Neuronal expression of Mgat1 rescues the shortened life span of Drosophila Mgat11 null mutants and increases life span

Abstract

The enzyme UDP-GlcNAc:alpha3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT1, encoded by Mgat1) controls the synthesis of paucimannose N-glycans in Drosophila. We have previously reported that null mutations in Drosophila Mgat1 are viable but exhibit defects in locomotion, brain abnormalities, and a severely reduced life span. Here, we show that knockdown of Mgat1 in the central nervous system (CNS) of wild-type flies decreases locomotor activity and life span. This phenotype is similar to that observed in Drosophila Mgat1(1) null mutants, demonstrating that Mgat1 is required in the CNS. We also found that neuronal expression of a wild-type Mgat1 transgene rescued the shortened life span of Mgat1(1) null mutants and resulted in a dramatic 135% increase in mean life span relative to genetically identical controls. Neuronal expression of a wild-type Mgat1 transgene in wild-type flies resulted in a modest 9% increase in mean life span relative to genetically identical controls. In both Mgat1(1) null mutants and wild-type flies, neuronal expression of wild-type Mgat1 transgene resulted in a significant increase in GnT1 activity and resistance to oxidative stress. Whereas dietary restriction is not absolutely essential for the increased life span, it plays a role in the process. Interestingly, we observe a direct correlation between GnT1 activity and mean life span up to a maximum of appropriately 136 days, showing that the ability of GnT1 activity to increase life span is limited. Altogether, these observations suggest that Mgat1-dependent N-glycosylation plays an important role in the control of Drosophila life span.

Fig. 1.

Targeted gene expression. (A) Targeted RNAi knockdown of Mgat1 in the wild-type CNS affects Drosophila life span. The conditional ELAV-GeneSwitch line was used to knock down Mgat1 in the CNS of wild-type flies. Transgene expression from ELAV-GeneSwitch was induced in the presence of the drug RU486 to down-regulate an Mgat1 RNAi line (no. 13) and life span was measured as previously described (5). The complete genotype and life span of the line are indicated in Table 1. (B) Targeted expression of Mgat1 in the CNS of Mgat1¹ null Drosophila affects life span. Mgat1¹ null mutants have a maximum life span of 15 days whereas wild-type flies live to 93 days. Targeted expression of Mgat1 to the CNS of Mgat1¹ null mutants [Mgat1 (+RU)] increases the mean and maximum life spans by 73 and 77%, respectively, relative to wild-type flies and by 135% relative to Mgat1¹ null mutant flies that were not treated by drug [Mgat1 (−RU)]. All life span plots were derived from a single population of flies.

Fig. 2.

Life span studies on Mgat1 rescued Mgat1¹ null flies. (A) The effect of oxidative stress on the life span of Mgat1 rescued flies. Resistance to oxidative stress was measured in adult Mgat1¹ null mutant flies that expressed a wild-type Mgat1 transgene in the CNS [Mgat1 (+RU)] compared with genetically matched controls that did not express Mgat1 [Mgat1 (−RU)]. Briefly, flies were fed normal food containing 3% hydrogen peroxide and their life span was measured over a 180-h period. The Mgat1 rescued flies [Mgat1 (+RU)] lived significantly longer under conditions of oxidative stress (mean life span = 127.9 ± 2.4 h) than control flies (mean life span = 80.8 ± 1.7 h). (B) The effect of percentage of yeast in the diet on the life span of Mgat1 rescued flies. The effect of dietary restriction on the life span of Mgat1 rescued flies [Mgat1 (+RU)] compared with Mgat1¹ null mutant flies that were not treated by drug [Mgat1 (−RU)] and to wild-type controls was determined by varying the amount of dietary yeast from 2 to 8% as described (34). Shown is mean life span with SE bars. Asterisks above the line for Mgat1 rescued flies [Mgat1 (+RU)] indicate that survival differed significantly between the Mgat1 rescued flies and controls that were not treated by drug [Mgat1 (−RU)] at yeast concentrations from 2 to 8% using the log-rank rest (P < 0.001). Asterisks between the lines for controls that were not treated by drug [Mgat1 (−RU)] and wild-type flies indicate that survival differed significantly between Mgat1 rescued flies [Mgat1 (+RU)] and wild-type flies only at 2% yeast, using the log-rank rest (P < 0.001). All life span plots were derived from a single population of flies. The standard media used for the data shown in Table 1 contained 2% yeast.

Fig. 3.

Life span studies on wild-type flies with neuron-specific overexpression of Mgat1. (A) Targeted expression of Mgat1 in the CNS of wild-type flies increases Drosophila life span by a relatively small amount. Targeted expression of Mgat1 to the CNS of wild-type flies increases the mean and maximum life spans by 8.8 and 12.7%, respectively, relative to wild-type flies. The genotypes, life spans, and GnT1 activities are shown in Table 1. (B) Wild-type flies overexpressing Mgat1 are resistant to oxidative stress. Resistance to oxidative stress was measured in adult wild-type flies that express a wild-type Mgat1 transgene in the CNS [Mgat1 (+RU)] compared with genetically matched controls that do not express Mgat1 [Mgat1 (−RU)]. Flies were fed normal food containing 3% hydrogen peroxide and their life span was measured over a 180-h period. The Mgat1 expressing flies [Mgat1 (+RU)] lived significantly longer under conditions of oxidative stress than control flies. All life span plots were derived from a single population of flies.