Randal Tibbetts - US grants

[unreadable] DESCRIPTION (provided by applicant): The major goals of this proposal are to delineate the structural motifs that are required for the checkpoint signaling functions of the ATM and Rad3-related protein, ATR and to elucidate the mechanisms of ATR recruitment to sites of DNA damage. ATR is a member of the phosphoinositide 3-kinase-related kinase gene superfamily that has been implicated as an essential regulator of mammalian DNA damage responses. ATR and the related protein ATM (ataxia-telangiectasia-mutated) are large molecular mass protein kinases that function atop signaling cascades that regulate cell cycle checkpoint activation, DNA repair, and transcriptional responses in the face of genetic damage. Recent advances have led to the identification of cellular substrates that are required for the checkpoint signaling functions of ATR and have pointed toward a critical role for ATR in the signaling of DNA damage incurred during S phase. Consistent with genome surveillance functions of ATR, recent results have shown that ATR targets to sites of DNA damage and/or stalled replication forks following cellular exposure to DNA damaging agents or DNA replication inhibitors. However, outside of its conserved carboxyl-terminal catalytic domain, the structural motifs that are required for the DNA damage-signaling functions of ATR have not been identified, nor have the underlying mechanisms of ATR regulation been elucidated. Within this context, we propose to: (1) delineate functional motifs that are required for the DNA replication checkpoint functions of ATR; (2) map and functionally characterize a nuclear foci-targeting domain in ATR; and (3) identify the cellular protein(s) that mediate the targeting of ATR to nuclear foci. The accomplishment of these objectives will be a strong first step toward understanding the mechanisms of ATR function and regulation. We hope that knowledge gained from these studies can be translated into a more fundamental understanding of how DNA damage is converted into cell regulatory cells, and ultimately, how genetic instability arises during tumor development.

DESCRIPTION (provided by applicant): The principle objective of this application is to discover novel disease pathways in a Drosophila melanogaster model of amyotrophic lateral sclerosis (ALS). Recently, dominant mutations in the RNA binding protein TDP-43 (43 kDa TAR DNA-binding protein) have been causally linked to ALS. In addition, ubiquitin-positive, insoluble aggregates of TDP-43 are frequently observed in degenerating motor neurons of ALS patients, suggesting that deregulation of TDP-43 through mutation or epigenetically is a precipitating event in this disease. We have generated a fruit-fly model of TDP-43 proteinopathy in which the expression of human TDP-43 in the motor neurons of flies leads to age- dependent paralysis and death. In this model, TDP-43 retains a nuclear expression pattern, suggesting that TDP-43 need not aggregate to cause motor neuron dysfunction. Based on these findings we hypothesized that TDP-43-dependent changes in nuclear gene expression are responsible for neurodegeneration. Consistent with this idea, gene expression analysis of TDP-43 transgenic flies identified deregulation of cellular pathways with plausible links to neurodegeneration. The goal of the present proposal is to use the Drosophila TDP-43 model to gain new insights into TDP-43-dependent neurodegeneration. The grant encompasses two specific aims. In Aim 1 we will perform microarray and RIP-Chip studies to identify TDP-43 RNA targets in motor neurons. In Aim 2 we will perform screens for genetic modifiers of TDP-43-dependent neurodegeneration, using both candidate and unbiased approaches. The combined studies should illuminate mechanisms of neurodegeneration in ALS and related proteinopathies that may lead to new therapeutic options. PUBLIC HEALTH RELEVANCE: ALS is an intractable condition that exerts severe tolls on affected patients, their families, and caregivers. Comprehensive approaches aimed at identifying the operative mechanisms in this disease are urgently needed. We believe that the fruit fly model of ALS to be explored in this proposal will provide important clues regarding ALS pathogenesis that may lead to new therapies.

