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

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Abstract

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.

Research Highlights

►Dube3a regulates GCH1 in a non-ubiquitin ligase dependent manner. ►Expression of a Dube3a mutant that cannot ubiquitinate can regulate GCH1. ►Endogenous Dube3a can ubiquitinate ectopically expressed Dube3a proteins in vivo. ►Activity is dependent on Dube3a levels and directly proportional to dopamine levels. ►The transcriptional co-activation function of Dube3a may be regulating GCH1 activity.

Introduction

The regulation of neurotransmitters like dopamine and serotonin in the brain is critical for proper synaptic function as well as plasticity. These neurotransmitters may be inappropriately regulated in the brains of autistic individuals accounting for at least some of the synaptic stability defects observed in autism (Makkonen et al., 2008). Although the underlying genetic heterogeneity of idiopathic autism has complicated the analysis of neurotransmitter levels, it has become apparent that at least some individuals with autism have altered tetrahydrobiopterin (THB) levels in cerebrospinal fluid (Tani et al., 1994). THB is a regulatory cofactor for the synthesis of monoamines including both dopamine and serotonin, neurotransmitters that have been linked to many of the behavioral traits manifested in ASD. Resperidone, an atypical neuroleptic that blocks dopamine and serotonin receptors has been widely employed to ameliorate monoamine-related behaviors in autistic individuals (c.f., Canitano and Scandurra, 2008). Treatment of the anxiety and hyperactivity associated with autism often involves the use of selective serotonin reuptake inhibitors (SSRIs), which inhibit the presynaptic re-uptake of serotonin thereby increasing postsynaptic activation (Kolevzon et al., 2006). Consistent with mounting evidence of monoamine variation in ASD are reports of haplotype associations in monoamine-related genes. For example, Hettinger et al. (2008) reported over-transmission in ASD families of two D1 dopamine receptor haplotypes (Hettinger et al., 2008). Thus, the improper regulation of synaptic monoamine levels may be a key aspect of autism pathogenesis.

The most common cytogenetic abnormalities detected in autistic individuals are maternally derived DNA duplications of the 15q11.2–q13 region, also known as the Prader–Willi/Angelman syndrome critical region (reviewed in (Hogart et al., 2010). The primary candidate gene in this region, UBE3A, encodes an E3 ubiquitin ligase protein that targets other proteins for degradation by the ubiquitin proteasome system (Kishino et al., 1997, Schroer et al., 1998). In the majority of cases maternally derived deletions of UBE3A result in the devastating neurogenetic disorder Angelman syndrome (AS), but other molecular defects that can turn off maternal UBE3A also result in an AS phenotype (Jiang et al., 1999). Mechanisms of UBE3A regulation may not be so simple, however, since UBE3A can also act as a transcriptional co-activator of steroid hormone receptors (Ramamoorthy and Nawaz, 2008) and mono-ubiquitination by UBE3A may be involved in the trafficking of synaptic proteins (Haas and Broadie, 2008, Hicke, 2001). The observation that UBE3A displays maternal allele-specific expression in the mammalian brain clearly implicates it in the pathology of both AS and the autism phenotype found in 15q11.2–q13 duplications (Albrecht et al., 1997, Lalande and Calciano, 2007, Rougeulle et al., 1997). In addition, individuals with Prader–Willi syndrome (PWS) who have two maternal copies of the 15q11.2–q13 region due to uniparental disomy for chromosome 15 also present with autistic features not typically found in PWS patients who have inherited paternal deletions of 15q11.2–q13 (Descheemaeker et al., 2006, Veltman et al., 2005). Finally, UBE3A levels are indeed elevated in lymphocytes from individuals with larger isodicentric 15q duplications, again implicating elevations of UBE3A in the pathogenesis of autism (Baron et al., 2006).

Using the powerful genetic tools available in Drosophila melanogaster we have been investigating the molecular etiology of both duplication 15q autism and AS. Our approach is to elevate fly and human UBE3A levels in the brains of Drosophila and then use proteomic profiling to identify potential UBE3A target proteins. Using this method we successfully identified a UBE3A-interacting protein involved in actin cytoskeletal rearrangements and neurogenesis, the Pbl/ECT2 protein (Reiter et al., 2006). Here we describe the identification of another Dube3a-regulated protein that is a key regulator of monoamine synthesis in flies as well as humans. In this report we show that expression of both wild type and ubiquitination defective Dube3a is able to regulate the activity of the Punch protein, an enzyme that produces tetrahydrobiopterin (THB), the rate-limiting co-factor in monoamine synthesis. This is the first report of a UBE3A target involved in neurotransmitter regulation and has broad implications for how the function of this target may contribute to the pathogenesis of AS, duplication 15q autism as well as idiopathic autism linked to UBE3A regulated pathways. We propose that the regulation of dopamine/serotonin levels by UBE3A may be responsible for some of the anxiety and hyperactivity observed as part of the clinical phenotype in AS and duplication 15q autism providing a mechanistic basis for therapeutic interventions that target the monoamine synthesis pathway.

Section snippets

Drosophila stocks and crosses

All fly crosses were performed at 25 °C on standard corn meal based media. The following stocks were obtained from the Bloomington Stock Center (Bloomington, IN): HS-GAL4, gmr-GAL4, elav-GAL4, Pur1, PuEY4414 and PuEY2616. The UAS-RNAi-Dube3a (6190-R3) line was obtained from the National Institute of Genetics RNAi stock center (Mishima, Shizuoka, Japan). The UAS-FLAG-Dube3a lines were constructed by PCR cloning the insert from Gold Collection cDNA LD21888 into the Gateway (Invitrogen) acceptor

The Punch protein is elevated in fly neurons expressing human or fly UBE3A

Our approach to the identification of UBE3A protein targets using proteomic profiling in Drosophila head extracts has been described previously (Reiter et al., 2006). In short, using the Drosophila GAL4/UAS system (Brand and Perrimon, 1993, Duffy, 2002) we expressed high levels of hUBE3A protein using the Heatshock-GAL4 driver (HS-GAL4). We then made cytoplasmic protein extracts from HS-GAL4 alone and HS-GAL > UAS-hUBE3A fly heads. Using this method, we identified a protein spot at approximately

Discussion

Finding proteins or genes regulated by UBE3A that result in neurological defects is a daunting task. Unlike the analysis of mutants for a developmental pathway which exhibit obvious phenotypic endpoints, it is clear from phenotypic variability in both AS and duplication 15q autism, that disruption of UBE3A pathway members may result in subtle synaptic or biochemical changes in the brain that are difficult to detect. For example, it has only recently been determined that loss of Ube3a results in

Conclusions

Here we show that Drosophila Dube3a is able to regulate dopamine levels through GCH1 with direct behavioral consequences even in the absence of ubiquitin ligase function. We conclude that this regulation may occur through the transcriptional co-activation domain of the Dube3a protein.

The following are the supplementary materials related to this article.

. Primers and probes used for qRT-PCR analysis.

Acknowledgments

We are grateful for primary antibodies supplied by J. Fischer and the Developmental Studies Hybridoma Bank. We also thank the UTHSC Molecular Resource Center for use of the Roche real-time PCR machine and the UTHSC Cancer Center for use of the Odessy IR imaging system. This work was funded in part by grants from Autism Speaks, the Angelman Syndrome Foundation and NIH NINDS R01NS059902 to L.T.R. Additional support was provided to J.M.O. by the University of Alabama. The National Institutes of

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