The nervous system is composed of two major cell types, neurons and glia. While previously regarded as passive support cells for neurons, glia’s active roles in nervous system development and function have recently gained appreciation. Glia have elaborate cell shapes across which they asymmetrically localize neuron-regulatory proteins. Thus, to fully understand glial roles in nervous system dynamics, we must determine how glial morphology and polarity are regulated. To investigate this, we use the amphid sheath (AMsh) glia of Caenorhabditis elegans. AMsh glia exhibit apical-basal polarity, with apical-protein-marked membranes contacting neurons at the cell’s anterior, and basal membranes extending posteriorly toward the cell body. A striking feature of the apical membrane is a discrete projection within the anterior glial process, which we term the Glial Apical Boundary or “GAB”. We find that the GAB localizes many glial cues which regulate neuronal properties. Upon comparing GABs of different apical proteins expressed by a single cell, we discovered they all overlayed. However, GABs of bilateral glia can be out of register, suggesting that the GAB is independently localized on a cell-to-cell basis. Because AMsh glia derive from neuroepithelial progenitors, we then asked if mechanistic regulation of the GAB is analogous to that of epithelial apical domains. Surprisingly, canonical epithelial polarity regulators PAR-3 and PAR-6 do not localize to AMsh apical membranes. Furthermore, junctional markers AJM-1 and DLG-1, which demarcate epithelial apical-basal domains, are absent either from the GAB or from the cell altogether. RNAi knockdown of these and other polarity genes does not impact GAB integrity or morphology. Thus, the GAB is a novel polarity feature of AMsh glia not governed by canonical apical-basal polarity mechanisms. Our current work focuses on elucidating how the GAB develops and is maintained, with overall importance to understanding how glia localize regulatory proteins in health and disease.

The mammalian brain contains neurons and glia in equal numbers. Glia contribute to proper neuronal communication by removing unnecessary synapses via a process known as pruning. Pruning plays a critical role in brain development, learning, and memory. How do neurons communicate which synapses must be pruned by glia? One way is through cell surface exposure of the lipid phosphatidylserine (PS) which serves as an “eat me” signal to glia. In other contexts, like apoptosis, flippases and scramblase enzymes regulate PS exposure. Flippases are membrane transporters that restrict exposure of lipids like PS on the extracellular leaflet, while scramblases translocate lipids bidirectionally, thereby promoting PS exposure. We don’t know if these molecules also regulate PS exposure during glial pruning. The Singhvi Lab previously established conservation of glial pruning in C. elegans. This optically transparent model contains a stereotyped nervous system, making it ideal for studying in vivo pruning with single-cell resolution. We focus on a single neuron-glia pair, AFD-AMsh, and use widefield fluorescence microscopy and posthoc image analysis to quantify the number of neuron fragments pruned by glia. We previously found that mutants lacking the flippase TAT-1/ATP8A have more pruning, suggesting a novel inhibitory role for this protein. Here, I examine several candidate scramblases: SCRM-1/PLSCR1, ATG-9/ATG9, CED-8/XKR8, and ANOH-1/TMEM16F. I conduct genetic crosses to put mutants for these scramblases in a fluorescent background to visualize pruning and use the described methods to characterize any pruning defects. Specifically, I expect that relevant scramblase mutants will have less pruning, as the “eat me” signal is not properly exposed. Dysregulation of pruning contributes to neurodegenerative disorders like Alzheimer’s. Similarly, flippase and scramblase mutations are linked to human brain dysfunction. Thus, studying the role of these enzymes in pruning offers novel insight into human brain health and disease.