Brian Ackley - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cells in the nervous system use intercellular signaling cues extensively to properly the extend neurites, navigate to and recognize target cells and form and maintain synaptic contacts. The ability to predict secreted proteins from whole genomes has led to the conclusion that perhaps as much as one third of all proteins encoded are secreted into the extracellular space. Given the complexity of nervous systems it stands to reason that many of these secreted proteins act in the patterning of the nervous system. We have generated a library of predicted secreted proteins from the C. elegans genome. We will take a systematic approach using RNA interference (RNAi) to knockdown of all 7,460 predicted secreted proteins and evaluate the effect of this RNAi on multiple aspects of neural development. A preliminary characterization will be done to identify molecules that have an effect on neuronal patterning, and a secondary level quantitative analysis will be done on those molecules. We are using a synthetic lethal screening approach to characterize these molecules first, and then we will examine the outgrowth and synaptic morphology in mutants for genes that demonstrate an effect. Understanding how the nervous system is patterned has long been a goal of neurobiology. A multitude of studies have demonstrated the importance of extracellular proteins on all aspects of neuronal development. The ability to systematically evaluate the effect of loss of function of all secreted proteins has only recently become available. This will provide many novel insights into an extremely complicated part of development.

Voltage-gated calcium channels are the engines that drive the synapse. They are required for vesicle exocytosis, and it is now clear that these molecules are critically important to the dynamics of formation, maintenance, adaption and elimination that underlie changes in neural networks. Therefore, as we study these molecules and their mode of action, we will gain a much clearer understanding of the basic assembly of the nervous system. VGCCs have been linked to human diseases and disorders, and our goal is to further the understanding of how these proteins contribute to neuronal development. Using animals that have mutations that inactivate or hyperactive synaptic VGCCs we will obtain transcriptome profiles to identify genes that are transcriptionally regulated by VGCC functional status. We will then target those genes for knockdown by RNAi to find molecules that contribute to VGCC-dependent synapse addition. Finally we will seek to visualize how calcium may be dynamic during times when synapses are being modified during development to correlate intracellular levels of calcium with specific changes in synapses. The organization of the C. elegans neuromuscular system provides a powerful genetic and cell biological model to study development. The primary motorneurons have many similarities to vertebrate CNS neurons, which are more difficult to study in vivo. C. elegans may provide important insights into the mechanisms that underlie the formation and spacing of these types of synapses in vivo.