Karl Obrietan, Phd - US grants

DESCRIPTION (Adapted from applicant's abstract): The circadian organization of behavior plays a critical role in an organisms's response to social and light/dark cycles encountered on a daily and seasonal basis. Alterations in the normal function of the mammalian biological clock, located in the suprachiasmatic nucleus (SCN), leads to a variety of neurological abnormalities including sleep disorders, depression, and mental fatigue. A feedback regulator of circadian rhythmicity is the neurohormone melatonin. Changes in environmental light information received by the SCN are converted into the nocturnal synthesis and release of melatonin from the pineal gland. An important regulatory of melatonin synthesis and circadian clock function is cAMP. cAMP regulates the transcription of several proteins critical for the circadian expression of melatonin, including serotonin N-acetyltransferase (NAT), which is responsible for catalyzing the synthesis of melatonin from serotonin. Additionally, cAMP may also play an important role in circadian timekeeping in the SCN by regulating gene transcription. By integrating different intracellular signal transduction pathways, the Ca2+/calmodulin-sensitive adenylyl cyclases may play an important role regulating circadian rhythmicity. With the two unique tools developed in the laboratory of Dr. Storm (mutant mice lacking Ca2+/calmodulin-sensitive adenylyl cyclases and a CRE-lacZ transgenic mouse strain), I propose to elucidate the roles Ca2+/calmodulin-sensitive adenylyl cyclases play in melatonin synthesis and in the modulation of SCN circadian rhythmicity and to determine whether CRE-mediated transcription in the SCN and pineal gland is regulated in a circadian manner.

DESCRIPTION (Adapted from applicant's abstract): The circadian organization of behavior plays a critical role in an organisms's response to social and light/dark cycles encountered on a daily and seasonal basis. Alterations in the normal function of the mammalian biological clock, located in the suprachiasmatic nucleus (SCN), leads to a variety of neurological abnormalities including sleep disorders, depression, and mental fatigue. A feedback regulator of circadian rhythmicity is the neurohormone melatonin. Changes in environmental light information received by the SCN are converted into the nocturnal synthesis and release of melatonin from the pineal gland. An important regulatory of melatonin synthesis and circadian clock function is cAMP. cAMP regulates the transcription of several proteins critical for the circadian expression of melatonin, including serotonin N-acetyltransferase (NAT), which is responsible for catalyzing the synthesis of melatonin from serotonin. Additionally, cAMP may also play an important role in circadian timekeeping in the SCN by regulating gene transcription. By integrating different intracellular signal transduction pathways, the Ca2+/calmodulin-sensitive adenylyl cyclases may play an important role regulating circadian rhythmicity. With the two unique tools developed in the laboratory of Dr. Storm (mutant mice lacking Ca2+/calmodulin-sensitive adenylyl cyclases and a CRE-lacZ transgenic mouse strain), I propose to elucidate the roles Ca2+/calmodulin-sensitive adenylyl cyclases play in melatonin synthesis and in the modulation of SCN circadian rhythmicity and to determine whether CRE-mediated transcription in the SCN and pineal gland is regulated in a circadian manner.

Lay abstract

Daily, or circadian, rhythms of biochemistry, physiology and behavior are observed in a wide variety of organisms. In mammals, the capacity to generate an endogenous circadian rhythm is dependent upon a discrete ensemble of neurons referred to as the suprachiasmatic nuclei (SCN). The SCN, located deep within the brain, form a "biological clock" that is entrainable to external stimuli, such as light. Over the past several years, advances at the molecular and genetic level have revealed that SCN rhythmicity results from autoregulatory feedback loops of so-called "clock genes". Through as yet unidentified molecular events, these feedback loops couple to output pathways that in turn initiate circadian rhythms of physiology and behavior. Importantly, external stimuli, such as light, are thought to entrain the circadian clock by resetting these loops. The main goal of this study is to determine the cellular signaling events that mediate rhythmic gene expression in the SCN. Towards this end, mouse SCN tissue will be utilized to 1) identify intracellular signaling pathways activated by light, 2) determine how light-activated pathways affect rhythmic expression of core clock genes, and 3) determine how genetic feedback loops initiate events that drive output from the clock.

