TY - JOUR
AU - Marobbio, Carlo M T
AB - Abstract Friedreich ataxia (FRDA) is an inherited recessive disorder caused by a deficiency in the mitochondrial protein frataxin. There is currently no effective treatment for FRDA available, especially for neurological deficits. In this study, we tested diazoxide, a drug commonly used as vasodilator in the treatment of acute hypertension, on cellular and animal models of FRDA. We first showed that diazoxide increases frataxin protein levels in FRDA lymphoblastoid cell lines, via the mammalian target of rapamycin (mTOR) pathway. We then explored the potential therapeutic effect of diazoxide in frataxin-deficient transgenic YG8sR mice and we found that prolonged oral administration of 3 mpk/d diazoxide was found to be safe, but produced variable effects concerning efficacy. YG8sR mice showed improved beam walk coordination abilities and footprint stride patterns, but a generally reduced locomotor activity. Moreover, they showed significantly increased frataxin expression, improved aconitase activity, and decreased protein oxidation in cerebellum and brain mitochondrial tissue extracts. Further studies are needed before this drug should be considered for FRDA clinical trials. Introduction Friedreich ataxia (FRDA, OMIM 229300) is an autosomal recessive disorder due to an increase in the number of GAA trinucleotide repeats within the first intron of the FXN gene on chromosome 9 (1). Normal individuals have 5–30 GAA repeats, whereas affected individuals have 70 to >1000 GAA triplet expansions, causing decreased gene expression. FRDA occurs with a prevalence of ∼1/50 000 in Caucasian populations and it is characterized clinically by an onset in the first two decades of life with limb ataxia, cerebellar dysarthria, lack of deep-tendon reflexes, pyramidal signs, and sensory loss. Most patients have degeneration of the sensory neurons in the dorsal root ganglia (DRG) and cerebellum, together with skeletal deformities and hypertrophic cardiomyopathy. There is also an increased incidence of blindness, deafness, and diabetes mellitus, suggesting that the disorder is not limited to the nervous system. The severity of clinical features in FRDA is related to the GAA trinucleotide repeat length (2). Symptoms begin usually in childhood (5–15 years), but onset may vary from infancy to adulthood (3). The FXN gene encodes the mitochondrial protein frataxin, which is involved in mitochondrial iron metabolism, assembly of iron–sulphur proteins and haeme synthesis (4). The exact physiological function of frataxin is unknown, but there is general agreement that it is involved in cellular iron homeostasis and that its deficiency results in multiple enzyme deficits, mitochondrial dysfunction and oxidative damage (5–7). There is no available effective treatment for FRDA, especially for neurological features. In fact, antioxidant agents, such as idebenone, a short-chain analogue of coenzyme Q10, and iron chelators, which have been used as potential therapeutic agents, ameliorate mitochondrial oxidative stress but have no effects on neurological symptoms (8). Since the disorder is caused by deficiency of FXN gene expression, a possible therapeutic strategy could be the stimulation of frataxin expression with exogenous substances to increase its level (9). Several molecules are reported to increase frataxin mRNA or protein levels. However, no clear results supporting a benefit of any of these drugs have so far been obtained in randomized-controlled trials (10). Here, we study the effect of diazoxide on cell lines from FRDA patients and on the YG8sR FRDA mouse model (11). Diazoxide is a potassium channel opener, active mainly on the mitochondrial ATP-sensitive potassium channel (mito-KATP channel) (12), which has been in use for over 50 years as a vasodilator in the treatment of acute hypertension (13). It is the active ingredient in Proglycem, a nondiuretic benzothiadiazine derivative taken orally for the management of symptomatic hypoglycaemia (14). Particularly, we studied the effect of diazoxide, to increase frataxin expression levels and to ameliorate the functional and biochemical features of a FRDA mouse model. Results Diazoxide induces frataxin expression in FRDA patient lymphoblastoid cells We analysed three lymphoblastoid cell lines from FRDA patients (P119, P200 and P338) with different GAA repeat expansions representing: (i) a mild phenotype with late onset and 170/230 GAA repeats (P338); (ii) a canonical phenotype with 500/800 GAA repeats (P119) and (iii) a severe phenotype with early onset and 1100/1100 GAA repeats (P200). As shown in Figure 1A, the amount of frataxin protein was virtually undetectable in each of the cell extracts (20 µg). First, we test the effect of different diazoxide concentrations on cell viability and we found no toxicity of diazoxide for all cell lines unless they were incubated in the presence of 400 µm or higher concentrations, for at least 4 days (Fig. 1B). Frataxin expression was measured upon incubating cell cultures in presence of 100 μm diazoxide (D), 100 μm diazoxide and 20 mm glucose (GD) or vehicle alone (0.1% dimethyl sulfoxide, DMSO) (NT). Cells were collected after 4 days of treatment. We used glucose in combination with diazoxide because the effect of mitoKATP channel openers is exerted only on the ATP-bound potassium channel and depends by the energetic state of the cells (12). Immunoblots of total cell lysates revealed a significant frataxin increase for all cell lines with D and DG treatments, compared with vehicle-treated cells (NT), ranging from 80 to 300% induction (Fig. 2A and B). The addition of glucose produced minimal increases in frataxin expression compared with diazoxide alone (Fig. 2A). Immunoblots of frataxin from total cell lysates of healthy control cell line revealed no significant differences (Fig. 2C). Figure 1. View largeDownload slide  100 µm diazoxide is not toxic for FRDA lymphoblastoid cells. (A) Immunoblot of FXN from 20 µg total cell lysates of lymphoblasts of three FRDA patients (P119, P200 and P338) and healthy control (C). Subunit β of ATP synthase was used as loading control. (B) Cell viability of lymphoblast cell lines. Lymphoblasts from three FRDA patients (P119, P200 and P338) were treated for 4 days with different diazoxide concentrations. Results demonstrated that 100 µm diazoxide is not toxic for cells. Results expressed as mean ± SD (*P < 0.05, **P < 0.01 Student’s t test, n = 4). Figure 1. View largeDownload slide  100 µm diazoxide is not toxic for FRDA lymphoblastoid cells. (A) Immunoblot of FXN from 20 µg total cell lysates of lymphoblasts of three FRDA patients (P119, P200 and P338) and healthy control (C). Subunit β of ATP synthase was used as loading control. (B) Cell viability of lymphoblast cell lines. Lymphoblasts from three FRDA patients (P119, P200 and P338) were treated for 4 days with different diazoxide concentrations. Results