Lynn Wecker, Ph.D. - US grants

Experimental evidence suggests that while the administration of choline may not alter the steady-state concentration or turnover of acetylcholine (ACh) in brain under "normal" physiological conditions, it does enhance the synthesis of ACh under conditions of increased neuronal demand for or a decreased endogenous supply of the neurotransmitter precursor. Although the specific mechanisms involved in the utilization of exogenous choline for the synthesis of ACh during conditions of increased neuronal activity have not yet been elucidated, results indicate that the mobilization of free choline from specific pools of bound choline plays a significant role. Furthermore, evidence suggests that the mechanisms regulating the metabolism of ACh may depend on the functional state of cholinergic neuronal activity. Hence, it is the specific aim of this research proposal to investigate further the relationships among neuronal activity, the disposition and utilization of administered choline and central cholinergic mechanisms. These studies will involve measurements of: synaptosomal choline uptake; steady-state concentrations of ACh, choline, phosphorylcholine, CDP-choline, glycerophosphorylcholine, lysophosphatidylcholine and phosphatidylcholine; and the incorporation of choline into and release from choline-containing compounds. Choline will be administered through injection and by dietary supplementation and cholinergic neuronal activity in various brain regions will be altered by pharmacological and physical manipulations. Results from these studies will provide valuable information on the relationship between neurotransmitter precursor availability and the activity of central cholinergic neurons which is essential for understanding the intrinsic mechanisms involved in the regulation of the metabolism of choline and ACh in brain. Furthermore, results will provide a basis to better assess the therapeutic rationale for and possible merits of the use of choline for the treatment of neuropsychiatric disorders postulated to involve hypocholinergic activity such as tardive dyskinesia, mania, Huntington's disease and Alzheimer senile dementia.

Experimental evidence to date indicate that although supplementation with choline does not alter the steady-state concentration of acetylcholine (ACh) in brain under normal biochemical and physiological conditions, it does support the synthesis of ACh during drug-induced increases in neuronal demand. Although the specific mechanisms involved in the utilization of supplemental choline for the synthesis of ACh during states of increased neuronal activity have not yet been elucidated, results indicate that this effect is not due to increased levels of free choline in brain, but rather, may involve alterations in the concentration or metabolism of choline-containing compounds. Hence, it is the overall objective of this research proposal to investigate the neurochemical mechanisms mediating the effects of choline supplementation. Neurochemical and neuropharmacological techniques will be used to address the following questions: 1) Do chronic alterations in the availability of choline alter the dynamics of ACh metabolism? 2) Is the utilization of supplemental choline for the synthesis of ACh dependent on the level of activity of cholinergic neurons? 3) What are the sources of choline mediating the effects of choline supplementation? 4) What are the mechanisms regulating the metabolism of choline at nerve terminals? 5) What is the nature and physiological significance of the serum factor whose activity or concentration is induced by choline supplementation and whose role may be to regulate the transport of choline across the blood-brain barrier? 6) What are the functional consequences of choline supplementation? and 7) Are the effects of choline supplementation observed in brain from adult rats manifest in brain from aged animals? Results from these studies will provide information on the relationship between neurotransmitter precursor availability and central cholinergic mechanisms which is essential for understanding the intrinsic mechanisms regulating the metabolism of choline and ACh in brain. Furthermore, results may provide a basis to better assess the therapeutic rationale and possible merits of the use of choline for the treatment of neuropsychiatric disorders postulated to involve hypocholinergic activity such as Alzheimer's Disease.