An understanding of the molecular events that drive SCN rhythmicity should provide new insights into how circadian-controlled behaviors, such as the sleep-wake cycle, are regulated. Furthermore, given the commonality of signaling events thought to mediate long-term adaptive changes in nervous system physiology, the data collected and approaches used in this study should be of relevance to neurobiologists examining a wide array of behavioral processes.

DESCRIPTION (provided by applicant): In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus function as the major biological clock. SCN-dependent rhythms of physiology and behavior are regulated by changes in the environmental light cycle. Recent work has revealed that a program of rhythmic transcriptional regulation is required for endogenous SCN timekeeping and that light-induced changes in circadian timing result from alterations in this transcriptional program. Given the transcriptional basis of circadian rhythm generation, a characterization of the intracellular signaling pathways and downstream transcription factors involved in biological timing will be critical for understanding the functional properties of the circadian clock. Our preliminary studies reveal that photic stimulation and endogenous pacemaker activity regulate the activation state of the p42/44 mitogen-activated protein kinase (MAPK) signal transduction pathway in the SCN. The MAPK signal transduction pathway is a potent regulator of numerous classes of transcription factors and has been shown to play a role in certain forms of neuronal plasticity. These observations lead us to hypothesize that the MAPK pathway couples photic input to clock entrainment and that signaling via the MAPK pathway functions as an output pathway from the clock. In Aim 1 we address whether the MAPK signaling pathway is required for endogenous clock timing. We will also investigate the expression of circadian-regulated genes after disruption of MAPK signaling and identify transcription factors regulated by the MAPK signaling pathway. In Aim 2 we investigate whether MAPK signaling couples photic stimulation to phase shifting of the circadian clock. We will also investigate whether the MAPK pathway couples photic stimulation to transcriptional activation in the SCN. In Aim 3 we investigate cellular mechanisms that activate and inactivate the MAPK pathway in the SCN. Identification of the signaling and transcriptional pathways that regulate SCN rhythm generation and light-entrainment of the clock will provide new targets for therapeutic treatment of circadian-related ailments.

DESCRIPTION (provided by applicant): Seizure-induced alterations in synaptic architecture may be an underlying mechanism in the development of some forms of epilepsy. Of particular prominence is the remodeling of dentate gyms mossy fiber connections in patients with temporal lobe epilepsy. What are the signaling events elicited by excessive excitatory neurotransmission that trigger synaptic reorganization in the dentate gyrus? Although the cellular events that underlie this process are not well characterized, the similarities in the paradigms used to produce mossy fiber sprouting (and recurrent seizures) and those used to produce long-term neuronal plasticity raise the possibility that the same set of intracellular signaling pathways underlie these distinct physiological processes. Thus, we propose to examine whether signaling via the CREB/CRE transcriptional pathway couples temporal lobe seizures to mossy fiber sprouting. Interest in this plasticity-associated transcriptional pathway also comes from our preliminary data showing that seizures trigger activation of the CREB/CRE pathway, and that over-expression of activated CREB leads to robust neurite outgrowth. Thus, we hypothesize that seizures trigger CREB/CRE pathway activation, which in turn drives the expression of genes responsible for mossy fiber sprouting. In Aim 1 will determine the temporal profile of seizure-induced CREB activation and CRE-mediated transcription in the dentate gyms. Activation will be monitored from seizure onset, through the silent period, and on into the period of recurrent seizures. The role of modulatory transcription factors and upstream kinases will also be examined. In Aim 2 we will investigate the causal relationship between CRE-dependent transcription and mossy fiber sprouting. In Aim 3 we will examine the expression pattern of CREB-regulated cell survival and plasticity genes and examine the role of CREB as a regulator of seizure induced neuronal precursor cell differentiation. We will also determine whether cognitive deficits resulting from status epilepticus are associated with aberrant regulation of the CREB/CRE transcriptional pathway. An understanding of the intracellular signaling events that couple seizures to synaptic remodeling should allow for the development of therapeutic approaches designed to block the development of some forms of epilepsy.

