Keith W. Kelley - US grants

hormone regulation /control mechanism; thymus; aging;

DESCRIPTION: The present application proposes to explore the role of proinflammatory cytokines in the brain. The investigators contend that although a great deal of knowledge regarding the role of proinflammatory cytokines in cachexia exists, there has been little investigation into systemic effects of CNS produced or administered cytokine. The investigator's working hypothesis is that a major component of the wasting of AIDS is caused directly by cytokines produced within the CNS. The studies proposed contain three objectives: 1) to compare the wasting properties of the three proinflammatory cytokines (TNF alpha, IL-1 beta and IL-6) injected into the lateral ventricle of the brain; 2) to identify and localize the receptors for TNF, IL-1 and IL-6 in the brain responsible for the cachexia; and 3) to employ novel approaches to block cytokine induced wasting by administering to the CNS cytokine antagonists. In this manner, the investigators state that they will address critically important, but as yet relatively unexplored area in the pathogenesis of wasting by carefully defining the role of cytokines and their receptors in the CNS.

Many infectious diseases, including AIDS, are accompanied by profound neuropsychological and behavioral alterations, even when the causal agent is not neurotrophic. AIDS patients experience neurological symptoms such as memory loss, dementia, drowsiness, weakness and motivational defects. Recent evidence indicates that these symptoms are caused by the neural effects of proinflammatory cytokines of which the predominant one is interleukin- 1 (IL-1). These data have led to the idea that peripheral cytokines released during inflammation lead to development of a new motivational state which we have designated as sickness behavior. The next major challenge in this field is to develop an understanding of how cytokines released in the periphery communicate this signal to the brain. Here we hypothesize that sensory afferent nerves are activated by peripheral immune stimuli which subsequently lead to synthesis of cytokines in distinct structures of the CNS. These central cytokines induce sickness behavior by acting on their receptors which are located on specific neuronal nuclei. Our first objective is designed to investigate the novel hypothesis that peripheral immune stimuli activate sensory afferent nerves which relay this message to the brain. Specifically, we will test whether transection of afferent nerves from the abdominal cavity abrogates the reduction in social exploration caused by peripheral activation of the immune system. We will then try to confirm that peripheral immune stimulation activates sensory afferent nerves by measuring tachykinins in the afferent nerves. We have recently shown that induction of sickness caused by activation of the peripheral immune system leads to the synthesis of IL-1 in the CNS. In the second objective, we will extend these findings by quantitatively measuring the amount of IL- 1alpha and IL-1beta transcripts (competitive RT-PCR) that are expressed in specific brain structures (hippocampus, hypothalamus, cortex, striatum, thalamus) in response to peripheral immune activation. We will then assess the consequences of transection of afferent nerves on expression of these cytokines within the CNS. Next, we will determine whether changes in IL-I expression in the CNS are responsible for the sickness-inducing properties of peripheral IL-1 by microinjecting intact mice with anti-IL-1alpha and IL-1beta neutralizing antibodies into the lateral ventricle of the brain. Based on our newest findings that mRNA for both isoforms of IL-1 receptors are present in mouse brain, the third objective is designed to characterize the nature and cellular localization of both central and peripheral IL-1 receptor proteins that mediate the behavioral effects of this cytokine. These experiments will employ both knock-out mice for the type I IL-1 receptor and i.c.v. administration of specific blocking antibodies directed against each receptor subtype. A major strength of this proposal is that we have developed all of the techniques and also obtained IL-1 receptor knock out mice to successfully test our hypothesis that peripheral nerves are used as an afferent pathway for the induction of sickness caused by activation of the peripheral immune system. These data are needed to understand and perhaps prevent the behavioral alterations and disturbances in mental health that accompany a wide variety of infectious diseases.

