Localized translational dysregulation in an autism spectrum disorder model using in situ cryo-EM - ABSTRACT/PROJECT SUMMARY
We propose to leverage innovative in situ cryo-electron microscopy (cryo-EM) techniques to study
dysregulated protein synthesis, termed translation, in an autism spectrum disorder (ASD) model. Dysregulated
translation disrupts normal neuronal plasticity, leading to various disorders including autism spectrum disorder
(ASD), major depressive disorders, chronic pain, and other pathologies affecting millions worldwide. However,
a comprehensive understanding of location-dependent ribosome regulation, which plays a crucial role in
plasticity, remains elusive. Current methods to study single events at a high resolution in cells are limited,
hindering the acquisition of this essential knowledge. We will overcome this limitation using correlative
fluorescence microscopy and in situ cryo-EM.
The loss of the Fragile-X mental retardation 1 (FMR1) gene product, known as FMRP, is responsible for
the most prevalent form of heritable ASD. FMRP interacts with polysomes within two types of RNA granules:
fragile X granules (FXGs) and neuronal RNA granules (nRNAgs), also known as "transport granules." Excessive
translation resulting from the absence of FMRP has been associated with long-term memory impairment,
supported by the notion that neuronal granules contribute to memory and synaptic plasticity. Despite its broad
importance in neuronal function, the pleiotropic nature of FMRP has prevented the field from developing a unified
understanding of its functions and its underlying role in local translation. To address this, we will develop novel
high-resolution approaches to unravel the functions of FMRP in translation within neuronal models. These
advances will pave the way for investigating FMRP's involvement in translational regulation using disease
models derived from human induced pluripotent stem cells (iPSCs).
We propose in this project to apply cutting-edge cryo-EM methods and develop effective workflows in
two Specific Aims: In Aim 1, we will develop a protocol to determine the localization, activity, and structure of
FMRP granule-associated ribosomes in iPSCs. This aim will establish a workflow and reveal how ribosome
localization and activity are regulated by FMRP in neurites. In aim 2, we will determine the localization, activity
and structures of ribosomes associated with nRNAgs and FXGs. This aim will reveal how ribosome inactivation
differs between granule types. More broadly, these investigations will lay the foundation for mechanistic studies
aimed at understanding how translation contributes to human disease in a location-specific manner.
Our work will provide structural insights into translational regulation, a fundamental process for proper
neuron function. Furthermore, it will enable the application of this analysis to patient-derived human iPSC models
of neurodevelopmental diseases, aligning with the missions of the National Institute of Neurological Disorders
and Stroke.
