Susan L. Ackerman - US grants

? DESCRIPTION (provided by applicant): Neurodegenerative disorders affect many millions of people around the world, particularly in the aging population. The vast majority of these diseases are not familial and the mutations that have been associated with rare familial forms of these disorders underscore the complexity of this group of diseases. To begin to understand this complexity, we have used forward genetic approaches to pinpoint the molecular pathways that maintain neuronal homeostasis in the aging mammalian brain. Using this approach we recently demonstrated that unresolved ribosome stalling is a novel mechanism for neurodegeneration. Despite the fundamental importance of translation, the cellular consequences of ribosome stalling in mammalian cells had been unknown until our discovery that a mutation in a novel mammalian ribosome rescue factor Gtpbp2 causes ataxia and degeneration of cerebellar granule cells, cortical and hippocampal neurons, and multiple retinal neurons. Importantly we demonstrated that loss of Gtpbp2 epistatically interacts with a mutation in a CNS- specific, cytoplasmic tRNAArgUCU in the widely used C57BL6/J (B6J) mouse strain to cause neurodegeneration. Our ribosome footprinting experiments revealed that loss of this tRNA led to low levels of ribosome stalling at Arginine AGA codons that was not associated with neurodegeneration. However, stalling was dramatically increased in the absence of Gtpbp2, demonstrating that this protein normally resolves ribosomal stalls. In this application we propose to determine the function of these and other ribosome rescue factors in neuron survival, the impact of increasing age on ribosome stalling in the brain, and additional molecular mechanisms which cause ribosome stalling in mammalian neurons. In Aim 1 we will determine the effects of loss of the ribosome rescue factors Gtpbp1, Hbs1l, and Pelo with- and without- tRNA deficiency. These studies will be complemented by novel computational methods to infer ribosomal locations at increased precision and ascertain mechanisms that distinguish strains using parameter-dependent simulations of the translation process. In Aim 2 we will determine the effects of aging on ribosome stalling and neurodegeneration in the brains of aged wild type and ribosome rescue mutant mice without the tRNA mutation and generate and analyze ribosome footprinting and RNA-Seq data from cerebella of aged mice. In Aim 3 we will investigate pathways that lead to cell death and determine their uniqueness for neurons. We will determine if deficiency of ubiquitously expressed tRNAs induces ribosome stalling and pathology in other organs, analyze the effects of the GCN2/ATF4 and P53 pathways on neurodegeneration, and identify additional modifier genes of neurodegeneration in Gtpbp2-/- mice. Together, we expect these studies to reveal the mechanisms by which dysregulation of translation elongation leads to cellular death and their specificity for neurodegenerative disease.

