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Angelman syndrome - a neurogenetic disorder caused by defective ubiquitination
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Drosophila genes associated with
Angelman syndrome
Related terms

Neuromuscular junction
Overview of the disease

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe mental retardation, lack of speech, ataxia, susceptibility to seizures, and unique behavioral features such as easily provoked smiling and laughter and autistic features. The disease is primarily caused by deletion or loss-of-function mutations of the maternally inherited UBE3A gene located within chromosome 15q11-q13. The UBE3A gene encodes a 100 kDa protein that functions as ubiquitin ligase and transcriptional coactivator. dUBE3A, the Drosophila homologue of human UBE3A, is deleted imprecisely such that the corresponding protein is not formed. Lack of dUBE3A is not lethal and the flies born show no morphological abnormality. However, they do show motor abnormalities when tested on motor specific tasks. They have impaired long-term memory formation and abnormal circadian rhythms (Jana, 2012 and references therein).

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Relevant studies of Angelman syndrome

Valdez, C., Scroggs, R., Chassen, R. and Reiter, L.T. (2015). Variation in Dube3a expression affects neurotransmission at the Drosophila neuromuscular junction. Biol Open 4: 776-782. PubMed ID: 25948754

Changes in UBE3A expression levels in neurons can cause neurogenetic disorders ranging from Angelman syndrome (AS) (decreased levels) to autism (increased levels). This study investigated the effects on neuronal function of varying UBE3A levels using the Drosophila neuromuscular junction as a model for both of these neurogenetic disorders. Stimulations that evoked excitatory junction potentials (EJPs) at 1 Hz intermittently failed to evoke EJPs at 15 Hz in a significantly higher proportion of Dube3a over-expressors using the pan neuronal GAL4 driver C155-GAL4 (C155-GAL4>UAS-Dube3a) relative to controls (C155>+ alone). However, in the Dube3a over-expressing larval neurons with no failures, there was no difference in EJP amplitude at the beginning of the train, or the rate of decrease in EJP amplitude over the course of the train compared to controls. In the absence of tetrodotoxin (TTX), spontaneous EJPs were observed in significantly more C155-GAL4>UAS-Dube3a larva compared to controls. In the presence of TTX, spontaneous and evoked EJPs were completely blocked and mEJP amplitude and frequency did not differ among genotypes. These data suggest that over-expression of wild type Dube3a, but not a ubiquitination defective Dube3a-C/A protein, compromises the ability of motor neuron axons to support closely spaced trains of action potentials, while at the same time increasing excitability. EJPs evoked at 15 Hz in the absence of Dube3a (Dube3a15b homozygous mutant larvae) decay more rapidly over the course of 30 stimulations compared to w1118 controls, and Dube3a15b larval muscles have significantly more negative resting membrane potentials (RMP). However, these results could not be recapitulated using RNAi knockdown of Dube3a in muscle or neurons alone, suggesting more global developmental defects contribute to this phenotype. These data suggest that reduced UBE3A expression levels may cause global changes that affect RMP and neurotransmitter release from motorneurons at the neuromuscular junction. Similar affects of under- and over-expression of UBE3A on membrane potential and synaptic transmission may underlie the synaptic plasticity defects observed in both AS and autism (Valdez, 2015).


  • Deficiency for Dube3a protein in Dube3a15b homozygous mutants causes a decrease in EJP amplitude and RMP during rapid stimulation.
  • Knockdown of Dube3a in either muscle or neurons alone does not recapitulate the Dube3a15b loss of function EJP and RMP phenotypes.
  • Elevated Dube3a expression results in fewer active zones per bouton and a decrease in vesicle area.

