Kristy Welshhans, Ph.D. - US grants

DESCRIPTION (provided by applicant): The proper development of the nervous system is dependent on a number of cellular processes occurring at the appropriate time and location. One such cellular process that is required for proper nervous system development is the appropriate localization and translation of certain mRNA transcripts. The localization of these transcripts is dependent on mRNA binding proteins, a class of molecules responsible for the transport, and in some cases, translation of their mRNA cargo. Zipcode binding protein 1 (ZBP1) is an mRNA binding protein that is responsible for the transport and regulates the translation of [unreadable]-actin mRNA. [unreadable]-actin is important for the development of the nervous system because it is one of the major structural proteins that composes the growth cone, the pathfinding structure of a developing neuron. The aim of the current proposal is to determine how the loss of ZBP1 affects nervous system development and specifically, the growth cone. It is of great importance to study the function of ZBP1 because it is essential for appropriate gross development. The current proposal has two main goals: 1) to examine the role of ZBP1 in the regulation of axon outgrowth and guidance and 2) to examine the role of ZBP1 in the transport and translation of [unreadable]-actin mRNA. To determine the role of ZBP1 in these processes, lentiviral vector-mediated shRNA interference to ZBP1 will be employed, as well as a ZBP1 knockout mouse model. This proposal also uses innovative fluorescent proteins and reporters in primary neuronal cultures in order to achieve the above stated aims. It is essential to study the localization and translation of [unreadable]-actin mRNA because it encodes a structural protein that is required for proper growth cone motility and guidance, and therefore is required for the correct connectivity of the nervous system to be established. The long-term objective of this proposal is to gain an understanding of the basic mechanisms that are required for axon outgrowth and growth cone guidance, such as local translation of mRNA transcripts within the growth cone;this information will further our knowledge about the appropriate development of the nervous system as a whole. The development of the nervous system is a process during which neurons must find their way to and make connections with their appropriate targets. If this does not happen properly, a number of disease states may result. This proposal investigates the role of the protein ZBP1, which is responsible for transporting mRNA during development;the loss of this protein may result in inappropriate development of the nervous system and thus, a neurological disease state.

PROJECT SUMMARY The long-term goal of this research is to understand the molecular mechanisms underlying the establishment of neuronal connectivity during development. Specifically, this research examines how the growth cone, which is the pathfinding structure of the developing neuron, migrates to and connects with its appropriate targets. Neuronal connectivity is directed via both external and internal cues, which results in the activation of various cellular signaling pathways and ultimately, cytoskeletal reorganization. This proposal focuses on understanding the molecular mechanisms linking cellular signaling and local translation in the developing nervous system. It has only recently been discovered that local translation in axonal growth cones is necessary for the processes of axon growth and guidance. As such, our understanding of local translation is very limited. This study focuses on a multifunctional scaffolding protein, RACK1, which likely provides a critical link between cellular signaling and the regulation of local translation. We have preliminary data that this local translation takes place at point contacts, which are adhesion sites within growth cones that are essential for appropriate axon growth and guidance. Thus, our preliminary data suggests that point contacts are not just adhesion sites, but also critical signaling centers. Here, we hypothesize that point contacts within growth cones serve as a strategic location for targeted local translation and RACK1 is critical to this process. This proposal will provide valuable research opportunities for both undergraduate and graduate students to study fundamental processes that take place during neural development. Using advanced live cell imaging, fluorescent translation reporters, shRNA and axon growth and guidance assays, here we will determine if local translation occurs at point contacts in growth cones, and examine how RACK1 regulates point contact dynamics and axon growth and guidance. Taken together, this research will enable a broad, mechanistic understanding as to how mRNA trafficking and local translation contributes to the establishment of neuronal connectivity, thereby increasing our knowledge about the complex nature of brain development.