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Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

Besson MT, Alegría K, Garrido-Gerter P, Barros LF, Liévens JC - PLoS ONE (2015)

Bottom Line: We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration.Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss.Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3 protective effects, whereas, in addition, the PPP provides increased protection to oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Aix-Marseille Université, CNRS, CRN2M-UMR7286, 13344 Marseille cedex 15, Marseille, France.

ABSTRACT
Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK) which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3 protective effects, whereas, in addition, the PPP provides increased protection to oxidative stress.

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Survivals of flies overexpressing hGluT3 or G6PD or PFK with mitochondrial dysfunctions in neurons.(A): RT-qPCR assays for E1-PDH and ND23 transcripts showing reduced levels of each transcript in flies expressing the RNAi under the Elav-Gal4 driver. Transcripts levels (filled bars) were expressed in percent relative to controls (no transgene; open bars). They represent the means + SEM of at least 3 separate experiments prepared from heads at the first day of adult age. The one-tailed Mann-Whitney test indicates a difference in mRNA levels between the RNAi-induced and control genotypes (**, p = 0.0076 for E1-PDH; **, p = 0.0083 for ND23). (B, C, D): Survival curves of flies expressing each mitochondria-targeting RNAi alone or together with either hGluT3, G6PD or PFK in the respective figure under the control of Elav-Gal4. Only the first fourty days of the lifespan were presented. The survival curves of flies expressing only the RNAis specifically-targeted to E1-PDH (filled squares; n = 51) and ND23 subunits (filled triangles; n = 53) were in common for the figures B, C and D. The survival for control flies (Elav; hGluT3 or Elav; G6PD or Elav; PFK) was indicated by open circles in each corresponding graph (n = 75; 81; 131, respectively). In each figure, the log-rank test indicates a p-value <0.0001 (***) between the survival curves of the flies overexpressing hGluT3 or G6PD or PFK and the RNAi and the survival curves of the flies expressing each RNAi only. (B): Survival curves of flies expressing the RNAis or co-expressing each respective RNAi and hGluT3 (open symbols; n = 84 and 73) under Elav-Gal4. (C): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and G6PD (open symbols; n = 84 and 60 respectively) under Elav-Gal4. (D): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and PFK (open symbols; n = 79 and 111 respectively) under Elav-Gal4.
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pone.0118765.g006: Survivals of flies overexpressing hGluT3 or G6PD or PFK with mitochondrial dysfunctions in neurons.(A): RT-qPCR assays for E1-PDH and ND23 transcripts showing reduced levels of each transcript in flies expressing the RNAi under the Elav-Gal4 driver. Transcripts levels (filled bars) were expressed in percent relative to controls (no transgene; open bars). They represent the means + SEM of at least 3 separate experiments prepared from heads at the first day of adult age. The one-tailed Mann-Whitney test indicates a difference in mRNA levels between the RNAi-induced and control genotypes (**, p = 0.0076 for E1-PDH; **, p = 0.0083 for ND23). (B, C, D): Survival curves of flies expressing each mitochondria-targeting RNAi alone or together with either hGluT3, G6PD or PFK in the respective figure under the control of Elav-Gal4. Only the first fourty days of the lifespan were presented. The survival curves of flies expressing only the RNAis specifically-targeted to E1-PDH (filled squares; n = 51) and ND23 subunits (filled triangles; n = 53) were in common for the figures B, C and D. The survival for control flies (Elav; hGluT3 or Elav; G6PD or Elav; PFK) was indicated by open circles in each corresponding graph (n = 75; 81; 131, respectively). In each figure, the log-rank test indicates a p-value <0.0001 (***) between the survival curves of the flies overexpressing hGluT3 or G6PD or PFK and the RNAi and the survival curves of the flies expressing each RNAi only. (B): Survival curves of flies expressing the RNAis or co-expressing each respective RNAi and hGluT3 (open symbols; n = 84 and 73) under Elav-Gal4. (C): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and G6PD (open symbols; n = 84 and 60 respectively) under Elav-Gal4. (D): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and PFK (open symbols; n = 79 and 111 respectively) under Elav-Gal4.