This study demonstrates that both over-expression and deficiency for Dube3a, the fly orthologue of human UBE3A, alters neurotransmission at the neuromuscular junction in Drosophila melanogaster 3rd instar larvae. In a significant proportion of larvae expressing elevated levels of Dube3a in neurons, rapid stimulation of motor nerves intermittently fails to evoke an EJP, and spontaneous depolarizations resembling evoked EJPs are frequently observed in the absence of TTX. However, the amplitude of the first EJP in the train of evoked EJPs and the amplitude and frequency of mEJPs does not vary between any of the genotypes, indicating that this is an axonal rather than vesicle recycling issue. Also, in over-expressors that do not exhibit evoked EJP failure, EJP amplitude does not change more than controls during rapid stimulation. Finally, the spontaneous depolarizations are not observed in larvae over-expressing a ubiquitination defective form of Dube3a (Dube3a-C/A) indicating that the phenomena is dependent on the ubiquitin ligase function of the Dube3a protein. These data could be explained by assuming that evoked EJP failure and spontaneous depolarizations result from regulation of Dube3a ubiquitin target(s) in motor neuron axons rather than directly on the release of neurotransmitter at the synapse. One possible explanation for the spontaneous depolarizations and failures is that Dube3a over-expression results in a depolarization of the RMP of the motor neurons. It is possible that a depolarized membrane potential could result in inactivation of Na+ channels, which could lead to inability of the axons to conduct closely spaced action potentials. At the same time, depolarization of the membrane potential could increase excitability of the axon by bringing it closer to the potential where large numbers of Na+ channels begin to activate. Under this condition any minor perturbation of the axon membrane potential could trigger an action potential in the motor neuron and subsequent EJP in the targeted muscle. The spontaneous depolarizations often appear as bursts, the termination of which might be also be explained by Na+ channel inactivation, similar to the intermittent failures observed at rapid stimulation rates. There was no significant difference in muscle RMP in Dube3a over-expressors versus controls, which is expected since the C155-GAL4 driver employed selectively targets neurons and not muscle (Valdez, 2015).

Complete loss of Dube3a expression in the mutant resulta in a different pattern of effects from over-expression. In w1118; Dube3a15b/Dube3a15b larvae, which make no functional Dube3a protein, the EJP decreases more rapidly in response to rapid stimulation compared to their w1118 controls. This is typically referred to as short term depression (STD). The observation of apparent STD in Dube3a15b larvae could be related to the observation that short term facilitation (STF) is less frequently observed in Dube3a15b versus their w1118 controls. STD is thought to be due to a depletion of the readily releasable pool of synaptic vesicles, while STF is thought to be the result of Ca2+ build up in the terminal due to rapid successive depolarizations. At the stimulation rate of 15 Hz, the overall change in EJP amplitude could be a balance between STF and STD. Possibly, a deficit in STF in Dube3a15b larvae could have led to an overall faster decrease in EJP amplitude relative to w1118 controls (Valdez, 2015).

Also, the RMP in the muscles of Dube3a15b mutants is significantly more negative than their w1118 controls. These data may reflect a deficit in one or more of the processes or elements involved in maintenance of the RMP. A recent study suggests that Na+/K+ ATPase is ubiquitinated in a Dube3a dependent manner. One might expect that if the loss of Dube3a is causing the more negative RMP in muscle via an effect on Na+/K+ ATPase levels or activity, then the motor neurons may also be affected because regulation of muscle and nerve cell membrane potential both depend on the Na+/K+ ATPase. Nevertheless, changes in resting K+ levels due to leakage across the membrane could also explain these findings. However, it may be more than a coincidence that the effects of over-expression of Dube3a results in increased evoked EJP failures and increased spontaneous, both of which may be indications of a depolarized RMP in motor neurons in the corresponding larvae. Over-expression of Dube3a may have the opposite effects on RMP as loss of Dube3a via opposing actions on this ubiquitin target (Valdez, 2015).

The data on the structure of the synaptic active zones suggests that C155>Dube3a-27 and C155>Dube3a-51 larvae have fewer active zones and that C155>Dube3a-51 also have smaller synaptic vesicles relative to the other genotypes. It was also shown that there is a slight increase in synaptic zone density by NC82 staining, however these results did not reach significance despite the large dataset analyzed. These effects of altered Dube3a expression do not seem to explain the effects of over-expression or deficiency on the electrophysiological paradigms employed. However, they may later prove to be important observations that explain deficits in synaptic transmission not tested in the present study. In mouse models of both Angelman syndrome (decreased Ube3a) and Duplication 15q autism (elevated Ube3a) there are defects in glutamatergic synaptic transmission. This study shows that these defects in glutamatergic signaling can be recapitulated in the fly models for both syndromes as well, validating the fly model system for both syndromes. Thus, in a simple and easy to manipulate model system, the Drosophila NMJ, one can now investigate the downstream effects of changes in Dube3a levels on potential ubiquitin targets in the context of neuronal function. Some putative Ube3a protein targets such as Arc and CamKII have been known for some time, while an entirely new set of potential Dube3a targets has been recently identified through a proteomic screen in flies. It can be anticipated that by manipulating the putative targets of Dube3a in the fly NMJ system through shRNAi knock down or mutations in these genes one can begin to unravel the molecular mechanism behind the neurological defects observed in humans with both AS and Duplication 15q autism (Valdez, 2015).