demonstrated that 100 µm diazoxide is not toxic for cells. Results expressed as mean ± SD (*P < 0.05, **P < 0.01 Student’s t test, n = 4). Figure 2. View largeDownload slide  Diazoxide induces FXN in cultured FRDA lymphoblastoid cells. (A) Lymphoblasts from three FRDA patients (P119, P200 and P338) were incubated in presence of 100 μm diazoxide (D), 100 μm diazoxide and 20 mm glucose (GD), or vehicle alone (0.1% DMSO) (NT). Total protein was collected after 4 days, and lysates (80 µg) were probed by western blot analysis for FXN expression and normalized to subunit β of ATP synthase. The plotted data represent the mean fold change in FXN protein in drug-treated cells, normalized to vehicle control (*P < 0.05, **P < 0.01 Student’s t test, n = 4). (B and C) Representative blots are shown for FRDA patient (B) or healthy control (C) lymphoblast cell lines treated as above. Figure 2. View largeDownload slide  Diazoxide induces FXN in cultured FRDA lymphoblastoid cells. (A) Lymphoblasts from three FRDA patients (P119, P200 and P338) were incubated in presence of 100 μm diazoxide (D), 100 μm diazoxide and 20 mm glucose (GD), or vehicle alone (0.1% DMSO) (NT). Total protein was collected after 4 days, and lysates (80 µg) were probed by western blot analysis for FXN expression and normalized to subunit β of ATP synthase. The plotted data represent the mean fold change in FXN protein in drug-treated cells, normalized to vehicle control (*P < 0.05, **P < 0.01 Student’s t test, n = 4). (B and C) Representative blots are shown for FRDA patient (B) or healthy control (C) lymphoblast cell lines treated as above. Diazoxide induces frataxin expression and decreases oxidative cell damage in YG8sR FRDA mouse model To test the effect of diazoxide on living organism, we used the YG8sR FRDA mouse model (11). This model is a new line of GAA-repeat-expansion-based FRDA mice derived from YG8R breeding, which contains a single copy of the human FXN transgene and a single pure GAA repeat expansion mutation, which was 120 GAA repeats in size in the founder mouse. These mice exhibit an age-dependent GAA repeat expansion accumulation in the CNS, particularly the cerebellum, progressive decrease in motor coordination and significant functional locomotor deficits, features similar to those observed in FRDA patients. In addition, the YG8sR mice exhibit the presence of pathological vacuoles within neurons of the DRG, together with reduced levels of brain aconitase activity, in line with an FRDA-like phenotype. Therefore, these YG8sR mice currently represent the most suitable GAA-repeat-based YAC transgenic mouse model to explore potential FRDA therapies. First, we observed that treatment of YG8sR mice p.o. daily for 5 days with 10 mg/kg diazoxide was safe and no adverse effects were observed with any mice. Since short-term treatment did not produce neither down-stream effects (data not shown), nor an increase of frataxin expression (data not shown) we extended the period of diazoxide treatment to 3 months. Groups of 20 YG8sR mice (10 male and 10 female, 4-months old) were treated p.o. daily for 3 months with either vehicle (1% DMSO) or 3 mg/kg diazoxide, followed by collection of snap-frozen brain, cerebellum, heart, liver and pancreas tissues. Quantitative RT-PCR (qRT-PCR) analysis of FXN mRNA expression of cerebellum and heart tissues from YG8sR mice revealed statistically significant differences between diazoxide-treated and vehicle-treated groups of mice (P < 0.05) (Fig. 3A), which translated into 2.6- and 1.6-fold increases in FXN expression levels, respectively, in diazoxide-treated mice (Fig. 3B). Figure 3. View largeDownload slide  Diazoxide induces FXN and NRF2 expression in YG8sR cerebellum and heart tissues. QRT-PCR analysis of transgenic FXN and NRF2 mRNA levels in YG8sR cerebellum and heart tissues. Expression data (A) and fold changes (B) of FXN and NRF2 mRNA levels of diazoxide-treated tissues (D) relative to those of vehicle-treated tissues (V), using the formula x = 2(ΔCt) and adjusting for input cDNA concentrations. Figure 3. View largeDownload slide  Diazoxide induces FXN and NRF2 expression in YG8sR cerebellum and heart tissues. QRT-PCR analysis of transgenic FXN and NRF2 mRNA levels in YG8sR cerebellum and heart tissues. Expression data (A) and fold changes (B) of FXN and NRF2 mRNA levels of diazoxide-treated tissues (D) relative to those of vehicle-treated tissues (V), using the formula x = 2(ΔCt) and adjusting for input cDNA concentrations. Similarly, significant 1.4- to 2.8-fold increases in frataxin protein levels were detected by western blot densitometry analysis of mitochondrial extracts from heart (P < 0.01), cerebellum (P < 0.05) and brain (P < 0.05) tissues from YG8sR mice (Fig. 4A and B). We also tested the activity of mitochondrial aconitase, an iron–sulphur protein involved in iron homeostasis that is deficient in FRDA cells. A significant increase in aconitase activity, was detected in YG8sR brain tissue (P < 0.05), together with trends to increase in other tissues (Fig. 5A). Furthermore, oxyblot analysis revealed a significant reduction (P < 0.05) in protein oxidation levels in YG8sR brain, pancreas and liver tissues and trends to decrease in other tissues (Fig. 5B). Figure 4. View largeDownload slide  Diazoxide induces FXN in YG8sR cerebellum, brain and heart tissues. (A) Tissues from a group of treated (n = 8) or untreated (n = 8) mice were analysed for the FXN protein expression. A total of 20 µg mitochondrial protein extracts from cerebellum (C), brain (B), heart (H), pancreas (P) and liver (L) tissues of YG8sR mice treated with p.o. daily for 3 months with either vehicle (1% DMSO) (open bars) or 3 mg/kg diazoxide (grey bars) were probed by western blot analysis for FXN expression and normalized to subunit β of ATP synthase. The plotted data represent the mean fold change in FXN protein in drug-treated tissues, normalized to vehicle control in three independent experiments (**P < 0.01, *P < 0.05 Student’s t test, n = 3). (B) A representative blot is shown for cerebellum (C), brain (B), heart (H), pancreas (P) and liver (L) tissues of YG8sR mice treated as above. Figure 4. View largeDownload slide  Diazoxide induces FXN in YG8sR cerebellum, brain and heart tissues. (A) Tissues from a group of treated (n = 8) or untreated (n = 8) mice were analysed for the FXN protein expression. A total of 20 µg mitochondrial protein extracts from cerebellum (C), brain (B), heart (H), pancreas (P) and liver (L) tissues of YG8sR mice treated with p.o. daily for 3 months with either vehicle (1% DMSO) (open bars) or 3 mg/kg diazoxide (grey bars) were probed by western blot analysis for FXN expression and normalized to subunit β of ATP synthase. The plotted data represent the mean fold change in FXN protein in drug-treated tissues, normalized to vehicle control in three independent experiments (**P < 0.01, *P < 0.05 Student’s t test, n = 3). (B) A representative