Characterizing the regulatory roles of microRNA on mitochondria in the mammalian CNS

PROJECT SUMMARY/ABSTRACT Neuronal tissue is the most energy consumptive tissue type of the entire body, and as such, depends heavily on the health and proper regulation of its mitochondria, its main source of energy. Thus, mitochondrial dysfunction is linked to a number of neuropsychiatric diseases, including the neurodevelopmental disorder Fragile X Syndrome (FXS). FXS is characterized by loss of the Fragile X Messenger Ribonucleoprotein (FMRP), an RNA- binding protein that has been shown to interact with microRNAs (miRNAs) and components of the RNA-induced silencing complex (RISC). Interestingly, using an optimized Ago-CLIP method that unambiguously identifies miRNA:target RNA interactions within the RISC, our lab has identified prevalent binding of miRNAs to mitochondrial transcripts (mt-RNA) in the mammalian forebrain. Though mitochondrial dysfunction in FXS has been identified, a connection with RISC and targeting of mt-RNA has not yet been explored. This proposal seeks to further characterize the molecular underpinnings of these observed miRNA:mt-RNA interactions, as well as their impact in both health and disease (FXS). Specific Aim 1 proposes to use the same optimized Ago-CLIP method, termed CIMERAseq, to determine the subcellular compartment in which the miRNA:mt-RNA interactions are occurring, as well as use a targeted approach to identify cell-type specificity of the transcript interactions. Specific Aim 2 explores the possible functional effect of the observed binding by altering levels of select miRNAs identified in our data and recording the effect in three ways: i) transcript abundance (via sequencing and qPCR) ii) protein abundance (via Western blot and BN-PAGE), and iii) mitochondrial function (via assessment of respiratory function, ATP synthesis, and reactive oxygen species (ROS) production). Aim 2 also uses the same effect readouts to investigate mitochondrial perturbations in a mouse model of FXS (Fmr1 KO). If successful, the proposed specific aims will aid in understanding the role of miRNAs as mitochondrial gene regulators in the mammalian forebrain, both in health and disease. In addition to completion of the proposed aims, this proposal outlines a training plan formulated specifically for my long-term goal of becoming a well- rounded independent scientist. Training received through the course of this fellowship will focus on enhancing my skills in molecular biology methods, bioinformatic methods, and scientific communication and writing. My sponsor and university, the Johns Hopkins University School of Medicine, are outstandingly equipped to not only allow for completion of the proposed experiments but also to provide the resources needed to receive exceptional training in the areas described. The Biochemistry, Cellular and Molecular Biology (BCMB) program in which I am enrolled is dedicated to providing an exceptional training environment and will support completion of the research plan as well as career training opportunities. This fellowship will support not only impactful research toward understanding miRNA and mitochondria in FXS pathology, but also my development into a capable and independent neuroscientist.