Mutant human torsinA, responsible for early-onset dystonia, dominantly suppresses GTPCH expression, dopamine levels and locomotion in Drosophila melanogaster.

Dystonia represents the third most common movement disorder in humans with over 20 genetic loci identified. TOR1A (DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. Most cases of DYT1 dystonia are caused by a 3 bp (ΔGAG) deletion that results in the loss of a glutamic acid residue (ΔE302/303) in the carboxyl terminal region of torsinA. This torsinAΔE mutant protein has been speculated to act in a dominant-negative manner to decrease activity of wild type torsinA. Drosophila melanogaster has a single torsin-related gene, dtorsin. Null mutants of dtorsin exhibited locomotion defects in third instar larvae. Levels of dopamine and GTP cyclohydrolase (GTPCH) proteins were severely reduced in dtorsin-null brains. Further, the locomotion defect was rescued by the expression of human torsinA or feeding with dopamine. Here, we demonstrate that human torsinAΔE dominantly inhibited locomotion in larvae and adults when expressed in neurons using a pan-neuronal promoter Elav. Dopamine and tetrahydrobiopterin (BH4) levels were significantly reduced in larval brains and the expression level of GTPCH protein was severely impaired in adult and larval brains. When human torsinA and torsinAΔE were co-expressed in neurons in dtorsin-null larvae and adults, the locomotion rates and the expression levels of GTPCH protein were severely reduced. These results support the hypothesis that torsinAΔE inhibits wild type torsinA activity. Similarly, neuronal expression of a Drosophila DtorsinΔE equivalent mutation dominantly inhibited larval locomotion and GTPCH protein expression. These results indicate that both torsinAΔE and DtorsinΔE act in a dominant-negative manner. We also demonstrate that Dtorsin regulates GTPCH expression at the post-transcriptional level. This Drosophila model of DYT1 dystonia provides an important tool for studying the differences in the molecular function between the wild type and the mutant torsin proteins.

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

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.

Catecholamines up integrates dopamine synthesis and synaptic trafficking.

The highly reactive nature of dopamine renders dopaminergic neurons vulnerable to oxidative damage. We recently demonstrated that loss-of-function mutations in the Drosophila gene Catecholamines up (Catsup) elevate dopamine pools but, paradoxically, also confer resistance to paraquat, an herbicide that induces oxidative stress-mediated toxicity in dopaminergic neurons. We now report a novel association of the membrane protein, Catsup, with GTP cyclohydrolase rate-limiting enzyme for tetrahydrobiopterin (BH(4)) biosynthesis and tyrosine hydroxylase, rate-limiting enzyme for dopamine biosynthesis, which requires BH(4) as a cofactor. Loss-of-function Catsup mutations cause dominant hyperactivation of both enzymes. Elevated dopamine levels in Catsup mutants coincide with several distinct characteristics, including hypermobility, minimal basal levels of 3,4-dihydroxy-phenylacetic acid, an oxidative metabolite of dopamine, and resistance to the vesicular monoamine transporter inhibitor, reserpine, suggesting that excess dopamine is synaptically active and that Catsup functions in the regulation of synaptic vesicle loading and release of dopamine. We conclude that Catsup regulates and links the dopamine synthesis and transport networks.

Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism.

The underlying defects in Angelman syndrome (AS) and autism spectrum disorder (ASD) may be in part due to basic defects in synaptic plasticity and function. In some individuals serotonin reuptake inhibitors, which decrease pre-synaptic re-uptake of serotonin, can ameliorate symptoms, as can resperidone, which blocks both dopamine and serotonin receptors. Loss of maternal UBE3A expression causes AS, while maternal duplications of chromosome 15q11.2-q13 that include the UBE3A gene cause ASD, implicating the maternally expressed UBE3A gene in the ASD phenotype. In a Drosophila screen for proteins regulated by UBE3A, we identified a key regulator of monoamine synthesis, the gene Punch, or GCH1, encoding the enzyme GTP cyclohydrolase I. Here we show that Dube3a, the fly UBE3A orthologue, regulates Punch/GCH1 in the fly brain. Over-expression of Dube3a elevates tetrahydrobiopterin (THB), the rate-limiting cofactor in monoamine synthesis while loss of Dube3a has the opposite effect. The fluctuations in dopamine levels were associated with hyper- and hypoactivity, respectively, in flies. We show that changes in Punch/GCH1 and dopamine levels do not depend on the ubiquitin ligase catalytic domain of Dube3a. In addition, both wild type Dube3a and a ubiquitination-defective Dube3a-C/A form were found at high levels in nuclear fractions and appear to be poly-ubiquitinated in vivo by endogenous Dube3a. We propose that the transcriptional co-activation function of Dube3a may regulate GCH1 activity in the brain. These results provide a connection between monoamine synthesis (dopamine/serotonin) and Dube3a expression that may explain why some individuals with ASD or AS respond better to selective serotonin reuptake inhibitors than others.