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.

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): 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.

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): 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.

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): 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.