Abstract
BackgroundNeuromuscular (NM) synaptogenesis is a tightly regulated process. We previously showed that in flies, Drosophila Nedd4 (dNedd4/dNedd4S) is required for proper NM synaptogenesis by promoting endocytosis of commissureless from the muscle surface, a pre-requisite step for muscle innervation. DNedd4 is an E3 ubiquitin ligase comprised of a C2-WW(x3)-Hect domain architecture, which includes several splice isoforms, the most prominent ones are dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo).Methodology/Principal FindingsWe show here that while dNedd4S is essential for NM synaptogenesis, the dNedd4Lo isoform inhibits this process and causes lethality. Our results reveal that unlike dNedd4S, dNedd4Lo cannot rescue the lethality of dNedd4 null (DNedd4T121FS) flies. Moreover, overexpression of UAS-dNedd4Lo specifically in wildtype muscles leads to NM synaptogenesis defects, impaired locomotion and larval lethality. These negative effects of dNedd4Lo are ameliorated by deletion of two regions (N-terminus and Middle region) unique to this isoform, and by inactivating the catalytic activity of dNedd4Lo, suggesting that these unique regions, as well as catalytic activity, are responsible for the inhibitory effects of dNedd4Lo on synaptogenesis. In accord with these findings, we demonstrate by sqRT-PCR an increase in dNedd4S expression relative to the expression of dNedd4Lo during embryonic stages when synaptogenesis takes place.Conclusion/SignificanceOur studies demonstrate that splice isoforms of the same dNedd4 gene can lead to opposite effects on NM synaptogenesis.
Highlights
Neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) is an E3 ubiquitin ligase that belongs to the Hect family [1]
Our previous work showed that dNedd4 is involved in regulating neuromuscular synaptogenesis, and revealed two isofoms of dNedd4 expressed in the body wall muscle [19]
Nedd4Lo are involved in its regulation of neuromuscular synaptogenesis Since the Drosophila Akt (dAkt) phosphorylation site in the unique N-terminal region of dNedd4Lo did not explain the functional difference between dNedd4Lo and dNedd4S, we studied the role of the N-terminal (Nterm) region as well as the unique middle (Mid) region of dNedd4Lo to determine if either region is involved in the adverse function of dNedd4Lo in NM synaptogenesis
Summary
Neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) is an E3 ubiquitin ligase that belongs to the Hect family [1]. Nedd proteins share common domain architecture: a C2 domain, 3–4 WW domains and a ubiquitin ligase Hect domain. Nedd is known to regulate stability of ion channels, such as ENaC, which has PY motifs that interact with the Nedd4-2 -WW domains to promote ENaC endocytosis [7,8,9,10]. We previously showed that in flies, Drosophila Nedd (dNedd4/dNedd4S) is required for proper NM synaptogenesis by promoting endocytosis of commissureless from the muscle surface, a pre-requisite step for muscle innervation. DNedd is an E3 ubiquitin ligase comprised of a C2-WW(x3)-Hect domain architecture, which includes several splice isoforms, the most prominent ones are dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo)
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