DESCRIPTION (provided by applicant): In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus function as the major biological clock. Recent work has revealed that a program of rhythmic transcriptional regulation is required for endogenous SCN timekeeping and that light-induced changes in circadian timing result from alterations in this transcriptional program. Given this central role of inducible gene expression, a characterization of the intracellular signaling pathways and downstream transcription factors involved in biological timing and entrainment is critical for understanding the functional properties of the circadian clock. In our prior round of funding we collected a wealth of data supporting the hypothesis that the MARK pathway couples photic input to the clock timing process. What is lacking is an understanding of the precise routes by which the MARK pathway regulates the clock. We hypothesize that the MARK signaling cascade regulates timing at both a transcriptional and translational level, and we have assembled an array of novel tools to test this hypothesis. In Aim 1 we will determine whether the MARK pathway-regulated kinases MSK1/2 and RSK2 function as cellular signaling intermediates that couple light to clock entrainment. Our published reports reveal that both kinases are activated by light in the SCN, and preliminary data indicate that MSK1 and RSK2 regulate clock entrainment. Behavioral studies will be complimented by studies that examine chromatin structure and gene expression in MSK and RSK null mice. In Aim 2 we investigate the role of the CREB/CRE- transcription pathway (a target of the MARK pathway) in clock entrainment and clock rhythm amplitude. The CREB/CRE pathway has been implicated as a principal route by which entrainment cues impinge upon the core transcriptional timing process. However, there has yet to be an effective set of tools to test this idea in vivo. To this end, we have developed a novel set of CREB repressor and CREB activator transgenic mice that will be used to examine this question at a behavioral, biochemical and genetic level. In Aim 3, we investigate the role of the MARK pathway as a regulator of light- and clock- dependent mRNA translation in the SCN. Given recent work implicating dysregulation of circadian timing/entrainment in disorders such as obesity, cardiovascular disease and cancer, there is a clear need to identify the intracellular signaling events that play a fundamental role in pacemaker entrainment.

The goal of this proposal is to elucidate the role of the p42/44 mitogen-activated protein kinase (MAPK) pathway as a regulator excitotoxic cell death and aberrant structural remodeling in the hippocampus. Traumatic brain injury-induced cell death and pathophysiological alterations in synaptic architecture are likely to be underlying events leading to profound, long-term, mental disability. Importantly, there is a fundamental unresolved question regarding the signaling pathway(s) that regulate brain injury-induced cell death and structural remodeling. Based on recent work by others, our published findings, and the preliminary data reported here, we propose that the MAPK pathway is both neuroprotective and couples excitotoxic stimuli to structural plasticity. To both test these hypotheses and begin to identify potential therapeutic approaches to target MAPK signaling, we have assembled a novel set of transgenic mice and an array of screening assays. In Aim 1, we will examine the role of the MAPK pathway as a regulator of cell viability. Importantly, the precise contribution of MAPK signaling to neuronal survival in vivo is not known. Along these lines, a number of in vitro studies have reported that MAPK signaling can either contribute to or attenuate neuronal death, depending on the experimental paradigm. In this aim, we will characterize the temporal and cell-type specific expression of status epilepticus-(SE) induced MAPK pathway activation in the hippocampus, and then determine whether MAPK signaling confers protection against SE-induced cell death. We will also test potential molecular mechanisms by which MAPK signaling modulates cell viability. In Aim 2, we will determine whether MAPK signaling couples excitotoxic stress to aberrant structural plasticity. A good deal of work has implicated the MAPK pathway as a regulator of developmentally-dependent dendrite and axon growth, however, the role of the MAPK pathway in pathophysiologically-induced structural remodeling has not been rigorously addressed. Given its robust reorganization, emphasis will be placed on the granule cell layer of the dentate gyrus. Our research will provide insights into the potential therapeutic value of targeting MAPK Signaling to avert traumatic brain injury cell-death and aberrant structural plasticity.