The major goal of this research proposal is to elucidate the mechanisms regulating the synthesis of the neurotransmitter acetylcholine (ACh) in brain, with an emphasis on determining how alterations in the availability of the precursor choline modify these processes. Evidence has supported the hypothesis that an increased supply of choline to the brain provides substrate for ACh synthesis that is of functional significance only when neurotransmitter synthesis is increased as a consequence of stimuli that increase ACh release. While this effect is manifest following the acute parenteral administration of choline, it has not been demonstrated following chronic dietary supplementation, despite evidence that both treatments increase choline availability in the brain. Furthermore, when fire choline is excluded from the diet, although steady-state levels of choline in brain are unaltered, the mobilization of free chorine from esterified sources decreases, with a concomitant reduction in the synthesis of ACh. Thus, the specific aim of this proposal is to elucidate the neurochemical mechanisms regulating the synthesis of ACh in brain, and determine, at the subcellular level, how alterations in choline availability modulate these processes. The studies outlined will use a combined in vivo/in vitro approach and investigate the effects of acute chorine administration, as well as the consequences of chronic dietary alterations; for the latter, rats will be maintained on chorine-deficient or chorine-supplemented diets for one month. Brain slices from these animals will be used for neurochemical investigations in vitro. The synthesis and release of ACh, the release and production of free chorine, and the esterified sources of chorine that provide precursor for ACh synthesis will be characterized in subcellular fractions from brain regions that contain a dense population of cholinergic nerve terminals, viz., striatum, hippocampus, and cerebral cortex. Specifically, the experiments will investigate: 1) the subcellular mechanisms responsible for the increased synthesis of ACh in brain slices from chorine-injected rats when these slices are exposed to stimuli that increase the demand for precursor by increasing neurotransmitter release; 2) whether chronic (dietary) supplementation with chorine has a direct effect on cholinergic neurons or whether observed in vivo effects are secondary to generalized membrane phospholipid perturbations; 3) the mechanism mediating the decreased synthesis of ACh in brain from rats fed a choline-deficient diet; and 4) the interactions among neuronal activity, the demand for choline, and phospholipid and ACh metabolism. Results from these studies will determine the nature and localization of the esterified choline pool that supplies free choline for ACh synthesis, and how this source is modulated by altering the availability of precursor. This knowledge is essential for a basic understanding of brain function and how such function can be impaired by the dietary restriction of an essential nutrient such as choline. Furthermore, results will provide a basis for the development of possible therapeutic strategies for the treatment of neuropsychiatric disorders postulated to involve central hypocholinergic activity such as Alzheimer's disease.

The objective of the research is to elucidate the role and mechanism of action of cytokines mediators of the acute phase response in regulation of cytochrome P450 (P450), a family of hemoproteins responsible for biotransformation of many drugs, other chemicals, and certain endogenous substrates. The cytokines to be studied are interleukin 6 (IL-6), interleukin 1 (IL-1), interferon (INF), and tumor necrosis factor (TNF). Cytokine effects on P450 may explain altered biotransformation occurring during infection and inflammation. In addition, effects on P450 by cytokines when used in cancer immunotherapy or other immune disorders may result in drug-drug interaction, influencing the disposition and toxicity of concurrently used drugs. Although effects on P450 have been shown during the acute phase response, or after the administration of cytokines, because of the complex interplay among the cytokines to affect each others synthesis, release, and biological activity it is unclear from in vivo studies which cytokines directly regulate P450. The project will use cultured rat hepatocytes to determine which cytokines directly regulate P450, and if P450 isoforms are differentially affected by cytokines. Rat hepatocytes will be cultured on Matrigel matrix in Williams E medium containing insulin and dexamethasone. Recently, such rat hepatocyte cultures been shown to be inducible by phenobarbital (PB) for P450IIB 1/2 to levels obtained in vivo. Preliminary results with hepatocyte culture demonstrated a direct effect of IL-6 to inhibit the PB induction of P450IIB 1/2. Hepatocyte cultures will be used to further elucidate the concentration response and temporal relationship for IL-6 to alter PB induction of benzyloxyresorufin O-deethylase activity, a measure of P450IIB1/2 enzymatic activity. Western blot analysis using antibodies to P450IIB1/2 will be used to study effects of IL-6 on the level and turnover of P45011B 1/2 protein. Solution hybridization on total RNA of hepatocytes will be performed using a labelled cDNA oligonucleotide probe for P450IIB1 and P450IIB2 mRNA to evaluate effects of IL-6 on mRNA level and turnover. Transcription rate will be determined using nuclear run-off assay. Similar experiments will be used to evaluate the ability of IL-1, TNF and INF to inhibit P45011B 1/2 induction. Thirdly, the project will determine if IL-6, IL- 1, TNF, and INF affect the induction of other P450 isoforms in rat hepatocyte cultures. Effects of cytokines on 3- methylcholanthrene induction of P450IA1/2 and ethanol induction of P450IIE1 will be examined. Ethoxyresorufin O-deethylase activity, and p-nitrophenol hydroxylase activity will be assayed as indices of P450IA1/2, and P450IIE enzymatic activity, respectively. Specific antibodies and labelled cDNA oligonucleotide probes for P450IA1/2, and P450IIE1 will be used in Western blot analysis and solution hybridization assays, respectively, to assess cytokine effects on level and turnover of protein and mRNA. Knowledge of the effects of cytokines on P450 is of utmost importance for understanding alterations in pharmacokinetics during infection or inflammation, as well as during cancer immunotherapy.

