Manganese-based superoxide dismutase mimics modify both acute and long-term outcome severity in a Drosophila melanogaster model of classic galactosemia

Abstract

Aims: The goal of this study was to use two manganese (Mn)-based superoxide dismutase (SOD) mimics to test the hypothesis that reactive oxygen species contribute to both acute and long-term outcomes in a galactose-1P uridylyltransferase (GALT)-null Drosophila melanogaster model of classic galactosemia.

Results: We tested the impact of each of two Mn porphyrin SOD mimics, MnTnBuOE-2-PyP(5+), and MnTE-2-PyP(5+), (i) on survival of GALT-null Drosophila larvae reared in the presence versus absence of dietary galactose and (ii) on the severity of a long-term movement defect in GALT-null adult flies. Both SOD mimics conferred a significant survival benefit to GALT-null larvae exposed to galactose but not to controls or to GALT-null larvae reared in the absence of galactose. One mimic, MnTE-2-PyP(5+), also largely rescued a galactose-independent long-term movement defect otherwise seen in adult GALT-null flies. The survival benefit of both SOD mimics occurred despite continued accumulation of elevated galactose-1P in the treated animals, and studies of thiolated proteins demonstrated that in both the presence and absence of dietary galactose MnTE-2-PyP(5+) largely prevented the elevated protein oxidative damage otherwise seen in GALT-null animals relative to controls.

Innovation and conclusions: Our results confirm oxidative stress as a mediator of acute galactose sensitivity in GALT-null Drosophila larvae and demonstrate for the first time that oxidative stress may also contribute to galactose-independent adult outcomes in GALT deficiency. Finally, our results demonstrate for the first time that both MnTnBuOE-2-PyP(5+) and MnTE-2-PyP(5+) are bioavailable and effective when administered through an oral route in a D. melanogaster model of classic galactosemia.

FIG. 1.

The Leloir pathway of galactose metabolism. Galactose 1-phosphate uridylyltransferase (GALT), the middle enzyme in the pathway, is profoundly impaired in patients with classic galactosemia. The three enzymes of the Leloir pathway, galactokinase (GALK), GALT, and UDP-galactose 4′-epimerase (GALE), are indicated together with the names of their encoding genes in Drosophila melanogaster.

FIG. 2.

Impact of small molecule superoxide dismutase mimics MnTnBuOE-2-PyP⁵⁺ and MnTE-2-PyP⁵⁺ on survival of control and GALT-null Drosophila larvae exposed to galactose. Relative survival of control (open bars) and GALT-null (shaded bars) Drosophila larvae reared on foods that contained both 555 mM glucose and 200 mM galactose in the absence versus presence of the indicated levels of MnTnBuOE-2-PyP⁵⁺ (A) and MnTE-2-PyP⁵⁺ (B). Values plotted represent average±standard error of the mean. The number of cohort replicates for each experimental condition is indicated in parenthesis above the corresponding bar. Relative survival of each cohort was calculated as the ratio of the number of larvae surviving to adulthood from that cohort over the number of control (GALT+/+) larvae surviving to adulthood in the same food lacking additive. Significant differences are indicated. Corresponding data for larvae raised in the absence of galactose are presented in Supplementary Figure S1.

FIG. 3.

Small molecule superoxide dismutase mimic, MnTE-2-PyP⁵⁺, partially rescues a long-term movement defect in adult GALT-null Drosophila independent of galactose exposure. Control (open bars) and GALT-null (shaded bars) Drosophila were reared on foods containing either zero or 10 μM MnTE-2-PyP⁵⁺ (TE) in either the presence or absence of 50 mM galactose, and adult animals were tested for climbing ability at 2 days posteclosion after 25 min at 39°C immediately before testing (see “Materials and Methods” section). Among control flies close to 70% of each cohort climbed above a designated mark regardless of the presence or absence of TE. In contrast, only between 30% and 40% of each cohort of GALT-null flies climbed above the designated mark in the absence of TE, while close to 60% climbed above the mark in the presence of TE. This difference was significant and occurred independent of the presence or absence of 50 mM galactose in the food. Values plotted represent average±standard error of the mean, n≥14. The number of cohort replicates for each experimental condition is indicated in parenthesis above the corresponding bar.

FIG. 4.

Impact of MnTnBuOE-2-PyP⁵⁺ and MnTE-2-PyP⁵⁺ on the accumulation of gal-1P in control and GALT-null Drosophila larvae reared in the presence versus absence of dietary galactose. Gal-1P was extracted from control (open bars) and GALT-null (shaded bars) late-stage larvae reared on foods either with or without 200 mM added galactose, and spiked with the indicated levels of MnTE-2-PyP⁵⁺ or MnTnBuOE-2-PyP⁵⁺. Significant differences are indicated. Values plotted represent average±standard error of the mean, n≥3, as indicated. The number of cohort replicates for each experimental condition is indicated in parenthesis above the corresponding bar.

FIG. 5.

Impact of MnTE-2-PyP⁵⁺ on the abundance of protein-bound glutathione and cysteine in control and GALT-null Drosophila early larvae (L2) reared in the presence of dietary galactose. Protein thiolation is a marker of oxidative stress in biological systems. Samples tested were from control (open bars) and GALT-null (shaded bars) second instar (L2) larvae harvested from foods containing 200 mM galactose (in addition to 555 mM glucose) that did versus did not contain 10 μM MnTE-2-PyP⁵⁺, as indicated. Values plotted represent average±standard error of the mean, n=6. (A) Levels of protein-bound cysteine, and (B) levels of protein-bound glutathione, are plotted. Significant differences are indicated.