blot is shown for cerebellum (C), brain (B), heart (H), pancreas (P) and liver (L) tissues of YG8sR mice treated as above. Figure 5. View largeDownload slide  Diazoxide decreases oxidative stress in YG8sR mice. (A) Mitochondria purified from brain, heart, pancreas and liver tissues of YG8sR mice treated with p.o. daily for 3 months with either vehicle (1% DMSO) (open bars) or 3 mg/kg diazoxide (grey bars) were tested (A) for aconitase activity (**P < 0.01, Student’s t test, n = 3), or (B) for oxyblot analysis (**P < 0.01, Student’s t test, n = 3). Figure 5. View largeDownload slide  Diazoxide decreases oxidative stress in YG8sR mice. (A) Mitochondria purified from brain, heart, pancreas and liver tissues of YG8sR mice treated with p.o. daily for 3 months with either vehicle (1% DMSO) (open bars) or 3 mg/kg diazoxide (grey bars) were tested (A) for aconitase activity (**P < 0.01, Student’s t test, n = 3), or (B) for oxyblot analysis (**P < 0.01, Student’s t test, n = 3). Diazoxide effect on functional behaviour of the YG8sR FRDA mouse model To investigate the ability of oral diazoxide to ameliorate the functional behaviour of the YG8sR FRDA mouse model, groups of 20 mice (10 male and 10 female) were given either 3 mg/kg/d diazoxide or vehicle (1% DMSO) p.o. ad libitum in drinking water for a period of 3 months from ∼4 months of age. This dose of diazoxide was well tolerated and no adverse effects were observed with any mice. Furthermore, there were no significant differences in weight detected between diazoxide-treated (D) and vehicle-treated (V) groups of YG8sR mice (Supplementary Material, Fig. S1). Functional behavioural studies were performed on all mice, including rotarod analysis (monthly), beam breaker locomotor open field testing (monthly), beam walking (start and end of study) and footprint analysis (start and end of study). Rotarod analysis did not reveal any significant improvement in the performance of YG8sR mice throughout 3 months of treatment with diazoxide compared with vehicle treatment (Fig. 6A). Beam breaker locomotor analysis identified a general slowing down of movement of diazoxide-treated YG8sR mice compared with vehicle-treated mice from 5–7 months of age, as exemplified by the relative reductions of average velocity (two-way analysis of variance (ANOVA) P < 0.01), no change in the amount of jumping (two-way ANOVA P < 0.01), but an increase in the amount of rearing up onto hind limbs, as shown by a relative increase in vertical counts (two-way ANOVA P < 0.001) and in vertical time on diazoxide treatment (two-way analysis of variance (ANOVA) P < 0.01), from months 1 to 3 of the study (Fig. 6B–E). Beam walk analysis revealed the most significant diazoxide-induced functional effect for YG8sR mice (Fig. 7A). The time taken for mice to cross a 22-mm diameter 90-cm long beam significantly increased over the 3-month test period for vehicle-treated YG8sR mice, indicating an increased difficulty in performing this task with age. Interestingly, the time taken to cross the beam significantly decreased over the 3-month time period for diazoxide-treated YG8sR mice and there were significant differences between diazoxide- and vehicle-treated groups at the 3 months time point, indicating much improved coordination abilities of YG8sR mice (Fig. 7A). Footprint analysis revealed a significant reduction in the stride length of diazoxide-treated YG8sR mice compared with vehicle-treated YG8sR mice over the 3-month test period (Fig. 7B, Supplementary Material, Fig. S2), whereas the hind limb base width for YG8sR mice was not affected by diazoxide treatment (Supplementary Material, Fig. S2 and S3). Figure 6. View largeDownload slide  Diazoxide reduces locomotor activity of YG8sR mice. (A) Rotarod performance of mice, measured at monthly intervals. Beam breaker locomotor analysis at monthly intervals, measured as fold changes in performance from months 1 to 3. (B) average velocity; (C) jump counts; (D) vertical counts; (E) vertical time. Each parameter was measured four times for each mouse (*P < 0.05, ***P < 0.001, Student’s t test, n = 20). Figure 6. View largeDownload slide  Diazoxide reduces locomotor activity of YG8sR mice. (A) Rotarod performance of mice, measured at monthly intervals. Beam breaker locomotor analysis at monthly intervals, measured as fold changes in performance from months 1 to 3. (B) average velocity; (C) jump counts; (D) vertical counts; (E) vertical time. Each parameter was measured four times for each mouse (*P < 0.05, ***P < 0.001, Student’s t test, n = 20). Figure 7. View largeDownload slide  Diazoxide improves YG8sR mice coordination. (A) Beam walk analysis, determining the average times taken for mice to cross a 22-mm diameter beam, before and after diazoxide or vehicle treatment. (B) Footprint analysis, determining the average stride length of mice, before and after diazoxide or vehicle treatment. Each parameter was measured four times for each mouse (*P < 0.05, ***P < 0.001, Student’s t test, n = 20). Figure 7. View largeDownload slide  Diazoxide improves YG8sR mice coordination. (A) Beam walk analysis, determining the average times taken for mice to cross a 22-mm diameter beam, before and after diazoxide or vehicle treatment. (B) Footprint analysis, determining the average stride length of mice, before and after diazoxide or vehicle treatment. Each parameter was measured four times for each mouse (*P < 0.05, ***P < 0.001, Student’s t test, n = 20). Diazoxide activates the mTOR pathway and promotes nuclear Nrf2 translocation Literature data showed the ability of diazoxide to activate mTOR kinases (15). To investigate the involvement of the mTOR pathway in the FXN expression, we first analysed the activation of mTOR kinases in our cell lines. The amount of phosphorylated form of S6K, a target of mTOR kinase (16), was increased after diazoxide treatment (Fig. 8A), confirming the positive effect of 100 µm diazoxide on mTOR pathway. This increase was almost abolished by the addition of 2 nm rapamycin (Fig. 8A), a well-known inhibitor of mTOR kinases (17). To link this effect to the FXN expression, we analysed the amount of FXN in diazoxide-induced cell cultures, by using rapamycin. We found that treatment with 2 nm rapamycin completely inhibited the frataxin-increasing effect elicited by diazoxide (Fig. 8B). The antioxidative response exerted by the activation of mTOR pathway has been recently linked to the activation of nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor ubiquitously expressed that regulates cellular redox homeostasis (18). We observed that induction of frataxin expression is mediated by NRF2 transcription factor, because diazoxide treatment also increased the NRF2 expression in cerebellum tissues of mouse model (Fig. 3). In order to elucidate the effect of diazoxide on the activation of antioxidative pathways exerted by nuclear NRF2 in lymphoblastoid cell lines, we performed an analysis of Nrf2 nuclear translocation. Immunoblotting analysis, 24 h after diazoxide treatment, demonstrated an increase of Nrf2 signal in the nuclear fraction of lymphoblastoid cells treated with diazoxide (D) or with diazoxide and glucose (GD) (Fig. 8C and D). We observed a significant increase (P < 0.05) of 1.62 ± 0.20 and 1.66 ± 0.28% in the relative nNRF2/cNRF2 signals in D and GD samples, respectively (Fig. 8C and D). Figure 8. View largeDownload slide  Diazoxide induces FXN expression through mTOR pathway and NRF2 nuclear translocation in cultured FRDA lymphoblastoid cells. (A) The induction of mTOR pathway exerted by diazoxide was tested by treating lymphoblast cell lines from FRDA patient (P200) with 100 μm diazoxide (D), 100 μm diazoxide and 20 mm glucose (DG), 100 μm diazoxide, 20 mm glucose and 2 nm rapamycin (DGR), or vehicle alone (0.1% DMSO) (NT). 