Mollie K. Meffert, M.D./Ph.D. - US grants

DESCRIPTION (Adapted From The Applicant's Abstract): Neuronal apopotosis and degeneration is common to a wide range of severe diseases in the nervous system including traumatic brain injury, focal ischemia, Parkinsons's disease and Alzheimers disease. In addition to the control of basic physiological processes, the NF-kB family of transcription factors have emerged as major regulators of cell life and death in many systems. Since their recent discovery in,the nervous system, both pro- and anti-apoptotic actions have been attributed to NF-kB. Our proposal will address the possibility that different mechanisms of neuronal stimulation may result in differences in the time course, degree, and subunit composition of activated neuronal NF-kB. These differences, in turn, could explain contrasting physiologic functions of neuronal NFkB in disparate settings and might lead to the development of strategies to therapeutically regulate NF-kB activation. We have selected the hippocampus as our model system for studying neuronal NF-kB because it is a well-defined area of both physiological (learning and memory) as well as pathological (stroke, Alzheimers disease) neuronal function. Our studies will use hippocampal tissue from both adult mice and neonates as well as several transgenic and knockout lines. We will identify the NF-kB family members present in hippocampal neurons and define the types of stimulation which lead to their activation. We will investigate representative neurotransmitters, neurotrophins, and cell adhesion molecules for their ability to activate neuronal NF-kB using electromobility shift assays and a kB-reporter construct. In addition, we will use a fluorescently labeled NF-kB subunit to examine the subcellular localization of NF-kB and determine its ability to translocate to the nucleus from neuronal processes following different stimulation parameters. We will examine if levels of synaptic activity within the normal physiological range functionally regulate NFkB, or if significant activation occurs only in response to stressful stimuli, Can the pattern of NFkB activation encode information on the physiological versus toxic nature of a stimulus? Electrical stimulation will be used to assess the pattern of NFkB activation and kBdependent gene expression in response to stimuli of precisely varied intensity, duration, and frequency. Our investigations will contribute new knowledge by specifically examining the roles of NF-kB family members in the regulation of neuronal gene expression by physiological or pathophysiological stimuli. As an M.D.- Ph.D. specializing in the neurosciences, I hope to have a career as an independent investigator studying transcriptional regulation and the pathogenesis of central nervous system disease.

DESCRIPTION (provided by applicant): Healthy cognitive function depends upon the correct regulation of specific targeted genes in response to incoming stimuli. Brain-derived neurotrophic factor (BDNF) is a neurotrophin with well-established roles in neuronal survival, differentiation, and synaptic plasticity. BDNF can regulate gene expression at the levels of both transcription and translation. Several functions of BDNF, including dendrite outgrowth and long-term synaptic plasticity, are known to explicitly depend upon the ability of BDNF to regulate protein synthesis. BDNF modestly increases total neuronal protein synthesis by enhancing the activity of translation initiation and elongation factors to globally induce the protein synthesis machinery. However, BDNF demonstrates an extraordinary degree of transcript specificity and strongly upregulates the translation of a small percentage of targets, while leaving some targets unaffected and downregulating the translation of others. This striking transcript selectivity is critical to the control of neuronal protein composition by BDNF and its role as a trophic factor. We recently delineated a pathway by which BDNF controls specificity in protein synthesis through both positively and negatively regulating the biogenesis of mature miRNAs to determine whether specific gene transcripts are repressed or undergo enhanced translation. The focus of this proposal is to examine the molecular mechanisms by which BDNF controls miRNA biogenesis, the spatial and temporal aspects of this regulation, and the role of these novel pathway components in cognitive function. Results from our investigations wil reveal previously unknown mechanisms controlling the specificity of gene expression and offer potential new therapeutic targets for the treatment of brain disorders with particular relevance to processes, such as Autism, Fragile X syndrome, depression, and neurodegenerative disease, with known links to BDNF, dysregulated translation, or both.

Neuropathic pain is a poorly treated medical condition of enormous public health importance. One mechanism by which nerve injury leads to neuropathic pain is by altering the expression of numerous proteins that regulate neuronal excitability. MicroRNAs (miRNAs) are key modulators of protein synthesis, and nerve injury alters the neuronal expression of many individual miRNAs. miRNAs function by reducing the stability and/or translation of target messenger RNAs (mRNAs) that contain binding sequences with partial complementarity to the miRNAs. A single miRNA can thereby repress the synthesis of many proteins. However, miRNAs themselves are coordinately regulated by upstream control pathways. One such pathway involves Lin28a and Lin28b, RNA binding proteins that selectively and coordinately suppress the biogenesis of the Let-7 family of miRNAs, which are amongst the most abundant miRNAs in differentiated tissues. Since many Let-7 miRNA targets encode proteins involved in growth and regeneration, increased Lin28 signaling promotes pro-growth programs of protein synthesis. Indeed, Let-7 miRNAs have been implicated as possible regulators of axon growth and nerve regeneration. Yet, the directions and cell type specificity of these effects remain unclear, as does the potential involvement of Lin28. Furthermore, the possibility that the Lin28/Let-7 pathway contributes to the severity or duration of neuropathic pain remains entirely unexplored. Here, we outline plans to directly tackle these questions, through a multidisciplinary approach. In Aim 1, we will map the spatiotemporal patterns and cell type specificity of changes in the expression of Let-7 miRNAs and Lin28 in three complementary mouse models of neuropathic pain. In Aim 2, we will utilize mouse genetics and adeno- associated virus mediated gene transfer, along with behavioral and in vivo electrophysiological approaches, to increase or ablate Let-7 miRNAs or Lin28 protein expression selectively in peripheral neurons or glia, to assess the functional importance of the Lin28/Let-7 pathway to the initiation, maintenance, and reversal of neuropathic pain. Finally, in Aim 3, we will combine bioinformatics and proteomic approaches to define the programs of altered protein expression following nerve injury that depend upon the Lin28/Let-7 pathway, and that might underlie the contributions of this pathway to neuropathic pain. Together, these studies will help to define the roles of the Lin28/Let-7 pathway in neuropathic pain and provide important information regarding when, where, and in what cell type this master regulatory pathway might be therapeutically targeted to alleviate pain.