microRNA-Mediated Mechanisms Essential for the Structural Plasticity of Drosophila Glutamatergic Synapses

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PROJECT SUMMARY / ABSTRACT The molecular and cellular mechanisms underlying the plasticity of excitatory synapses have fascinated biologists for many decades. In addition to the importance of these processes in the acquisition and storage of memories, as well as other adaptations of neural circuits to sensory input or other changing conditions, many of the effector genes that participate in such mechanisms have recently been associated with a wide range of neurological, psychiatric and other disorders of the human nervous system. Thus, it is little surprise that synapse formation, plasticity and structural remodeling are under tight control at many levels. To better understand this, we have investigated small, non-coding microRNA genes that serve as versatile yet selective regulators of dynamic gene expression changes that underly the morphological plasticity of the synapse. Through multiple rounds of genetic tool development, screening, and tissue-specific analysis, we have identified several highly conserved microRNAs that are required in the postsynaptic cell to allow coordinated remodeling of the synapse in response to acute stimulation. Because each microRNA controls the expression of specific target mRNAs, our studies have led us to several key proteins whose expression must be downregulated to allow synapse remodeling. In particular, our unpublished analysis of miR-219 suggests that it controls expression of a guanine nucleotide exchange factor (GEF) specific to the Ral GTPase. Although this GEF (dRalGPS) is very highly conserved, there are no peer reviewed publications on the Drosophila ortholog. Moreover, while fly miR-219 is perfectly conserved with human miR-219a, and the miR-219 response element (MRE) in RalGPS is also conserved across species, this relationship has escaped study by other labs. Prior work on Ral at the Drosophila larval neuromuscular junction (NMJ) delineated a postsynaptic pathway that mediates morphogenesis of a dendritic-spine like membrane array called the subsynaptic reticulum (SSR) by recruiting Sec5 and other Exocyst components in response to neural activity. Analysis of our unpublished null mutation, expression transgenes, and antibodies against dRalGPS show that, like Ral, this Ral GEF is both necessary and sufficient to control the postsynaptic recruitment of key determinants of SSR structure. These and other observations described in this proposal suggest a working model where synapse plasticity depends on convergent microRNA regulation of dRalGPS and other effectors to reprogram the synaptic proteome for synapse addition rather than stability. We propose to rigorously test this model with a combination of site-directed mutagenesis and tissue-specific analysis (Aim 1), genetic epistasis and protein localization studies (Aim 2), and thorough regulatory analysis of the target genes to address their dependence on miR-219 and other postsynaptic microRNA activities (Aim 3).