Neurons communicate with each other by transmitting electrical impulses down a long cellular process called the axon. The central question of this project is how does a neuron coordinate the synthesis of the many structural proteins that are needed to make an axon during development? This coordination had long been thought to occur as genes are first read out (i.e., during transcription), but new information indicates that much of it also happens afterward, as the proteins themselves are being synthesized. Loss of a single protein that regulates protein synthesis, called hnRNP K, leads selectively to the loss of not only one of the most abundant axonal structural proteins (a neurofilament protein) but also of the entire axon. Because loss of neurofilaments does not normally lead to loss of entire axons, hnRNP K is hypothesized to be a global regulator of the synthesis of multiple structural proteins that cooperate in building the axon. The first objective of this project is to use high throughput biochemical assays to identify these proteins. The second objective is to use mutated versions of hnRNP K that can be visualized in living neurons to study how the actions of hnRNP K are regulated by cell signaling pathways during axon outgrowth. The potential impact of this work is to provide new insights into how neurons coordinate the expressions of multiple, functionally interrelated proteins during development. This project will provide research training for two PhD students and undergraduates at a public institution with a culturally diverse student body. Spinoffs from this work will have an impact on undergraduate student laboratory exercises in Developmental Biology and on courses taught by the PI in Molecular Biology, Developmental Neurobiology, and Molecular Neurobiology.
Intellectual Merit: The long term goal of this research is to understand how neurons make an axon, which neurons use to send information in the form of action potentials and secreted chemicals to other neurons. By defining the circuitry of the brain and how rapidly information flows from one neuron to another, the length, branching, and diameter of the axon provide the structural underpinnings of sensation, movement, learning, and memory. These morphological parameters are ultimately governed through the axonal cytoskeleton composed of highly organized polymers of distinct proteins whose expression and transport are tightly regulated. Historically, studies of the cytoskeleton of developing axons have focused on how the dynamics of polymer composition, assembly, and transport direct outgrowth, branching, and caliber expansion, but have paid less attention to how axonal cytoskeletal composition is regulated. This regulation is nonetheless very important, since having either too little, too much, or the wrong mix of proteins leads to serious defects in neural circuitry and to neurodegeneration. Our laboratory studies this question in neurons of the South African claw-toed frog, Xenopus laevis. Xenopus is a good animal model to use because embryonic neurons are readily accessible and easily manipulated at even the earliest stages and because its central nervous system (CNS) axons successfully regenerate after injury. These characteristics allow developmental and regenerative axonal outgrowth to be compared to identify molecular mechanisms controlling axonal cytoskeletal composition in growing axons. Earlier, NSF-funded work established that the subunit composition of neurofilaments, the most abundant cytoskeletal polymer of vertebrate myelinated axons, is controlled at multiple checkpoints from gene to protein. Injured retinal neurons, for example, make more neurofilament RNA during regeneration than normal, but retain most of it within the nucleus. Subsequent export of the RNA and translation into protein in the cytoplasm fine tune the amount of neurofilaments supplied to the growing axon, and these later steps require a particular protein that specifically binds neurofilament RNAs, hnRNP K. Moreover, hnRNP K is also required for axon outgrowth itself. In exploring this phenomenon further, this project made several important discoveries about hnRNP K. First, it regulates nuclear export and translation of not just neurofilament RNAs, but also those of additional cytoskeletal-related proteins that collectively function to organize the axonal cytoskeletal polymers. Second, hnRNP K performs similarly in both embryonic and regenerative axon outgrowth. It is thus a master regulator of the synthesis of cytoskeletal protein used to make an axon. Third, phosphorylation of hnRNP K by the kinase, c-Jun N-terminal kinase, regulates the loading of hnRNP K and its associated RNAs onto the cellular machinery that directly synthesizes proteins, linking the synthesis of key axonal cytoskeletal proteins with a cell signaling pathway that is activated by extracellular cues that govern axonal outgrowth . Broader Impacts: This project contributed to the development of scientific resources by creating a database of RNAs that associate with hnRNP K in the brain and the development of a new technique to knockdown expression of specific proteins in retinal neurons of an intact eye. The project further contributed to the development of scientific infrastructure by directly supporting the education and training of six PhD students and four undergraduates. One PhD student recently became an assistant professor at Morehead State University in Kentucky, one is now a postdoc at Harvard Medical School, and a third is about to start a postdoc at CalTech; three others remain in the lab. One undergraduate is in medical school, and another a high school science teacher, while two others will return to the lab next year. Four PhD students and three undergraduates were women; two undergraduates were African-American. Thus, the project also served to broaden participation of under-represented groups in science. The project supported international outreach activities. I visited Huazhong University of Science and Technology (2011) and Shanghai Medical College (2013), where I discussed this research with faculty and students. Also, the lab hosted a visiting PhD student from Fudan University, who after completing her research on this project, returned to join the faculty there. Another international MS student in education interned in the laboratory to learn firsthand what happens in a science lab; she is now developing secondary school curricula in Wuxi, China. The project also enhanced undergraduate and graduate courses at our own University. Research from this project was discussed in two courses I teach in full – molecular biology and neurochemistry – and in another capstone course I participate in as part of our Neuroscience minor. The project also provided Xenopus embryos and exercises for undergraduate laboratory courses run by Christine Gervasi, who also manages our animal colony. Finally, this project supported participation by me and my students in our University’s RNA Institute, which facilitates research, training, and commercial development of applications in RNA science to contribute to the economic development of upstate New York.
