Dtorsin, the Drosophila ortholog of the early-onset dystonia TOR1A (DYT1), plays a novel role in dopamine metabolism

Abstract

Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.

Figure 1. dtorsin loss-of-function mutant flies exhibit pigmentation phenotypes.

A–D, Pigmentation phenotype of dtorsin mutants. Adult males of the genotype y w/Y (A), y w dtorsin^KO13/Y (B), Oregon R (C), and w dtorsin^KO78/Y (D) flies. The pictures for A–D were taken one day after eclosion to ensure pigmentation reached the maximum level. Since dtorsin^KO78 male has y+ genotype, it was compared to the Oregon R, which is also y+.

Figure 2. dtorsin mutant larvae have mobility defects.

A. dtorsin mutants have reduced larval mobility. Peristaltic frequencies were counted manually during the wandering stage of third instar larvae of the genotype y w (n = 33), y w dtorsin^KO13/Y (n = 29), and y w dtorsin^KO13/Y; GDT101-2 (n = 21). Results are the mean ± S.E.M. ** p<0.0001, very significant difference between the wild type and the dtorsin mutant. B–E. Representative crawling patterns of the wild type (B, C) and the dtorsin mutant male (D, E) larvae. Locations of larval heads (blue line) and tails (red line) were captured by video recording for 60 seconds and visualized by the Prairie Dog software. Tracking of larval movement was stopped when the larva reached the edge of the plate. The genotypes of larvae were y w (B, C) and y w dtorsin^KO13/Y (D, E). Most of the wild type larvae reached the edge of the dish within 30–40 seconds when they started from the center of the dish (B, C). Many of the dtorsin mutant male larvae circled around the same location without much net distance gain from the starting point (D, E). The numbers on x- and y- axes are arbitrary numbers indicating x- and y- coordinates to define locations of the larval head and tail.

Figure 3. Rescue of dtorsin mutant male larvae mobility defects by cDNA expression.

Peristaltic frequencies were counted for the wandering stage third instar larvae of the genotype wild type y w/Y male (n = 15), w elavGAL4/Y (n = 9), y w/Y; TH-GAL4(III) (n = 12), w elavGAL4 dtorsin^KO13/Y (n = 32), w elavGAL4 dtorsin^KO13/Y; UAS-dtorsinB5(II)/+ (n = 20), w elavGAL4 dtorsin^KO13/Y; UAS-htorA#8(II)/+ (n = 20), y w dtorsin^KO13/Y (n = 28), and y w dtorsin^KO13/Y; TH-GAL4(III)/+; UAS-dtorsinB5(II)/+ (n = 12). Results are mean ± S.E.M. *** p<0.0001, very significant difference between the dtorsin mutant male (w elavGAL4 dtorsin^KO13/Y) and the dtorsin mutant male with elavGAL4/UAS-dtorsin. *** p<0.0001, very significant difference between the dtorsin mutant male (w elavGAL4 dtorsin^KO13/Y) and the dtorsin mutant male with elavGAL4/UAS-htorA. ** p<0.001, significant difference between the dtorsin mutant (y w dtorsin^KO13/Y) and the dtorsin mutant male with TH-GAL4/UAS-dtorsin.

Figure 4. Rescue of dtorsin mutant larvae mobility defects by dopamine feeding.

Peristaltic frequencies were counted for the wandering stage third instar larvae of the genotype wild type (y w) (n = 15), wild type (y w) (+20 mM dopamine) (n = 10), wild type (y w) (+20 mM octopamine) (n = 8), wild type (y w) (+10 mM serotonin) (n = 9), y w dtorsin^KO13/Y (n = 28), y w dtorsin^KO13/Y (+20 mM dopamine) (n = 15), y w dtorsin^KO13/Y (+20 mM octopamine) (n = 10), and y w dtorsin^KO13/Y (+10 mM serotonin) (n = 13). Results are mean ± S.E.M. *** p<0.0001, very significant difference between the dtorsin mutant with no drug supplementation and the dtorsin mutant with 20 mM dopamine supplementation. There was no significant difference between the dtorsin mutant males with no drug supplementation and the dtorsin mutant males with 20 mM octopamine (p = 0.4240) or the dtorsin mutant male with 10 mM serotonin (p = 0.8148). There was no significant difference between the wild type with no drug supplementation and the wild type with drug (+20 mM dopamine, +20 mM octopamine, or +10 mM serotonin).

Figure 5. Effect of dopamine on the larval crawling patterns.

Video tracking of the representative crawling patterns of the wild type (A, B), the wild type with 50 mM dopamine supplementation (C, D), the y w dtorsin^KO13 mutant male (E, F), and the y w dtorsin^KO13 mutant male with 50 mM dopamine supplementation (G, H). Locations of larval heads (black lines) were tracked by the ICARUS software. Tracking was stopped when the larva reached the edge.

Figure 6. Effect of dtorsin mutation on dopamine in larval brains and adult heads.

