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 a defect in neocortical plasticity, despite the fact that this mouse model was generated over ten years ago (Yashiro et al., 2009
). Just generating these AS animal models is not enough, one must also take maximum advantage of the particular strengths of these models. For example, behavior and neuroanatomical studies are more suited to the mouse model while genetic pathway and biochemical analysis is better suited to the fly model. Here we have taken a strictly biochemical approach to the identification of Dube3a targets in Drosophila
. We have identified a protein that not only changes expression as a result of changes in Dube3a but also has a direct effect on brain neurochemistry related to monoamine pools. We also have demonstrated, for the first time in neuronal tissues that this regulation is at the transcriptional level and is not dependent on the ubiquitin ligase function of Dube3a. We also demonstrated that endogenous Dube3a can ubiquitinate ectopically expressed Dube3a proteins in vivo
, a phenomenon that was assumed from in vitro
work but never actually demonstrated in animals. Our results with the FLAG-tagged form of Dube3a also suggest that this transgenic construct can increase expression at the endogenous Dube3a
locus (i.e. Dube3a may regulate its own expression) ().
It is not surprising to find Dube3a in the nucleus, per se
, since it has been known for some time that Ube3a antibodies show a nuclear signal and that at least two splice-forms of Ube3a
localize to the nucleus in the mouse (Dindot et al., 2008
; Reiter et al., 2006
). In this case, we see changes in both transcript and protein levels in Punch-RB, as well as downstream up-regulation of dopamine when we over-express a ubiquitination-defective form of Dube3a
in neurons. These results are bolstered by the observations that Punch transcription levels decrease in a homozygous Dube3a
mutant, but are still detectable, suggesting that Dube3a may act as a transcriptional co-activator in fly neurons just as it does in cultured cells with regards to the human steroid hormone receptor (Ramamoorthy and Nawaz, 2008
). A recent survey of gene expression changes in the cerebellum of Ube3a
deficient mice also supports the argument that transcriptional regulation may play an important role the pathogenesis of AS. Low et al.
found that 89% of transcripts that were differentially expressed in Ube3a
deficient versus wild type mice were down-regulated in the Ube3a
deficient brain consistent with the idea that transcriptional co-activation by Dube3a may be just as critical as ubiquitination (Low and Chen, 2010
). The possibility that transcriptional change may be at least partially responsible for the human AS phenotype are bolstered by the identification of individuals with AS-like features who have mutations in TCF4
(Takano et al., 2010
), which encodes a transcription factor protein, and MeCP2
(Milani et al., 2005
; Watson et al., 2001
), which encodes a transcriptional repressor protein. The possibility that UBE3A is a transcriptional co-activator in conjunction with TCF4 in humans has not yet been investigated, but the exploration of the interaction between Dube3a and the fly orthologue to TCF4, the daughterless transcription factor, could be an interesting avenue of research in flies, leading to a better understanding of the cadre of genes regulated at the transcriptional co-activation level by UBE3A in humans.
In principle, the elevation in transcription of the Punch locus could occur through one of two mechanisms. The ubiquitination function of Dube3a could act to remove a transcriptional silencer of Punch, indirectly stimulating elevated Punch expression. Alternatively, the transcriptional co-activator function of Dube3a could directly, or indirectly, lead to stimulation of Punch transcription. Since over-expression of the ubiquitination-defective Dube3a-C/A mutant led to elevated transcription (and subsequent elevation of translation) of Punch mRNA, we conclude that the ubiquitination function of the Dube3a enzyme plays no detectable role in the regulation of GTP cyclohydrolase expression in flies.
The modulation of GTP cyclohydrolase synthesis has direct consequences for the production of the monoamines, dopamine and serotonin, and therefore, in synaptic function and downstream behaviors. The GTP cyclohydrolase catalytic function, the conversion of GTP to the pteridine dihydroneopterin triphosphate, is the rate-limiting step in the production of THB. THB is a redox cofactor that is absolutely required by the rate-limiting enzyme in dopamine biosynthesis, tyrosine hydroxylase (TH), for the conversion of tyrosine to 3, 4-dihydroxyphenylalanine (L-Dopa), which is subsequently converted to dopamine (Axelrod, 1971
; Nagatsu et al., 1964
TH, encoded by pale
) (Neckameyer and White, 1993
), shares 60% amino acid similarity with human TH (Neckameyer et al., 2005
). TH catalytic activity in Drosophila
, is tightly regulated by availability of the THB cofactor, and therefore by GTP cyclohydrolase modulation, as it is in mammals. In heterozygous Punch
mutants, reductions in THB pools are closely mirrored by similar deficits in TH activity and in dopamine pools (Chaudhuri et al., 2007
; Krishnakumar et al., 2000
; Mackay et al., 1985
). Similarly, mutations in the human GCH1
locus lead to the hereditary diseases hyperphenylalaninemia and Dopa-responsive dystonia (reviewed in (Thony et al., 2000
)). This protein, like TH, is also highly conserved: the human and Drosophila
GTPCH proteins share 80% similarity within the catalytic core, diverging only in N-terminal domains that serve to regulate catalytic activity (Funderburk et al., 2006
; McLean et al., 1993
The human GCH1
gene encodes several isoforms of GTP cyclohydrolase I, only one of which is enzymatically active. The remaining forms are truncated at the C-terminus and are thought to have regulatory functions. In contrast, the Punch
locus of Drosophila
encodes at least 3 isoforms of GTP cyclohydrolase, all sharing identical C-terminal catalytic domains and therefore, all are catalytically active. Each isoform has a unique N-terminal domain originating through a combination of alternative RNA splicing and alternative promoter use. Interestingly, Pu-RB
(originally designated as GTPCH isoform A in McLean et al., 1993
) is transcribed from a different promoter than the remaining forms, and this promoter must therefore possess target sequences for a transcription factor capable of functionally interacting with Dube3a or that is itself regulated by Dube3a.