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Lee, S.Y., Ramirez, J., Franco, M., Lectez, B., Gonzalez, M., Barrio, R. and Mayor, U. (2014). Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10. Cell Mol Life Sci 71: 2747-2758. PubMed ID: 24292889

Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. This study has developed a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, it was found that Ube3a directly ubiquitinylates three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, it was found that Ube3a and the C-terminal part of Rpn10 interact genetically in vivo. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells (Lee, 2014).


  • A new protocol to monitor ubiquitination in a neuronal cell culture.
  • Identification of direct ubiquitination substrates of Ube3a in a neuronal cell culture.
  • Ube3a and Rpn10 interaction in vivo.

The lack of a neuronal system for both in vivo identification and validation of ubiquitination targets has restricted many candidate ubiquitin substrates of UBE3A to be validated only in vitro. Evidence for the ubiquitination of those substrates by UBE3A in vivo, or even in cell culture, is lacking. One report has even redefined a previously suggested substrate as not being ubiquitinated by UBE3A when tested in cells. A recent in vivo proteomic approach for the identification of Ube3a direct and indirect targets in the Drosophila brain did produce a list of 49 candidates for validation, some of which overlapped with another list of ubiquitinated proteins in the fly brain. Ubiquitination by Ube3a of one out of the 49 candidates, ATPα, has been validated in vitro, but when tested in vivo was found not to be ubiquitinated by Ube3a (Lee, 2014).

This study identified Rpn10 and other regulators of the proteasome as direct targets for Ube3a in a neuronal cell culture system, while confirming their genetic interaction in vivo. These results open a new perspective for interpretation of previous identifications of UBE3A substrates based on changes in protein levels. In this new paradigm, UBE3A-dependent changes in the levels of proteins can be interpreted not as direct ubiquitination substrates of this E3 ligase, but as the result of a downstream effect caused by proteasomal regulation by UBE3A (Lee, 2014).

UBE3A has been reported to be associated in the brain both to synaptic and cytosolic proteasomes, but the nature of this association is not clear. It has also been reported that UBE3A associates with proteasome complexes in HEK 293 cells, HeLa cells, and rat muscle. Direct interaction of Ube3a with the proteasome subunit PSMD4 (RPN10) has been reported as part of intact proteasomes, as well as in a smaller complex of approximately 200 kDa, interpreted as containing the shuttling pool of PSMD4. Having identified in neuronal cells that three proteasomal proteins are direct ubiquitination substrates of Ube3a, it can be speculated now that one of the major roles of Ube3a is actually to regulate proteasomal function. Indeed, the suppression of Ube3a-WT overexpression phenotypes in the eye by co-expression with Rpn10DN strongly supports the view of Ube3a being a key regulator of neuronal proteasome function (Lee, 2014).

Expression of UBE3A is induced in response to a variety of stress conditions, but the regulation of the proteasome itself by Ube3a has never been suggested previously. In cultured neuronal cells, this study identified four novel substrates of Ube3a, including two proteasome-interacting receptors as well as one proteasome-associated DUB. Furthermore, Ube3a seems to target protein homeostasis via ubiquitination of the ribosomal protein RpS10b. While proteasomes are ubiquitously distributed in the cell, it appears that a significant subpopulation is recruited to dendritic spines following synaptic stimulation. Translocation and recruitment of proteasomes into spines seems to be highly regulated, presumably due to the requirement for coordinated proteolysis at the synapse. In addition, proteasomes are known to regulate dendritic spine growth, with proteasomal inhibition being reported to acutely reduce new spine outgrowth. Very recently, a specific centrosomal pool of PSMD4 has been shown to regulate dendrite development in the mammalian brain. Since loss of maternal UBE3A in transgenic mice causes a reduction in spine density and spine length, while spine densities are greater on pyramidal cells in the cortex of ASD subjects than in controls, it can be speculated that UBE3A regulation of stability or function of proteasomal components might be an essential mechanism for synaptic plasticity (Lee, 2014).

Interestingly, only Rpn10 appears to be itself targeted for degradation upon ubiquitination by Ube3a. In flies, Rpn10 null mutants are lethal, but survive until pupal stages thanks to the large amount of maternal proteasomes available from early embryonic stages. However, the brain of wandering larvae of these mutants is reduced in size. PSMD4 knockout in mice also results in early embryonic lethality. Regarding the other identified Ube3a substrates, RpS10b has been identified to be differentially expressed in patients with schizophrenia. UcH-L5 is a proteasome-associated DUB enzyme responsible for cleavage of K48 ubiquitin chains, and it is essential in most cells, so it will require some further work to dissect the specific effect its misregulation would cause in brain development. CG8209 is the Drosophila homologue of UBXN1/SAKS, a component of the complex required to couple deglycosylation and proteasome-mediated degradation of misfolded proteins retrotranslocated into the cytosol from the endoplasmic reticulum (Lee, 2014).

