Glial involvement in neuronal synaptic bouton formation implicates pak3 and draper function. Emily F. Ozdowski, Nina T. Sherwood. Dept Biol, Duke Univ, Durham, NC.

   Neurons require cytoskeletal regulators, such as the microtubule-severing protein Spastin, to produce proper axonal branching and functional synaptic connections. When Spastin function is compromised in humans, the motor neuron disease Hereditary Spastic Paraplegia (HSP) results. This disorder is characterized by degeneration of long axons within the corticospinal tracts and ultimately loss of mobility in the lower limbs. Similarly, when spastin function is lost in Drosophila, neuronal signaling at the larval neuromuscular junction (NMJ) is diminished, and adult flies are not able to walk normally, jump, or fly. In spastin null mutants, larval axons form unique grape-like bunches of small synaptic boutons at the NMJ, and microtubules are missing from the distal tips. These aberrant structures are useful in searching for regulators of spastin function in neurons, and we discovered that the actin regulator, p21-activated kinase 3 (pak3) is a bypass suppressor of the spastin phenotype. pak3 hypomorphic mutations have little effect on wild-type NMJ synapses but strongly suppress the bunched bouton morphology of spastin null mutants. In addition, neuronal overexpression of pak3 results in numerous actin-rich filopodial projections, as observed in cell culture. However, we found that Pak3 is expressed primarily in glia, and glial-specific reduction of pak3 also suppresses spastin bunches. Glia have previously been linked to synaptic bouton number and synaptic debris clearance via Draper receptor function. We found that both the draper null mutation and glial-specific draper knockdown by RNAi suppress spastin bunches, suggesting that draper and pak3 work in a similar pathway. We are currently examining mutations in Draper ligands and effectors for suppression of spastin null bouton bunches. We are also imaging the physical interactions between glia and neurons during normal development compared to these mutants. Understanding the mechanism by which glia influence synaptic bouton formation could ultimately instruct us on potential methods for disease amelioration in humans.