Vitamin B5, a coenzyme A precursor, rescues TANGO2 deficiency disease-associated defects in Drosophila and human cells

Abstract

Mutations in the Transport and Golgi Organization 2 (TANGO2) gene are associated with intellectual deficit, neurodevelopmental delay and regression. Individuals can also present with an acute metabolic crisis that includes rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, the latter of which are potentially lethal. While preventing metabolic crises has the potential to reduce mortality, no treatments currently exist for this condition. The function of TANGO2 remains unknown but is suspected to be involved in some aspect of lipid metabolism. Here, we describe a model of TANGO2-related disease in the fruit fly Drosophila melanogaster that recapitulates crucial disease traits. Pairing a new fly model with human cells, we examined the effects of vitamin B5, a coenzyme A (CoA) precursor, on alleviating the cellular and organismal defects associated with TANGO2 deficiency. We demonstrate that vitamin B5 specifically improves multiple defects associated with TANGO2 loss-of-function in Drosophila and rescues membrane trafficking defects in human cells. We also observed a partial rescue of one of the fly defects by vitamin B3, though to a lesser extent than vitamin B5. Our data suggest that a B complex supplement containing vitamin B5/pantothenate may have therapeutic benefits in individuals with TANGO2-deficiency disease. Possible mechanisms for the rescue are discussed that may include restoration of lipid homeostasis.

Keywords: Drosophila; TANGO2; coenzyme A; membrane traffic; metabolic crisis; neurodevelopment; vitamin B5.

Figure 1.. The Drosophila TANGO2^G517 mutants display behavioral defects that are rescued by vitamin B5 supplementation.

(A) Female fly populations were placed into empty plastic vials and survival was examined every hour until all flies were dead. (B), (C) Female fly populations were subjected to elevated temperatures for 120 seconds as detailed in the Methods section. The percentage of flies experiencing a seizure (B) and the time to the first seizure (C) are shown. (D) Female fly populations were subjected to a learning assay as described in the Methods section. The percentage of flies that learned to avoid the quinine-laced light side of the setup were recorded. Where indicated, supplements of either vitamin B5or B3 were included in the food for at least one generation prior to the assays. The concentrations of vitamin B5 used were 4 mM in (A) and 2 mM in (B), (C) and (D). N values for each experiment shown were 75 in (A) and (B), and (C), and 30-40 in (D). The asterisks for statistical analyses indicate p≤0.05 (*), p≤0.01 (**) and p≤0.0001 (****). CTRL represents Ore^R flies and TM represents TANGO2^G517 flies. Results obtained with male flies are shown in Figure S1.

Figure 2.. Motor defects associated with the TANGO2^G517 mutants are rescued by vitamin B5 supplementation.

(A) Female flies were placed into an empty vial, gently knocked to the bottom of the vial and were examined as they climbed. The percentages of flies that reached a line 5 cm from the bottom of the vial within 10 seconds are shown. (B) Third instar larvae were observed as they crawled over 9 minutes on a dish containing 2% agar. The total distance moved is shown. (C) Adult females were placed in the center of a 10 cm diameter petri dish and the distance moved over 10 minutes is displayed, as well as the time spent in the central area of the dish (D). Vitamins B5, B3, B9 or C as indicated were included in the food for at least one generation prior to the assays. The concentrations of vitamin B5 used were 4 mM in (A), (C) and (D), and 2 mM in (B). N values for each experiment shown were 50-75 in (A), 15-35 in (B) and 15 in (C) and (D). The asterisks for statistical analyses indicate p≤0.01 (**) and p≤0.0001 (****). CTRL represents Ore^R flies and TM represents TANGO2^G517 flies. Results for male flies are shown in Figure S2.

Figure 3.. Vitamin B5 supplementation shows a time-dependent rescue of phenotypes in the TANGO2^G517 mutants.

TANGO2^G517 female flies were grown either on food without vitamin B5, or grown for at least one generation either on regular food or on vitamin B5-supplemented food. A portion of the untreated TANGO2^G517 adult flies were placed on vitamin B5-containing food either 1, 2 or 3 days prior to the assays. Flies were then assayed for their climbing ability (A), survival during starvation (B) and resistance to heat-induced seizures (C). The concentrations of vitamin B5 used were 4 mM in (A) and (C), and 2 mM in (B). N values for each experiment shown were 50-75 in (A), 75 in (B) and 75 in (C). The asterisks for statistical analyses indicate p≤0.05 (*), p≤0.01 (**), p≤0.001 (***) and p≤0.0001 (****). TM represents TANGO2^G517 flies. Results obtained with male flies are shown in Figure S3. Note that although blue colouring has been used for vitamin B5 supplementation in all figures, different colouring has been used in panel B to ensure ease of visualization of the curves.

Figure 4.. Vitamin B5 rescues an endoplasmic reticulum-to-Golgi trafficking defect in TANGO2-deficient human fibroblasts.

(A) Wild type (CTRL) fibroblasts or fibroblasts harboring the compound heterozygous exon3-9 del/exon 6 del allele (TANGO2-deficient cells) were transfected with a plasmid expressing sialyltransferase-GFP-streptavidin binding protein which is retained in the ER by association with an ER-localized hook composed of streptavidin. After release of the GFP-tagged protein by biotin, fluorescence was monitored in the Golgi over time and quantified. Solid lines represent CTRL cells and dashed lines represent the TANGO2 mutant cells. Black colouring represents untreated cells, red colouring represents vitamin B5-treated cells, blue colouring represents vitamin B3-treated cells, gray colouring represents vitamin B9-treated cells and green colouring represents vitamin C-treated cells. Note that the only dashed line that clusters with the solid lines is from the vitamin B5-treated cells. (B) Lysates from CTRL and TANGO2-deficient cells either untreated or treated with vitamin B5 for 4 days were probed for TANGO2 and tubulin as a loading control. Note that the protein product of the exon 6 del allele would be expected to appear at ~15kD if it was produced and stable.