Kari R. Hoyt, Ph.D. - US grants

DESCRIPTION (provided by the applicant): Huntington's disease (HD) is a hereditary neurodegenerative disease characterized by selective basal ganglia damage and movement disorders. The genetic defect responsible for HD is an expanded poly-glutamine repeat in the huntingtin protein in excess of that found in unaffected individuals. The mechanism by which the expanded poly-glutamine repeats in huntingtin causes neuronal degeneration and motor dysfunction is unknown. Based on findings in HD patients and animal models of HD it has been suggested that the genetic defect leads to metabolic compromise with consequent increased sensitivity to glutamate-induced neuronal injury (excitotoxicity). This excitotoxic injury may involve elevated levels of [Ca2+] mitochondrial dysfunction and oxidative stress. Recently, several transgenic mouse models of HD (HDTg) which express the mutant huntingtin gene have been created and exhibit neurologic symptoms and pathology similar to that seen in HD. Recent results from this laboratory and others suggest that HDTg mice do have deficits in metabolic enzyme activity and that neuronal responses to glutamate receptor activation are substantially altered by the expression of the mutant huntingtin protein. We propose to test the hypothesis that mutant huntingtin expression leads to mitochondrial dysfunction and ionotropic glutamate receptor mediated neurotoxicity. This glutamatergic dysfunction may be a consequence of alterations in neuronal metabolism or direct effects on receptor function caused by mutant huntingtin expression. As a model of HD, we will use fluorescence imaging techniques in primary neurons cultured from HDTg mice and their non-transgenic (WT) littermates to test this hypothesis. Our specific aims are to identify the mechanism(s) underlying the potentiation of 1) Glutamate-receptor mediated [Ca2]i responses in HDTg neurons. 2) Glutamate-receptor stimulated mitochondrial depolarization in HDTg neurons. 3) Excitotoxic neuronal death in HDTg neurons. These studies address our long-term goal of evaluating the role of metabolic compromise on glutamate toxicity as it relates to the neuronal dysfunction and death evident in HD. The cell culture models of HD proposed in this work should also provide a convenient means for testing relevant therapies for HD.

DESCRIPTION (provided by applicant): The overall goal of this R15 AREA proposal is to test whether Antioxidant Response Element (ARE)-mediated gene expression is neuroprotective in Huntington's disease (HD). The are drives the expression of phase II detoxifying/antioxidant enzymes, and as such, may be an effective therapeutic target to attenuate reactive oxygen species (ROS)-induced cell toxicity, which is an underlying element in the development of HD. To this end, we have generated a novel transgenic mouse strain where the are transcription factors Nrf2 and MafK are driven in a tetracycline-inducible manner within forebrain neurons or glia. We will cross these new transgenic mice with a well-established transgenic mouse model of HD (the R6/2 line) to drive cell-type specific expression of Nrf2 and MafK and thereby increase the expression of phase II detoxifying/antioxidant enzymes. In Aim 1, we will test the neuroprotective effects of ARE-mediated gene expression in the R6/2 mouse model of HD. In Aim 2, we will perform a systematic analysis of basal and transgenically-induced Nrf2-ARE transcriptional pathway activation in the R6/2 mouse. Overall, the approach we have outlined in this proposal will allow us to discover whether up-regulation of ARE-mediated gene expression is a viable preventative or therapeutic approach for the treatment of HD. Our results should also lead to the identification of new, potentially cytoprotective, targets for future drug design. PUBLIC HEALTH RELEVANCE: Our work is based on the idea that oxidative stress contributes to neuronal damage in Huntington's disease, and our goal is to discover whether it is possible to activate endogenous antioxidant defense systems and achieve protection from neuronal damage. The results of this work will contribute to the future design of neuroprotective therapies for HD.

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.