DESCRIPTION (provided by applicant): A major advance in understanding the neurobiological basis of normal and pathological behavior was the discovery that proinflammatory cytokines act in the CNS to alter behavior and induce affective and cognitive disorders. Proinflammatory cytokines can act in the neuroimmune system to alter behavior by regulating synthesis and release of several neurotransmitters. We now have data to show that TNFalpha can also act in conjunction with classical neurotransmitters to change behavior. In both cases, very little is known about endogenous protective molecules that regulate actions of proinflammatory cytokines in the brain and hence their behavioral effects. We have exciting new preliminary evidence in vivo showing that IGF-1 inhibits both sickness behavior induced by TNFalpha and spatial memory disorders that are induced by glutamate/kainate in a TNFalpha-dependent manner. We have extended these in vivo findings by showing that TNFalpha exacerbates neuronal stress only in the presence of glutamate and kainate and that IGF-I protects neurons from these insults in vitro. In Objective 1, we will further assess the possibility that IGF-I ameliorates the affective and cognitive effects of TNFalpha. Mice deficient in TNF receptors and a critical intracellular TNF receptor signaling protein known as FAN will be used. Changes in social exploration, immobility and rearing activities will used to quantify sickness behavior following i.c.v, injection of TNFalpha Objective 2 will use both TNFalpha- and TNF receptor-deficient mice to determine the in vivo contribution of TNFalpha to kainate-induced loss of spatial memory. This objective will then confirm the role of IGF-I in preventing this form of cognitive disorder. Objective 3 will explore the novel possibility that IGF-I-induced signaling proteins directly target glutamate receptors and increase expression of survival proteins to reduce excitation-induced neuronal stress. Experiments in Objective 4 will delineate the molecular signals by which TNFalpha promotes neuronal dysfunction and loss of spatial memory by using mice that lack TNF receptors or FAN and determining if TNFalpha increases glutamate receptor activation. This objective will also determine how IGF-I counterbalances TNFalpha-induced neuronal stress. These studies are needed to learn how TNFalpha interacts with an endogenous protective factor in the brain. These innovative experiments will advance our understanding of brain and immune communication systems that control the neurobiological basis of behavioral disorders

DESCRIPTION (provided by applicant): The anemia, neutropenia, loss of lean body mass and mortality of AIDS patients with wasting are associated with elevated levels of the proinflammatory cytokines TNFalpha and IL-1beta. AIDS patients with wasting are often given a 12- week therapy with very high doses of recombinant human growth hormone to increase plasma IGF-I, lean muscle mass and quality of life. However, the responsiveness of both hematopoietic and muscle cells to IGF-I has been documented to be defective in these patients. The central hypothesis of this application is that TNFalpha and IL-lbeta are responsible for inducing a state of IGF-I receptor resistance, which contributes to not only muscle wasting but also to the anemia and neutropenia of AIDS. IGF-I targets both hematopoietic myeloid progenitor cells and muscle myoblasts, and here we hypothesize that the molecular mechanism for IGF-I receptor resistance in wasting AIDS patients is caused by proinflammatory cytokines. Objective 1 will test the idea that IGF-I promotes promyeloid cell survival by blocking activation of the caspase family of serine proteases and whether this is inhibited by TNFa. Objective 2 focuses on the survival promoting activity of the tyrosine phosphorylated IGF-I receptor, including insulin-receptor substrate- 1 (IRS-1), IRS-2, PI 3-kinase and Akt. Objective 3 will determine if proinflammatory cytokines inhibit key IGF-I proliferative signals, including Shc, EFK1/2, AFX forkhead transcription factors and the cyclin-dependent kinase inhibitor 27. Finally, objective 4 will extend these results with myeloid progenitor cells to muscle myoblasts. Preliminary results indicate that low blood concentrations of TNFalpha and IL-1beta found in wasting AIDS patients inhibit the ability of IGF-I to promote both protein synthesis and differentiation into myotubes. This objective will also test the new idea that ceramide, a mediator of the actions of both TNFalpha and IL-1beta, induces resistance of the IGF-I receptor in muscle myoblasts. These studies are needed to understand how clinically-relevant concentrations of proinflammatory cytokines in wasting AIDS patients impair the functional ability of a major hormone receptor on both immune and muscle myoblast cells.

