This proposal concerns spastin and blue cheese beached (bchs), two genes identified in screens we conducted for genes involved in motor axon guidance and synaptogenesis in Drosophila larvae. We selected these genes for further study because they encode members of highly conserved but poorly understood protein families that have not been previously implicated in neural development. The two genes are not related to each other, but both encode proteins likely to be involved in protein trafficking within neurons and can be studied using similar methods. Both genes have orthologs or relatives affected in human genetic diseases. (1) The first gene, spastin, is the ortholog of a human gene affected in autosomal dominant spastic paraplegia (ADSP). spastin encodes an AAA ATPase. These ATPases are involved in catalyzing assembly and disassembly of protein complexes involved in vesicle trafficking, protein degradation, microtubule dynamics, and other processes. Analysis of an AAA ATPase sequence does not allow definition of the cellular process(es) in which it participates, however, so the targets of Spastin function are still unknown. Neuronal overexpression of spastin causes convergence of central nervous system (CNS) axons onto the midline, spastin loss-of-function (LOF) null mutant larvae display altered synaptic morphologies at their neuromuscular junctions (NMJs). They also have reduced evoked junctional potential (EJP) amplitudes, indicating that their NMJ synapses are abnormal. Spastin-null animals that survive to adulthood are unable to fly, walk poorly, and have a shortened lifespan. To further analyze Spastin function, we will complete the morphological and electrophysiological analysis of larval NMJs. We will also perform electrophysiological tests to examine why the mutant adults cannot fly and examine the adult brain for structural defects and neurodegeneration. We will examine the mechanisms involved in human ADSP by introducing mutations that cause spasticity in humans into the fly gene and determining if these act as dominant negatives in Drosophila. To define other components of the pathways(s) in which Spastin acts, we will perform an enhancer/suppressor genetic screen using a spastin gain-of-function (GOF) eye phenotype. Candidate genes emerging from the eye screen will be tested for modification of the spastin GOF axonal phenotype and for interaction with spastin LOF mutations. (2) The second gene, bchs, encodes a protein closely related to the founding member of the BEACH domain protein family: the human protein whose loss causes Chediak-Higashi syndrome (CHS), a lethal genetic disease characterized by immunological and neurological defects. Cells from CHS patients contain abnormal giant lysosomes, bchs overexpression in neurons produces a unique phenotype in which bulges form at the junctions between motor axon trunks and side branches. bchs LOF mutations cause adult neurodegeneration phenotypes in the brain and eye, and bchs flies have short lifespans. In bchs larvae, some motor axon pathways are abnormally thickened, suggesting that individual axons are swollen or that additional axons have joined the pathways. Bchs contains a FYVE domain, which binds to phosphatidylinositol-3-phosphate (Ptdlns3P). It is a vesicular protein that occasionally colocalizes with a fluorescent marker (GFP-2XFYVE) for Ptdlns3P-containing endosomes; however, most Bchs vesicles are distinct from GFP-2XFYVE vesicles, suggesting that they represent different compartments. To study Bchs, we will analyze its subcellular localization and determine the vesicular compartment(s) in which it functions. We will examine bchs LOF and gain-of-function (GOF) phenotypes in the larval neuromuscular system using antibody staining, electron microscopy, and electrophysiology. We will also search for enhancers and suppressors of a bchs GOF eye phenotype. Candidate genes from the eye screen will be tested for modification of the bchs GOF neuromuscular phenotype and for interaction with bchs LOF mutations.