Stuart Maudsley, PhD - US grants

Research Summary My proposed study involves investigation of age- and disease-dependent alteration of three distinct levels of receptor-mediated signal transduction control. These three levels of control are: receptor accessory protein modulation of signal transduction;scaffolding protein functionality in neuronal cells;receptor microdomain environment and its impact on receptor function. To these multiple ends myself and my research fellows have been undertaking the following research to illuminate these areas. Receptor Accessory protein modulation We have generated two sets of stably expressing human neuronal cell lines (SH-SY5Y) expressing an epitope-tagged (3x haemagglutinins) human m1 and m2 muscarinic acetylcholine G protein-coupled receptors (GPCRs). These receptors have been chosen as they represent one of the most important and tractable therapeutic targets for the treatment of Alzheimers disease (AD), e.g. the only currently prescribed AD therapeutic, AriceptTM, works by facilitating stimulation of these targets. We have chosen 24 clones of each of these cell lines and have been employing these for proteomic research. Our need to generate these clones resides in the purification techniques required to isolate large amounts of the receptor proteins so that their associated accessory proteins can be identified by tandem mass spectrometry. Using conventional antibody-based techniques using sera directed against functional regions of the receptor it is likely that subtle ultrastructural changes between receptor isoforms may be disregarded. Our hypothesis is that during aging and disease there are changes of the stoichiometry of the receptor with its accessory proteins and that this then affects the interaction of the receptor with its cognate ligand. Therefore our need to unambiguously purify transmembrane receptors was paramount. We have successfully purified high numbers of muscarinic receptors and demonstrated that large numbers of accessory proteins can associate with the GPCR. We intend now to compare these associated proteins to the groups of proteins that interact with the receptor in a disease state model, i.e. addition of exogenous amyloid beta peptide or oxidative stress in the form of either hydrogen peroxide addition or glucose deprivation. Scaffolding protein functionality modulation Scaffolding proteins are large proteins often associated with GPCRs at the cell surface but also with cytoskeletal entities. These proteins assemble multiple signaling factors into coherent cascades of functional relevance. These scaffolding proteins are thought to be the basis of GPCR selectivity of function. The ability of these scaffolding proteins to still function in the face of disease-mimicking stressors (see above) has yet to the thoroughly investigated. We have been developing technologies to isolate and purify these scaffolding molecules both from clonal human neuronal cells and also from primary animal tissue from disease models. The two GPCR-interacting scaffolding proteins we have been investigating are the G protein coupled receptor kinase-interacting transcript (GIT) and spinophilin. The first protein has been demonstrated to link GPCR desensitization to the ability of the stimulated receptor to affect cytoskeletal dynamics. This will be of great importance at the functional synapse. We are currently generating stable SH-SY5Y cell lines expressing epitope tagged (FLAG and haemagglutinin) and GFP-tagged forms of these proteins for live confocal microscopy. In addition we have created our own affinity matrix purification systems to isolated specifically large amounts of GIT from cells and tissues through chemical combination of GIT antisera to an immobilized agarose phase. This process has also bee repeated for the second scaffolding protein, spinophilin. This protein, specifically enriched in the dendritic spines of neurons (of particular importance for control of synaptic transmission) also can act as a novel scaffolding protein than becomes recruited to the GPCR upon ligand activation. We are currently analyzing the functional signaling proteins that interact with these scaffolding factors in the presence or absence of disease mimetics. Receptor microdomain study The interactions that GPCRs make with other functional transmembrane proteins are primarily controlled via their association with microdomain regions of the plasma membrane. Such microdomain regions include focal adhesions, clathrin-coated pits and lipid rafts. We have begun studying how GPCR-interacting proteins, such as the beta-arrestins, can control the presence of GPCRs in these microdomains is affected by neurodegenerative disorders such as AD. To this end we are in the process of generating SH-SY5Y cell lines overexpressing various forms of the beta-arrestin molecules. With purification of both clathrin pits and lipid raft structures we are intending to identify how the presence of the GPCRs in various different plasma membrane compartments can affect their pharmacology, with respect to ligand interaction and also their downstream signaling ability. In addition to this we have been elucidating the nature of the connection between GPCR activity and the enzymatic function of the gamma-secretase complex that is specifically enriched in the same compartment as many GPCRs, e.g. the lipid raft. We have been employing both proteomic identification and in vitro enzymatic assays to demonstrate how acute receptor stimulation may control minute-to-minute beta amyloid production.

Summary Cognitive impairment places a severe burden upon both the sufferers and their carers. While approximately 5-10% of patients displaying cognitive impairment possess a genetic predisposition, such as a Flemish or Swedish mutation in the amyloid pre-cursor protein, the majority of patients do not possess such an obvious cause of their malady. The causes of age-dependent cognitive decline in the general population are likely to extremely diverse and therefore multifactorial. However there is one phenomenon that is generally expressed in the population, cognitive performance in tasks, that appears to be positively correlated to a prophylactic action against age-dependent neurodegeneration. This underlies the anecdotal adage of the use it or lose it phenomenon. Hence in this study we are attempting to understand not only the natural basis for cognitive capacity but also hopefully elucidate signaling mechanisms that are linked to this capacity that possess an intrinsic neuroprotective action. We are already nearing the completion of the first phase of this study in which we have used a large genetically identical murine pool of both male and female animals. We are in the process of generating a semi-quantitative relationship between protein expression in the cortex and hippocampus of these animals and their maze solving capacity. We feel that the data derived from this study will illuminate the field of cognition and provide a novel insight into the molecular basis of individual intellectual variation.

Summary[unreadable] [unreadable] For many of the major neurodegenerative disorders alteration of protein function and cell physiology by changes in oxygen availability or the presence of damaging oxygen radicals may underlie many of the deleterious effects of these diseases. Therefore a true in-depth understanding of how the oxidative environment affects cellular responses to protective or detrimental stimuli is of great importance. At this present time, we are only now, understanding that the modes in which we perform cell and receptor signaling investigation may be biased towards a non-physiological paradigm, i.e. investigating receptor pharmacology under atmospheric oxygen tensions. We are therefore initially undertaking a painstaking analysis first of how central nervous system tissue responds, at the genomic and proteomic level, to multiple oxygen tensions. Our results from this primary study will facilitate subsequent studies to identify how transmembrane receptor-mediated signaling paradigms, one of the most important therapeutic targets, act at these various tensions. It is highly likely, as we have seen with our preliminary data, that there are wholescale reactive genomic and proteomic cellular changes in response to altered oxygen tension environments. With these complex and widespread changes it is highly likely that receptor signaling systems may be affected and thus potentially alter the efficacy of pharmacotherapeutics. Our research will aim to generate pharmacotherapeutics that will counteract these deficits and therefore maintain pharmacological efficacy in a variety of pathophysiological or aging states.