The main role of Rpn10/PSMD4 is to shuttle polyubiquitinated proteins to the proteasome for their degradation. Overexpression of Rpn10DN results in some of its client proteins being trapped, so an accumulation of polyubiquitinated material is observed. Endogenous Rpn10, however, will remain shuttling non-trapped polyubiquitinated proteins to the proteasome. When Ube3a is overexpressed, a strong eye phenotype is observed, this could be due to the persisting activity of some proteins that would otherwise have been targeted -via Rpn10- to the proteasome for degradation. Those proteins might remain ubiquitin-conjugated, or processed by DUBs into their normal forms. If both Rpn10DN and Ube3a are overexpressed, the reduction of shuttling endogenous Rpn10 is compounded by the trapping of its client ubiquitinated proteins by Rpn10DN, which would no longer be processed by DUBs resulting in a much higher level of polyubiquitinated proteins accumulating. As those proteins are trapped by Rpn10DN, they would not retain activity, and the eye phenotype associated with their misregulation would no longer be detectable (Lee, 2014).

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Geier, B., Neely, L., Coronado, E. and Reiter, L. T. (2023). Drosophila UBE3A regulates satiety signaling through the Piezo mechanosensitive ion channel. Res Sq. PubMed ID: 37461494


Angelman syndrome (AS) is a rare neurogenetic disorder characterized by developmental delays, speech impairments, ataxic movements, and in some cases, hyperphagic feeding behavior. Loss of function mutations, loss of expression from the maternal allele or absence of maternal UBE3A result in AS. Recent studies have established a connection between UBE3A and the mechanosensitive ion channel PIEZO2, suggesting the potential role of UBE3A in the regulation of PIEZO channels. This study investigated the role of Drosophila UBE3A (Dube3a) in Piezo associated hyperphagic feeding behavior. A novel assay was developed using green fluorescent protein (GFP) expressing yeast to quantify gut distention in flies with Piezo and Dube3a mutations. It was confirmed that Dube3a (15b) loss of function flies displayed gut distention to almost identical levels as Piezo (KO) flies. Further analysis using deficiency (Df) lines encompassing the Dube3a locus provided proof for a role of Dube3a in satiety signaling. Endogenous Piezo expression was examined across the fly midgut and tracheal system. Piezo protein could be detected in both neurons and trachea of the midgut. Overexpression of Dube3a driven by the Piezo promoter resulted in distinct tracheal remodeling within the midgut. These findings suggest that Dube3a plays a key role in the regulation of Piezo and that subsequent dysregulation of these ion channels may explain the hyperphagic behavior observed in 32% of cases of AS. Further investigation will be needed to identify the intermediate protein(s) interacting between the Dube3a ubiquitin ligase and Piezo channels, as Piezo does not appear to be a direct ubiquitin substrate for UBE3A in mice and humans.

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Jensen, L., Farook, M.F. and Reiter, L.T. (2013). Proteomic profiling in Drosophila reveals potential Dube3a regulation of the actin cytoskeleton and neuronal homeostasis. PLoS One 8: e61952. PubMed ID: 23626758

The molecular defects associated with Angelman syndrome (AS) and 15q duplication autism are directly correlated to expression levels of the E3 ubiquitin ligase protein UBE3A. This study used Drosophila melanogaster to screen for the targets of this ubiquitin ligase under conditions of both decreased (as in AS) or increased (as in dup(15)) levels of the fly Dube3a or human UBE3A proteins. Using liquid phase isoelectric focusing (IEF) of proteins from whole fly head extracts, a total of 50 proteins were identified that show changes in protein, and in some cases transcriptional levels, when Dube3a fluctuates. Head extracts from cytoplasmic, nuclear and membrane fractions for Dube3a regulated proteins were analyzed. Data indicate that Dube3a is involved in the regulation of cellular functions related to ATP synthesis/metabolism, actin cytoskeletal integrity, both catabolism and carbohydrate metabolism as well as nervous system development and function. Sixty-two percent of the proteins are >50% identical to homologous human proteins and 8 have previously be shown to be ubiquitinated in the fly nervous system. Eight proteins may be regulated by Dube3a at the transcript level through the transcriptional co-activation function of Dube3a. One autism-associated protein, ATPα, was investigated and it was found that it can be ubiquitinated in a Dube3a dependent manner. It was also found that Dube3a mutants have significantly less filamentous actin than wild type larvae consistent with the identification of actin targets regulated by Dube3a. The identification of UBE3A targets is the first step in unraveling the molecular etiology of AS and duplication 15q autism (Jensen, 2013).


