Search

Dopamine (DA) regulates movement, cognition, attention and reward (Calabresi et al., 2007; Palmiter, 2007; Sillitoe and Vogel, 2008), while dysfunction of dopaminergic signaling has been implicated in numerous neurological diseases and abnormal behaviors. The highly reactive nature of DA itself places dopaminergic neurons in the substantia nigra at heightened risk of oxidative damage, the primary route to cellular death in Parkinson’s disease (PD) (Goedert, 2001; Lotharius and Brundin, 2002, Maries et al., 2003). Moreover, elevated levels of DA or BH4, a cofactor required for DA synthesis, are neurotoxic in several model systems (Berman and Hastings, 1999; Choi et al., 2003; Gomez-Santos et al., 2006). Recently, it has been reported that DA in Drosophila PD models similarly enhances neurodegeneration (Bayersdorfer et al., 2010). In an earlier study, we found that tyrosine hydroxylase (TH; EC1.14.16.2), the rate-limiting enzyme in DA biosynthesis, is dominantly activated by a post-translational mechanism in Drosophila strains heterozygous for loss-of-function Catecholamines up (Catsup) mutations (Stathakis et al., 1999). Subsequently, we investigated the effects of Catsup and other genes that regulate DA synthesis and homeostasis on sensitivity to the oxidative toxin, paraquat (PQ), which we found to selectively trigger dopaminergic neuron degeneration and parkinsonian-like movement disorders (Chaudhuri et al., 2007). Surprisingly, loss-of-function Catsup mutations, despite having strongly elevated levels of DA and tetrahydrobiopterin (BH4), were dominantly neuroprotective against PQ exposure (Chaudhuri et al., 2007). Here, we report the results of our investigation of the mechanisms by which Catsup mutations can enhance BH4 and DA synthesis and yet simultaneously confer neuroprotection against PQ-induced oxidative insult. We find that Catsup acts to negatively regulate BH4 and DA synthesis pathways through physical interactions with the rate-limiting enzymes of both pathways, GTP cyclohydrolase I (GTPCH; EC3.5.4.16) and TH, respectively. Moreover, we provide evidence that Catsup is localized to synaptic termini and acts as a negative regulator of vesicular monoamine transporter (VMAT), which is responsible for synaptic vesicle uptake of DA. We conclude that Catsup has the ability to regulate DA homeostasis by effectively integrating the synthesis of both the BH4 and DA pathways through their respective rate-limiting enzymes, with the transport and synaptic release of DA.

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.

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.

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.