Hoau-Yan Wang - US grants

Signal transduction for adrenergic receptors is regulated by a distinct array of guanine nucleotide binding proteins (G proteins). Our preliminary data indicate that in aorta, (alpha 1b-adrenoceptors (ARs) are linked to both Go and Gq, while (alpha 1d)ARs couple exclusively to Gq. In addition, the levels of various a1 AR subtypes in different blood vessels were different. These results suggest that differential coupling of a1 AR subtypes to G proteins may occur in different blood vessels. These data furthermore suggest that age-related altering the interaction of specific receptor and its associated G protein(s) may significantly affect the receptor-mediated signaling and ultimately the function of the organ. Changes in alpha-and B-AR functions in blood vessels have been reported both in Fisher-344 rats and human. IN addition, an age-related alterations in expression of (alpha)1AR subtypes has also been observed in rat aorta. As aging plays an significant role in mediating the pathogenesis of cardiovascular disease, such as hypertension, orthostatic hypotension and atherosclerosis, it is important to study the molecular mechanism (s) contributing to altered vascular function during aging. The proposed study aims to test the hypothesis that different compositions of alpha (1)AR/G proteins regulate diverse arrays of transmembrane signalling pathways n vasculature which are differentially affected by age. We are proposing a series of experiments to fully characterize the contribution of altered (alpha 1)ARv subtypes/G proteins coupling to the age-related changes in (alpha 1) AR- mediated functions i vasculature. Specifically, we will directly assess linkage of (alpha1) adrenoceptors with G proteins in aortas and tail arteries from young and old rats. Membranes prepared for blood vessels will be used to determine the coupling of (alpha1)-adrenoceptor subtypes to G proteins by an established immunoprecipitation procedure using specific anti-G(alpha) or anti (alpha1 AR antibodies. In addition, we will define the molecular mechanisms responsible for the changes in alpha1 AR transduction systems during aging and test the specificity of these alterations with respect to subclasses of (alpha1 AR, G proteins and blood vessels. Results from the present proposal will greatly enhance our knowledge of the role of the signal transduction pathways in the pathophysiology of age-related cardiovascular diseases and ultimately help in designing specific strategies for more effective pharmacotherapy of those disorders in geriatric patients.

Project Summary (Significance) Multiple lines of evidence have implicated mGluR5 signaling for the pathophysiology and treatment of schizophrenia; yet possible dysregulation of mGluR5 signaling and their pathophysiologic roles are presently unknown. Recently, we found direct evidence for reduced mGluR5 signaling in the postmortem dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia. mGluR5 is physically connected with NMDA receptor (GluN) complexes in the postsynaptic density (PSD) and these two pathways facilitate the activity of each other. (Preliminary Studies) We examined the agonist-induced activation of mGluR5 signaling in the postmortem dorsolateral prefrontal cortex (DLPFC) derived from 17 matched pairs of patients and controls. We found a striking reduction in three pathways downstream to mGluR5 receptor activation; namely, Gq/11 activation and recruitment of PI3K and scaffolding proteins compared to controls (P<0.01). This dysregulation was accompanied by alterations in phosphorylation of mGluR5, important for receptor desensitization, and in mGluR5 association with RGS4, Preso 1, norbin and tamalin, which are critical for surface expression and clustering. Importantly, we find evidence supporting disruption of reciprocal facilitation between mGluR5 and GluN signaling in schizophrenia and thus hypofunction of one can further compromise the other. (Scientific Premise) Schizophrenia is associated with mGluR5 signaling hypoactivity mediated by altered mGluR5 phosphorylation and protein ? protein interactions (PrPrIs) in the receptor complexes. mGluR5 hypofunction can contribute to GluN signaling hypoactivity and vice versa via disruption of reciprocal facilitation. PrPrIs in the interactome may be a point of convergence for etiologic factors. (Specific Aims) To further characterize mGluR5 hypoactivity and its interplay with GluN signaling in schizophrenia, we will examine molecular underpinnings for mGluR5 hypoactivity on GluN signaling and vice versa in the DLPFC of patients compared to controls. (Aim 1). Our preliminary data and recent studies point to PrPrIs as substrates upon which various molecular alterations converge and precipitate glutamatergic dysregulation. Combining discovery and quantitative proteomics, we will analyze PrPrI alterations in the mGluR5-GluN interactome in patients at the basal level and in response to receptor activation (Aim 2). PrPrI alterations in the interactome can be traced to their etiologic underpinnings at the genomic, transcriptomic and proteomic levels. Cutting edge systems biology algorithms permit us to leverage largest genomics data sets presently available and impute DLPFC transcriptomics data of the same number of subjects. Genetic variants, imputed transcriptomic results, along with quantitative PSD proteomics results will be projected onto the interactome using heat diffusion based algorithms (Aim 3). This will identify etiologically significant and potentially targetable subnetworks in the interactome. (Impact) We will establish mGluR5 hypofunction as integral to glutamatergic dysregulation in schizophrenia and identify etiologic underpinnings and potential points of intervention.

Li, Christine Project Summary Deposition of beta-amyloid (A?)-rich dense plaques and the presence of hyperphosphorylated tau neurofibrillary tangles are two postmortem criteria used in the diagnosis of Alzheimer's disease (AD). Although A? is derived from a larger amyloid protein precursor (APP), the function of APP and its various cleavage products and the targets/pathways in which they act are still poorly understood. A family of APP-related proteins is present in mammals. Knockout of the APP family in mice leads to postnatal lethality and type II lissencephaly, indicating that the APP family has essential functions during development. sAPP? can modulate BACE activity and levels of tau phosphorylation, suggesting that sAPP? can modulate levels two markers of AD pathology. Like the mammalian APP family, the Caenorhabditis elegans ortholog apl-1 is critical for survival; loss of apl-1 leads to larval lethality, which can be rescued by germline transformation with an apl-1 genomic fragment or constructs encoding only the extracellular domain of APL-1. We will leverage the strengths of C. elegans and mouse to identify the function and cellular pathways in which APP acts and regulates. We have performed biochemical and mutagenesis screens to identify protein interactors and suppressors of the apl- 1 lethality, respectively; our goal is to verify interacting proteins and identify the molecular identity of the suppressor genes. Our second goal is to look at global changes in the whole animal C. elegans transcriptome when APL-1 levels are perturbed in mutant and transgenic strains. The C. elegans findings will be translated into the mouse system to determine whether homologues have similar functions in APP biology. By using data uncovered in the C. elegans system and testing them in the mouse system, we have a unique opportunity to identify pathways in which APL-1/APP act, including pathways involved in the survival and proper functioning of neurons. Our results may provide novel insights into how alterations in these pathways may contribute to the pathologies seen in AD and highlight ways to formulate new therapeutic strategies to effectively treat AD.