DESCRIPTION (provided by applicant): The chromosome abnormality in Down syndrome (DS) results from a triplication in a portion of human chromosome 21 (Hsa21), but how this chromosomal anomaly causes the DS phenotype is not clear. The current proposal will directly address this issue, with an emphasis on a novel class of endogenous gene regulators, microRNAs (miRNAs). MiRNAs are generally regarded as negative regulators of gene expression that inhibit translation and/or promote messenger RNA (mRNA) degradation by base-pairing to complementary sequences within protein-coding mRNA transcripts. Our recent bioinformatic analyses established that Hsa21 harbors five miRNA genes. Importantly, miRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments demonstrated that all five Hsa21-derived miRNAs are over-expressed in brain and heart specimens from individuals with DS. We now hypothesize that the over-expression of the five Hsa21-derived miRNAs results in the under-expression of a number of important protein targets which contribute, in part, to the DS phenotype. Bioinformatic analyses demonstrated that several thousand proteins may be regulated by these miRNAs. Because combinatorial targeting of multiple miRNAs with a single mRNA may lead to a more pronounced down-regulation relative to mRNAs targeted by a few miRNAs, all of the Hsa21-derived miRNA/mRNA pairs were re-analyzed for the presence of multiple Hsa21-derived miRNA binding sites. This list of candidate targets was subsequently prioritized with respect to the potential clinical relevance of an individual target gene in playing a role in DS. Based on these criteria, we chose to investigate the methyl-CpG-binding protein (MeCP2), a transcription factor, as a potentially important Hsa21- derived miRNA target since its 34-untranslated region harbors at least one putative recognition site for all of the Has21-derived miRNAs. Additionally, MeCP2 is a provocative miRNA target since mutations in this gene contribute to Rett syndrome, a neurodevelopmental disorder that shares some of the neurologic abnormalities observed in DS. Our preliminary data now demonstrate that MeCP2 mRNA is a direct target of Hsa21-derived miR-155 and that MeCP2 is under-expressed in human fetal and adult DS brain specimens and in a mouse model of DS. As a consequence of attenuated MeCP2 expression, transcriptionally-activated and -silenced MeCP2 target genes are aberrantly regulated in these DS brain specimens. To begin to substantiate a causal role of Hsa21-derived miRNAs in DS, in vivo silencing of endogenous mature miR-155 expression by intra- ventricular injection of antagomir-155 resulted in the normalization of miR-155 and MeCP2 expression levels in the DS mouse brains. Taken together, these preliminary data suggest that improper repression of MeCP2, secondary to trisomic over-expression of miR-155, result in the aberrant regulation of MeCP2 target genes. This dysregulation subsequently results in the destabilization of important "regulatory circuits" that contribute, in part, to the cognitive defects that occur in DS individuals. PUBLIC HEALTH RELEVANCE: This project represents a novel line of inquiry regarding the molecular mechanisms of DS. This study will provide "proof of concept" that Hsa21-derived miRNAs inhibit the expression of critical regulatory proteins, which in turn, results in aberrant expression of a number of factors critical for neurodevelopment. Our approach includes a comprehensive and multi-disciplinary approach and includes human tissues, cell lines, and a DS mouse model. Our project will define miRNA/mRNA targets responsible for DS and will potentially lead to novel therapeutic strategies to treat DS individuals in the perinatal period to change the course of pathogenesis.

