Drosophila UBE3A regulates satiety signaling through the Piezo mechanosensitive ion channel

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. In this study, we investigated the role of Drosophila UBE3A (Dube3a) in Piezo associated hyperphagic feeding behavior. We developed a novel assay using green fluorescent protein (GFP) expressing yeast to quantify gut distention in flies with Piezo and Dube3a mutations. We confirmed that Dube3a15b loss of function flies displayed gut distention to almost identical levels as PiezoKO flies. Further analysis using deficiency (Df) lines encompassing the Dube3a locus provided proof for a role of Dube3a in satiety signaling. We also investigated endogenous Piezo expression 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.


Introduction
Angelman syndrome (AS) is a rare neurogenetic disorder with an incidence of 1/15,000 births and characterized by severe developmental delays, speech impairments, ataxic movements, epilepsy, and frequent laughter [1]. AS arises from the loss of expression of a single paternally imprinted copy of the UBE3A gene, a HECT domain E3A ubiquitin ligase, also known as E6AP [2,3]. While the majority of AS cases occur as a result of deletion of the maternal 15q11.2-q13.1 locus [4], loss of function (LoF) mutations that exclusively affect UBE3A on the maternal allele will also result in AS [5]. Biochemical studies investigating point mutations in UBE3A reveal that any loss or reduction of catalytic activity plays a critical role in the development of AS [6]. Large maternal deletions comprise 75% of all AS cases, point mutations of maternal UBE3A comprise 20% of cases, imprinting center defects make up 3%, and paternal uniparental disomy (pUPD) causes up to 2% of causes [7]. Previous work suggested that, unlike the major deletion class, only pUPD patients display hyperphagic feeding behavior [8,9]. However, a newer large-scale AS cohort study suggests this hyperphagic phenotype may be present in all AS subtypes [10].
A new connection between UBE3A and the mechanosensitive ion channel PIEZO2 has recently been established. In this study, loss of Ube3a in an AS mouse model and human AS cell lines resulted in decreased PIEZO2 activity and protein expression [11]. Piezo proteins are pore-forming subunits of ion channels that activate in response to mechanical stimuli [12]. The human genome encodes two channel isoforms, PIEZO1 and PIEZO2, which are expressed in a myriad of tissues (kidneys, vasculature, Merkel cells, and others) [13][14][15][16][17]. In D. melanogaster there is only one copy of the Piezo ion channel gene, but it shares equal homology for both mammalian isoforms [18]. Like mammals, Drosophila express Piezo in the crop (stomach) and intestine. Knockout of Piezo in Drosophila results in a severe gut distention phenotype, indicative of hyperphagia [19,20]. The high homology between y and mammalian Piezo, in addition to the connection between UBE3A and PIEZO2 recently described in Ube3a de cient mice, prompted us to look for a connection between Dube3a and Piezo in ies in order to investigate hyperphagia associated with AS.
Here we investigated the role of Dube3a (the y UBE3A homolog) in hyperphagic feeding behavior and its role in altering expression of Piezo in cells of the midgut. We established a novel feeding assay incorporating green uorescent protein (GFP) expressing yeast for quantitative analysis of gut distention.
Using this new assay, we show clear connections between Dube3a, Piezo and hyperphagia. Furthermore, to assess changes in Piezo expression within the y midgut, we generated a new y that expresses GFPtagged Piezo and also overexpresses Dube3a. By driving expression of our new ies under the Piezo promoter, we were able to identify morphological changes in Piezo positive terminal tracheal cells (TTC) within the y midgut. The identi cation of Dube3a as a Piezo regulator provides a new avenue for potential therapeutics of AS related hyperphagia.

