High-content chemical and RNAi screens for suppressors of neurotoxicity in a Huntington's disease model.

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.

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 (RNAi) to deplete specific myosin motors in cultured Drosophila neurons and quantified the effects on mitochondrial motility.

Automatic robust neurite detection and morphological analysis of neuronal cell cultures in high-content screening.

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.

DMob4/Phocein regulates synapse formation, axonal transport, and microtubule organization.

The monopolar spindle-one-binder (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 primarily 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.

Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.

We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown.

Identification of neural outgrowth genes using genome-wide RNAi.

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.

Reciprocal interactions between neurons and glia are required for Drosophila peripheral nervous system development.

A major developmental role of peripheral glia is to mediate sensory axon guidance; however, it is not known whether sensory neurons influence peripheral glial development. To determine whether glia and neurons reciprocally interact during embryonic development, we ablated each cell type by overexpressing the apoptosis gene, grim, and observed the effects on peripheral nervous system (PNS) development. When neurons are ablated, glial defects occur as a secondary effect, and vice versa.

RhoA and Rac1 GTPases mediate the dynamic rearrangement of actin in peripheral glia.

Peripheral glial cells in both vertebrates and insects are born centrally and travel large distances to ensheathe axons in the periphery. There is very little known about how this migration is carried out. In other cells, it is known that rearrangement of the Actin cytoskeleton is an integral part of cell motility, yet the distribution of Actin in peripheral glial cell migration in vivo has not been previously characterized.