DESCRIPTION (provided by applicant): MicroRNA is a recently characterized class of small, non-coding, RNA that repress mRNA translation. Work over the past several years has revealed important roles for microRNA in a vast array of developmental and disease-related processes. Within the developing mammalian central nervous system, results from dicer null mice support a role for microRNAs in neuronal morphogenesis and neuronal survival. However, relatively little is known about how neuronal activity regulates microRNA expression patterns in the mature nervous system and, importantly, whether microRNA regulate neuronal plasticity and cell viability. Based on recent work by a number of investigators, and on the preliminary data reported here, we propose that microRNA plays a key role in activity-dependent structural plasticity in the mature nervous system. To test this hypothesis we have assembled a novel set of genetically modified mouse models, and an array of genetic and functional screening assays. In Aim 1, we propose to utilize the Solexa deep sequence method to examine activity-dependent expression of non-coding RNA in the hippocampus. We will also examine the contribution of transcriptional networks that underlie activity-dependent neuronal plasticity and perform a series of experiments to identify functionally relevant microRNA targets. In Aim 2 we propose to determine the contribution of microRNA to adult neuronal structural plasticity and neuroprotection. To this end, we will employ an inducible form of Cre-recombinase to disrupt Dicer expression. The effects on both physiological and pathophysiological levels of neuronal activity will be examined. In Aim 3, we propose to determine the role of the microRNA-132 locus in activity-induced structural remodeling in vivo. A combination of knockout and tet-inducible microRNA mouse strains will be used to test this question. The data generated here should provide a wealth of new insights regarding how neuronal activity sculpts microRNA expression patterns, and, in turn, how these changes affect key aspects of neuronal plasticity and pathology.

CORE B: GENETICS CORE The purpose of this Core is to provide support for transgenic mouse and zebrafish research. This Core will facilitate current NINDS-funded research and stimulate synergistic Interactions between diverse Investigators using different animal model systems. This support will benefit all investigators by subsidizing costs and providing advanced genome editing services and aquaculture services. The Genetics Core has two components, each with its own faculty Director. I. Transgenic Mice Core Overview The aim of this Core is to support the development of novel mouse models that will facilitate NINDS-funded researchers and stimulate synergistic interactions between Pis in the OSU Neuroscience Center. Because the Core will subsidize the cost of making transgenic and knockout mice, it will support new investigators to develop mouse models which otherwise could be cost prohibitive. This support will be provided by subsidizing costs of generating transgenic and knockout mouse lines and subsidizing the per diems during the initial expansion and characterization phase of the mouse line. Over the 4.5 years of the grant 43 transgenic mice were generated using Core Support benefiting four Pis (Oberdick, Burghes, Obrietan, Yoon). This Core component was cited in twelve papers and led to two new R01s. The projected need for the renewal is forty two transgenics and ten knock outs benefiting ten Pis. Thus, this is a valued, productive Core.

Deep within the brain resides a set of neurons that have inherent time-keeping capacity. The electrical firing properties of this cell population (referred to as the suprachiasmatic nucleus, or SCN) generates a 24 hour oscillation (referred to as a circadian rhythm) that functions as a timing cue to ancillary neuronal oscillator populations found throughout the rest of the brain. This circadian timing circuit has remarkable power over the nervous system. For example, key functions of the nervous system, such as the sleep/wake cycle, and complex cognitive processes (e.g., learning, memory and critical thinking skills) are modulated by this circadian rhythm. Further, the disruption of this circadian timekeeping system has profound effects on mood, cognitive capacity and sleep. Notably, the disruption of circadian timing can result from alterations in one's work schedule (often seen in night shift workers), or from a number of acquired and congenital disorders of the brain (e.g., depression, Alzheimer's disease and Huntington's disease). In fact, the disruption of circadian timing in individuals with Alzheimer's disease is considered to be one of the most pressing issues for health care workers. This rich series of observations raises questions about the functional relationship between the SCN and ancillary neuronal oscillator populations, and, relatedly, about the underlying neuronal circuits that modulate cognitive capacity over the circadian cycle. In this application, the researchers propose to employ a wide array of innovative interdisciplinary approaches to determine the functional significance and mechanistic underpinnings by which circadian clock timing in brain circuits modulate complex cognitive processes, such as learning and memory. The research will also provide an opportunity for direct student involvement in research and the data from the study will be used to generate an interactive website for outreach to the public and included in the Brain Awareness Program that presents research findings to elementary and middle schools.