Results
Dube3a loss of function ies display gut distention phenotype indicative of hyperphagia To explore the connection between UBE3A and PIEZO2, we generated a novel feeding assay using GFP expressing Saccharomyces cerevisiae (brewer's yeast). Flies were starved prior to the assay and then fed GFP expressing yeast for uorescent quanti cation of gut distention. The GFP signal can be detected in the crop and gut of ies after consumption for accurate quanti cation. To determine if loss of Dube3a affects feeding behavior, we used a previously published Dube3a LoF mutant (Dube3a 15b ) [21] to compare to ies that have a known gut distention phenotype due to loss of Piezo [19]. Homozygous Piezo KO mutants display signi cant gut distention, whereas w 1118 animals feed at a basal levels and do not show distention of the crop region. Quantitative comparison of abdomen sizes for Piezo KO mutants to w 1118 ies showed a signi cant difference in uorescent signal and abdomen size in a region of interest (ROI) surrounding the crop (Fig. 1A). Using Dube3a 15b mutants, we compared feeding behavior among w 1118 , Piezo KO (Fig. 1B). These ndings suggest a possible role of Dube3a in the regulation of satiety signaling through Piezo regulation.
De ciencies that uncover the Dube3a locus con rm Dube3a as a regulator of satiety signaling To con rm Dube3a's role in the gut distention phenotype using a different genetic background, we tested several Bloomington Drosophila Stock Center (BDSC) de ciency (Df) lines. The Df lines have small and large deletions across the entirety of the Drosophila genome [22,23]. To ensure a direct effect of loss of Dube3a on satiety signaling, we performed the GFP feeding assay on three Df lines on Drosophila 3L. Two lines, Df 9355 and Df 8977 ank the Dube3a gene, while Df 24403 is lacking the Dube3a gene ( Fig. 2A). To eliminate other background effects, we removed CyO and TM6B, Tb, Hu balancers by outcrossing all Df lines to w 1118 ies prior to testing. Comparison of quantitative gut distention revealed a signi cant increase in feeding behavior of Df 24403 (Dube3a LoF) but not for anking Df lines (Fig. 2B). While Df 8977 showed an elevated uorescent intensity signal, it was not signi cantly different from w 1118 . An unpaired t-test comparing Df 24403 and Df 8977 showed a signi cant increase in distention for Dube3a LoF mutants, con rming the relationship between Dube3a and hyperphagic feeding behavior ( Fig. 2B), regardless of genetic background. These results suggest that loss of UBE3A could cause hyperphagia though down regulation of PIEZO2, parallel to their roles in the ataxia phenotype in the AS mouse model [11].
Fluorescent imaging of Piezo:GFP ies reveals distinct Piezo patterning across Drosophila midgut The gastrointestinal (GI) system of Drosophila is a tubular structure consisting of a foregut, midgut, and hindgut, responsible for the intake, breakdown, and absorption of nutrients ( Fig. 3A) [24]. Before assessing the relationship of Dube3a and Piezo in the y gastrointestinal (GI) system, we determined which cells in the gut normally express Piezo. We performed immuno uorescent (IF) staining on whole Drosophila intestines for both Piezo > Piezo:GFP and w 1118 ies. Staining of w 1118 intestines with antibodies against GFP, neurons (elav), and counterstained for nuclei (DAPI) revealed both neuronal and nuclear staining in the anterior and distal midgut but no GFP signal detected (Fig. 3B). Using a Piezo speci c GAL4 driver [25] to drive expression of UAS-Piezo:GFP, we found that Piezo > Piezo:GFP ies showed GFP signal in the proventriculus, crop, and all sections of the midgut. Additionally, neuronal and nuclear staining showed strongly colocalization of a-GFP with a-elav, suggesting speci city for Piezo in gut neurons (Fig. 3C).
Overexpression of Dube3a results in tracheal remodeling of the anterior midgut Along with elav-positive cells, imaging of Piezo > Piezo:GFP ies showed GFP-positive cells with elaborate arborization patterns (red arrows Fig. 3C). These cells displayed a similar morphology to TTCs [26]. To con rm that these are TTC and not neurons, we drove the expression of Piezo:GFP using the tracheal GAL4 driver breathless (btl-GAL4). Fluorescent imaging of btl-GAL4 > Piezo:GFP revealed a similar morphological pattern in the midgut to Piezo > Piezo:GFP ies (white arrows Fig. 4A). These results imply that Piezo is also expressed within the tracheal system.