A. Effect of dtorsin heterozygous mutation on dopamine pools in larval brains (nanograms per brain). Monoamines were extracted from third instar larval brains and dopamine was separated and quantified by HPLC. dtorsin mutation significantly reduced dopamine levels. The genotypes of larvae were y w females (n = 3 independent replications) and w dtorsin^KO78/+ females (n = 3 independent replications). Error bars indicate S.E.M. ** p<0.001, significant difference between the wild type and the dtorsin heterozygous mutant. B.-D. Effect of dtorsin heterozygous mutation on dopamine levels (nanograms per head). (B), DOPAC levels (nanograms per head) (C), and DOPAC/dopamine ratio (D) in adult heads. The genotypes of adults were y w females (n = 3 independent replications), y w dtorsin^KO13/+ females (n = 3 independent replications), and w dtorsin^KO78/+ females (n = 3 independent replications). Error bars indicate S.E.M. ** p<0.001, significant difference in dopamine levels between the wild type and the dtorsin heterozygous mutant females (B). No statistically significant difference was observed in DOPAC levels between the wild type and the dtorsin heterozygous mutant females, although the mutant levels were slightly lower (C). * p<0.01, significant difference in DOPAC/dopamine ratio between the wild type females and the dtorsin heterozygous mutant females (D).

Figure 7. The third instar larval brains of dtorsin mutants have TH-positive cells.

A and B. Comparison of patterns of TH immunostaining (in green) in the third instar larval central nervous system of the wild type male (A) and the dtorsin mutant male (B). A: A projection of twenty-seven confocal z-sections of wild type (y w) third instar male brain is shown. B: A projection of thirty confocal z-sections of y w dtorsin^KO13/Y third instar male larval brain is shown. The projection was made from a series of confocal Z-sections (optical thickness 2 µm) that covered the whole brain regions using the NIH Image-J program with the Max intensity method. vg: ventral ganglion.

Figure 8. The double heterozygous combinations for dtorsin and Punch or pale mutants show reduced mobility in third instar larvae.

A. Peristaltic frequencies were counted for the wandering stage third instar larvae of the genotype y w female (n = 31), Pu^Z22/+ female (n = 23), dtorsin^KO13/+ female (n = 22), and dtorsin^KO13/+; Pu^Z22/+ female (n = 30). Results are the mean ± S.E.M. **p<0.0001, significant difference between the wild type females (yw, Pu^Z22/+, or dtorsin^KO13/+) and the double heterozygous females (dtorsin^KO13/+; Pu^Z22/+). No significant difference was observed between the wild type females (y w) and the single heterozygous females (Pu^Z22/+ or dtorsin^KO13/+). B. Peristaltic frequencies for the wandering stage third instar larvae of the genotype y w female (n = 20), ple²/+ female (n = 20), dtorsin^KO13/+; female (n = 31), and dtorsin^KO13/+; ple²/+ female (n = 30). Results are the mean ± S.E.M. *p<0.05, significant difference between the wild type females (y w, ple²/+, or dtorsin^KO13/+) and the double heterozygous females (dtorsin^KO13/+; ple²/+). No significant difference between the wild type females (y w) and the single heterozygous females (ple²/+ or dtorsin^KO13/+).

Figure 9. Effect of dtorsin mutation on TH activity and GTPCH activity in adult heads.

A. Effect of dtorsin heterozygous mutation on TH activity in adult heads (L-dopa nmoles/min/mg protein). The genotypes of adults were y w (n = 3 independent replications), y w dtorsin^KO13/+ (n = 3 independent replications), and w dtorsin^KO78/+ (n = 3 independent replications). Error bars indicate S.E.M. No significant difference between the wild type and the dtorsin heterozygous mutant females. B. Effect of dtorsin heterozygous mutation on GTPCH activity (neopterin nmoles/min/mg protein). The genotypes of adults were y w (n = 3 independent replications), y w dtorsin^KO13/+ (n = 3 independent replications), and w dtorsin^KO78/+ (n = 3 independent replications). Error bars indicate S.E.M. ** p<0.01, significant difference in dopamine levels between the wild type females and the dtorsin heterozygous mutant females.

Figure 10. Effect of dtorsin mutation on TH and GTPCH protein levels.

Total adult head extracts from the wild type males (y w) (lanes 1 and 4), y w dtorsin^KO13/+ females (lanes 2 and 5), or y w dtorsin^KO13/Y males (lanes 3 and 6) were analyzed by Western blots. The membrane was probed with anti-GTPCH B/C (lanes 1–3, upper panel) and reprobed with anti-Tubulin (lanes 1–3, lower panel). The second membrane was probed with anti-TH (lanes 4–6, upper panel) and reprobed with anti-Tubulin (lanes 4–6, lower panel). The location of GTPCH (43–45 kDa), TH (58 kDa), and alpha-Tubulin (50 kDa) are indicated. Twenty µg of proteins were loaded in each lane.