# Simultaneous multiplexed in situ fluorescence imaging of neuronal proteins and messenger RNAs

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2021 · $400,900

## Abstract

PROJECT SUMMARY
The development, functional activity, and plasticity of neuronal circuits rely critically on the spatially controlled
expression and regulation of synaptic proteins and their messenger RNAs (mRNAs). Genome-wide association
studies have revealed extensive polygenic variation in synaptic proteins in association with diseases including
autism, schizophrenia, and Alzheimer's. A molecular understanding of how these complex genetic variations
impact neuronal synapse development, plasticity, and homeostasis is crucial for the development of new
therapies to treat these diseases. Fluorescence imaging offers the potential to characterize neuronal synapse
protein and mRNA levels and localizations in situ; however, current imaging approaches can simultaneously
interrogate no more than four of the several dozen molecules of interest in any given neuronal sample. To
overcome this obstacle, we propose to develop a transformative fluorescence imaging assay that enables
simultaneous, highly multiplexed, high-throughput molecular characterization of protein and mRNA expression
levels and localizations in intact neurons. To this end, we will develop an innovative labeling strategy that
exploits transiently binding fluorescent nucleic acids to enable multiple rounds of imaging of intact specimens,
using both standard and super-resolution microscopy. In conjunction, we will develop ultra-bright fluorescent
probes based on hybridization chain reaction and structured nucleic acids for visualization of single mRNA
molecules. We will apply both standard confocal and super-resolution imaging to characterize spatial
distributions and molecular interactions of synaptic proteins and regulatory mRNA-binding proteins, including
Fragile-X Mental Retardation Protein (FMRP) in both mouse and human induced pluripotent stem cell models.
Using this approach, we will characterize the impact of gene deletions associated with autism on the levels and
localizations of more than 10 synaptic and cytoskeletal proteins, as well as examine the interactions of FMRP
with dozens of mRNAs in intact dendritic arbors, spines, and synapses. We intend our technique to become
broadly useful as a platform technology for the study of the molecular impacts of genetic variations in
psychiatric diseases, including autism and schizophrenia. Consequently, we will develop our imaging platform
in close collaboration with the Stanley Center at the Broad Institute of MIT and Harvard. The high-throughput
nature of our imaging approach ensures that it will be useful for development of novel methods of treating
psychiatric diseases using small-molecule and gene-editing approaches.

## Key facts

- **NIH application ID:** 10107847
- **Project number:** 5R01MH112694-05
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Mark Bathe
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $400,900
- **Award type:** 5
- **Project period:** 2017-04-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10107847

## Citation

> US National Institutes of Health, RePORTER application 10107847, Simultaneous multiplexed in situ fluorescence imaging of neuronal proteins and messenger RNAs (5R01MH112694-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10107847. Licensed CC0.

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