Abstract
O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, we generated catalytically inactive and precise knockout Oga alleles (OgaD133N and OgaKO, respectively) in Drosophila melanogaster. Adult OgaD133N and OgaKO flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionarily conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected a number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, we report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.
Highlights
O-GlcNAcylation is an abundant post-translational modification in neurons
Earlier studies have uncovered a link between O-GlcNAcase and learning in mouse and rat models [30, 31]
Heterozygous Ogaϩ/Ϫ mice with increased O-GlcNAc levels exhibited hippocampal-dependent spatial learning and memory defects [31], whereas rats treated with an OGA inhibitor, thiamet-G, showed reduced performance in novel object and placement tests [30]
Summary
It has never been investigated before whether OGA possesses any nonenzymatic roles. We developed tools to dissect enzymatic or nonenzymatic functions of Oga in normal neuronal development and cognition/learning. We generated rationally designed catalytically inactive Oga (OgaD133N) and novel Oga knockout (OgaKO) alleles by exploiting the CRISPR/Cas gene editing toolbox, resulting in elevated levels of protein O-GlcNAcylation in homozygous flies. We discovered that a loss of O-GlcNAcase activity affects locomotion and causes deficits in habituation learning, thereby demonstrating a conserved role of Drosophila Oga in cognitive function. We showed that synaptic bouton counts at the larval neuromuscular junctions are altered in OgaKO flies, indicating a novel role for Oga in synaptic development. Our phenotypic characterization of OgaD133N and OgaKO lines revealed that the primary role of Oga in these processes is O-GlcNAcase enzyme activity
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