The aim of this grant is to develop the first -omics level approach for the cell type specific identification and nanoscale localization of synaptic mRNA transcripts and proteins. The transport of mRNA to synaptic locations and their localized translation has long been an intriguing phenomenon, recognized to be of immense importance for understanding synaptic function, and yet hitherto studied using only a few select systems. Recent discoveries identifying a pivotal role for translation dysfunction in neurodevelopmental disorders underscore the importance of gaining a comprehensive understanding of synaptic translation. We propose to combine and focus at the synapse, two state-of-the-art methods, Translating Ribosome Affinity Purification (TRAP) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) for the novel identification and localization of synaptic transcripts and locally translated proteins. The power of TRAP, using cell specific expression of eGFP tagged ribosomes, would allow us to specifically purify translating ribosomes and associated mRNA from synaptic sites, away from the cell body (Synap-TRAP). Analysis of captured mRNA would provide us with the first unbiased genome wide analysis of synaptically translated proteins. Further, STORM will be developed to provide nanoscale localization of RNA and proteins identified from Synap-TRAP, providing orthogonal validation of candidates as well as providing new ultrastructural information of synaptic translation. Our combined approaches are likely to have significant impact on our understanding of normal synaptic function in the context of learning and memory, as well as in specific synaptopathies.
Translation of new proteins in the synapses of neurons is thought to be an important component of normal learning and memory, and dysregulation of this translation may be involved in the etiology autism spectrum disorders. Little is known about which proteins are translated at the synapses of different types of neurons of the brain, and about the nanoscale molecular architecture of this process. This proposal aims to generate new methods to address these questions, which can then be applied to gain a better understanding of these disorders.
|Maloney, Susan E; Khangura, Eakta; Dougherty, Joseph D (2016) The RNA-binding protein Celf6 is highly expressed in diencephalic nuclei and neuromodulatory cell populations of the mouse brain. Brain Struct Funct 221:1809-31|
|Li, Peiyao; Miao, Yong; Dani, Adish et al. (2016) Î±-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels. Mol Biol Cell 27:2542-53|
|Kopp, Nathan; Climer, Sharlee; Dougherty, Joseph D (2015) Moving from capstones toward cornerstones: successes and challenges in applying systems biology to identify mechanisms of autism spectrum disorders. Front Genet 6:301|
|Ouwenga, Rebecca L; Dougherty, Joseph (2015) Fmrp targets or not: long, highly brain-expressed genes tend to be implicated in autism and brain disorders. Mol Autism 6:16|
|Wells, Alan; Kopp, Nathan; Xu, Xiaoxiao et al. (2015) The anatomical distribution of genetic associations. Nucleic Acids Res 43:10804-20|
|Park, Dongkook; Li, Peiyao; Dani, Adish et al. (2014) Peptidergic cell-specific synaptotagmins in Drosophila: localization to dense-core granules and regulation by the bHLH protein DIMMED. J Neurosci 34:13195-207|
|Suleiman, Hani; Zhang, Lei; Roth, Robyn et al. (2013) Nanoscale protein architecture of the kidney glomerular basement membrane. Elife 2:e01149|