20 µg of proteins from cellular or nuclear fractions were separated by SDS-PAGE and transferred to nitrocellulose membranes and probed for phosphorylated form of S6K and normalized to subunit β of ATP synthase. (B) To test if the mTOR kinases are involved in the mechanism of FXN induction, lymphoblast cell lines from FRDA patient (P200) were treated with 100 μm diazoxide and 20 mm glucose (GD), 100 μm diazoxide, 20 mm glucose and 2 nm rapamycin (GDR), 100 μm diazoxide (D), 100 μm diazoxide and 2 nm rapamycin (DR), or vehicle alone (0.1% DMSO) (NT). Total protein was collected after 4 days, and lysates were probed by western blot analysis for FXN expression and normalized to subunit β of ATP synthase. (C) For the analysis of Nrf2 nuclear translocation, lymphoblast cell lines from FRDA patient (P200) were treated with 100 μm diazoxide (D), 100 μm diazoxide and 20 mm glucose (DG), or vehicle alone (0.1% DMSO) (NT) for 24 h. Cellular and nuclear fractions were normalized to subunit β of ATP synthase and lamin A, respectively. (D) The ratio of Nrf2 nuclear and cellular content of samples treated as above was calculated and displayed in the histograms (*P < 0.05, Student’s t test, n = 3). Figure 8. View largeDownload slide  Diazoxide induces FXN expression through mTOR pathway and NRF2 nuclear translocation in cultured FRDA lymphoblastoid cells. (A) The induction of mTOR pathway exerted by diazoxide was tested by treating lymphoblast cell lines from FRDA patient (P200) with 100 μm diazoxide (D), 100 μm diazoxide and 20 mm glucose (DG), 100 μm diazoxide, 20 mm glucose and 2 nm rapamycin (DGR), or vehicle alone (0.1% DMSO) (NT). 20 µg of proteins from cellular or nuclear fractions were separated by SDS-PAGE and transferred to nitrocellulose membranes and probed for phosphorylated form of S6K and normalized to subunit β of ATP synthase. (B) To test if the mTOR kinases are involved in the mechanism of FXN induction, lymphoblast cell lines from FRDA patient (P200) were treated with 100 μm diazoxide and 20 mm glucose (GD), 100 μm diazoxide, 20 mm glucose and 2 nm rapamycin (GDR), 100 μm diazoxide (D), 100 μm diazoxide and 2 nm rapamycin (DR), or vehicle alone (0.1% DMSO) (NT). Total protein was collected after 4 days, and lysates were probed by western blot analysis for FXN expression and normalized to subunit β of ATP synthase. (C) For the analysis of Nrf2 nuclear translocation, lymphoblast cell lines from FRDA patient (P200) were treated with 100 μm diazoxide (D), 100 μm diazoxide and 20 mm glucose (DG), or vehicle alone (0.1% DMSO) (NT) for 24 h. Cellular and nuclear fractions were normalized to subunit β of ATP synthase and lamin A, respectively. (D) The ratio of Nrf2 nuclear and cellular content of samples treated as above was calculated and displayed in the histograms (*P < 0.05, Student’s t test, n = 3). Discussion A number of laboratories have focussed on small molecule activators of FXN gene expression as potential therapeutic agents. There are several molecules that increase frataxin mRNA or protein levels (9). Due to chromatin structural changes and histone deacetylation, HDAC inhibitors may revert heterochromatin activating the right conformation and restoring the normal function of silenced frataxin gene (19–21). A different strategy is the increase of the amount of frataxin protein by a post-transcriptional mechanism. Recombinant human erythropoietin has been reported to increase the level of frataxin in lymphocytes from FRDA patients and in various human cell lines (22). The molecular basis for this observation is unknown, but it has been proposed to depend on increased stabilization of frataxin protein because the level of frataxin mRNA is the same in treated and untreated control cells. In this study we investigated the possible role of diazoxide on frataxin expression. Diazoxide is a pharmacological agent derived from benzothiazine and it is an activator of the mitochondrial K+ channels, causing the opening of channels, and in medical practice it is a drug normally used for acute hypertension therapy. Diazoxide activates the mitochondrial ATP-dependent potassium channels (mitoKATP) composed by a potassium channel subunits (Kir6.2) together with the regulatory subunit sulphonylurea receptor (12). These channels can potentially modulate the tightness of coupling between respiration and ATP synthesis, ROS production, mitochondrial dynamics and mitophagy (12 and references therein). MitoKATP is involved in the regulation of mitochondrial ionic homeostasis and, importantly, its activation has been observed to provide cytoprotection against ischemic damage (23,24). In our study, we tested the effect of diazoxide in vitro, using lymphoblastoid cell lines from FRDA patients and in vivo, using the YG8sR mouse model. Cells derived from FRDA patients carry the complete frataxin locus together with GAA repeat expansions and regulatory sequences. They are easily accessible and constitute one of the most relevant frataxin-deficient cell models. We used three different cell lines with different number of GAA expansions (170/230 GAA repeats for P338, 500/800 GAA repeats for P119 and 1100/1100 GAA repeats for P200). We used a diazoxide concentration of 100 µm, which is the approximate human blood concentration of diazoxide after an oral administration of 600 mg (25), and we treated lymphoblastoid cells derived from FRDA patients with diazoxide, alone or in presence of glucose, because the effect of mitoKATP channel openers on mitochondrial function is dependent by the cell’s energetic state, activating only the ATP-bound potassium channel (12). We found that diazoxide, alone or in combination with glucose, was able to induce the expression of frataxin protein in all three FRDA cell lines, after 4 days. Preliminary results show that the effect of diazoxide in increased frataxin protein in cell lines is higher after 4 days. We did not observe any increase of protein amount after 1 day, but mRNA increase of FXN expression was detectable after 24 h (data not shown). The probable mechanism involves activation