Phenotypes ofDrosophilaBrain Neurons in Primary Culture Reveal a Role for Fascin in Neurite Shape and Trajectory

Abstract

Subtle cellular phenotypes in the CNS may evade detection by routine histopathology. Here, we demonstrate the value of primary culture for revealing genetically determined neuronal phenotypes at high resolution. Gamma neurons of Drosophila melanogaster mushroom bodies (MBs) are remodeled during metamorphosis under the control of the steroid hormone 20-hydroxyecdysone (20E). In vitro, wild-type ␥ neurons retain characteristic morphogenetic features, notably a single axon-like dominant primary process and an arbor of short dendrite-like processes, as determined with microtubule-polarity markers. We found three distinct genetically determined phenotypes of cultured neurons from grossly normal brains, suggesting that subtle in vivo attributes are unmasked and amplified in vitro. First, the neurite outgrowth response to 20E is sexually dimorphic, being much greater in female than in male ␥ neurons. Second, the ␥ neuron-specific "naked runt" phenotype results from transgenic insertion of an MB-specific promoter. Third, the recessive, panneuronal "filagree" phenotype maps to singed, which encodes the actin-bundling protein fascin. Fascin deficiency does not impair the 20E response, but neurites fail to maintain their normal, nearly straight trajectory, instead forming curls and hooks. This is accompanied by abnormally distributed filamentous actin. This is the first demonstration of fascin function in neuronal morphogenesis. Our findings, along with the regulation of human Fascin1 (OMIM 602689) by CREB (cAMP response element-binding protein) binding protein, suggest FSCN1 as a candidate gene for developmental brain disorders. We developed an automated method of computing neurite curvature and classifying neurons based on curvature phenotype. This will facilitate detection of genetic and pharmacological modifiers of neuronal defects resulting from fascin deficiency.

Common Proteomic Changes in the Hippocampus in Schizophrenia and Bipolar Disorder and Particular Evidence for Involvement of Cornu Ammonis Regions 2 and 3

“…Fascin 1 is an actin-bundling protein that has roles in neurite outgrowth. 104 These findings are in keeping with the presence of altered cytoskeletal dynamics 105 in these disorders. 46 Our third main finding is that our study implicates the process of CME in psychotic disorders by showing altered expression of proteins involved in or regulating this pathway, namely synaptotagmin, heat shock cognate 71-kDa protein, 106 44 These changes are in keeping with studies that found similar changes in the expression of SPTAN1, 45 heat shock cognate 71-kDa protein, 114 cathepsin D, 90 and the clathrin coat assembly protein AP180 in schizophrenia.…”

A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions

“…1A) were significantly different ; Fig. 1E (Kraft et al, 2006). This is consistent with the relative function among the genotypes.…”

“…development (R.K. and L.L.R., unpublished data), but not for viability (Bender, 1960). We discovered that singed mutations cause a neuronal morphogenesis defect, 'filagree' , when developing mutant CNS neurons are cultured in vitro (Kraft et al, 2006). Filagree neurite arbors have exaggerated clockwise curvature and erratic variation in neurite caliber, but no reduction in length, branching or axon-dendrite ratio (Kraft et al, 2006).…”

The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway

“…The genes whose transcription is selectively regulated by CREB following 20E treatment include the yolk protein precursor genes with CREB as a potent transcriptional repressor (37) and Fascin1 in D. melanogaster mushroom body remodeling with CREB as an up-regulated transcriptional coactivator (70). We found 20E induced CREB binding to CRE via the 20E-triggered non- , and U73122 (10 M) were added to the cells for 30 min, and DMSO was used as solvent control.…”