Significant work is ongoing to reveal how different cell types in the brain contribute to behavior and pathology, and how they change in plasticity and disease, empowered by new genetic, optical, and physiological tools. However, the functional activity and dysregulation of neuronal circuits relies critically on the in situ molecular composition of neuronal synapses. Although it is clear that the properties of a given synapse are determined by, amongst other things, the specific types of cells that are thus connected, far less is known about the diversity of synapse types in the brain than cell types, perhaps because this is an intrinsically proteomic problem: a given neuron might make many different kinds of synapse with different targets, and thus transcriptomics (which is prevailing as a method for cell type analysis) may not suffice for synapse typing. High- throughput in situ proteomic tools are needed to characterize synapse molecular composition at the single-cell level in the context of whole brains or brain regions, and thus to connect the currently distant topics of neuronal activity and genetic aberrations associated with disease pathology. Here, we propose a high-risk, high-payoff, and as far as we know entirely novel agenda: to develop tools capable of resolving the molecular proteomic composition of synapse types, testing them in cultured neurons and intact brain tissues. To achieve this transformative goal of establishing a broadly useful tool for in situ synapse proteomics, we will build on our recent breakthrough in developing the DNA-based highly multiplexed, quantitative super-resolution imaging method DNA- PAINT (Points Accumulation for Imaging in Nanoscale Topography). DNA-PAINT exploits the transient binding of short fluorescently labeled DNA-probes for simple and easy-to-implement quantitative, highly multiplexed, super-resolution imaging with sub-10 nm resolution. In this application, we plan to develop and apply DNA-PAINT to enable quantitative, ultra-multiplexed, in situ characterization of neuronal synapse proteins for understanding synaptic types and studying cell type-specific synaptic functions. The outcome of our work will be a broadly useful in situ proteomic tool for quantification of neuronal synapse composition that can be used by diverse neurobiology laboratories to study single cell-level synapse properties in fixed tissues from whole brains or cell culture assays.
The proposed research is relevant to public health because understanding the molecular structure and composition of synapses is key to understanding neurological and psychiatric disorders where information transfer between neurons is impaired. Many neurological and psychiatric disorders are associated with mutations or deficits in synaptic protein availability, morphology, or physiology. Our technology will enable the proteins within a synapse to be understood, providing fundamental information that will support development of new brain therapeutics.
|Oran, Daniel; Rodriques, Samuel G; Gao, Ruixuan et al. (2018) 3D nanofabrication by volumetric deposition and controlled shrinkage of patterned scaffolds. Science 362:1281-1285|
|Beliveau, Brian J; Kishi, Jocelyn Y; Nir, Guy et al. (2018) OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes. Proc Natl Acad Sci U S A 115:E2183-E2192|
|Agasti, Sarit S; Wang, Yu; Schueder, Florian et al. (2017) DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging. Chem Sci 8:3080-3091|
|Freifeld, Limor; Odstrcil, Iris; Förster, Dominique et al. (2017) Expansion microscopy of zebrafish for neuroscience and developmental biology studies. Proc Natl Acad Sci U S A 114:E10799-E10808|
|Schueder, Florian; Lara-Gutiérrez, Juanita; Beliveau, Brian J et al. (2017) Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT. Nat Commun 8:2090|
|Zhao, Yongxin; Bucur, Octavian; Irshad, Humayun et al. (2017) Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy. Nat Biotechnol 35:757-764|
|Schueder, Florian; Strauss, Maximilian T; Hoerl, David et al. (2017) Universal Super-Resolution Multiplexing by DNA Exchange. Angew Chem Int Ed Engl 56:4052-4055|
|Zhang, Yu Shrike; Santiago, Grissel Trujillo-de; Alvarez, Mario Moisés et al. (2017) Expansion Mini-Microscopy: An Enabling Alternative in Point-of-Care Diagnostics. Curr Opin Biomed Eng 1:45-53|
|Beliveau, Brian J; Boettiger, Alistair N; Nir, Guy et al. (2017) In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT. Methods Mol Biol 1663:231-252|
|Wang, Yu; Woehrstein, Johannes B; Donoghue, Noah et al. (2017) Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues. Nano Lett 17:6131-6139|
Showing the most recent 10 out of 18 publications