Richard S. Jope - US grants

This project is designed to study the cholinergic system in the blood of patients with Alzheimer's Disease and in the brain of a rat model of the disease, aluminum poisoning. There have been brief reports of changes in the erythrocyte choline levels, erythrocyte choline transport, erythrocyte acetylcholinesterase activity and plasma acetylcholinesterase pseudocholinesterase activities in patients with Alzheimer's Disease. The present study will be unique in that we will make all of these measurements in each sample. Subjects will be well documented cases from University Hospital and will consist of controls, aged non-demented, and demented with and without the diagnosis of Alzheimer's Disease. These results should indicate the specificity and degree of biochemical changes occurring in the blood of patients with Alzheimer's Disease. We will examine the data for relationship among the biochemical measures and between biochemical and clinical data. Patients will be re-examined and blood samples obtained at least yearly to investigate the effects of the progressive deterioration of Alzheimer's Disease on these biochemical measures. Confirmation of preliminary evidence of changes in these blood samples will be followed by investigations of possible biochemical mechanisms causing the changes. Among these studies will be measurements of the kinetics of cholinesterase, ion requirements of choline transport, choline transport in erythrocyte ghosts and in "young" and "old" fractions of erythrocytes. We will also examine the effects of aluminum on cholinergic activity rat brain because aluminum has been implicated as a causative factor in Alzheimer's Disease. We will test this proposal by treating rats acutely and chronically with aluminum followed by measurements of 14C-glucose conversion to 14C02, 14C-acetylcholine and 14C-lipids in brain slices, high affinity choline transport in synaptosomes, choline acetyltransferase, muscarinic receptors and phosphatidylinositol turnover. We will emphasize measurements that indicate the dynamics of the cholinergic system, such as phosphatidylinositol turnover, choline uptake and acetylcholine synthesis and release. We will also test the effects of drugs that alter cholinergic activity on aluminum toxicity. These experiments should clarify whether or not aluminum causes deficits in the cholinergic system similar to those reported in patients with Alzheimer's Disease.

Lithium-is-the-drug of choice in the treatment-of-bipolar- affective-disorder, although its mechanism of action remains unclear. Our major working hypothesis is that one effect of lithium is enhancement of CNS cholinergic activity. We have identified three potentially important effects of lithium and our goals are now to identify the mechanisms accounting for these effects. 1) Presynaptically, lithium enhances the synthesis and release of acetylcholine. We hypothesize that this is due to enhancement by lithium (i ) of intracellular calcium or (ii) of cyclic AMP, either of which enhances acetylcholine release. To test this hypothesis, we will measure the effects of lithium on calcium influx, efflux, sequestration and ATPase in synaptosomes. Second, we will measure the effects of lithium on cyclic AMP and cyclic AMP modulation of acetylcholine release. 2) We have found that chronic treatment with lithium depresses the muscarinic agonist-induced hydrolysis of phosphoinositides in rat cortical slices. We hypothesize that lithium reduces the response of this major second messenger-producing system by influencing either the receptor or the two primary second messengers, diacylglycerol and inositol trisphosphate. Therefore, we will measure the specificity of this effect and the effects of lithium on protein kinase C activity and inositol polyphosphate metabolism. 3) Lithium potentiates the in vivo response of the CNS to the muscarinic agonist, pilocarpine, as seen by the seizures generated by these two drugs. We have hypothesize that both presynaptic and postsynaptic effects of lithium play a role in this response. Presynaptic effects are discussed in #1 above and postsynaptic effects in #2 above. We will continue to test our major hypothesis by investigating whether lithium potentiates the effects of other cholinomimetics, including arecoline, physostigimine (an inhibition of acetylcholinesterase) and carbachol, an agonist which stimulates a much greater response by the phosphoinositide system than does pilocarpine. These studies are designed to test the hypothesis that lithium enhances cholinergic activity in the brain and to test the stated hypotheses as to the specific mechanisms of these effects of lithium. Attainment of these goals will increase our understanding of the effects of lithium that may be related to its therapeutic effect in mania and may generate hypotheses as to the underlying causes of bipolar affective disorders.

This project uses electrophysiological and biochemical methods to study a new model of status epilepticus. This model uses pretreatment with lithium followed by administration of subthreshold doses of convulsants. The basic model entails treatment with LiCl (3 mEq/kg; ip) and pilocarpine (30 mg/kg; sc) which invariably produces generalized convulsive status epilepticus which lasts for several hours and is fatal. There are three major goals of this proposal. (1) We will test the hypothesis that lithium lowers the seizure threshold to other convulsive stimuli, including ECS, bicuculline, picrotoxin, pentylenetetrazole, kainic acid and chemical (carbachol) and electrical kindling. (2) We will test the hypothesis that function of the GABAergic system is impaired during status epilepticus by measuring the state of the GABA/benzodiazepine/picrotoxin chloride channel complex during seizures. (3) We will test the hypothesis that alteration of calcium homeostatis plays a role in status epilepticus by testing the anticonvulsant and proconvulsant actions of calcium antagonists and a calcium agonist, respectively, and the effects of seizures and drug treatments on calcium homeostasis in synaptosomes.

