Although previously thought to be a feature unique to the pUPD class of AS [8], more recent research has identified 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 flies, resembling hyperphagia in PiezoKO flies 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 fly 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 fluorescent intensities were detected between Dube3a15b and PiezoKO flies in this assay, suggesting a putative role for Dube3a in satiety signaling. This connection between satiety and Dube3a was confirmed using Df lines that uncover the Dube3a locus (Fig. 2B). We previously demonstrated that Dube3a15b mutants have significantly fewer actin filaments than their wild type counterparts [27]. Actin is essential for the trafficking 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 filaments, via loss of Dube3a, inhibits Piezo trafficking/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 flies 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 pathfinding and angiogenesis [31–33]. Mice deficient for Piezo1 also die during mid gestation stage emphasizing the role of Piezo channels in proper development [34]. Consistent with these findings, attempts to analyze Piezo > Piezo:GFP; Dube3a-FLAG flies were hindered by low eclosure rates, suggesting an effect on Piezo expression and or function after Dube3a overexpression.
In Piezo > Piezo:GFP; Dube3a-FLAG flies, 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 flies 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 fly 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) unidentified, current evidence suggest cofilin as a potential Dube3a polyubiquitination substrate that could affect Piezo function in the membrane [11]. The findings here strengthen the connection between Dube3a and Piezo and their potential role in the regulation of hyperphagia in AS.