of the mTOR pathway, since rapamycin treatment of cell cultures completely blocked the ability of diazoxide to significantly enhance the frataxin expression. The relationship between frataxin expression and the mTOR pathway has recently been revealed by Franco et al. (26). They found that frataxin stimulation by insulin-like growth factor 1 (IGF-1) was mediated by the PI3K/Akt/mTOR pathway. Using either the PI3kinase inhibitor Ly294002, which prevents Akt activation, or rapamycin, the stimulatory action of IGF-1 was blocked. In addition, IGF-1 increases levels of phosphorylated mTOR, an indirect measurement of its activity status, whereas other kinases downstream of the IGF-1 receptor, such as protein kinase C (PKC), are not involved in increasing frataxin expression after IGF-1 treatment (26). We then focused on determining the therapeutic efficacy of diazoxide to increase frataxin expression levels and to ameliorate the functional and biochemical disease effects of a FRDA mouse model. Although cell models rarely represent tissue-specific disease features, we used a mouse model with residual frataxin expression, the YG8sR mouse, to investigate the effect of diazoxide in different tissues. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy, as they present an increased somatic GAA repeat instability in the brain and cerebellum, together with a significantly reduced expression of frataxin, reduced aconitase activity and a presence of pathological vacuoles in DRG that suggests autophagy of damaged mitochondria (11). We treated the mice with diazoxide by oral administration, because it is known that oral diazoxide crosses the blood-brain barrier fairly rapidly in rats, reaching a plateau by the fourth hour as demonstrated by cerebrospinal fluid analysis (27). Daily dosage of diazoxide was well tolerated and no adverse effects were observed with any of the mice, and at the end of the study. However, unlike lymphoblastoid cell experiments, a 5-day treatment (short term) did not produce neither an increase of frataxin expression, nor other down-stream effects. In contrast, 3 months of treatment significantly improved frataxin expression in cerebellum, brain and heart mitochondrial tissue extracts, although the variability in frataxin increase is high amongst different samples. These data suggest that the effect of diazoxide on frataxin expression is not constant and this should be considered for future translational studies. Probably, single dosage of the treatment could impair the drug effect, and this could also explain different effects observed between short-term treated mice and FRDA cells. In humans e.g. diazoxide is metabolized by oxidation and sulphate conjugation and excreted in urine (28). The half-life of diazoxide is about 21 h, and usual maintenance dosage of 3–8 mg/kg is daily divided in 2 or 3 equal doses every 8–12 h (29 and references therein). Instead, diazoxide concentration in cell culture medium is constant, as it is not metabolized by lymphoblasts. YG8sR mice also showed an increase of aconitase activity, significant only in brain and a significantly decreased protein oxidation in brain, pancreas and liver mitochondrial tissue extracts, but only a trend to decrease in cerebellum and heart tissues. This suggests a different sensitivity of the tissues to oxidative stress, or a different half-life of frataxin that has been degraded through the proteasome (30). Further studies will be required to clarify these differences. Long-term treatment also revealed effects concerning efficacy of diazoxide on functional studies. Beam walk analysis, a test for the coordination abilities, revealed the most significant diazoxide-induced functional effect for YG8sR mice after 3 months of treatment. Also footprint analysis revealed a significant reduction in the stride length of diazoxide-treated YG8sR mice compared with vehicle-treated YG8sR mice over the 3-month test period. Both analyses suggest a positive effect of diazoxide on the coordination abilities. On the contrary, locomotor analysis identified a generally reduced locomotor activity of diazoxide-treated YG8sR mice compared with vehicle-treated mice, although treated mice showed an increase in the amount of rearing up onto hind limbs. This behaviour suggests the possible involvement of dopamine receptors that are known to be regulated by KATP channels (31). In fact, mice lacking dopamine D4 receptors show improved coordination abilities but decreased locomotor activity and rearing behaviour (32). According to the presence of functional KATP channels on dopaminergic axons, diazoxide treatment prevented single-pulse evoked dopamine release (31). This adverse effect of diazoxide should be considered for future therapeutic strategy. The hind limb base width and the weight of YG8sR mice was not affected by diazoxide treatment. We observed an increase in weight in all mice over 3 months. The increase in weight is observed in FRDA mice in comparison to the healthy control (33). Usually, the increase in weight may be attributed to the decreased locomotor activity of the mice, but Tomassini et al. (34) found that increase of FXN expression and amelioration in locomotor activity and motor coordination due to IFNg treatment, occurred independently of body weight changes. Our results suggest that the protective effects of diazoxide mediated by a stimulatory effect on frataxin expression, as observed in cell culture experiments and in brain and cerebellum of YG8sR tissues, are sufficient to ameliorate the coordination abilities. Neuroprotective effects of diazoxide have been well documented in conditions of ischaemia and hypoxia (35,36). It has been shown to promote myelination in the neonatal brain and attenuate hypoxia-induced brain injury in neonatal mice (37) as well as oligodendrocyte differentiation in neonatal mouse brain (38). The involvement of the mTOR signalling pathway as a possible mechanism by which diazoxide can induce frataxin expression is confirmed by the increase of S6K phosphorylation and by the treatment of rapamycin that completely inhibited the frataxin-increasing effect elicited by diazoxide (16). Kwon et al. (15) found that [3H]thymidine incorporation in adult rodent islets chronically exposed to glucose is mainly mediated through mTOR via the KATP channel, and 250 µm diazoxide enhanced the ability of elevated glucose to stimulate cell proliferation and S6K1 phosphorylation, a known target of mTOR kinases. The authors also demonstrated that 25 nm rapamycin completely inhibited the effect of diazoxide. A possible downstream target of mTOR signalling could be the NRF2 transcription factor-mediated pathway. Activation of the NRF2 pathway induces frataxin expression as demonstrated by Sahdeo et al. (39). They found seven potential Nrf2-binding sites (antioxidant responsive elements, AREs) between 20 kb upstream and 5 kb downstream of the FXN locus, three of them, located