Understanding the functional status of signal transduction systems in Alzheimer's disease (AD) and the endogenous regulatory processes for these signal transduction systems is vital for the treatment of this disorder. In AD, cholinergic neurons degenerate but postsynaptic muscarinic receptors remain intact, which led to the therapeutic use of cholinergic agonists, including cholinesterase inhibitors. however, the functional state of the receptors, which are coupled to phosphoinositide (PI) hydrolysis, is unknown. We recently developed a method to measure agonist-induced, GTP- dependent PI hydrolysis in postmortem human brain membranes. Therefore we can now study the functional status of the therapeutically targeted PI- linked receptors in AD and initial studies revealed impaired cholinergic- stimulated PI hydrolysis in AD. Therefore this is the focus of the first specific aim. Understanding how the activity of the PI second messenger system is regulated and is impaired in AD is of critical importance. Neither aging nor lesions cause decreased PI activity, as is seen in AD, but glucocorticoid hormones (e.g., cortisol), which are elevated in AD and which exacerbate age- and toxin-related neurodegeneration, impair the Pi response. Therefore identifying modulatory interactions of glucocorticoid hormones on signal transduction systems associated with PI hydrolysis is the focus of the second specific aim. The first specific aim is to test the hypothesis that agonist-induced PI metabolism can be measured in postmortem human brain, that it is impaired in AD, and that G-protein dysfunction contributes to the impairment. PI hydrolysis will be measured in response to activation of phospholipase C, G-proteins, and several receptor subtypes by agonists in SAD and age- matched control brain regions, using a variety of experimental methods. Western blots will b used to measure protein levels of components of this system, including phospholipase C, G-protein subtypes, and protein kinase C, and G-protein function will be measured. The second specific aim is to test the hypothesis that elevated glucocorticoid hormones impair rat brain signal transduction systems associated with the PI second messenger system, especially when coupled with excitotoxic lesions. Rats will be adrenalectomized, administered glucocorticoids and/or kainate. Cortical and hippocampal IP metabolism, G- protein levels and function, protein kinase C, and gene expression will be measured. The results of this project will greatly increase our knowledge of the function of the PI signal transduction system in AD, will identify specific sites impaired in AD, will identify how glucocorticoids modulate its activity in rat rain, and will identify consequences of glucocorticoid effects at the level of gene expression.

Because of the association of oxidative stress with widespread debilitating disorders of the CNS, it is imperative to identify how signaling systems are affected by oxidative stress. This project is focused on testing specific hypotheses concerning the effects of oxidative stress on signaling systems linked to cholinergic muscarinic receptors. These receptors are coupled to the phosphoinositide (PI) signal transduction system, increases in protein tyrosine phosphorylation, and downstream transcription factor modulation. Deficient PI signaling has been reported in Alzheimer's disease, which is centered on dysfunction of the G- proteins that mediate signal transduction. Human neuroblastoma SH-SY5Y cells provide an optimal model system to study because they express m3 muscarinic receptors which mediate robust stimulation of the PI signal transduction system, intracellular increases in protein tyrosine phosphorylation, and activation of transcription factors such as AP-1 and NFkappaB, each of which appears to be important in cellular responses to oxidative stress. The overall goal is to test the hypothesis that oxidative stress modulates specific sites in these three signaling components, PI hydrolysis, protein tyrosine phosphorylation, and transcription factor activation. Three complementary approaches will be used to model oxidative stress, exposure of SH-SY5Y cells to (a) H2O2, or (b) peroxynitrite, or (c) the use of "cybrid" SH-SY5Y cells in which endogenous mitochondria have been replaced with mitochondria from Alzheimer's disease or matched control subjects. The Alzheimer's disease-derived cybrid cells thus possess defective cytochrome c oxidase and produce excessive reactive oxygen species. Specific Aim 1 will test the hypotheses that oxidative stress impairs muscarinic receptor-induced PI hydrolysis and that inhibition of the G-protein Gq/11 is a critical site of action. Specific Aim 2 will test the hypotheses that oxidative agents impair the palmitoylation of cysteines on the G-protein Gq/11 and that inhibition of Gq/11 palmitoylation impairs PI hydrolysis. Specific Aim 3 will test the hypotheses that oxidative stress alters intracellular protein tyrosine phosphorylation, including substrates responding to muscarinic stimulation. Specific Aim 4 will test the hypothesis that oxidative agents inhibit the tyrosine phosphorylation of Gq/11 and will test if this is associated with inhibition of Gq/11 palmitoylation. Specific Aim 5 will terst the hypothesis that oxidative agents modulate transcription factor activation, including those activated by muscarinic receptor stimulation.