Within the central nervous system, rhythmicity is not restricted to the SCN, but rather, is found in a diversity of brain regions. In line with this, forebrain structures, including the cortex and hippocampus, express all of the molecular components required to drive clock timing. Here, the researchers propose to systematically test the contribution of forebrain clocks to the circadian regulation of learning and memory. This application is predicated on the hypothesis that the SCN clock works in a coordinated manner with ancillary hippocampal/forebrain oscillators to regulate cognitive capacity as a function of the time of day. To test this idea, the researchers propose to use an innovative set of transgenic animal models that will allow them to resolve clock timing at a single cell level, and thus, create a systems and cellular level blueprint of hippocampal timing (Objectives 1 and 2). This aim will allow the researchers to identify the cycling cell types (e.g., glutamatergic excitatory neurons, GABAergic inhibitory neurons, astrocytes and microglia) and to assess the phase relationship of discrete oscillator populations. Addressing these questions is key to the understanding of the tightly intertwined relationship between circadian timing and cognition. In Objectives 3 and 4, the researchers will use conditional knockout technology to disrupt forebrain circadian timing and assess the effects on learning and memory. Importantly, with this approach, SCN timing will be intact, and thus, the scientists will be able to specifically test the contribution of forebrain oscillators to learning and memory. These data sets will provide new insights into the complex processes by which the clock shapes cognition, and in turn provide a foundation for further experimental approaches designed to reveal clock functionality in a broad range of physiological and psychological processes.

DESCRIPTION (provided by applicant): Human physiology is modulated by an inherent 24-hr (circadian) clock. Central to this time-keeping process is the master circadian pacemaker located within the suprachiasmatic nucleus (SCN). This relatively small brain region provides a daily timing cue that orchestrates ancillary clock timing systems found in all organ systems of the body. Of note, within the central nervous system (CNS), the SCN appears to function in coordination with forebrain oscillators to modulate an array of complex cognitive processes, and the disruption of clock physiology as a result of the aging process, neurodegeneration or photic desynchrony has profound effects on mood, memory and executive function. These observations raise questions about the functional features of forebrain cellular oscillators, clock gated synaptic circuitry and rhythmic gene expression patterns. In this application we propose to employ a wide array of innovative interdisciplinary approaches to determine the functional significance and mechanistic underpinnings of clock physiology in the forebrain. This application is predicated on the central hypothesis that forebrain circadian clocks function in coordination with the SCN to modulate cellular plasticity as a function of the time-of-day. To maintain focus, our analysis of forebrain oscillatory activity will be centered on the pyramidal neurons of the hippocampal CA1 cell layer. In Aim 1, we propose to perform a cellular-level analysis of clock timing. For these studies, we will use a combination of innovative transgenic reporter mouse models to address the following questions: 1) does the CA1 cell layer consist of a homogenous or heterogeneous population of oscillators, and 2) is there a relationship between forebrain clock cell phase and the responsiveness of signaling pathways that contribute to neuronal plasticity. In Aim 2, we propose to test the role that forebrain clocks play in the generation of molecular rhythms. Although rhythmic activity has been reported in the forebrain, we do not know what role these forebrain oscillators play in driving these rhythms. Here, we propose to use a conditional knockout mouse line, where the circadian clock is deleted in forebrain excitatory neurons to assess how forebrain timing shapes kinase rhythms. Further, to assess how the forebrain clock shapes the transcriptional profile of the CA1 cell layer, we propose to employ an array-based transcriptome profiling approaches in combination with a newly developed in vivo RNA labeling and isolation approach which will allow us to selectively profile gene expression from discrete cell populations. In Aim 3 we will examine whether microRNA132 functions as a clock-gated regulator of cellular plasticity and cognition. For this study, we propose a novel set of transgenic and knockout mouse models designed to 'lock' microR132 to stable physiological levels across the circadian cycle. The combined use of these approaches will provide an unparalleled level of insight into the role that forebrain clock timing plays in shaping forebrain functionality from the molecular to the behavioral level.

? DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the roles of the p42/44 Mitogen-Activated Protein Kinase (MAPK) pathway effectors Ribosomal S6 Kinase (RSK) and Mitogen/Stress activated Kinase (MSK) as regulators of excitotoxic cell death and aberrant structural plasticity in the hippocampus. Traumatic brain injury-induced cell death and alterations in synaptic architecture are likely to be underlying events leading to an array of cognitive disorders and the development of epilepsy. Importantly, there is a fundamental unresolved question regarding the signaling events that couple traumatic brain injury to cell death and structural remodeling. Based on recent work by others, our published findings, and the preliminary data reported here, we propose that RSK and MSK are both neuroprotective and couple traumatic brain injury to structural remodeling. Furthermore, we hypothesize that RSK and MSK function through distinct transcriptional and post-translational mechanisms to regulate these processes. To test this hypothesis, we have assembled a novel set of transgenic mice, knockout mice, and an array of screening assays. In Aim 1 we will use the pilocarpine model of status epilepticus (SE) to systematically test the role of MSK as a regulator of SE-induced cell death. We propose that a MSK-CREB signaling cassette plays a key role in cell viability. Specific mechanisms of MSK-CREB- dependent neuroprotection, including the inducible expression of detoxifying enzymes and miRNAs will be examined. In Aim 2 we propose to test the role of RSK in neuroprotection against SE-induced cell death. At a mechanistic level, we will examine the role of RSK as a regulator of pro-apoptotic signaling pathways. In Aim 3, we propose to determine whether MSK and RSK signaling couple traumatic brain injury to aberrant structural plasticity in the hippocampus. A good deal of work has implicated the MAPK pathway as a regulator of activity- dependent dendrite and axon plasticity, however, the role of the MAPK pathway as a regulator of pathophysiologically-induced structural remodeling has not been systematically addressed in vivo. Here we propose to test the hypotheses that RSK stimulates injury-induced axon growth and that MSK couples injury to changes in dendrite structure. The proposed studies will provide novel and definitive data sets, which in turn, could lay the foundation for the development of new therapeutic approaches designed to 'uncouple' SE (and other forms of traumatic brain injury) from its long-term pathophysiological sequelae (e.g., epileptogenesis and cognitive impairments).

Recent work has established a clear connection between Alzheimer?s disease (AD) and the disruption of the circadian timing system. However, the mechanistic underpinnings of this relationship have not been clearly identified. Interestingly, if we attempt to deconstruct this relationship and place it within the context of the profound effects that Alzheimer?s disease has on cognition, several ideas begin to come into focus. First, data to date has revealed that circadian timing within cortico-limbic circuits modulates complex behavioral states, including cognition. Second, AD has marked effects on functional plasticity of these same circuits. These observations raise an interesting question: could the cognitive deficits in AD result, in part, from the dysregulation of circadian timing within cortico-limbic circuits? As an initial examination of this idea, we propose to test the following hypothesis: The cognitive deficits during early- to mid-stage of AD results in part from a systems-wide breakdown in the fidelity of the cortico-limbic circadian timing systems. To test this hypothesis, we have assembled an innovative set of transgenic mouse models and state-of-the-art imaging methods that will allow us to both profile and manipulate circadian timing over the course of disease progression. In Aim 1, the effects of amyloid ? peptide (A?) on the fidelity of cellular-and circuit-based time-keeping capacity will be examined. In Exp. 1A, we will use a cell-culture based profiling approach to test the effects of A? oligomer on the cell autonomous circadian timekeeping capacity of neurons isolated from the SCN (the locus of the master circadian clock), the cortex and the hippocampus. In Exp. 1B brain slice explant imaging will be used to test the effects of A? on circuit-based circadian rhythm generation. In Aim 2 we propose to profile clock timing and clock-gated gene expression in the 5XFAD mouse model of AD. In Exp. 2A, cranial window imaging (via multiphoton microscopy) of clock timing in the frontal cortex and the hippocampus will be used to generate a cellular- and systems-level profile of clock phasing, rhythm amplitude and oscillator synchrony over the course of the AD-like pathology. This study will be complemented by immunofluorescence-based clock gene profiling (Exp. 2B) and by transcriptomic profiling (Exp. 2C). In Aim 3, we will test the effects that disease progression in the 5XFAD model has on clock-gated (Exp. 3A) and activity-evoked (Exp. 3B) cellular signaling, as well as on dendritic spine formation. In Aim 4 we will test whether the desynchronization of cortico-limbic oscillators underlies the cognitive deficits in the 5XFAD mouse model of AD. Key to this aim will be to test whether the clock enhancing compound PF-670462 triggers the resynchronization of cortico-limbic oscillator populations, and if so, whether this effect underlies the capacity of PF-670462 to augment cognition. If our underlying hypothesis is validated, these data will provide an important starting point for new lines of inquiry (and potentially new therapeutic interventions) designed to further understand the mechanistic relationships (at a cellular, systems, and genetics level) between circadian timing and AD pathogenesis.