? DESCRIPTION (provided by applicant): This study will explore the hypothesis that hyperactivation of the cAMP response element-binding protein (CREB)-a transcription factor with diverse functions in the central nervous system and metabolic regulation-contributes to mitochondrial defects and pathologic reactive oxygen species (ROS) formation in ataxia-telangiectasia (A-T), a neurodegenerative disease caused by mutations in the ATM gene. ATM encodes a protein kinase with instrumental roles in the signaling and repair of DNA double-strand breaks, a highly carcinogenic form of DNA damage. ATM is also thought to play an important role in mitochondrial homeostasis and suppression of toxic ROS; however this aspect of ATM function is poorly understood. In published work we showed that ATM phosphorylates CREB on a conserved cluster of Ser residues that attenuates CREB transactivation potential in response to DNA damage and other forms of cellular stress. Of importance to this proposal, an independent study recently showed that the nuclear corepressor NCoR1 represses a large number of CREB target genes with mitochondrial function. Here we will explore the idea that ATM, CREB, and NCoR1 function in a common pathway to critically attenuate mitochondrial function and ROS generation in response to DNA damage and oxidative stress. Specifically, we propose that ATM-mediated phosphorylation of CREB recruits NCoR1 to silence mitochondrial target genes. The relevance of this hypothesis for A-T is that defective CREB phosphorylation may engender mitochondrial defects and oxidative stress that contribute to neuronal injury. We will test these ideas using a combination of biochemical and genetic approaches, including the use of gene-targeted mice expressing a mutant CREB allele (CREBS111A) refractory to phosphorylation by ATM. CREBS111A mice exhibit metabolic abnormalities and alterations in CREB- mediated gene expression, and fibroblasts and neurons from these mice will be used to explore the mechanisms of NCoR1-dependent CREB attenuation. In summary, the proposed work will define the impact of ATM-mediated CREB phosphorylation on transcriptional regulation and mitochondrial homeostasis. Results from this work may provide important new insights into how loss of ATM leads pathologic oxidative stress in A-T. The Specific Aims of the proposal are to: i) Assess ROS, mitochondrial dynamics, and cerebellar gene expression in CREBS111A mice; and ii) Define signal and phosphorylation-dependent functional relationships between CREB and NCoR1.

Project Summary The goal of this R21 project is to test whether enhancement of endogenous CREB (cAMP response element binding protein) signaling ameliorates disease phenotypes in a mouse model of the autism spectrum disorder, Rett syndrome (RTT). CREB is an evolutionarily conserved transcription factor that executes critical roles in metabolism, neuronal synaptic transmission, and cell growth regulation. Upregulation of CREB signaling has been linked to cancer and metabolic disease whereas reductions in CREB signaling are associated with age- dependent cognitive decline and a host of neurodegenerative disorders, including Alzheimer?s Disease, Huntington?s Disease and, of particular relevance to this proposal, RTT. CREB is activated by the second messenger cAMP through a two-hit mechanism involving its phosphorylation on S133 by protein kinase A (PKA), which recruits the transcriptional coactivator CREB-binding protein (CBP), and PKA-dependent nuclear import of CRTC proteins (cAMP/Ca2+-regulated transcriptional coactivators), which stabilize CREB-DNA interactions. We recently discovered that the critical S133 residue is dephosphorylated by protein phosphatase 2A (PP2A), which is recruited to CREB through short linear motifs (SLiMs) that are recognized by B56-type PP2A targeting subunits. Mutation of B56 binding sites in CREB strongly potentiated basal and stimulus dependent S133 phosphorylation and CREB transcriptional potential, informing a strategy for the genetic enhancement of CREB signaling in vivo. To this end, we used CRISPR/CAS9 to introduce a conservative E153D mutation that abolished B56-PP2A binding into the mouse Creb gene. Cells from homozygous CrebE153D mice exhibited increased S133 phosphorylation and upregulation of CREB-dependent gene expression, supporting further study of CrebE153D mice as a model for hypermorphic CREB signaling. In this study we will test whether CREB hyperactivation can reverse behavioral defects in a mouse model of RTT, a devastating neurodevelopmental disordered caused by X-linked mutations in the transcriptional repressor methyl-CpG binding protein (MeCP2). Previous work from the Chang laboratory revealed that CREB expression and S133 phosphorylation were downregulated in Mecp2- mutant neurons and that pharmacologic activators of CREB signaling partially reversed behavioral defects in Mecp2+/- mice. These findings set the stage for this proposal where we will use CrebE153D mice to test whether enhancement of endogenous CREB activity is sufficient for behavioral rescue in the RTT mouse model. The objectives of the proposal are to: (i) test the effect of CREB hyperphosphorylation on disease progression in male and female Mecp2 knockout (KO) mice; and (ii) determine impacts of B56-PP2A-CREB signaling on neuronal gene expression. In addition to testing genetic interaction between Creb and Mecp2, these studies, will define physiologic implications of the PP2A-B56-CREB signaling axis and develop the CrebE153D model as a tool for manipulating endogenous CREB signaling in other physiologic paradigms.