DESCRIPTION (provided by applicant): Glycogen synthase kinase-3beta (GSK3b) is linked to most key aspects of Alzheimer's disease. These include: GSK3b phosphorylates tau and amyloid precursor protein, Abeta peptide activates GSK3b and inhibition of GSK3b protects from Ab-toxicity, and presenilin-1 binds and regulates the activity of GSK3b, actions altered by mutant presenilin-l. Also, GSK3b impairs neural plasticity, facilitates apoptotic signaling cascades, and inhibits the activities of multiple transcription factors (CREB, AP-1, NFkB, myc, b-catenin, and others), all actions likely important in Alzheimer's disease. These actions and associations indicate that GSK3b may be an important modulator of neuropathological processes associated with Alzheimer's disease as well as other neurodegenerative conditions, but much remains to be learned about the actions of GSK3b. The overall goal of this project is to investigate mechanisms regulating GSK3b and to delineate its effects on cell function, especially neural plasticity and apoptosis. The aims are based on our findings that (i) thapsigargin, which increases intracellular calcium levels and causes endoplasmic reticulum (ER)-stress, conditions associated with Alzheimer's disease, activates GSK3b, and GSK3b is obligatory for thapsigargin-induced apoptosis, (ii) apoptotic stimuli cause intranuclear accumulation of GSK3b, and (iii) GSK3b inhibits the function of the key transcription factor CREB. Specific Aim 1 will test the hypothesis that GSK3b is activated by, and is a critical mediator of, toxicity induced by thapsigargin and other agents perturbing calcium or the ER, and will identify the mechanisms involved in GSK3b activation and assess the regulatory roles of GSK3b-binding proteins. Specific Aim 2 will test the hypothesis that apoptotic stimuli induce nuclear accumulation of GSK3b, identify the mechanisms controlling the intranuclear distribution of GSK3b, and test if nuclear GSK3b contributes to apoptotic signaling. Specific Aim 3 will test the hypothesis that GSK3b has dual functions in apoptosis, both attenuating antiapoptotic signals, with a focus on survival-promoting transcription factors, and facilitating proapoptotic signals connecting ER stress to caspase activation. Overall, these experiments will clarify mechanisms regulating GSK3b and its effects on neural plasticity and survival.

