Much has been learned about the signaling pathways and networks of proteins that function together to build and modulate synaptic connections. This rich molecular landscape is under the control of multiple classes of regulatory factors. MicroRNA (miR) are versatile posttranscriptional regulators capable of tuning levels of gene expression across a large number of target genes. Through genetic screens in Drosophila, we have discovered that synapse formation and growth are controlled by conserved microRNA genes that orchestrate different stages of synapse development through distinct sets of direct and indirect targets. Having recently created a means of selectively inhibiting the function of any microRNA with spatio-temporal precision in vivo, we are now equipped to survey the functions of all microRNAs in Drosophila in many aspects of neural development, connectivity, behavior, and neurodegeneration. Once this regulatory landscape has been mapped through comprehensive screens in this model organism, it will be possible for us to test the conservation of these mechanisms in mammalian neurons and circuits.
It has become increasingly clear that neuronal connectivity, function and plasticity all rely on the post-transcriptional regulation of gene expression. Recent studies reveal many hundreds of highly localized mRNAs in axons, dendrites and synapses. With an array of techniques, many of which we are developing with and/or learning from our collaborators, we will determine which MicroRNA (miR) are essential for activity-induced remodeling at the synapse. We will use new genetic tools to define the spatial and temporal logic for each miR function. We will then use a state-of-the-art combination of transcriptome sequencing and computational informatics, followed by use of in vivo activity sensors and functional validation, to discover the downstream mechanisms for each miR that intersects our coordinated screens.
Our studies of the LAR receptor phosphatase led us to the discovery that the LAR pathway regulates synaptic growth and the morphogenesis of the active zone – a structure that orchestrates neurotransmitter release at chemical synapses. We have defined factors upstream and downstream of LAR in this context, and the machinery appears to be highly conserved. Upstream, LAR interacts with synaptic heparan sulfate proteoglycans that control distinct aspects of synapse morphogenesis or function. Downstream, LAR activity is mediated by a pathway linking the phosphatase the intracellular tyrosine kinase Abl. Genetic screens for Abl effectors in Drosophila identified the both actin regulatory factors and the microtubule plus-tip interacting protein (MT+TIP) CLASP as a protein required for Abl function in vivo. Our subsequent biochemical and functional studies showed that CLASP associates with and is phosophorylated by CLASP in mammalian cells, suggesting conservation in the guidance machinery. We have used genetic and proteomic tools to define a network of functional partners for CLASP, and find not only additional MT+TIPs, but also MT-actin cross-linking factors suggesting that CLASP and Abl are involved in the coordination of the two major polymer systems. Our recent analysis of the Abl and CLASP-interactors revealed a novel growth cone-specific role for the MT polymerase (XMAP215) that appears to involve coordination and coupling between the MT and actin polymer networks. We are currently pursuing additional CLASP and Abl interacting components.