We next investigated how changes in Dube3a affect Piezo expression and distribution in the gut as well as gut morphology. We generated a double UAS line UAS-Piezo:GFP; UAS-Dube3a:FLAG to visualize Piezo while also elevating Dube3a levels. This line expresses the Piezo:GFP fusion protein at basal levels in conjunction with the overexpression of Dube3a-FLAG in Piezo expressing cells. Western blot analysis on y head extracts from w 1118 and glass multimer report (GMR) GMR > Piezo:GFP; Dube3a:FLAG animals con rmed the presence of Dube3a-FLAG in double UAS line (Fig. S1). Protein expression studies using a-FLAG antibody revealed a band at 120kDa exclusively in GMR > Piezo:GFP; Dube3a:FLAG ies, con rming the presence of Dube3a-FLAG expression in the recombinant ies. It became apparent that Piezo > Piezo:GFP; Dube3a:FLAG ies did not emerge at 25°C, so crosses were set at 18°C to reduce the level of Dube3a-FLAG overexpression in the Piezo > Piezo:GFP; Dube3a:FLAG offspring. Although there was signi cant lethality for Piezo > Dube3a-FLAG ies at 18°C, at least two Piezo > Piezo:GFP; Dube3a:FLAG ies could be generated for study (Fig. S2). Confocal imaging of these two Piezo > Piezo:GFP; Dube3a:FLAG ies showed no major changes in the proventriculus but a noticeable change in the TTC morphology within the anterior midgut (Fig. 4B & 4C). Piezo > Piezo:GFP; Dube3a:FLAG TTC projections exhibited a tortuous appearance when compared to the Piezo > Piezo:GFP counterparts. Additionally, overexpression of Dube3a-FLAG caused bloating of TTC cell bodies when compared to control lines (white arrows Fig. 4C). These results indicate that overexpression of Dube3a in normally Piezo expressing cells of the gut results in severe tracheal remodeling of the Drosophila midgut, potentially through the dysregulation of Piezo.

Discussion
Although previously thought to be a feature unique to the pUPD class of AS [8], more recent research has identi ed hyperphagia as a common symptom across all AS subtypes [10]. Collaborative work from our laboratory also recently established a connection between UBE3A and the mechanosensitive ion channel PIEZO2, leading to the hypothesis that UBE3A might indirectly regulate PIEZO channels, contributing to the both ataxia [11] and hyperphagia in AS. Here we investigated this hypothesis and establish a link between the loss of Dube3a and the emergence of hyperphagic feeding behavior in Dube3a mutant ies, resembling hyperphagia in Piezo KO ies and establishing that these two genes may act in the same pathway to control satiety. Additionally, intestinal imaging of Piezo driven overexpression of Dube3a revealed noticeable tracheal remodeling within the y midgut further supporting the role of Dube3a in the Drosophila satiety signaling.
We also developed a new assay utilizing GFP expressing yeast to quantify gut distention. Equivalent uorescent intensities were detected between Dube3a 15b and Piezo KO ies in this assay, suggesting a putative role for Dube3a in satiety signaling. This connection between satiety and Dube3a was con rmed using Df lines that uncover the Dube3a locus (Fig. 2B). We previously demonstrated that Dube3a 15b mutants have signi cantly fewer actin laments than their wild type counterparts [27]. Actin is essential for the tra cking of proteins to the membrane [28]. Studies have shown that a functional cytoskeleton is also required for proper Piezo activity [29,30]. Here we propose that the dysregulation of actin laments, via loss of Dube3a, inhibits Piezo tra cking/activity resulting in hyperphagia.
