Identification of Genetic Factors That Affect Neuronal Patterning, Function, and Disease in Drosophila melanogaster

摘要

Heparan Sulfate Proteoglycans (HSPGs) are required for normal synaptic development at the Drosophila melanogaster larval neuromuscular junction (NMJ). When enzymes required for biosynthesis of HSPGs are inhibited through mutations of RNA interference, a variety of morphological and electrophysiological defects are observed at the NMJ. These defects included changes in the post-synaptic specialization of the muscle (the SSR), loss of mitochondria from the sub-synaptic cytosol, and abnormal mitochondrial morphology. Identification of autophagic regulation as the mechanism by which HSPGs influenced synaptic properties was the foundation of this dissertation.;The present work more fully characterizes the influence of HSPG function on autophagic markers in muscle tissue. Confocal microscopic and Western blot analysis of both endogenous proteins and ectopically expressed reporter constructs was used to assess the level of autophagic degradation in muscle tissue. RNA inhibition of sfl and ttv, two HS biosynthetic enzymes, influenced these measures of autophagy in a direction that indicated an overall increase in autophagic flux.;HSPGs are ubiquitously expressed, and have a variety of critical functions throughout the organism. Similarly, autophagy is a globally important catabolic pathway. To better understand the significance of this novel regulatory role for HSPGs, we extended our analysis into a second tissue. Drosophila fat body tissue is an energetically sensitive tissue in which autophagy is commonly studied. Using many of the same assays, we identified a similar increase in autophagy in response to HSPG biosynthetic inhibition that was non-cell autonomous.;While autophagy is important for the maintenance of cellular homeostasis throughout the organism, certain cell types are more reliant on this pathway than others. Neurons are one such autophagy sensitive cell type, and failure of neuronal autophagy is closely associated with neurodegenerative disease. When HSPGs were inhibited in the brain of adult flies, an increase in autophagic degradation was once again identified. This increase in autophagy protected against the accumulation of oxidant-damaged cellular components after environmental exposure to hydrogen peroxide, and reduced neuronal death in a model of Alzheimer's disease. These findings indicate that the regulation of autophagy by HSPGs is of high clinical relevance to human health.;This thesis also describes an attempt to develop a Drosophila behavioral assessment protocol intended to provide biological validation of Autism Spectrum Disorder (ASD) candidate genes. Sequencing and microarray analysis of individuals with Autism spectrum disorders has implicated an overwhelmingly large pool of candidate genes. Thorough assessment of the functional contributions of these genes to neurobehavioral development in model organisms is most drastically hindered by the generally low rate of recurrence in genetic changes affecting each candidate gene.;Behavioral screening in the inexpensive and genetically facile model D. melanogaster was attempted using RNA interference (RNAi) to model genetic changes associated with human ASD and identify high-throughput behavioral methods suitable for providing the lacking biological validation for these genetic models. Three behavioral methods were selected based on their compatibility and straightforwardness, with consideration for evidence of previous successful use in established fly models of Autism. While the pilot run of this study was able to identify a few phenotypes of potential interest, the majority of the data indicated that these methods were not wellsuited to the use of RNAi.