? DESCRIPTION (provided by applicant): A mutagenesis screen to develop mouse models for neurological diseases led to our recent finding that neurodegeneration could arise from an epistatic mutation affecting transfer RNA (tRNA) biogenesis and function. Loss of function of GTPBPP2, a protein that we showed resolves stalled ribosomes caused by insufficiency of a tRNA, results in a severe phenotype only when present together with a single C50U mutation in a central nervous system (CNS)-specific tRNAArgUCU, n-Tr20. Mutation of this tRNA, which constituted almost 60% of the brain tRNAArgUCU pool, caused a dramatic decrease in processing and thus aminoacylation, and led to ribosomal pausing at AGA codons on cerebellar transcripts. Without GTPBP2 to recycle stalled ribosomes, disease developed with pronounced locomotor defects and neuron loss in the cerebellum beginning at four weeks of age. Neurons also degenerated in other brain regions beginning at 6 weeks of age. While the brain- specificity of phenotypes is consistent with the expression pattern of tRNAArgUCU, there were notable differences in the processing of the C50U tRNAArgUCU depending on the region (cerebellum < cortex, hippocampus; postnatal day 30 < day 0 or 10). Therefore, we postulate that tissue- or development-specific distinctions in the inventory of enzymes that acts on tRNAs are key determinants for disease etiology. It is in this regard that we focus on RNase P, which catalyzes removal of the 5?? leader of pre-tRNAs. Although mammalian liver RNase P functions as a ribonucleoprotein (RNP) consisting of a ribozyme and 10 protein cofactors, there is growing evidence that RNase P may be subject to remodeling especially in the brain. Thus, altered substrate recognition could stem from structural alterations either in the pre-tRNA substrate or in RNase P. These findings provide the framework for our hypotheses: (i) the C50U tRNAArgUCU mutant has a destabilized structure that disables 5?? processing by RNase P, and (ii) spatiotemporal variations in brain RNase P make-up engender defects in processing pre-tRNAs with mutations. We will test these ideas by pursuing two aims. First, we will test the activity of partially purified mouse cerebellar RNase P towards wild-type (WT) and C50U pre- tRNAArgUCU. We will also use chemical/enzymatic probing and high-resolution structural studies to map structural differences between the WT and mutant pre-tRNAArgUCU. Second, we will perform expression analysis and RNP affinity purification to investigate the make-up of brain RNase P from different regions at specific developmental stages. Collectively, our studies will lead to the first biochemical characterization of mammalian brain RNase P and test the novel idea that spatiotemporal variations in the catalytic repertoire that acts on brain tRNAs as well as other non-coding RNAs partly hold the key to delineating how defects in RNA metabolism might result in neurological disorders.

Project Summary/Abstract This proposal requests partial support for the meeting ?Translation Machinery in Health & Disease ? from Biology to Medicine? as a part of the Gordon Research Conference (GRC) series in conjunction with Gordon Research Seminar (GRS) that will be held in Hotel Galvez, Galveston, Texas, during March 18-24, 2017. To our knowledge, this GRC/GRS meeting is the first and only conference linking researchers in diverse fields of the translation process to experts in clinical sector. The purpose of this conference is to provide a platform in which researchers and clinicians can exchange and obtain state-of-the-art information on the relevance of diverse translational processes to human disease and health, and to open doors for the development of novel treatments and therapies. This conference will deal with subjects that are common interests for both clinicians and basic scientists including cancer, infectious and immunological diseases, hematological and vascular diseases, neurological diseases, and metabolic diseases. In addition it will also include talks on newly emerging technologies for translation research. The associated GRS will provide early career scientists (both graduate students and post-doctoral fellows) with opportunities to present their research on translation and disease and network with experts this field. This meeting will convene 40 leading experts who cover critical basic science and clinical topics in translation with a total of 200 participants for seven days. The GRS meeting is planned to coordinate with the GRC with four sessions including an opening keynote lecture (Dr. Christine Mayr at Sloan-Kettering Cancer Center). Subsequently, the GRC meeting will be organized into nine sessions including an opening session featuring two keynote lectures, followed by eight sessions, each of which will focus on a specific disease area. The keynote lecture will be given by Dr. Nahum Sonenberg who is one of the most renowned scientists for his seminal contributions to our understanding of translation, and notable for the discovery of the mRNA 5' cap-binding protein, eIF4E, the rate-limiting component of the eukaryotic translation apparatus. He will address the functional relevance of eIF4E to cancer and other diseases. In addition, five afternoon poster sessions including one during the GRS meeting will allow all participants to present their research. Following the spirit of Gordon Conferences, this meeting will seek for every possibility to provide young investigators, women, and minorities with the opportunities to present their research and interact with senior key leaders in the field. As the first and only disease-focused conference in the field of translation, this conference would significantly impact our current understanding of many important diseases?such as cancer, neurodegeneration, diabetes, anemia, and viral and bacterial infections?and their treatment.