upstream of the transcription start site for FXN, with excellent ARE scores. They demonstrated by chromatin Immunoprecipitation experiments that they are active and functional Nrf2-binding sites (39). The increase of frataxin protein may be achieved, at least in part, by a transcriptional mechanism, since qRT-PCR analysis of FXN mRNA revealed increases in expression of cerebellum and heart tissues from YG8sR mice, together with increases in Nrf2 expression. We also demonstrated that activation of the mTOR pathway in diazoxide-treated cell lines induces Nrf2 nuclear translocation, and this process is inhibited by rapamycin. Our data are in agreement with a recent study by Virgili et al. (40) who showed that diazoxide prevents endogenous oxidative damage of NSC-34 motoneurons after different neurotoxic insults and increases Nrf2 nuclear translocation. This study suggests that diazoxide is able to induce FXN expression in human cells from FRDA patients and in brain, cerebellum and heart tissues of YG8sR mice. Positive effects have also been observed on oxidative stress protection and coordination abilities of treated mice but further studies are needed to clarify the variable effects concerning functional studies. Materials and Methods Cell lines and culture conditions Human control and FRDA lymphoblastoid cell lines were maintained in RPMI 1640 supplemented with 8 mm glutamine, 2 mm sodium pyruvate, 1× non-essential amino acids, 15% foetal bovine serum, and 100 U/ml penicillin and 0.1 mg/ml streptomycin. All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2 in 25 cm2 culture flasks (kept in an upright position). Antibiotics were omitted during pharmacological compound assays. Cells were treated for 4 days with 100 μM diazoxide, alone or combined with 20 mm glucose or 2 nm rapamycin. Medium with reagents were changed every day during the treatment period. Nrf2 level was determined in nuclear protein extracts from lymphoblastoid cell cultures 24 h after treatments, using 4 wells from 24-well plates for each experimental condition. Cytotoxicity assay Potential cytotoxic effects of diazoxide were carried out on lymphoblastoid cell lines using MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide] assay. Cells were seeded into 6-well plates at a density of 2.5 × 105 cells per well in 1 ml of RPMI medium and incubated for 4 days in 5% CO2 environment at 37°C. The assay is based on the mitochondrial activity of living cells to convert water soluble MTT into water insoluble formazan crystals due to the reductive activity of mitochondria. Cells were incubated for 4 days in the presence of the test agent at four dilutions ranging from 50 to 400 µm. The assay was carried out as elsewhere described in (15,41). The experiment was carried out in triplicate, after 4 days. Tissue processing Homogenate of each tissue was prepared suspending 200 mg of tissue in a buffer containing 250 mm sucrose, 1 mm EGTA and 10 mm TRIS pH7.4 and using a Teflon glass potter. Cell debris was pelleted by centrifugation at 800g and discarded. Mitochondria from tissue homogenates were harvested by centrifugation at 13 000g for 12 min at 4°C. Mitochondria enriched pellets was then stored at −80°C until further processing. Quantitative reverse transcriptase PCR Total RNA was isolated from the mouse tissues by homogenisation with Trizol (Invitrogen), followed by DNase I treatment, and cDNA was then prepared by using AMV reverse transcriptase (Invitrogen) with oligo (dT)20 primers and quantified by Nanodrop (ThermoScientific) analysis (42). Levels of human transgenic FXN or mouse Nrf2 mRNA expression were assessed by qPCR using an Quant Studio 7 detection system and SYBR Green (Applied Biosystems) with the following primers: (i) FXN: FRT-I forward 5′-TTGAAGACCTTGCAGACAAG-3′ and RRT-II reverse 5′-AGCCAGATTTGCTTGTTTGG-3′, 121 bp amplicon size; (ii) Nrf2: forward 5′-TTTTCCATTCCCGAATTACAGT-3′ and reverse 5′- GGAGATCGATGAGTAAAAATGGT-3′, 126 bp amplicon size. For each sample, assays were performed in triplicate. Western blot analysis Lymphoblastoid cells were washed three times, lysed with cell lysis buffer (0.15 M NaCl, 5 mm EDTA, 1% NP-40, 10 mm Tris-Cl, pH 7.4) and transferred to a microcentrifuge tube. For Nrf2 analysis, cytoplasmic and nuclear fractions were separated by using the NE-Nuclear and Cytoplasmic Extraction kit (Thermo Fisher) by following the manufacturer’s protocol. Protein concentration was determined using the BCA protein assay reagent (Thermo Scientific). 20 µg of mouse mitochondrial protein or 80 µg of human cell proteins were separated on BOLT BIS-TRIS PLUS 12% polyacrylamide gels (43) and electroblotted onto nitrocellulose membranes (Thermo Fisher). Membranes were washed 20 min with TBST buffer and incubated with anti-frataxin (Santa Cruz Biotechnology, 1:500), anti-Nrf2 (Thermo Fisher, 1:500), anti-S6K1 (phospho T389; 1:500), anti-lamin A (BD Bioscience, 1:500), anti-βATPase (BD Bioscience, 1:10 000) primary antibodies. Anti-NRF2, anti-lamin A and anti-frataxin were incubated for 12 h at 4°C. Anti-βATPAse and anti-S6K were incubated for 1 h at RT. Membranes were washed three times with TBS for 15 min, and then incubated for 1h at RT with anti-mouse (Pierce, 1:10 000) and anti-rabbit (Pierce, 1:5000) HRP secondary antibodies (Thermo Fisher). Finally, membranes were washed three times with TBS and immunoreactive bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) (44). The intensity of protein bands was quantified by using QuantityOne 4.6.3 Image software (ChemiDoc XRS; Bio-Rad) and normalized against proper loading controls (45,46). Quantification of mitochondrial protein oxidation Carbonylated mitochondrial proteins were detected using the OxyBlot Protein Oxidation Detection Kit (MILLIPORE). 2, 4 dinitrophenylhydrazine- derivatized crude mitochondrial protein extracts (10 µg) were dot blotted, incubated with a primary antibody against DNP-hydrazone followed by a horseradish peroxidase-conjugated secondary antibody, visualized using the ECL chemiluminescent detection method and quantified by densitometry (47). Enzymatic assays Citrate synthase activity was determined following the rate of 5, 5’-dithiobis-(2-nitrobenzoic) acid (DTNB) consumption, as described in (48). The CoA produced in the reaction mix reacts with the DTNB to form 5-thio-2-nitrobenzoic acid (TNB), a yellow product observed spectrophotometrically by measuring absorbance at 412 nm. In total 10 µg of mitochondrial proteins from each tissue were used for spectrophotometric assay using Cary win 50 UV-Spectrophotometer. Aconitase activities were determined according to the method described previously in (23). Briefly, mitochondria were lysed in Tris-HCl (pH 7.4) containing 0.5% Triton X-100 and 2.5 mm sodium citrate, for 5 min on ice. Insoluble material was removed by centrifugation (14 000g, 40 s), and protein concentration was determined with Bradford reagent (Bio-Rad, Milan, Italy). 