  • IEF screen reveals fifty unique proteins regulated by changes in Dube3a or hUBE3A levels.
  • Quantitative real-time PCR reveals eight potential transcriptional targets of Dube3a.
  • Proteins identified are involved in nervous system development, actin cytoskeleton remodeling, ATP synthesis/metabolism and carbohydrate synthesis/metabolism.
  • ATPα is regulated by the ubiquitin ligase function of Dube3a.
  • Dube3a is involved in muscle actin structural integrity in fly larvae.

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Ferdousy, F., Bodeen, W., Summers, K., Doherty, O., Wright, O., Elsisi, N., Hilliard, G., O'Donnell, J.M. and Reiter, L.T. (2011). Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism. Neurobiol Dis 41: 669-677. PubMed ID: 21147225

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, this study identified a key regulator of monoamine synthesis, the gene Punch, or GCH1, encoding the enzyme GTP cyclohydrolase I. It was shown that Dube3a, the fly UBE3A ortholog, 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 are associated with hyper- and hypoactivity, respectively, in flies. It was found 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. The study proposes 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 (Ferdousy, 2011).


  • The Punch protein is elevated in fly neurons expressing human or fly UBE3A.
  • Punch mutations are able to enhance Dube3a over-expression phenotypes in the eye.
  • Punch protein isoform B levels change as a result of Dube3a over-expression or loss of function in the fly brain.
  • Endogenous Dube3a protein can trans-ubiquitinate catalytically inactive Dube3a-C/A in vivo.
  • Punch B transcript levels are modulated by changes in Dube3a.
  • Dube3a levels coincide with changes in dopamine levels in the brain.

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, loss of Ube3a results in a defect in neocortical plasticity, despite the fact that this mouse model is over ten years old. 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. This study uses a strictly biochemical approach for the identification of Dube3a targets in Drosophila. The study identifies 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. It also demonstrates, 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. Also, 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. 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) (Ferdousy, 2011).

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. In this case, changes in both transcript and protein levels in Punch-RB are seen, as well as downstream up-regulation of dopamine upon over-expressing a ubiquitination-defective form of Dube3a in neurons is observed. 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. A study 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. t has been shown that 89% of transcripts that are differentially expressed in Ube3a deficient versus wild type mice are down-regulated in the Ube3a deficient brain consistent with the idea that transcriptional co-activation by Dube3a may be just as critical as ubiquitination. 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, which encodes a transcription factor protein, and MeCP2, 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 (Ferdousy, 2011).

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 leads to elevated transcription (and subsequent elevation of translation) of Punch mRNA, it can be concluded that the ubiquitination function of the Dube3a enzyme plays no detectable role in the regulation of GTP cyclohydrolase expression in flies (Ferdousy, 2011).

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. Drosophila TH, encoded by pale (ple), shares 60% amino acid similarity with human TH. 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. Similarly, mutations in the human GCH1 locus lead to the hereditary diseases hyperphenylalaninemia and Dopa-responsive dystonia. 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 (Ferdousy, 2011).

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) 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 (Ferdousy, 2011).

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 500 kDa it would be exceptionally challenging. The consequence of these complex interactions is that it is not certain 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. Suppression of the Dube3a eye phenotype in Punch mutant backgrounds was expected, but instead it was found that the eye phenotype is enhanced. This result may be due to uncoordinated expression of the various Punch isoforms in the over-expression background (Ferdousy, 2011).

Another unexpected outcome 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 elevation of both the THB pathway components and dopamine pools were observed in neurochemical analysis and a functional consequence of these modulations in levels of dopamine was found, it could be inferred that the immunoblots are perhaps not as sensitive in quantifying expression levels. 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 was not observed. 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. 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 (Ferdousy, 2011).

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. Serotonin deficits associated with Punch mutations have been linked to developmental abnormalities including failure of ectodermal cell movements during gastrulation and in cuticular patterning, while diminished production of dopamine results in aberrant tracheal cell migration in Drosophila embryos. Subsequently, abnormalities in dopamine pools lead to variations in activity/locomotion, as well as to altered stress responses. 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 (Ferdousy, 2011).