Partial support is requested for the Translation Machinery in Health and Disease-Protein Synthesis and Pathway Integration Gordon Research Seminar and Gordon Research Conference to be held in Galveston, Texas on February 17-22, 2019. This biennial conference was inaugurated in 2015 and is the only meeting on translation per se in the GRC series. Furthermore, it is the first, and to our knowledge, the only, disease- focused, interdisciplinary meeting on mRNA translation. Unlike other conferences that mainly focus on the structure of the translational machinery and its function in protein synthesis, this focus of this meeting is on the role of translation in human disease and the pathological consequences of alterations in this process. This unique conference will provide a forum for scientists to discuss their findings with an ultimate long term goal of building collaborations toward understanding, and finding treatments, for disease. The specific aims of the Gordon Research Conference is to convene 52 speakers and an additional 125-150 participants who are experts in the fields of cancer, neurological disease, inflammation, infectious, and immunological diseases, hematological diseases, mitochondrial diseases and metabolic diseases to discuss basic science and therapeutic interventions. The accompanying Gordon Research Seminar will provide both a forum for scientists in early stages of their career to present their data on translation and disease and network working activity. Dr. Rachel Green, an expert in ribosome structure and function will give the keynote lecture at the Gordon Research Seminar and Dr. Aaron Gitler, who investigates translation dysfunction and neurodegeneration, will give the keynote at the Gordon Research Seminar. In addition to invited speakers and speakers chosen from the abstracts, four poster sessions will allow all participants to contribute to the meeting. Numerous opportunities for your investigators, women, and minorities to interact with senior leaders in the field including a GRC-sponsored ?power hour? which will provide a platform to discuss challenges that women and other underrepresented minorities face in science. As the first and only conference on translation and disease, this conference will significantly impact the participant's understanding of disease mechanisms.

PROJECT SUMMARY/ABSTRACT Several forms of neurodegeneration have been linked to perturbations in RNA homeostasis. In the case of pontocerebellar hypoplasia type 10 (PCH10), neurodegeneration is due to homozygous mutation of the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit 1), which phosphorylates the 5?OH of RNA. PCH10 is a rare pediatric disorder resulting in profound cognitive impairment, motor dysfunction, and epilepsy as a result of hypoplasia and/or atrophy of the brain and spinal cord. The causative mutation in CLP1 (R140H) leads to partial loss of function; however, why this results in neurodegeneration is unclear. In vitro experiments suggest that CLP1 may play roles in mRNA cleavage, tRNA splicing, and miRNA activation; however, at present, there is not enough evidence to conclude that it performs any of these functions in vivo. In order to discover the functions of CLP1 implicated in PCH10, we have generated a knock-in mouse line with the patient mutation that displays mild motor defects. We have also generated a Clp1 null allele which, in combination with the patient mutation allele, produces a severe motor phenotype in Clp1R140H/- mice. Our preliminary data demonstrates that Clp1R140H/- mice have degeneration of lower motor neurons. This proposal expands upon this finding in two ways. In Aim 1, we propose to extend our analysis of the Clp1R140H/- phenotype to the brain and sensory neurons. This will clarify which subtypes of neurons are sensitive to Clp1 mutation. We will first perform histological analyses to identify any gross abnormalities, and then focus specifically on cells and brain regions affected in patients. In Aim 2, we will examine the molecular phenotype associated with Clp1 mutation using FACS-sorted motor neurons from mutant and wild-type mice to make libraries for RNA profiling. CLP1 is a member of the mRNA polyadenylation and cleavage complex. Thus we will investigate the role of CLP1 in mRNA cleavage by generating 3? end mRNA libraries. This will indicate whether loss of CLP1 alters mRNA cleavage and whether this occurs in a sequence-specific or -independent manner. Second, we will use a set of miRNA libraries to determine whether CLP1 phosphorylates miRNAs in motor neurons. While most miRNAs are thought to be constitutively active, it has been demonstrated that one miRNA is maintained in a dephosphorylated state until application of a stimulus prompts 5? phosphorylation by CLP1. In conclusion, by exploring the functions of CLP1 in mRNA and miRNA processing in motor neurons in a mouse model of PCH10, we will elucidate the mechanism(s) by which Clp1 mutations lead to neurodegeneration.