100–300 µg of mitochondrial extract were added to a substrate mix (50 mm Tris/HCl pH 7.4, 0.2 mm NADP+, 5 mm Na citrate, 0.6 mm MgCl2, 0.1% (v/v) Triton X-100 and 1–2 U of isocitrate dehydrogenase) and the reactions were incubated at 25°C for 15 min, followed by spectrophotometric absorbance measurements at 340 nm. In samples of low aconitase activity, an initial lag in NADPH formation is seen, probably due to a delayed accumulation of cis-aconitate (49). Thus, the determinations of aconitase activity were determined using linear rates during the latter half of a 60-min assay. Animal procedures and behavioural assessments YG8sR mice were assessed from 4 months of age for a period of 3 months. Weight determination, rotarod performance and beam breaker locomotor performance were assessed monthly, while beam walk performance and footprint analysis were assessed at the start and end of the test period, using previously described methodology (11). All procedures were carried out in accordance with the UK Home Office Animals Scientific Procedures Act (1986) and with ethical approval from the Brunel University London Animals Welfare and Ethical Review Board. Statistical analysis Statistical analysis was performed using the paired Student’s t test when comparing two groups or a two-way ANOVA when comparing multiple groups. Differences with P < 0.05 were assumed to be significant. In the figures, P < 0.05, P < 0.01 and P < 0.001 were marked with *, ** and ***, respectively. Supplementary Material Supplementary Material is available at HMG online. Acknowledgements We are grateful to the Genomic and Genetic Disorders Biobank, Telethon Network of Genetic Biobanks (Telethon Italy grant GTB12001G), and to the EuroBioBank network for biospecimens banking. Conflict of Interest statement. Santoro A., Palmieri L. and Marobbio C.M.T. filed a U.S. patent: Diazoxide for the treatment of Friedreich’s Ataxia. US 8, 716, 250 B2, Università degli Studi di Bari. Merla G. is a paid consultant for Takeda Pharmaceutical Company. Funding This work was supported by funding from the Friedreich’s Ataxia Research Alliance (FARA); and the Italian Ministry of Health [Ricerca Corrente 2014–16] to G.M.; and ‘5 × 1000’ voluntary contributions to G.M. References 1 Campuzano V., Montermini L., Moltò M.D., Pianese L., Cossée M., Cavalcanti F., Monros E., Rodius F., Duclos F., Monticelli A. et al.   ( 1996) Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science , 271, 1423– 1427. Google Scholar CrossRef Search ADS PubMed  2 Filla A., De Michele G., Cavalcanti F., Pianese L., Monticelli A., Campanella G., Cocozza S. ( 1996) The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia. Am. J. Hum. Genet ., 59, 554– 560. Google Scholar PubMed  3 Mateo I., Llorca J., Volpini V., Corral J., Berciano J., Combarros O. ( 2003) GAA expansion size and age at onset of Friedreich’s ataxia. Neurology , 61, 274– 275. Google Scholar CrossRef Search ADS PubMed  4 Napier I., Ponka P., Richardson D.R. ( 2005) Iron trafficking in the mitochondrion: novel pathways revealed by disease. Blood , 105, 1867– 1874. Google Scholar CrossRef Search ADS PubMed  5 Dürr A., Cossee M., Agid Y., Campuzano V., Mignard C., Penet C., Mandel J.-L., Brice A., Koenig M. ( 1996) Clinical and genetic abnormalities in patients with Friedreich’s ataxia. N. Engl. J. Med ., 335, 1169– 1175. Google Scholar CrossRef Search ADS PubMed  6 Pandolfo M. ( 2006) Iron and Friedreich ataxia. J. Neural. Transm. Suppl ., 70, 143– 146. Google Scholar CrossRef Search ADS   7 Lu C., Cortopassi G. ( 2007) Frataxin knockdown causes loss of cytoplasmic iron–sulfur cluster functions, redox alterations and induction of heme transcripts. Arch. Biochem. Biophys ., 457, 111– 122. Google Scholar CrossRef Search ADS PubMed  8 Fogel B.L., Perlman S. ( 2007) Clinical features and molecular genetics of autosomal recessive cerebellar ataxias. Lancet Neurol ., 6, 245– 257. Google Scholar CrossRef Search ADS PubMed  9 Gottesfeld J.M. ( 2007) Small molecules affecting transcription in Friedreich ataxia. Pharmacol. Ther ., 116, 236– 248. Google Scholar CrossRef Search ADS PubMed  10 Pandolfo M. ( 2013) Treatment of Friedreich’s ataxia. Expert Opin. Orphan Drugs , 1, 221– 234. Google Scholar CrossRef Search ADS   11 Anjomani Virmouni S., Ezzatizadeh V., Sandi C., Sandi M., Al-Mahdawi S., Chutake Y., Pook M.A. ( 2015) A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia. Dis. Model. Mech ., 8, 225– 235. Google Scholar CrossRef Search ADS PubMed  12 Szabò I., Leanza L., Gulbins E., Zoratti M. ( 2012) Physiology of potassium channels in the inner membrane of mitochondria. Pflugers Arch ., 463, 231– 246. Google Scholar CrossRef Search ADS PubMed  13 Hutcheon D.E., Harman M.A., Schwartz M.l. ( 1962) Diazoxide in the treatment of hypertension. J. New Drugs , 2, 292– 297. Google Scholar CrossRef Search ADS PubMed  14 Samols E., Marks V. ( 1966) The treatment of hypoglycaemia with diazoxide. Proc. R. Soc. Med ., 59, 811– 814. Google Scholar PubMed  15 Kwon G., Marshall C.A., Liu H., Pappan K.L., Remedi M.S., McDaniel M.L. ( 2006) Glucose-stimulated DNA synthesis through mammalian target of rapamycin (mTOR) is regulated by KATP channels: effects on cell cycle progression in rodent islets. J. Biol. Chem ., 281, 3261– 3267. Google Scholar CrossRef Search ADS PubMed  16 Magnuson B., Ekim B., Fingar D.C. ( 2012) Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem. J ., 441, 1– 21. Google Scholar CrossRef Search ADS PubMed  17 Wullschleger S., Loewith R., Hall M.N. ( 2006) TOR signaling in growth and metabolism. Cell , 124, 471– 484. Google Scholar CrossRef Search ADS PubMed  18 Lim J.L., Wilhelmus M.M., de Vries H.E., Drukarch B., Hoozemans J.J., van Horssen J. ( 2014) Antioxidative defense mechanisms controlled by Nrf2: state-of-the-art and clinical perspectives in neurodegenerative diseases. Arch. Toxicol ., 88, 1773– 1786. Google Scholar CrossRef Search ADS PubMed  19 Bidichandani S.I., Ashizawa T., Patel P.I. ( 1998) The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure. Am. J. Hum. Genet ., 62, 111– 121. Google Scholar CrossRef Search ADS PubMed  20 Di Prospero N.A., Fischbeck K.H. ( 2005) Therapeutics development for triplet repeat expansion diseases. Nat. Rev. Genet , 6, 756– 765. Google Scholar CrossRef Search ADS PubMed  21 Sandi C., Pinto R.M., Al-Mahdawi S., Ezzatizadeh V., Barnes G., Jones S., Rusche J.R., Gottesfeld J.M., Pook M.A. ( 2011) Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model. Neurobiol. Dis ., 42, 496– 505. Google Scholar CrossRef Search ADS PubMed  22 Sturm B., Stupphann D., Kaun C., Boesch S., Schranzhofer M., Wojta J., Goldenberg H., Scheiber−Mojdehkar B. ( 2005) Recombinant human erythropoietin: effects on frataxin expression in vitro. Eur. J. Clin. Invest ., 35, 711– 717. Google Scholar CrossRef Search ADS PubMed  23 Bernardi P. ( 1999) Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol. Rev ., 79, 1127– 1155. Google Scholar CrossRef Search ADS PubMed  24 Costa A.D.T., Garlid K.D. ( 2009) MitoKATP activity in healthy and ischemic hearts. J. Bioenerg. Biomembr ., 41, 123– 126. Google Scholar CrossRef Search ADS PubMed  25 Calesnick B., Katchen B., Black J. ( 1965) Importance of dissolution rates in producing effective diazoxide blood levels in man. J. Pharm. Sci ., 54, 1277– 1280. Google Scholar CrossRef Search ADS PubMed  26 Franco C., Fernández S., Torres-Alemán I. ( 2012) Frataxin deficiency unveils cell-context dependent actions of insulin-like growth factor I on neurons. Mol. Neurodegener ., 7, 1– 10. Google Scholar CrossRef Search ADS PubMed  27 Kishore P., Boucai L., Zhang K., Li W., Koppaka S., Kehlenbrink S., Schiwek A., Esterson Y.B., Mehta D., Bursheh S. et al.   ( 2011) Activation of K(ATP) channels suppresses glucose production in humans. Clin. Invest ., 121, 4916– 4920. Google Scholar CrossRef Search ADS   28 American Society of Health-System Pharmacists ( 2015) Diazoxide. In McEvoy G.K. (ed.), AHFS Drug Information . Bethesda, United States. 29 Friciu M., Zaraa S., Roullin V.G., Leclair G., Agbor G. ( 2016) Stability of diazoxide in extemporaneously compounded oral suspensions. PLoS One , 11, 1– 12. Google Scholar CrossRef Search ADS   30 Rufini A., Fortuni S., Arcuri G., Condo I., Serio D., Incani O., Malisan F., Ventura N., Testi R. ( 2011) Preventing the ubiquitin-proteasome-dependent degradation of frataxin, the protein defective in Friedreich’s ataxia. Hum. Mol. Genet ., 20, 1253– 1261. Google Scholar CrossRef Search ADS PubMed  31 Patel J.C., Witkovsky P., Coetzee W.A., Rice M.E. ( 2011) Subsecond regulation of striatal dopamine release by pre-synaptic KATP channels. J. Neurochem ., 118, 721– 736. Google Scholar CrossRef Search ADS PubMed  32 Rubinstein M., Phillips T.J., Bunzow J.R., Falzone T.L., Dziewczapolski G., Zhang G., Fang Y., Larson J.L., McDougall J.A., Chester J.A. et al.   ( 1997) Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine. Cell , 90, 991– 1001. Google Scholar CrossRef Search ADS PubMed  33 Anjomani Virmouni S., Sandi C., Al-Mahdawi S., Pook M.A., Pastore A. ( 2014) Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich ataxia. PLoS One , 9, 1– 13. Google Scholar CrossRef Search ADS   34 Tomassini B., Arcuri G., Fortuni S., Sandi C., Ezzatizadeh V., Casali C., Condo I., Malisan F., Al-Mahdawi S., Pook M., Testi R. ( 2012) Interferon gamma upregulates frataxin and corrects the functional deficits in a Friedreich ataxia model. Hum. Mol. Genet ., 21, 2855– 2861. Google Scholar CrossRef Search ADS PubMed  35 Domoki F., Perciaccante J.V., Veltkamp R., Bari F., Busija D.W., Bryan R.M. ( 1999) Mitochondrial potassium channel opener diazoxide preserves neuronal–vascular function after cerebral ischemia in newborn pigs. Stroke , 30, 2713– 2718. Google Scholar CrossRef Search ADS PubMed  36 Shake J.G., Peck E.A., Marban E., Gott V.L., Johnston M.V., Troncoso J.C., Redmond J.M., Baumgartner W.A. ( 2001) Pharmacologically induced preconditioning with diazoxide: a novel approach to brain protection. Ann. Thorac. Surg ., 72, 1849– 1854. Google Scholar CrossRef Search ADS PubMed  37 Fogal B., McClaskey C., Yan S., Yan H., Rivkees S.A., Smith M.A. ( 2010) Diazoxide promotes oligodendrocyte precursor cell proliferation and myelination. PLoS One , 5, 1-9. Google Scholar CrossRef Search ADS   38 Zhu Y., Wendler C.C., Shi O., Rivkees S.A. ( 2014) Diazoxide promotes oligodendrocyte differentiation in neonatal brain in normoxia and chronic sublethal hypoxia. Brain Res ., 1586, 64– 72. Google Scholar CrossRef Search ADS PubMed  39 Sahdeo S., Scott B.D., McMackin M.Z., Jasoliya M., Brown B., Wulff H., Perlman S.L., Pook M.A., Cortopassi G.A. ( 2014) Dyclonine rescues frataxin deficiency in animal models and buccal cells of patients with Friedreich’s ataxia. Hum. Mol. Genet ., 23, 6848– 6862. Google Scholar CrossRef Search ADS PubMed  40 Virgili N., Mancera P., Wappenhans B., Sorrosal G., Biber K., Pugliese M., Espinosa-Parrilla J.F., Reddy H. ( 2013) K(ATP) channel opener diazoxide prevents neurodegeneration: a new mechanism of action via antioxidative pathway activation. PLoS One , 8, 1– 11. Google Scholar CrossRef Search ADS   41 Laquintana V., Denora N., Lopalco A., Lopedota A., Cutrignelli A., Lasorsa F.M., Agostino G., Franco M. ( 2014) Translocator protein ligand-plga conjugated nanoparticles for 5-fluorouracil delivery to glioma cancer cells. Mol. Pharm . 11, 859– 871. Google Scholar CrossRef Search ADS PubMed  42 Kishita Y., Pajak A., Bolar N., Marobbio C.M.T., Maffezzini C., Miniero D.V., Monné M., Kohda M., Stranneheim H., Murayama K. et al.   ( 2015) Intra-mitochondrial methylation deficiency due to mutations in SLC25A26. Am. J. Hum. Genet ., 97, 761– 768. Google Scholar CrossRef Search ADS PubMed  43 Giannuzzi G., Lobefaro N., Paradies E., Vozza A., Punzi G., Marobbio C.M.T. ( 2014) Overexpression in E. coli and purification of the L. pneumophila Lpp2981 protein. Mol. Biotechnol ., 56, 157– 165. Google Scholar CrossRef Search ADS PubMed  44 Marobbio C.M.T., Punzi G., Pierri C.L., Palmieri L., Calvello R., Panaro M.A., Palmieri F. ( 2015) Pathogenic potential of SLC25A15 mutations assessed by transport assays and complementation of Saccharomyces cerevisiae ORT1 null mutant. Mol. Genet. Metab ., 115, 27– 32. Google Scholar CrossRef Search ADS PubMed  45 Ersoy Tunalı N., Marobbio C.M.T., Tiryakioğlu N.O., Punzi G., Saygılı S.K., Onal H., Palmieri F. ( 2014) A novel mutation in the SLC25A15 gene in a Turkish patient with HHH syndrome: functional analysis of the mutant protein. Mol. Genet. Metab ., 112, 25– 29. Google Scholar CrossRef Search ADS PubMed  46 Lunetti P., Damiano F., De Benedetto G., Siculella L., Pennetta A., Muto L., Paradies E., Marobbio C.M.T., Dolce V., Capobianco L. ( 2016) Characterization of human and yeast mitochondrial glycine carriers with implications for heme biosynthesis and anemia. J. Biol. Chem ., 291, 19746– 19759. Google Scholar CrossRef Search ADS PubMed  47 Marobbio C.M.T., Pisano I., Porcelli V., Lasorsa F.M., Palmieri L. ( 2012) Rapamycin reduces oxidative stress in frataxin-deficient yeast cells. Mitochondrion , 12, 156– 166. Google Scholar CrossRef Search ADS PubMed  48 Blomstrand E., Rådegran G., Saltin B. ( 1997) Maximum rate of oxygen uptake by human skeletal muscle in relation to maximal activities of enzymes in the Krebs cycle. J. Physiol ., 501, 455– 460. Google Scholar CrossRef Search ADS PubMed  49 Gardner P.R., Nguyen D.D., White C.W. ( 1994) Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs. Proc. Natl. Acad. Sci. U.S.A ., 91, 12248– 12252. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
TI - Effect of diazoxide on Friedreich ataxia models
JF - Human Molecular Genetics
DO - 10.1093/hmg/ddy016
DA - 2018-01-08
UR - https://www.deepdyve.com/lp/oxford-university-press/effect-of-diazoxide-on-friedreich-ataxia-models-kL0Wp9rHDP
SP - 992
EP - 1001
VL - 27
IS - 6
DP - DeepDyve
ER -