[unreadable] DESCRIPTION (provided by applicant): Inflammation is now recognized to be responsible for major health problems of the aging population, contributing to costly diseases such as obesity, the metabolic syndrome, heart disease and insulin resistance in type-2 diabetes. The chronic process of even healthy aging is also associated with development of low grade inflammation. However, age-related changes in inflammation have mainly been considered in relation to systemic disorders. We and others have now collected substantial evidence to show that this inflammation status, as defined by the overexpression of proinflammatory cytokines, is not restricted to the periphery but is also found in the brain. Brain inflammation causes symptoms of sickness that are usually associated with microbial infections, which is likely to make an important contribution to the comorbid behavioral and psychological disturbances that occur in the elderly. Indeed, one in five individuals over the age of 65 suffer from depressive disorders, which is more than twice the prevalence found in the general population. A likely mechanism for the increased prevalence of depressive disorders during aging is a reduction in the synthesis of serotonin, a key neurotransmitter in the regulation of mood, caused by proinflammatory cytokines acting in the brain. This action is mediated by immune-induced activation of the tryptophan-degrading enzyme, indoleamine 2,3 dioxygenase (IDO). This process decreases the bioavailability of tryptophan for the synthesis of serotonin. Our preliminary data indicate that peripheral immune activation activates IDO and induces depressive-like behavioral alterations, and these effects are exacerbated in aged compared to adult mice. Based on this evidence, we propose that peripheral immune activation precipitates the occurrence of mood disorders in aged individuals. We propose to test this hypothesis in aged mice exposed to both acute and chronic peripheral immune activation, two events that we have already shown to increase brain IDO activity. In the first objective, we will determine whether the depressive-like behavioral alterations that develop in response to both acute and chronic peripheral immune activation are exacerbated in aged mice. In the second objective, we will assess the role of increases in peripheral and brain IDO, as well as brain tryptophan and serotonin, in these behavioral changes, whereas the third objective will determine the contribution of brain glial cells to the age-associated increase in brain IDO. We will then use novel pharmacological approaches to determine if targeting brain inflammation (Objective 4) or brain IDO (Objective 5) attenuates the functional consequences of aging on development of depressive like behavior. These exciting experiments will be the first to use integrative neuroimmune approaches to evaluate IDO as the critical mediator between the age-related increase in peripheral and brain inflammation and the increased prevalence of mood disorders in aged individuals. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The present application aims at using neuroimmune approaches to understand the neurobiological basis of behavior. The objective is to study how dysregulated interactions between the nervous and immune systems contribute to comorbidity of depression and pain. Depressive disorders and chronic pain represent two major health burdens in the Western world. Chronic pain predisposes to depression and vice versa, and 30-60% of the cases suffer from both depression and chronic pain. The broad question we seek to answer using a neuroimmune approach is: Why is chronic pain a risk factor for depression, and vice versa? Peripheral inflammation causes sickness behavior that can culminate into depressive behavior when the tryptophan metabolizing enzyme 2,3 indoleamine dioxygenase (IDO) is upregulated. We recently made the exciting discovery that an intracellular protein known as G protein coupled receptor kinase 2 (GRK2) possesses anti- inflammatory properties and acts as a molecular switch that regulates transition from acute to chronic pain. Our newest preliminary data indicate that low GRK2 enhances IDO expression by microglia in vitro. This proposal will test the completely novel concept that GRK2 is a critical molecule that explains the shared risk for developing depression and chronic pain. We have shown that chronic neuropathic pain or inflammation significantly reduces GRK2 in microglia from rodents. This finding is clinically important because low GRK2 in microglia is sufficient to transform transient inflammatory pain into chronic pain. We also showed that low GRK2 augments pro-inflammatory cytokine production and increases activation of p38 in vivo and in vitro. Moreover, we have preliminary data that p38 activity regulates IDO expression, which is key to development of inflammation-associated depressive-like behavior. We hypothesize that the reduction in microglial GRK2 caused by chronic neuropathic pain increases microglial p38 activity, pro-inflammatory cytokine production and IDO expression, thereby acting as risk factor for prolonged depressive-like and pain behaviors. To test this hypothesis, we will answer the following specific questions: 1. Is development of neuropathic pain and depressive-like behavior temporally related to the inflammation-induced reduction in GRK2 and the increase in IDO in CNS microglia/mF? 2. Is low GRK2 a risk factor for development of depressive-like behaviors and what is the mechanism? We will use mice with low GRK2 in microglia that we have generated using Cre-Lox technology. 3. Are comorbid depression and chronic pain both prevented by treatments that interfere with the loop of reduced GRK2, increase in p38 activity and upregulation of IDO? Collectively, these innovative experiments will identify low GRK2 as a completely novel risk factor for development of comorbid depression and chronic pain via a p38/cytokine/IDO-dependent pathway. Identification of these new molecular mechanisms underlying comorbid depression and pain is needed for effective development of novel prevention and therapeutic strategies.