variants: recent pathogenic mutations and ancient adaptive polymorphisms. To rapidly and cheaply evaluate both classes of single nucleotide variants (SNVs), we have developed an integrated system in which mtDNA SNVs are analyzed by multiplex primer extension using the SNaPshot system. A multiplex PCR amplification strategy was used to amplify the entire mtDNA, a computer program identifies optimal extension primers, and a complete global haplotyping system is also proposed. This system genotypes SNVs on multiplexed mtDNA PCR products or directly from enriched mtDNA samples and can quantify heteroplasmic variants down to 0.8% using a standard curve. With this system, we have developed assays for testing the common pathogenic mutations in four multiplex panels: two genotype the 13 most common pathogenic mtDNA mutations and two genotype the 10 most common Leber Hereditary Optic Neuropathy mutations along with haplogroups J and T. We use a hierarchal system of 140 SNVs to delineate the major global mtDNA haplogroups based on a global phylogenetic tree of coding region polymorphisms. This system should permit rapid and inexpensive genotyping of pathogenic and lineage-specific mtDNA SNVs by clinical and research laboratories

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L-L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination

Increased prevalence of val(66)met BDNF genotype among subjects with cervical dystonia

Abnormalities of cortical representational maps and their plasticity have been described in dystonia. A common polymorphism for BDNF has been associated with abnormal cortical plasticity, and thus might contribute to pathogenesis of dystonia in some subjects. As a first step towards this suggestion, the current study examined the prevalence of this polymorphism. BDNF genotype was examined in 34 subjects with cervical dystonia, 54 age-matched healthy controls, and 53 subjects with a different movement disorder, Parkinson\u27s disease. ApoE genotype, known to influence neurological outcome in some conditions, was also examined as a control. In subjects with cervical dystonia, the val(66)met polymorphism was approximately twice as prevalent when compared to either control group. This was not true of ApoE genotype, which was similarly distributed across subject groups. The current findings suggest that the BDNF val(66)met polymorphism might play a role in the pathogenesis of cervical dystonia in some subjects

Heme oxygenase 1 is differentially involved in blood flow-dependent arterial remodeling: role of inflammation, oxidative stress, and nitric oxide

Heme oxygenase 1 is induced by hemodynamic forces in vascular smooth muscle and endothelial cells. We investigated the involvement of heme oxygenase 1 in flow (shear stress)-dependent remodeling. Two or 14 days after ligation of mesenteric resistance arteries, vessels were isolated. In rats, at 14 days, diameter increased by 23% in high-flow arteries and decreased by 22% in low-flow arteries compared with normal flow vessels. Heme oxygenase activity inhibition using Tin-protoporphyrin abolished diameter enlargement in high-flow arteries and accentuated arterial narrowing in low-flow arteries (32% diameter decrease versus 22% in control). Two days after ligation, heme oxygenase 1 expression increased in high-flow and low-flow vessels, in association with a reduced mitochondrial aconitase activity (marker of oxidative stress) in high-flow arteries only. Inhibition of macrophage infiltration (clodronate) decreased heme oxygenase 1 induction in low-flow but not in high-flow arteries. Similarly, inhibition of NADPH oxidase activity (apocynin) decreased heme oxygenase 1 induction in low-flow but not high-flow arteries. However, dihydroethidium staining was higher in high-flow and low-flow compared with normal flow arteries. In arteries cannulated in an arteriograph, heme oxygenase 1 mRNA increased in a flow-dependent manner and was abolished by N(G)-nitro-l-arginine methyl ester, catalase, or mitochondrial electron transport chain inhibition. Furthermore, heme oxygenase 1 induction using cobalt-protoporphyrin restored altered high-flow remodeling in endothelial NO synthase knockout mice. Thus, in high-flow remodeling, heme oxygenase 1 induction depends on shear stress-generated NO and mitochondria-derived hydrogen peroxide. In low-flow remodeling, heme oxygenase 1 induction requires macrophage infiltration and is mediated by NADPH oxidase-derived superoxide

Iron deficiency without anemia is responsible for decreased left ventricular function and reduced mitochondrial complex I activity in a mouse model

BACKGROUND: Iron deficiency (ID), with or without anemia, is frequent in heart failure patients, and iron supplementation improves patient condition. However, the link between ID (independently of anemia) and cardiac function is poorly understood, but could be explained by an impaired mitochondrial metabolism. Our aim was to explore this hypothesis in a mouse model. METHODS AND RESULTS: We developed a mouse model of ID without anemia, using a blood withdrawal followed by 3-weeks low iron diet. ID was confirmed by low spleen, liver and heart iron contents and the repression of HAMP gene coding for hepcidin. ID was corrected by a single ferric carboxymaltose (FCM) injection (ID + FCM mice). Hemoglobin levels were similar in ID, ID + FCM and control mice. ID mice had impaired physical performances and left ventricular function (echocardiography). Mitochondrial complex I activity of cardiomyocytes was significantly decreased in ID mice, but not complexes II, III and IV activities. ID + FCM mice had improved physical performance, cardiac function and complex I activity compared to ID mice. Using BN-PAGE, we did not observe complex I disassembly, but a reduced quantity of the whole enzyme complex I in ID mice, that was restored in ID + FCM mice. CONCLUSIONS: ID, independently of anemia, is responsible for a decreased left ventricular function, through a reduction in mitochondrial complex I activity, probably secondary to a decrease in complex I quantity. These abnormalities are reversed after iron treatment, and may explain, at least in part, the benefit of iron supplementation in heart failure patients with ID

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