Mentions: An increasing number of studies have shown that mutant Htt action results in mitochondrial dysfunction [59, 60]. We tested whether or not increasing glucose metabolism could prevent effects of mitochondrial dysfunction. Therefore, we examined the impact of hGluT3 neuronal overexpression on fly lifespan after genetic inactivation of two key genes for mitochondrial activity: the pyruvate dehydrogenase complex and the mitochondrial respiratory system. In mitochondrial matrix, the pyruvate dehydrogenase complex (PDH) catalyses the conversion of pyruvate to acetyl-coA and constitutes the first step of the TCA cycle. The mitochondrial respiratory complex I contains evolutionary conserved NADH ubiquinone oxidoreductase complex components; it produces significant amounts of ROS and its dysfunction triggers oxidative impairment as observed in several neuropathological diseases [17, 61, 62]. Firstly, we verified by RT-qPCR analysis that RNA interference (RNAi) expressions in neurons have efficiently reduced the expression of their respective targets: the alpha-subunit of the acetyl-transferring component of the PDH complex (E1-PDH) and the 23kD subunit (ND23) in the complex I (Fig. 6A). The Fig. 6B shows that knockdown of these both genes in neurons led to a dramatic reduction of the lifespan with a expectancy mean of 5 and 4 days respectively; this shows the key role of mitochondria in neuronal functions. However, when the hGluT3 transgene was expressed together with each RNAi, life expectancies were very significantly rescued since the means reached up 56 days with the E1-PDH RNAi and 59 days in the case of the ND23 RNAi (Fig. 6B). These results indicate that hGluT3 overexpression was sufficient to rescue the survival of the flies presenting mitochondrial defects. Then, to test the respective roles of the glycolysis or PPP to counteract mitochondrial dysfunction, we overexpressed the key enzymes G6PD (Fig. 6C) or PFK (Fig. 6D) in neurons in the presence of each RNAi. Both enzymes rescued the survival of the E1-PDH or ND23 RNAi expressed in neurons. This suggests that an increase in glycolysis and/or in PPP was required to maintain cell survival in stress conditions following impairment of mitochondrial functions.


Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

Besson MT, Alegría K, Garrido-Gerter P, Barros LF, Liévens JC - PLoS ONE (2015)

Survivals of flies overexpressing hGluT3 or G6PD or PFK with mitochondrial dysfunctions in neurons.(A): RT-qPCR assays for E1-PDH and ND23 transcripts showing reduced levels of each transcript in flies expressing the RNAi under the Elav-Gal4 driver. Transcripts levels (filled bars) were expressed in percent relative to controls (no transgene; open bars). They represent the means + SEM of at least 3 separate experiments prepared from heads at the first day of adult age. The one-tailed Mann-Whitney test indicates a difference in mRNA levels between the RNAi-induced and control genotypes (**, p = 0.0076 for E1-PDH; **, p = 0.0083 for ND23). (B, C, D): Survival curves of flies expressing each mitochondria-targeting RNAi alone or together with either hGluT3, G6PD or PFK in the respective figure under the control of Elav-Gal4. Only the first fourty days of the lifespan were presented. The survival curves of flies expressing only the RNAis specifically-targeted to E1-PDH (filled squares; n = 51) and ND23 subunits (filled triangles; n = 53) were in common for the figures B, C and D. The survival for control flies (Elav; hGluT3 or Elav; G6PD or Elav; PFK) was indicated by open circles in each corresponding graph (n = 75; 81; 131, respectively). In each figure, the log-rank test indicates a p-value <0.0001 (***) between the survival curves of the flies overexpressing hGluT3 or G6PD or PFK and the RNAi and the survival curves of the flies expressing each RNAi only. (B): Survival curves of flies expressing the RNAis or co-expressing each respective RNAi and hGluT3 (open symbols; n = 84 and 73) under Elav-Gal4. (C): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and G6PD (open symbols; n = 84 and 60 respectively) under Elav-Gal4. (D): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and PFK (open symbols; n = 79 and 111 respectively) under Elav-Gal4.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4356621&req=5

