RNA and glia at the Drosophila neuromuscular junction: importance for synaptic plasticity

Glial cells are essential components of both human and invertebrate nervous systems, including in the fruit fly, Drosophila melanogaster. Historically, research has focused more on neurons, with glia and their role in synaptic plasticity being somewhat overlooked. It is generally accepted that synaptic plasticity requires messenger ribonucleic acid (mRNA) and proteins localising at synapses, but the role of glia and mRNA localisation within glia in this process has been comparatively overlooked, despite the knowledge of their close association with synapses.
In recent research from the Davis laboratory, high-resolution microscopy was utilised to investigate mRNA and protein expression patterns in the larval nervous system. One common single molecule fluorescence in situ hybridisation (smFISH) probe was designed against the Yellow Fluorescent Protein (YFP) mRNA sequence of 200 Drosophila YFP fusion lines, allowing for the simultaneous detection of the reporter YFP protein and its mRNA within them. This study identified 19 mRNAs believed to be localised in the neuromuscular junction (NMJ) glia. This thesis aims to determine if these mRNAs and their corresponding proteins are indeed in the NMJ glial cells protrusions and if they play a role in regulating synaptic plasticity of the nearby motor neurons.
In Chapter 3, I verified that mRNAs of 18 of these transcripts are localised in the NMJ glial protrusions. Some were predominantly glia-specific, while others were also found in surrounding muscle. In Chapter 4, I evaluated the effects of knocking down these 18 mRNAs in glia using an RNA interference (RNAi) candidate screen. Some knockdowns led to NMJ defects, anatomical abnormalities, and, in a few cases, lethality. A crawling impairment phenotype was observed only in the Lachesin (Lac)-RNAi larvae, making Lac a gene of significant interest.
In Chapter 5, I investigated the role of Lac in NMJ glia. Lac mRNA localises to specific and targeted NMJ glial structures, while in Lac-RNAi NMJs, the glial projections are disordered and aimless. I determined that the area occupied by Lac::YFP protein decreases after an assay which induces synaptic plasticity, similarly to glial area in control NMJs, and the axon terminal projection area stays constant. In Lac-RNAi larvae, the glial area does not decrease, and the neuronal area increases, suggesting a zero-sum game where the glial and neuronal projection areas at the NMJ exist in a conditional equilibrium. Lac mRNA preferentially associates with the glial and Blood-Brain Barrier (BBB) areas of the NMJ, and the knockdown of Lac in the subperineurial glia, which form the BBB in Drosophila, causes the most severe phenotype of all glial subtypes when comparing to the knockdown in all glia. Lac deficiency at the periphery of NMJ glial cells resulting in aberrant glial cell morphology could lead to altered synaptic plasticity in motor neurons and result in aberrant locomotor behaviour due to disruptions in glia-neuron communication, the BBB function and maintenance, altered neurotransmitter homeostasis, and changes in synaptic strength.
In summary, my thesis offers evidence suggesting that glial cell projections closely associated with synapses actively participate in the regulation of the synaptic plasticity happening at these synapses, and such control might be exerted through mRNA localisation of specific transcripts to the glial periphery. I anticipate that this thesis could serve as a valuable reference for subsequent studies on other proteins whose mRNAs might be localised to the glial periphery, possibly specifically ones related to BBB maintenance and formation.

Keywords: neuroscience
Drosophila
RNA
synaptic plasticity
glial cells
neurobiology
mRNA
glia
Advisor: Davis, I