There is at least some evidence that selective serotonin reuptake inhibitors can dampen the hyperactivity and anxiety in both AS deletion and duplication 15q autism individuals 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 have been reported, and deficits in dopamine-dependent behaviors have been recently in a mouse Ube3a knock-out model of AS. 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 (Ferdousy, 2011).

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Lu, Y., Wang, F., Li, Y., Ferris, J., Lee, J.A. and Gao, F.B. (2009). The Drosophila homologue of the Angelman syndrome ubiquitin ligase regulates the formation of terminal dendritic branches. Hum Mol Genet 18: 454-462. PubMed ID: 18996915

Angelman syndrome is a severe neurodevelopmental disorder mostly caused by loss-of-function mutations in the maternal allele of UBE3A, a gene that encodes an E3 ubiquitin ligase. Drosophila UBE3A (dUBE3A) is highly homologous to human UBE3A (hUBE3A) at the amino acid sequence level, suggesting their functional conservation. This study generated dUBE3A-null mutant fly lines and found that dUBE3A is not essential for viability. However, loss of dUBE3A activity reduces dendritic branching of sensory neurons in the peripheral nervous system and slows the growth of terminal dendritic fine processes. Several lines of evidence indicate that dUBE3A regulates dendritic morphogenesis in a cell autonomous manner. Moreover, overexpression of dUBE3A also decreases dendritic branching, suggesting that the proper level of dUBE3A is critically important for the normal dendritic patterning. These findings suggest that dendritic pathology may contribute to neurological deficits in patients with Angelman syndrome (Lu, 2009).


  • Generation of dUBE3A mutant flies.
  • Reduced dendritic growth and branching in dUBE3A mutant larvae.
  • dUBE3A regulates dendritic morphogenesis in a cell autonomous manner.
  • dUBE3A is required for the proper dendritic formation of other neurons.
  • Genetic interaction between dUBE3A and eff that encodes an E2 ligase.
  • The proper level of dUBE3A is critical for neuronal development.

Since the mental retardation disorder AS is primarily caused by loss of the UBE3A protein product, it is of great importance to understand the normal function of UBE3A in neuronal development and synaptic plasticity. This study used the P-element local hop-out approach to generate dUBE3A-null mutant fly lines. Although UBE3A knockout mice often die shortly after birth, dUBE3A mutant flies are viable to adulthood. The EP3214 line obtained from the Bloomington Stock Center is homozygous lethal, but the lethality is probably not due to the EP insertion in the dUBE3A locus itself, as the EP insertion could be segregated from the background lethal mutation(s) after outcrosses with wild-type flies (Lu, 2009).

To understand the role of dUBE3A in neuronal development, this study focused on the dendritic morphogenesis of DA neurons in the Drosophila PNS. One of the advantages of using DA neurons for phenotypic analysis is the ease of visualizing their dendrites in living animals. The dendritic trees of DA neurons are sandwiched between the epidermis and the body muscle wall and are essentially two-dimensional. Thus, the number of dendritic branches, especially their terminal fine processes, can be easily quantified at high resolution. Using this system, it was found that loss of dUBE3A reduces the formation of terminal dendritic branches. This finding is consistent with the notion that dendritic pathology contributes to the pathogenesis of AS, as shown by the reduced length and density of dendritic spines in cerebellar, cortical and hippocampal neurons and by the relatively normal appearance of dendritic trees stained with calbindin and examined by light microscopy. In dUBE3A mutant larvae, the major dendritic branches of ddaC neurons also appear to be normal. The finding that the development of terminal fine dendritic processes is affected by dUBE3A in Drosophila raises the possibility that this defect contributes to the neurological deficits in AS patients and mouse models. It is interesting to note that the overexpression of dUBE3A also decreases dendritic branching, which may have some implications for some forms of autism in which the genomic region containing UBE3A is duplicated. It is plausible that dendritic developmental defects of CNS neurons in dUBE3A mutants may underlie, at least in part, the behavioral abnormalities of these flies (Lu, 2009).

Another advantage of the Drosophila model system is the ability to examine the cell autonomous functions of a gene of interest. Genetic analyses provide strong evidence that dUBE3A influences dendritic morphogenesis in a cell autonomous manner. Dendritic pathology has been implicated in fragile X syndrome, Rett syndrome and autism. Although the genes mutated in these neurodevelopmental disorders are different, including FMR1, an RNA-binding protein, MeCP2, a transcription regulator, and UBE3A, an E3 ubiquitin ligase, their downstream targets may participate in the same genetic pathways that regulate the formation of dendritic branches and dendritic spines. Loss of MECP2 activity leads to a significant reduction in UBE3A expression in human brains. This finding may help explain the decreased dendritic branching and synaptogenesis caused by MECP2 deficiency. However, MECP2 knockout mice show normal levels of UBE3A; therefore, it seems that there is no direct genetic link between MECP2 and UBE3A (Lu, 2009).