Project Summary/Abstract Virtually every aspect of human physiology and behavior is modulated by an inherent 24 hour (circadian) timing process. At the center of this clock timing system is the suprachiasmatic nucleus (SCN) of the hypothalamus. A key feature of the SCN clock is the tight, time-of-day, dependent regulation of the MAPK (p44/42 mitogen-activated protein kinase) pathway. Two examples of this phenomenon are the daily oscillations in the activation state of the MAPK pathway, and the clock-gated regulation of the photic responsiveness of the pathway. Importantly, the clock-generated, temporally-delimited, regulation of MAPK signaling appears to play a central role in SCN timing and entrainment. Further, the daily gating of MAPK signaling may be an underlying design principal of all oscillator populations, and as such, MAPK rhythms could have profound and far-reaching effects on a range of physiological processes. Given these implications, it is surprising that we still know relatively little about the cellular mechanisms and synaptic circuits that confer circadian control over MAPK activity. Here, we hypothesize that the circadian regulation of MAPK signaling is an inherent (cell autonomous) feature of SCN cellular oscillators and that this MAPK rhythm is a key mechanistic building-block by which the circadian clock modulates both basic and complex physiological states. To test this hypothesis, we propose the following set of experimental goals. In Aim 1, we will identify the cellular and network properties of the SCN that give rise to the rhythmic regulation of the MAPK pathway. To this end, we will, A) Determine whether MAPK rhythms are cell autonomous or whether they result from an intercellular SCN network, and B) Determine the intracellular signaling events that generate MAPK activity rhythms. In Aim 2 we propose to characterize the molecular, cellular and systems-based mechanisms by which the SCN clock gates light-evoked MAPK pathway activation. To address this largely unexplored phenomenon, we will, A) determine when and how the molecular gate opens, and B), test whether the cytoplasmic ERK scaffold protein PEA-15 serves as the principal circadian gate on MAPK signaling. Of note, we recently identified PEA-15 as a modulator of MAPK signaling in the SCN, and its capacity to dynamically regulate ERK signaling makes it an attractive candidate for the gating of MAPK signaling. In Aim 3 we propose to employ a selective targeting approach to transgenically disrupt MAPK signaling within the SCN core and shell regions to address the roles of MAPK signaling in A) the generation of circadian rhythms, and B) the entrainment of the circadian clock. Further, conditional PEA-15 KO and point mutant PEA-15 transgenic mouse lines will be used to test a model in which PEA-15 phosphorylation leads to rapid ERK dissociation, which we posit to be a key step in the initiation of light-evoked phase-shifting. Together, these data will provide fundamental new insights into the relationship between MAPK signaling and the circadian clock, and point to potential ways in which the dysregulation of clock-gated MAPK signaling could contribute to disorders of the CNS.