pone.0118765.g006: Survivals of flies overexpressing hGluT3 or G6PD or PFK with mitochondrial dysfunctions in neurons.(A): RT-qPCR assays for E1-PDH and ND23 transcripts showing reduced levels of each transcript in flies expressing the RNAi under the Elav-Gal4 driver. Transcripts levels (filled bars) were expressed in percent relative to controls (no transgene; open bars). They represent the means + SEM of at least 3 separate experiments prepared from heads at the first day of adult age. The one-tailed Mann-Whitney test indicates a difference in mRNA levels between the RNAi-induced and control genotypes (**, p = 0.0076 for E1-PDH; **, p = 0.0083 for ND23). (B, C, D): Survival curves of flies expressing each mitochondria-targeting RNAi alone or together with either hGluT3, G6PD or PFK in the respective figure under the control of Elav-Gal4. Only the first fourty days of the lifespan were presented. The survival curves of flies expressing only the RNAis specifically-targeted to E1-PDH (filled squares; n = 51) and ND23 subunits (filled triangles; n = 53) were in common for the figures B, C and D. The survival for control flies (Elav; hGluT3 or Elav; G6PD or Elav; PFK) was indicated by open circles in each corresponding graph (n = 75; 81; 131, respectively). In each figure, the log-rank test indicates a p-value <0.0001 (***) between the survival curves of the flies overexpressing hGluT3 or G6PD or PFK and the RNAi and the survival curves of the flies expressing each RNAi only. (B): Survival curves of flies expressing the RNAis or co-expressing each respective RNAi and hGluT3 (open symbols; n = 84 and 73) under Elav-Gal4. (C): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and G6PD (open symbols; n = 84 and 60 respectively) under Elav-Gal4. (D): Survival curve of flies expressing the RNAis or co-expressing each respective RNAi and PFK (open symbols; n = 79 and 111 respectively) under Elav-Gal4.
Mentions: An increasing number of studies have shown that mutant Htt action results in mitochondrial dysfunction [59, 60]. We tested whether or not increasing glucose metabolism could prevent effects of mitochondrial dysfunction. Therefore, we examined the impact of hGluT3 neuronal overexpression on fly lifespan after genetic inactivation of two key genes for mitochondrial activity: the pyruvate dehydrogenase complex and the mitochondrial respiratory system. In mitochondrial matrix, the pyruvate dehydrogenase complex (PDH) catalyses the conversion of pyruvate to acetyl-coA and constitutes the first step of the TCA cycle. The mitochondrial respiratory complex I contains evolutionary conserved NADH ubiquinone oxidoreductase complex components; it produces significant amounts of ROS and its dysfunction triggers oxidative impairment as observed in several neuropathological diseases [17, 61, 62]. Firstly, we verified by RT-qPCR analysis that RNA interference (RNAi) expressions in neurons have efficiently reduced the expression of their respective targets: the alpha-subunit of the acetyl-transferring component of the PDH complex (E1-PDH) and the 23kD subunit (ND23) in the complex I (Fig. 6A). The Fig. 6B shows that knockdown of these both genes in neurons led to a dramatic reduction of the lifespan with a expectancy mean of 5 and 4 days respectively; this shows the key role of mitochondria in neuronal functions. However, when the hGluT3 transgene was expressed together with each RNAi, life expectancies were very significantly rescued since the means reached up 56 days with the E1-PDH RNAi and 59 days in the case of the ND23 RNAi (Fig. 6B). These results indicate that hGluT3 overexpression was sufficient to rescue the survival of the flies presenting mitochondrial defects. Then, to test the respective roles of the glycolysis or PPP to counteract mitochondrial dysfunction, we overexpressed the key enzymes G6PD (Fig. 6C) or PFK (Fig. 6D) in neurons in the presence of each RNAi. Both enzymes rescued the survival of the E1-PDH or ND23 RNAi expressed in neurons. This suggests that an increase in glycolysis and/or in PPP was required to maintain cell survival in stress conditions following impairment of mitochondrial functions.

Bottom Line: We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration.Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss.Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3 protective effects, whereas, in addition, the PPP provides increased protection to oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Aix-Marseille Université, CNRS, CRN2M-UMR7286, 13344 Marseille cedex 15, Marseille, France.

ABSTRACT
Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK) which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3 protective effects, whereas, in addition, the PPP provides increased protection to oxidative stress.

Show MeSH
Related in: MedlinePlus