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Wu, Y., Bolduc, F. V., Bell, K., Tully, T., Fang, Y., Sehgal, A. and Fischer, J. A. (2008). A Drosophila model for Angelman syndrome. Proc Natl Acad Sci U S A 105: 12399-12404. PubMed ID: 18701717

Angelman syndrome is a neurological disorder whose symptoms include severe mental retardation, loss of motor coordination, and sleep disturbances. The disease is caused by a loss of function of UBE3A, which encodes a HECT-domain ubiquitin ligase. This study generate a Drosophila model for the disease. The results of several experiments show that the functions of human UBE3A and its fly counterpart, dube3a, are similar. First, expression of Dube3a is enriched in the Drosophila nervous system, including mushroom bodies, the seat of learning and memory. Second, dube3a null mutants were generated, and they appear normal externally, but display abnormal locomotive behavior and circadian rhythms, and defective long-term memory. Third, flies that overexpress Dube3a in the nervous system also display locomotion defects, dependent on the ubiquitin ligase activity. Finally, missense mutations in UBE3A alleles of Angelman syndrome patients alter amino acid residues conserved in the fly protein, and when introduced into dube3a, behave as loss-of-function mutations. The simplest model for Angelman syndrome is that in the absence of UBE3A, particular substrates fail to be ubiquitinated and proteasomally degraded, accumulate in the brain, and interfere with brain function. Flies useful for genetic screens were generated to identify Dube3a substrates. These flies overexpress Dube3a in the eye or wing and display morphological abnormalities, dependent on the critical catalytic cysteine. It is concluded that dube3a mutants are a valid model for Angelman syndrome, with great potential for identifying the elusive UBE3A substrates relevant to the disease (Wu, 2008).

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Reiter, L. T., Seagroves, T. N., Bowers, M. and Bier, E. (2006). Expression of the Rho-GEF Pbl/ECT2 is regulated by the UBE3A E3 ubiquitin ligase. Hum. Mol. Genet. 15(18): 2825-35. Medline abstract: 16905559

Genetic tools available in Drosophila have been applied to identify candidate substrates of the UBE3A ubiquitin ligase, the gene responsible for Angelman syndrome (AS). Human UBE3A was expressed in Drosophila heads to identify proteins differentially regulated in UBE3A-expressing versus wild-type extracts. Using two-dimensional gel and MALDI-TOF analysis, 20 proteins were detected that were differentially regulated by over-expression of human UBE3A in Drosophila heads. One protein responsive to UBE3A was the Rho-GEF Pebble (Pbl). Three lines of evidence are presented suggesting that UBE3A regulates Pbl. First, genetic evidence is shown that UBE3A and the Drosophila de-ubiquitinase Fat facets (Faf) exert opposing effects on Pbl function. Secondly, it was found that both Pbl and ECT2, the mammalian orthologue of Pbl called epithelial cell transforming sequence 2 oncogene, physically interact with their respective ubiquitin E3 ligases. Finally, it was shown that Ect2 expression is regulated by Ube3a in mouse neurons, since the pattern of Ect2 expression is dramatically altered in the hippocampus and cerebellum of Ube3a null mice. These results suggest that an orthologous UBE3A post-translational regulatory pathway regulates neuronal outgrowth in the mammalian brain and that dysregulation of this pathway may result in neurological phenotypes including AS and possibly other autism spectrum disorders (Reiter, 2006).

Identifying neurologically relevant substrates of the ubiquitin E3 ligase UBE3A is essential to understand the phenotypes associated with AS. However, the results from this study may also have implications for other learning and behavior disorders. For example, maternally derived interstitial duplication of the 15q11-q13 region, which includes the UBE3A gene, consistently results in an ASD phenotype. Although there have been rare cases of paternally inherited 15q11-q13 that result in developmental impairments there is an overwhelming preference for maternal inheritance of this duplication. This implies that the UBE3A gene, which is maternally imprinted in both the hippocampus and cerebellum, is likely to be the key gene in this region responsible for the ASD phenotype. It is proposed, therefore, that at least a subset of idiopathic autism cases may be the result of dysregulation of UBE3A substrates. It may be possible to address this hypothesis by performing association studies on UBE3A substrates like ECT2 in autism families (Reiter, 2006).