While there is concordance between the effects of varying Dube3a expression on the Pu-RB transcript and protein isoform levels, the levels of Punch isoforms RA and RC appear to be elevated in parallel with the RB isoform, despite the apparent lack of RA/RC transcriptional response when the human UBE3A or the ubiquitination-defective form of Dube3a is expressed. Since the levels of Transcripts RA and RC do not change, one explanation for this observation is that the elevated levels of Isoform RB serve to stabilize the remaining isoforms in the GTP cyclohydrolase homodecamer complex. All isoforms have identical catalytic and homomultimer interaction domains, differing only in their N-terminal regulatory domains. Therefore, the excess RB polypeptides have the capacity to associate with RA and RC isoforms, and in consequence, could slow the turnover of isoforms that are normally highly sensitive to neural signaling. In principle, such hetero-isoform assemblies could be detected in native electrophoresis gels, but with a molecular mass approaching 500kDa it would be exceptionally challenging. The consequence of these complex interactions is that we cannot conclude that the observed elevation in THB pathway products or dopamine are due solely to the action of Dube3a in regulating RB transcription. These complex relationships between isoforms may also contribute to the enhanced Dube3a over-expression phenotype in the adult eye. We expected to observe suppression of the Dube3a eye phenotype in Punch mutant backgrounds, but instead found that the eye phenotype was enhanced. This result may be due to uncoordinated expression of the various Punch isoforms in the over-expression background.
Another unexpected outcome of our experiments is that pan-neuronal over-expression of wild type Dube3a
results in a 3.6 + 1.6 fold elevation in Punch-RB transcript (), while the wild type form has a modest effect on Punch RB protein levels (). Since we observe elevation of both the THB pathway components and dopamine pools in our neurochemical analysis and observe a functional consequence of these modulations in levels of dopamine, we infer that the immunoblots are perhaps not as sensitive in quantifying expression levels. We do not observe, nor did we expect, a precise correspondence between the transcriptional effects of over-expressing the wild type and ubiquitination-defective forms of Dube3a and the THB and dopamine endpoints. Under normal conditions, the expression of Punch
is rate-limiting for THB and dopamine production, but under over-expression conditions it is expected that other components of these biosynthesis pathways will become limiting to some extent. Moreover, the THB and dopamine pathways are very sensitively regulated by post-translational mechanisms that include end-product feedback inhibition and phosphorylation or dephosphorylation of both GTP cyclohydrolase and tyrosine hydroxylase (Axelrod, 1971
; Funderburk et al., 2006
; Neckameyer et al., 2005
; Thony et al., 2000
). These homeostatic mechanisms can be over-ridden by over-expression of Punch
only to a point, as sensitive regulation of these pathways is critical for neuronal function.
The consequences of mutations in the Punch
locus are varied as expected for the rate-limiting step in the biosynthesis of a cofactor that is not only required for dopamine synthesis, but for the synthesis of serotonin and nitric oxide, as well (Thony et al., 2000
). Serotonin deficits associated with Punch
mutations have been linked to developmental abnormalities including failure of ectodermal cell movements during gastrulation and in cuticular patterning (Colas et al., 1999
), while diminished production of dopamine results in aberrant tracheal cell migration in Drosophila
embryos (Hsouna et al., 2007
). Subsequently, abnormalities in dopamine pools lead to variations in activity/locomotion, as well as to altered stress responses (Chaudhuri et al., 2007
). There are clear parallels in these functions with those ascribed to these neurotransmitters in mammals, and suggest that the effect of changes in Dube3a
expression in Drosophila
will be an important model for identifying the underlying molecular framework of syndromes associated with altered Ube3a
gene dosage in humans.
There is at least some evidence that selective serotonin reuptake inhibitors can dampen the hyperactivity and anxiety in both AS deletion (Pelc et al., 2008
) and duplication 15q autism individuals (Hogart et al., 2010
) indicating that altered serotonin levels contribute to the phenotype in these conditions. Significantly, associations of dopamine-related variation such as dopamine D1 receptor haplotypes, in ASD families (Canitano and Scandurra, 2008
) have been reported, and deficits in dopamine-dependent behaviors have been recently in a mouse Ube3a
knock-out model of AS (Mulherkar and Jana, 2010
). It is likely that both monoamine classes, which are both dependent upon GCHI activity, are altered is ASD individuals. This study is the first step in connecting UBE3A levels with changes in brain neurochemistry, but subsequent studies of THB levels in cerebrospinal fluid from both AS and duplication 15q autism subjects will be required in the future to establish the regulation of GCH1 by UBE3A in the brain extends to humans.