Kai Zhang - US grants

Project Summary/Abstract Transcription factors drive dynamic, cell-type specific, gene expression to define cell fate and functionality. Current optical microscopy technologies now enable direct visualization of transcription factors in live cells but cannot modulate transcription factor activity, which is required for delineating the contribution of genotypic modulation and phenotypic response. The emerging non-neuronal optogenetics provides a new strategy to regulate gene transcription, either by recruiting a transcription activation domain to a specific promoter or by photo-uncaging a sequestered transcription factor. Unlike native transcription factors, which regulates hundreds and thousands of target genes, the current optogenetic strategy only works for single- or a few gene targets and could suffer from high basal activity in the dark. Controlling multiplexed gene transcription with a larger library of transcription factors, thus, calls for an alternative strategy that empowers new modalities of optical control of gene transcription. The goal of this project is to fill this gap by developing a strategy based on the controlled rescue of protein degradation. In this strategy, base-level protein activities are suppressed by constant protein degradation until light triggers a burst of protein production. This strategy does not depend on the activation mechanism of the protein of interests and will significantly enhance the capacity of non-neuronal optogenetics. In this project, we present a plan within a four-year budget period to develop and validate the control native transcription factors. We will demonstrate blue-light-controlled T cell factor (TCF) downstream of the well- established Wnt signaling pathway (Aim 1) and develop an orthogonal optogenetic system to regulate the Notch intracellular domain (NICD)-mediate transcription with red light (Aim 2). Using our recently developed spatial light modulator, we will achieve precise multiplexing transcription control in space and time and ultimately achieve controlling the native transcription factors at the single-cell level (Aim 3). Our recent success in developing optogenetic tools for mammalian cells and Xenopus embryos well positions the applicant to carry out the proposed project. Results of this project will provide valuable assets to researchers who are interested in dissecting the spatial and temporal regulation of signal transduction during early embryonic development.

The project brings together material scientists and electrical engineers who build synthetic 3D scaffolds with embedded electronic and optoelectronic devices, neuroscientists who culture neural cells on the 3D scaffold to form biological neural networks with precisely defined 3D topology and integrated multimodal information interfaces, chemists and chemical engineers who synthesize functional molecules for controlling the neural cell placement, development, and activity, and computer scientists who operate the neurocomputer prototype and extract its information-coding and processing algorithms with machine-learning methods. The goals of the project are to contribute to the grand challenge of reverse engineering the brain and open up new computing paradigms based on cultured biological neural networks to propel machine learning and artificial intelligence to the next level.

The neurocomputer prototype pursued in the project employs biological neuronal circuits engineered into well-defined 3D topologies reminiscent of deep-neural-network models as the information-processing units. Electronic and optoelectronic devices will be integrated with each neural cell to administer and monitor the neuronal and synaptic activities based on electrophysiology, optogenetics, and neurochemistry. The fabricated neurocomputer prototype will then be utilized to perform various learning and computing tasks such as image recognition and space navigation. Neural code and learning algorithms will be extracted using a combination of experiment and simulations based on spike generation models and recurrent neural network models. The results will help reveal how complex living neural networks function and provide a technologically transformative approach to information-processing machines.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY/ABSTRACT Adaptation of living organisms to constantly changing environments depends on the plasticity of the nervous system. Neuronal plasticity often requires activity-dependent translation to rapidly supply selected proteins, for example, through activation of Group 1 metabotropic glutamate receptors (Gp1 mGluRs). Gp1 mGluRs, including mGluR1 and mGluR5, mediate translation-dependent synaptic plasticity, including long-term synaptic depression (LTD). Dysregulated Gp1 mGluR signaling is observed with various neurological and mental disorders, including Fragile X Syndrome (FXS) and autism spectrum disorders (ASDs). Although pharmacological correction of Gp1 mGluR activity reverses many of the phenotypes in animal models of those diseases, the molecular and cellular mechanisms underlying Gp1 mGluR-mediated synaptic plasticity have been elusive. Our published and preliminary data introduce the ubiquitin E3 ligase Murine double minute-2 (Mdm2) as a novel translational repressor and a ?switch? that permits Gp1 mGluR-induced protein translation (Liu et al., Hum Mol Genet., 2017). In our proposed research, we aim to characterize the role of Mdm2 in Gp1 mGluR- dependent synaptic plasticity (Aim 1) and determine the mechanism by which Mdm2 mediates activity-dependent protein translation (Aim 2). Our new data also show that Mdm2 is molecularly altered and unresponsive to Gp1 mGluR activation in the Fmr1 knockout (KO) mouse, the commonly used animal model for studying FXS (Tsai et al., Hum Mol Genet., 2017). In Aim 3 we will characterize the mechanism by which Fmr1 interconnects Gp1 mGluR signaling to permit translational activation through de-repressing Mdm2. Successful completion of this proposal will greatly facilitate the understanding of Gp1 mGluR-mediated synaptic plasticity through a novel mechanism of translational control. Building on the deep knowledge of Mdm2 in cancer biology, our research will also open a new avenue for the study of neurological disorders associated with abnormal Gp1 mGluR signaling.