Gut imaging experiments provided insights not only into the normal expression pattern of Piezo within the Drosophila midgut, but also the effects of Dube3a overexpression on normal gut morphology. Piezo expressing neurons were detected in both the anterior and posterior midgut. These Piezo expression patterns were identical to other studies investigating Piezo within the adult midgut but were extended in this study to include co-localization with neuronal and tracheal markers [25]. Notably, Piezo:GFP expression under the breathless promoter (btl-GAL4) showed a pattern similar to Piezo > Piezo:GFP ies for patterning across the midgut, indicating the presence of Piezo channels within the tracheal system. The Drosophila tracheal system, akin to vasculature in humans, serves as a complex tubular system that provides oxygen to cells [26]. Similarly, mammalian Piezo1 channels are present in vasculature, playing a role in path nding and angiogenesis [31][32][33]. Mice de cient for Piezo1 also die during mid gestation stage emphasizing the role of Piezo channels in proper development [34]. Consistent with these ndings, attempts to analyze Piezo > Piezo:GFP; Dube3a-FLAG ies were hindered by low eclosure rates, suggesting an effect on Piezo expression and or function after Dube3a overexpression.
In Piezo > Piezo:GFP; Dube3a-FLAG ies, the midgut displayed a highly disorganized tracheal phenotype. While tracheal branching is not fully understood, trachea increase their arborization under both physical and cellular stress conditions to meet the metabolic demands of surrounding cells [35,36]. Although the direct cause of tracheal remodeling in these ies is unknown, swelling of tracheal cells implies stressed intestinal conditions due to Dube3a overexpression. The Drosophila midgut contains enteroendocrine cells (EC) and enteric neurons, which play roles in satiety signaling [37,38]. Furthermore, Piezo is expressed in subsets of intestinal stem cells (ISCs), contributing to EC differentiation [25]. The negative effect of Dube3a on Piezo channels may disrupt proper satiety signaling through dysregulation of enteric neurons, ISCs and ECs, but further studies will be needed to narrow down the exact cell types where Dube3a can regulate Piezo in the gut.
In summary, these studies show a clear connection between Dube3a loss of function and hyperphagic feeding behavior. Imaging analysis also revealed a tortuous appearance of Piezo positive trachea within the y midgut supporting the role of Dube3a in altering Piezo expression and or function during development. Although the intermediate protein(s) involved in regulating Piezo expression remain(s) unidenti ed, current evidence suggest co lin as a potential Dube3a polyubiquitination substrate that could affect Piezo function in the membrane [11]. The ndings here strengthen the connection between Dube3a and Piezo and their potential role in the regulation of hyperphagia in AS. Dube3a 15b has been previously published as a Drosophila model for AS [21]. UAS-Dube3a-FLAG was constructed in the Reiter lab and is available upon request.

Gut distention assay
Adult female ies were collected post-eclosion and stored at 25°C for 3-5 days. Before testing, ies were transferred to empty vials containing only a damp kimwipe. Flies were starved for 15-18 hours at 25°C prior to feeding. GFP expressing yeast (The ODIN) were cultured at 33°C for 48 hours on G418 (gibco) yeast extract peptone dextrose (YPD) agar. GFP + yeast were scrapped and layered on top of y food in vials. Starved ies were transferred to newly created GFP + yeast food vials for one hour. After feeding, ies were incapacitated using FlyNap (Carolina Biological Supply). Lower legs were removed and ies were placed with the abdomen facing up for imaging on a Lieca uorescent dissecting microscope.
Images were collected and uploaded into FIJI (imageJ) for analysis. The crop region of the most distended Piezo KO /Piezo KO y was traced, generating a normalization region of interest (ROI). The normalization ROI was then applied to all ies in the analysis to quantify distention through uorescent intensity.
Images were acquired using a Zeiss 710 confocal microscope (Zeiss) located in the UTHSC neuroscience imaging core. All imaging settings remained constant between control and experimental conditions. GFP signal was captured using a 488 nm laser, and elav signal was captured using a 568 nm laser. Z-stacks were acquired using 1 µm optical sections. All z-stack projections were transformed using maximum intensity projections using ZEN software (Zeiss). Images were acquired with ZEN software (Zeiss).

Data analysis
All data analysis was performed using Prism 9 (GraphPad). Gut distention analysis was performed using one-way ANOVA with Dunnett's multiple comparison test. For all statistical tests we set the α to 0.05. All gures were generated using Adobe Illustrator (Adobe).