Paul Davies - US grants

Our long-term goal is the prevention and reversal in the microcirulation of the structural remodeling causing pulmonary hypertension (PH). An intrinsic feature of this remodeling is change in pericyte phenotype. In the rat model of hypoxic PH, three events are critical to this change: proliferation, production of pericellular elastin and differentiation. The first two produce lumen narrowing, while the last may increase wall rigidity. We will establish the timing of these events as a pointer to their cause and control. The contribution of hypoxia and of pericyte interaction with matrix will be analyzed. We will use antibodies of known purity and specificity to determine, by immunocytochemistry, the distribution of new elastin, basement membrane laminin and type I collagen. Ultrastructural studies of microdissected arteries will quantify adhesion plaques, through which the pericyte interacts with matrix, and junctions that connect the pericyte with the endothelium or with other pericytes. In cell culture, we will characterize the pericyte from normal rats, "new" pericyte from hypoxic-exposed rats and compare them with the corresponding arterial smooth muscle cells. We will determine their (a) proliferation, (b) elastin production and (c) adhesion-as a marker of differentiation, and analyze whether hypoxia changes these functions directly or through new matrix. Because increased adhesion is a likely factor contributing to PH, we will determine if adhesion molecules, on the cell and in the matrix, are altered or increased, with particular reference to three that are markers of the smooth muscle cell - elastonectin, thrombospondin and a 38kD glycoprotein. Cell culture studies will thus provide markers of phenotype for each cell type. Overall, the results will identify the events in remodeling critical to hypertension and susceptible to the goals of prevention or reversal; in addition they will indicate ways that these goals can be achieved.

Our program of research is predicated on the conviction that physics can provide deep new insights into the origin and nature of cancer. Importantly, we see physics as providing not merely a support role in diagnosis and analysis, but as a crucial component in the conceptual basis on which a full understanding of the living cell in general, and cancer in particular, must be built. The conceptual driver of the program will be a Cancer Forum, a think tank that will critically examine the foundational assumptions of cancer research, and help develop fundamentally new hypotheses and research strategies. The Forum will serve as a scaled-down yet diversified follow-up of the NCI workshop series that preceded this announcement, and will draw upon expertise from within the Network and the broader scientific community. It will be hosted by The Beyond Center for Fundamental Concepts in Science, which has a track record of facilitating ground-breaking research strategies in a multidisciplinary context. Forum workshops will be made available through webcasts and podcasts via the PS-OC website. The Forum will work closely with three interlinked experimental projects organized around the theme of mapping the physical correlates of cancer across a range of size scales from chromatin to cell clusters, and as a function of cancer progression. Thus for the first time it will be possible to systematically and accurately parameterize cancer cells at various stages of development and from a variety of micro-environments, according to their physical properties. The projects capitalize on the unique cluster of powerful new instrumentation being installed at Arizona State University, including three dimensional optical and elastic tomography, and will build on the long-standing collaboration between ASU and the Fred Hutchinson Cancer Research Center, who will provide common cell lines for all the experimental projects. The cells will be complemented by tissue samples from the Mayo Clinic, Scottsdale. The experimental program will be paralleled by an advanced computational modeling system developed by ASU's Center for Biophysics. A placement scheme and graduate seminar program will be designed to foster interdisciplinary education and training, and a journalism fellowship program created to improve public outreach