ECT2 is the first candidate substrate of UBE3A with an obvious relevance to the neurological phenotypes observed in AS and ASD patients. While the dysregulation of UBE3A substrates like ECT2 in the hippocampus may explain the general learning and behaviour defects observed in both AS and ASD patients, the findings of a Purkinje cell phenotype may provide yet another link between AS and ASD. For example, AS patients exhibit ataxia and motor control problems, which could be explained by the dysregulation of ECT2 and/or other UBE3A substrates in cerebellum. Similarly, a strong correlation has been found between ASD and non-progressive congenital ataxia, whereas a link has been found between sensory-motor deficits and ASD. Perhaps, more telling in terms of understanding ASD pathology is the possibility that cerebellar defects may explain some of the emotion recognition and expressive language problems observed in ASD individuals (Reiter, 2006).

It is hypothesized that UBE3A may play a role in regulating growth of neuronal processes or synapse formation through the degradation or cellular localization of various proteins, such as Pbl/ECT2. The observation that the intracellular distribution of Ect2 is controlled by Ube3a parallels previous studies in which it was observed that Ect2 undergoes a cell cycle-dependent redistribution from the nucleus to cytoplasm, which is controlled by N-terminal sequences distinct from the Rho-GEF domain. Furthermore, mutations in pbl have also been shown to adversely affect neuronal outgrowth in post-mitotic cells. Thus, gross dysregulation of Pbl may lead to defects in neuronal pathfinding and/or synaptogenesis. Perhaps, UBE3A regulates the sub-cellular localization of ECT2 in post-mitotic neurons to ensure that ECT2 is delivered to the tips of growing axons or dendrites only under appropriate conditions. Given the critical role that Pbl and the Rho/Rac/Cdc42 system plays in axonal navigation and synapse formation in Drosophila, it seems highly likely that the gross dysregulation of this exquisitely dosage sensitive regulator in the hippocampus and cerebellum of Ube3a null mice would result in aberrant neuronal development, connectivity, or function. Such primary phenotypes in turn may underlie part of the observed learning defects and central nervous system features of this murine model for AS. These data are also consistent with growing evidence that the ubiquitin pathway is a key regulator of synaptic growth/stabilization and function (Reiter, 2006).

It has been reported that activity of ECT2 during G2/M phase of the cell cycle is regulated by phosphorylation, however, the data provides the first evidence that ECT2 may also be regulated through the ubiquitin proteasome system via its interaction with UBE3A. One unresolved question is whether ubiquitination of Ect2 would act primarily by marking this protein for degradation, modulating its function or cellular distribution, or whether it acts in both of these capacities. Interestingly, ubiquitination has been implicated in the regulation of both cellular trafficking and intercellular signalling in addition to protein stability. These recent observations are intriguing in light of the results that cellular distribution of Ect2 was altered in response to deletion of Ube3a in the murine brain. However, it is still not clear whether the levels of Ect2 protein per cell increase substantially overall in Ube3a–/– brains since in specific regions, such as the cerebellum or hippocampus, there appears to be substantial redistribution of Ect2 protein into regions in which Ect2 is not detectable in wild-type littermates. Therefore, it is possible that the primary defect in Ect2 regulation in these mice is the cellular relocalization of the UBE3A candidate substrate from the perinuclear region of the cell body to axonal or dendritic processes, rather than control of total protein levels. Further investigation of the role of ubiquitination in regulating Ect2 stability, activity and subcellular localization will be necessary in order to discriminate between these possible mechanisms (Reiter, 2006).

In summary, the combined approach that was taken, which exploits the strengths of both Drosophila and mouse models, strongly suggests that Pbl/ECT2 is a direct substrate of the ubiquitin ligase UBE3A and that ECT2 is the most compelling putative substrate identified to date that could be relevant to neurological disorders. Given that increased levels of UBE3A have also been implicated in the pathogenesis of ASD, continued identification and characterization of the multiple substrates regulated by UBE3A in the brain could have far reaching clinical impact for the most common forms of learning defects in humans (Reiter, 2006).

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Jana, N.R. (2012). Understanding the pathogenesis of Angelman syndrome through animal models. Neural Plast 2012: 710943. PubMed ID: 22830052

Gatto, C.L. and Broadie, K. (2011). Drosophila modeling of heritable neurodevelopmental disorders. Curr Opin Neurobiol 21: 834-841. PubMed ID: 21596554

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