DESCRIPTION (provided by applicant): Nicotine is a potent central nervous system stimulant whose effects are mediated through interactions with neuronal nicotinic receptors, a family of receptors composed of a or a and 13 subunits. Receptors containing a4 and 132 subunits are the most abundant receptors in the brain, and exhibit high affinity for agonist. These receptors inactivate and exhibit an increased density following sustained exposure to nicotine, although the mechanisms mediating these effects are not clearly unders aboutod. Based on studies indicating that a4132 receptors either isolated from rat brain or expressed in Xeno pus oocytes are phosphorylated by protein kinases, that the surface expression and function of a4B2 receptors stably expressed in HEK 293 cells are modified by activators and inhibitors of protein kinases, and that nicotine enhances receptor phosphorylation, it is likely that these receptors may be regulated by phosphorylation/ dephosphorylation mechanisms, post-translational processes that regulate other ligand-gated ion channels. The goal of these studies is to elucidate the role of phosphorylation/dephosphorylation mechanisms in mediating the effects of the chronic administration of nicotine or nicotinic agonists. This goal will be achieved by testing the hypothesis that a4 subunits of a4132 neuronal nicotinic receptors are phosphorylated/dephosphorylated by specific protein kinases/phosphatases and that alterations in the phosphorylation state of the a4 subunit following sustained exposure to nicotine or nicotinic agonists alters the function of the receptor. To test this hypothesis, cellular and molecular studies will be carried out using Xenopus oocytes expressing rat a4B2 receptors, SH-EPI cells stably expressing human a4B2 receptors, transiently transfected SH-EP-1 cells expressing mutant a4 subunits with wild-type 132 subunits, primary neuronal cultures, fusion proteins corresponding to the major cytoplasmic domain of the a4 subunit, and synthetic peptides corresponding to sequences within the cytoplasmic domain of the a4 subunit that contain putative phosphorytation sites. These studies will determine: I) whether changes in the phosphorylation state of the receptor affect its function; 2) which amino acids are phosphorylated/dephosphorylated and wnich enzymes are involved; 3) how nicotine and nicotinic agonists alter the phosphorylation/ dephosphorylation of the receptor; and 4) whether phosphorylationidephosphorylation alters receptor function. Results will provide knowledge on the regulation of a4132 receptors which is necessary for understanding both normal brain function and the long-term consequences of exposure to nicotine and nicotinic agonists, pharmacological agents that have been and continue to be abused, as well as used therapeutically.

DESCRIPTION (provided by applicant): There is no current pharmacological treatment to alleviate the imbalance and lack of voluntary muscle coordination manifest by individuals with ataxia. Ataxic movements affecting balance and coordination may be a consequence of either hereditary diseases or non-hereditary causes, and typically may be ascribed to a loss of function in the cerebellum or its associated afferent or efferent pathways. Although knowledge of the genetic basis of several cerebellar ataxias is increasing at a rapid pace and may lead to gene therapy approaches for treatment, this area is in its infancy. In addition, because many ataxias are a consequence of a cerebellar insult and do not involve genetic alterations, there is a need for the development of therapeutic agents to alleviate the symptoms associated with these disorders. Clinical studies have indicated that varenicline, a partial agonist at a4b2 and full agonist at a7 neuronal nicotinic receptors, improves balance and coordination in patients with ataxias of distinct pathogenic etiology, and recent preclinical studies in our laboratory have provided proof-of-principle that neuronal nicotinic receptor agonists prevent the progression of and/or improve motor behavior in an animal model of olivocerebellar degeneration. Based on these findings, the overall goal of this proposal is to further characterize the ability of neuronal nicotinic receptor agonists to alleviate ataxia in animal models and identify the cellular mechanisms involved. The overall hypothesis to be tested is that the partial activation of a4b2 and/or full activation of a7 neuronal nicotinic receptors in the cerebellum or inferior olive leads to the increased expression and release of insulin-like growth factor (IGF-1) in the cellular milieu, which shifts the balance between pro-apoptotic and anti-apoptotic signaling to favor the latter. Through a complementary and parallel series of in vivo and in vitro studies involving both diverse animal models and tissue and cell-based assays, this translational proposal will use a classical pharmacological approach to identify the specific receptor subtypes mediating the anti-ataxic effects of neuronal nicotinic receptor agonists and determine whether these compounds can alleviate ataxias resulting from different chemical and genetic insults. Further, through measures of key molecules involved in apoptotic signaling, studies will ascertain whether nicotinic receptor agonists promote cell survival, and whether this action is a consequence of a nicotinic receptor-mediated increased expression of IGF-1. Results will lead to the development of new therapeutic agents for the treatment of these disorders for which there is no current efficacious pharmacological therapy.