DESCRIPTION (provided by applicant): Acute alcohol exposure in humans results in memory impairments similar to the impairments caused by hippocampal damage. Imaging studies reveal a smaller hippocampus in those people who abuse alcohol as compared to non-abusers;however, the mechanisms underlying such alcohol-mediated damage to the hippocampus are poorly understood. The zinc activated channel (ZAC) is a novel member of the alcohol sensitive Cys-loop ligand-gated cation channel superfamily and is gated by the transition metals, zinc and copper. The gene encoding for ZAC is a non-functional pseudo-gene in rodents. However, in humans the gene encodes ZAC mRNA in fetal brain and in areas of the adult brain that are innervated by zinc and copper enriched hippocampal neurons. My preliminary data show that ethanol, at intoxicating concentrations, potentiates copper-evoked currents from cells expressing ZAC channels. Based on structural similarities with 5-HT3A and nicotinic alpha 7 subunits, ZAC is predicted to be calcium permeable. Physiologically, an enhancement by ethanol of a ZAC- mediated signal would increase post-synaptic calcium signaling. Excessive stimulation would expose the neuron to risk of excitotoxicity. Based on these data I have formed my central hypothesis: ZAC is a calcium permeable ion channel and ethanol stabilizes the open channel state, resulting in increased agonist sensitivity and slower deactivation and desensitization kinetics. To test this hypothesis I will pursue three specific aims: 1) Establish the sensitivity of ZAC channels to ethanol. 2) Characterize ZAC channel kinetics and their alteration by ethanol. 3) Determine the calcium permeability of ZAC channels. This research is highly significant as it will elucidate the mechanisms of alcohol-mediated damage to human hippocampal neurons in areas known to be innervated by zinc and copper neurons. PUBLIC HEALTH RELEVANCE: In humans, alcohol abuse results in memory impairments thought to be caused by damage to a part of the brain called the hippocampus. The mechanisms of alcohol mediated damage to the hippocampus are poorly understood. This project looks at a new alcohol sensitive protein found in the areas of the hippocampus that is most at risk from alcohol damage and examines the actions of alcohol on this protein.

Core 1 is the conceptual driver for the entire Center. It is founded on the conviction that the physical sciences in general, and physics in particular, offer new and penetrating insights into the origin, behavior and management of cancer. By asking disruptive, provocative and challenging questions, the Core will facilitate the sort of major conceptual leap that will transform understanding ofthe subject. Core 1 will exploit the highly successful brainstorming ethos ofthe Beyond Center for Fundamental Concepts in Science at Arizona State University, a think< tank dedicated to grappling with the deepest foundational questions ranging across the sciences. It will be responsible for the overall scientific mission, and will take responsibility for the convergence, integration and interpretation ofthe three experimental projects. A key component of Core 1 is the Cancer Forum, the main part of which is a workshop program that will critically examine challenging new hypotheses, and transform them into implementable research projects. The workshops will focus on conceptual issues surrounding the physical correlates of cancer, including its origin, evolution, diversification and progression, viewed as physical processes. They will capitalize on the creative synergy of bringing biologists and physical scientists together to think outside the box and examine the hidden assumptions that underlie existing research themes. Consideration will be given equally to experimental, clinical, theoretical and mathematical modeling aspects ofthe subject. The goal will always be to ask, Are we thinking about this the wrong way?

: The Education and Training Unit will address three primary objectives that will serve to encourage inter- and transdisciplinary experience in the context of cancer research. The first objective of this unit is designed to initiate, cultivate, and maintain transdisciplinary experiences for postdoctoral researchers and junior faculty through a placement scheme that exchanges these individuals between participating departments and institutions within the Center. The Unit will also create a graduate seminar course that will be open to all graduate students at Arizona State University (ASU), but be required of graduate students participating in the Center's research. Finally, the Unit will work with Core 1 (the Beyond Center) to facilitate and coordinate a Cancer Forum workshop that emphasizes the transdisciplinary opportunities in Physical Sciences, Biology, and Medicine. This Unit will be integrated with the Dissemination and Outreach Unit in that the graduate seminars and parts ofthe workshop will be video recorded and available on the Center website.

