Karen Mruk - US grants

Human spinal cord injuries (SCIs) affect multiple cell types and after the initial injury, a self-propagating cascade of cellular and biochemical mechanisms lead to additional tissue loss that exacerbates the SCI. The zebrafish central nervous system (CNS) shares many organizational, cellular and molecular pathways with mammals; however, functional regeneration occurs in zebrafish even after complete transection of the spinal cord. Our understanding of how the CNS of zebrafish responds to injury and subsequently regenerates remains incomplete. Our long-term goal is to advance our understanding of the cellular response to neural injury and subsequent process of regeneration using optogenetic tools and zebrafish models. The scientific aims of this research plan are to: (1) identify the cellular events that occur after different types of injury and (2) determine changes in the bioelectric network as the basis for locomotor recovery. This project will establish a framework to utilize the zebrafish larvae as a model for studies in neural injury and neurodegeneration. Upon completion, the proposed research is expected to provide a more complete picture of the biological basis for locomotor recovery and provide new therapeutic intervention points.

Project Summary RNA-based technologies have enabled perturbation and observation of gene function in multicellular organisms, resulting in major discoveries in biomedical research. However, gaining mechanistic insights into developmental and regenerative processes requires the manipulation of gene functions with spatial and temporal precision, a task that remains a major challenge in vertebrate animal models. The overall goal of this exploratory technology development proposal is to develop an innovative and widely applicable molecular toolset, which will enable control of gene expression with spatial and temporal precision. Specifically, we will introduce a new class of versatile RNA-based genetic conditional switches to regulate translation within the developing zebrafish. In our first aim, we will develop RNA switches that are controlled through non-invasive administration of non-toxic small molecules. These chemically-induced switches enable dynamic, titratable, and conditional control of a target gene. We will establish a rapid mammalian- based prototyping platform to prioritize ideal switch candidates for development in the zebrafish model. In our second aim, we will establish a conceptually new optogenetic switch that can be turned on and off using blue light. These novel tools will be developed in zebrafish and benchmarked against existing optogenetic tools including light-activated transcriptional tools. The successful execution of this project will provide a streamlined pipeline to develop new RNA switches that respond to a diversity of orthogonal non-invasive inputs. Given the species-independent machinery, the toolkit of RNA switches developed are likely to be broadly applicable to animal models. By specifically focusing the development of the switches in zebrafish, we will we will contribute a new approach to reprogram and interrogate developmental and regenerative biology.