[unreadable] DESCRIPTION (provided by applicant): Oxidative stress may be the single most prevalent cause of neuronal dysfunction in neurodegenerative disorders, and its prevalence underscores the need to clarify mechanisms causing and attenuating the deleterious effects, the overall goals of this project. We report exciting and novel results: (1) DNA damaging agents that elevate p53 cause a novel mechanism of activation of the pro-apoptotic glycogen synthase kinase-3b (GSK3b). (2) Oxidative stress induces RGS2 (Regulator of G-protein Signaling 2) expression, a G-protein GTPase-activating protein, providing a mechanistic basis for impaired signaling. (3) Stimulation of muscarinic receptors greatly attenuates oxidative stress-induced apoptosis, remarkably as effectively as a general caspase inhibitor. These results provide important new insights about mechanisms that contribute to oxidative stress-induced impairments and about mechanisms capable of attenuating the deleterious effects. Specific Aim 1 will test the hypothesis that oxidative stress and DNA damage activate p53-mediated signaling encompassing recruitment of GSK3b by a novel activation mechanism. We will test the hypotheses that p53-induced activation of GSK3b leads to inhibition of survival-promoting transcription factor substrates of GSK3b, and promotes responses to p53, identify the p53-binding domain on GSK3b, determine if p53 binding alters the association of GSK3b with other proteins, identify the GSK3b-binding domain on p53 and determine if GSK3b binding alters p53 functions. Specific Aim 2 will test the hypothesis that oxidative stress and DNA damage induce the expression of RGS2 which attenuates muscarinic receptor-coupled signaling and facilitates oxidative stress-induced apoptosis. We will identify the signal mediating H202-induced increases in RGS2, Determine if H202-induced increases in RGS2 impair muscarinic receptor-coupled signaling, and test if IGS2 expression is pro-apoptotic role after oxidative stress. Specific Aim 3 will test the hypothesis that stimulated muscarinic receptors protect cells from oxidative stress, identify the blocked site in -1202-inducedsignaling, test the hypothesis that muscarinic receptors provide protection from other apoptotic conditions, identify the signaling pathways activated by muscarinic receptors providing protection, and test the hypothesis that activation of Rho family small G-proteins is protective.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to identify cellular mechanisms of action of lithium and other mood stabilizers in order to contribute to understanding the underlying pathophysiology and to enhance treatment of mood disorders. To achieve this, four specific aims are to be addressed. 1. Lithium is a direct inhibitor of glycogen synthase kinase-3 (GSK3), and recently we showed this action is amplified in vivo in mouse brain by phosphorylation of select pools of GSK3, substantially increasing the inhibitory effect of a therapeutic level of lithium. Specific Aim 1 will examine this amplification mechanism inhibiting the activity of GSK3, the influence of lithium, and this mechanism will be examined in Aims 2-4. 2. We recently discovered that serotonergic activity inhibits GSK3 in mouse brain in vivo, revealing a direct link between serotonergic activity and lithium's target. Specific Aim 2 will examine the in vivo mechanisms by which serotonergic activity regulates GSK3, the serotonin receptor subtypes that are involved, the effects of mood stabilizers and antidepressants, and signaling pathways involved. 3. The goal of the third aim is to establish an in vivo model of pathological activation of GSK3 that is counteracted by mood stabilizers and antidepressants. We will follow-up our recent findings that In mouse brain in vivo hypoxia rapidly and robustly activates GSK3 and this is blocked by in vivo administration of mood stabilizers or imipramine. Specific Aim 3 will test the hypothesis that hypoxia activates GSK3 and mood stabilizers and antidepressants attenuate this GSK3 activation and will investigate mechanisms underlying these interactions in cultured cells, since this provides the only model of pathophysiological GSK3 activation in vivo that is attenuated by these therapeutic agents. 4. Substantial evidence indicates that neurogenesis may be impaired in mood disorders and be promoted by therapeutic agents. Specific Aim 4 will test the hypothesis that GSK3, which often promotes apoptosis, is activated during apoptosis of neural precursor cells and that inhibition of GSK3 promotes survival. This aim will also test if inhibition of GSK3 promotes precursor cell differentiation or survival of differentiated cells. Overall, this project will continue to provide leading-edge insight into mechanisms that may underlie the pathology of mood disorders and the actions of mood stabilizers. [unreadable] [unreadable] [unreadable]

Modified Project Summary/Abstract Section: RO1 MH095380 Novel targets need to be identified to develop new therapies for depression, a prevalent and debilitating disease often not adequately treated. The immune system is one such novel target, as substantial evidence demonstrates its involvement in depression. While previously the CNS was considered insulated from the immune system, it is now well-established that immune cells in the CNS modulate multiple processes, such as neurogenesis and cognition. The immune system includes a rapid-response innate immune system that produces cytokines and chemokines that activate and recruit the adaptive immune system, which includes T cells that possess the capacity of memory. Many CNS functions, such as cognition and mood, are affected by cytokines, but less is known about the regulation and functional effects of T cells in the CNS associated with depression. The overall hypothesis of this project is that T cells have subtype-specific regulatory effects on the susceptibility to depression and responses to antidepressants. We will examine the influences of T cell subtypes on susceptibility to depressive-like behavior, and examine the role of glycogen synthase kinase-3 (GSK3) as a regulatory mechanism linking immune responses with susceptibility to depression. These goals will identify new mechanisms by which the adaptive immune system may contribute to depression and be a novel target for therapeutic intervention. Specific Aim 1 will test the hypothesis that T cell subtype-selective actions in the brain modify depressive-like behavior. T cell depletion and transfer approaches will be used to identify consequences of T cell subtypes on susceptibility to depression-like behavior. Our Preliminary Results show accumulation of inflammatory Th1 and Th17 cells in the brains of mice exhibiting depression-like behavior, that Th1 cell depletion exacerbates depression-like behavior, that Th17 cell depletion ameliorates depression-like behavior, and that Treg repletion has antidepressant effects. Specific Aim 2 will test the hypothesis that glycogen synthase kinase-3 (GSK3) is a critical link between immune activation and susceptibility to depressive-like behavior. Our Preliminary Results show that mice expressing constitutively active GSK3 (GSK3 knockin mice) display increased susceptibility to depression-like behavior and that GSK3 drives the production of inflammatory Th17 cells. Because GSK3 promotes depression-like behavior, is linked to human depression, regulates the generation of T cell subtypes, and promotes inflammatory responses, we will apply molecular and pharmacological manipulations of GSK3 to test if GSK3's immune effects contribute to its promotion of susceptibility to depression.