Neuroactive steroids (NASs) such as allopregnanolone (ALLO) play a central role in regulating behavior via their potent anxiolytic, anticonvulsant, sedative, and hypnotic actions. Accordingly, modifications in the levels of NASs contribute to anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome. Classically, NASs are thought to act by rapidly boosting neuronal inhibition by positive allosteric modulation of the activity of ?-aminobutyric acid type A receptors (GABAARs). In addition to their allosteric actions, we have recently shown that NASs act via a protein kinase C-dependent mechanism to enhance the phosphorylation of residues including Serine?s 408 and 409 in the ?3 subunit (S408/9), a process that increases GABAAR number on the plasma membrane leading to a sustained increase in the efficacy of GABAergic inhibition. Although we have shown that NASs do not directly activate PKC, the mechanism by which NASs lead to changes in phosphorylation of GABAAR subunits are unknown. It is emerging that in addition to their positive allosteric modulation of GABAARs, NASs can directly activate membrane progesterone receptors (mPRs); G-protein coupled receptors that regulate PKC signaling. However, no information is available on the role that mPRs play in regulating GABAAR activity. Likewise, the behavioral significance of the sustained mPR-mediated metabotropic actions of NASs remains unexplored. To address these issues we have created mice in which S408/9 in the ?3 subunit have been mutated to alanines, mutations that are predicted to reduce the metabotropic actions of NASs on GABAAR function. Preliminary studies using these tools have allowed us to formulate a central hypothesis that will be tested here; NASs activate mPRs to enhance the phosphorylation of GABAARs on residues including S408/9 in the ?3 subunit, a mechanism that underlies their anticonvulsant efficacy. In contrast, their anxiolytic efficacy is mediated via allosteric potentiation of GABAAR activity, a process dependent upon Q241 in the ?2 subunit. Our experiments will focus on the following aims. Aim 1. To test the hypothesis that the ability of NAS to induce sustained effects on GABAergic inhibition is dependent upon S408/9A in the ?3 subunit. Aim 2. To test the hypothesis that the anxiolytic, and anticonvulsant efficacy of NASs is dependent upon S408/9 in the ?3 subunit. Aim 3. To test the hypothesis that NASs mediate their metabotropic effects on GABAARs via the activation of mPRs. Collectively, our proposal will identify the molecular mechanisms by which NAS exert their therapeutic actions. This information may aid the development of new therapeutic strategies to alleviate the burdens of anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome.

Cancer Biology; physical science;

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and a major cause for a diagnosis of autism spectrum disorders. The symptoms of FXS include hypersensitivity to sensory stimuli, and seizures. The cause of FXS is a loss of the fragile X mental retardation protein (FMRP) yet the underlying pathways and mechanisms of these symptoms are poorly described. GABAA receptors (GABAARs) are major inhibitory ion channels in the brain. Studies from both FXS patients and animal models have revealed altered expression levels of GABAAR ?4 subunits with a concomitant reduced efficacy of tonic inhibition, a non-synaptic type of inhibition important for determining the gain of the neuronal output, thus regulating the excitability and activity of neuronal circuits. Our preliminary data suggest that in Fmr1 KO mice there is a decrease in the phosphorylation of ?4 subunits leading to decreased tonic current, and an increased excitation. The deficits of tonic inhibition are due to a disruption in trafficking of ?4-containing GABAARs from extrasynaptic to synaptic sites. Both FXS patients and Fmr1 KO mice show auditory processing deficits making them hypersensitive to sound. Acoustic information is processed by thalamic auditory neurons in the medial geniculate body (MGB) as it ascends to the auditory cortex. Excitation of auditory thalamic neurons is controlled by tonic inhibition dependent upon GABAARs containing ?4 subunits. Reduced tonic inhibition in MGB of FXS patients could explain the increase in auditory sensitivity and be a target for therapy to reduce sensory hypersensitivity. Studies from our laboratory have revealed that neuro-active steroids (NASs) act via a metabotropic, kinase dependent mechanism to increase the phosphorylation of serine 443 in the ?4 subunit to promote GABAAR insertion into the plasma membrane leading to sustained elevations in their accumulation on the cell surface and increases in the efficacy of tonic inhibition. These preliminary studies have allowed us to formulate a central hypothesis that will be tested here; Hypersensitivity to sensory stimuli in FXS is caused by reduced tonic inhibition in the central auditory system due to mis-trafficking of ?4 subunits to GABAergic synapses. Exposure to neuroactive steroids will increase the trafficking of ?4 subunit containing GABAARs to the membrane to boost tonic inhibition and diminish the severity of the disorder. Aim 1. Characterize the phosphorylation and sub-cellular localization of ?4 subunit containing GABAARs in Fmr1 KO mice. Aim 2. Examine the electroencephalographic (EEG) activity of Fmr1 KO mice. Aim 3. Determine the efficacy of NAS treatment to reverse deficits in GABAergic inhibition and neuronal excitability. The result of this study will provide new insights into the mechanisms that regulate the efficacy of tonic inhibition and if that alterations in these contribute to the pathophysiology of FXS. Such insights may promote the development of new therapeutics to alleviate the burdens of FXS.