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1.  High-Content Chemical and RNAi Screens for Suppressors of Neurotoxicity in a Huntington's Disease Model 
PLoS ONE  2011;6(8):e23841.
To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.
PMCID: PMC3166080  PMID: 21909362
2.  Intelligent Interfaces for Mining Large-Scale RNAi-HCS Image Databases 
Recently, High-content screening (HCS) has been combined with RNA interference (RNAi) to become an essential image-based high-throughput method for studying genes and biological networks through RNAi-induced cellular phenotype analyses. However, a genome-wide RNAi-HCS screen typically generates tens of thousands of images, most of which remain uncategorized due to the inadequacies of existing HCS image analysis tools. Until now, it still requires highly trained scientists to browse a prohibitively large RNAi-HCS image database and produce only a handful of qualitative results regarding cellular morphological phenotypes. For this reason we have developed intelligent interfaces to facilitate the application of the HCS technology in biomedical research. Our new interfaces empower biologists with computational power not only to effectively and efficiently explore large-scale RNAi-HCS image databases, but also to apply their knowledge and experience to interactive mining of cellular phenotypes using Content-Based Image Retrieval (CBIR) with Relevance Feedback (RF) techniques.
PMCID: PMC3028207  PMID: 21278820
3.  Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening 
Neuroinformatics  2010;8(2):83-100.
Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutamine-mediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington’s Disease (HD) model.
PMCID: PMC3022421  PMID: 20405243
High content screening; Neurite detection; Neuromeres; Gabor filter; Phase symmetry; Huntington’s Disease
4.  Evidence that Myosin Activity Opposes Microtubule-based Axonal Transport of Mitochondria 
Neurons transport and position mitochondria using a combination of saltatory, bidirectional movements and stationary docking. Axonal mitochondria move along microtubules (MTs) using kinesin and dynein motors, but actin and myosin also play a poorly-defined role in their traffic. To ascertain this role, we have used RNA interference to deplete specific myosin motors in cultured Drosophila neurons and quantified the effects on mitochondrial motility. We produced a fly strain expressing the C. elegans RNA transporter SID-1 in neurons to increase the efficacy of RNAi in primary cultures. These neurons exhibited significantly increased RNAi-mediated knockdown of gene expression compared to neurons not expressing this transporter. Using this system, we observed a significant increase in mitochondrial transport upon myosin V depletion. Mitochondrial mean velocity and duty cycle were augmented in both anterograde and retrograde directions, and the fraction of mitochondrial flux contained in long runs almost doubled for anterograde movement. Myosin VI depletion increased the same movement parameters, but was selective for retrograde movement, while myosin II depletion produced no phenotype. An additional effect of myosin V depletion was an increase in mitochondrial length. These data indicate that myosin V and VI play related but distinct roles in regulating MT-based mitochondrial movement: they oppose, rather than complement protracted MT-based movements and perhaps facilitate organelle docking.
PMCID: PMC2904968  PMID: 20592219
Axonal transport; Drosophila; Mitochondria; Myosin; Neuron; RNAi
5.  DMob4/Phocein regulates synapse formation, axonal transport, and microtubule organization 
The Mob family of kinase-interacting proteins regulate cell cycle and cell morphology, and their dysfunction has been linked to cancer. Models for Mob function are largely based on studies of Mob1 and Mob2 family members in yeast. In contrast, the function of the highly conserved metazoan Phocein/Mob3 subfamily is unknown. We identified the Drosophila Phocein homolog (DMob4) as a regulator of neurite branching in a genome-wide RNAi screen for neuronal morphology mutants. To further characterize DMob4, we generated null and hypomorphic alleles and carried out in vivo cell biological and physiological analysis. We find that DMob4 plays a prominent role in neural function, regulating axonal transport, membrane excitability and organization of microtubule networks. DMob4 mutant neuromuscular synapses also show a profound overgrowth of synaptic boutons, similar to known Drosophila endocytotic mutants. DMob4 and human Phocein are >80% identical, and the lethality of DMob4 mutants can be rescued by a human phocein transgene, indicating a conservation of function across evolution. These findings suggest a novel role for Phocein proteins in the regulation of axonal transport, neurite elongation, synapse formation and microtubule organization.
PMCID: PMC2862384  PMID: 20392941
Drosophila; knockout; Axonal Transport [Axoplasmic Transport]; Microtubule; Dendrite; Synapse
6.  Identification of Neural Outgrowth Genes using Genome-Wide RNAi 
PLoS Genetics  2008;4(7):e1000111.
While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system.
Author Summary
Development and function of the brain requires the coordinated action of thousands of genes, and currently we understand the roles of only a small fraction of them. Recent advances in genomics, such as the sequencing of entire genomes and the discovery of RNA-interference as a means of testing the effects of gene loss, have opened up the possibility to systematically analyze the function of all known and predicted genes in an organism. Until now, this type of functional genomics approach has not been applied to the study of very complex cells, such as the brain's neurons, on a full-genome scale. In this work, we developed techniques to test all genes, one by one in a rapid manner, for their potential role in neuronal development using neurons isolated from fruit fly embryos. These results yielded a global perspective of what types of genes are necessary for brain development; importantly, they show that a large variety of genes can be studied in this way.
PMCID: PMC2435276  PMID: 18604272

Results 1-6 (6)