Bernard Agranoff - US grants

Experiments are directed at identifying biochemical events in the teleost retinal ganglion cell that mediate regrowth of the optic nerve and lead to recovery of visual function. The nature of altered radiolabeling of axonally transported proteins, including tubulin, as well as glycerolipids, terpenes and nucleotides will be investigated. A major effort is directed at biochemical characterization of explanted retinal cultures, as well as single ganglion cells and elucidation of the mechanism of enhanced outgrowth of neurites which results from prior crush of the optic nerve. These studies are directed at a better understanding of nerve regeneration in general, and in particular, as it appears within the central nervous system of both lower and higher vertebrates.

It has become increasingly apparent that the stimulated labeling of phosphatidate (PA) and phosphatidylinositol (PI), upon addition of muscarinic ligands to various tissues, is a reflection of lipid turnover in which the receptor-ligand interaction initiates the breakdown of inositide, especially of phosphatidylinositol 4,5 bisphosphate (PIP2). Inositol trisphosphate (IP3), the product of PIP2 cleavage, has been implicated as a second messenger. Our laboratory is particularly interested in the CNS muscarinic receptor in nerve endings (synaptosomes). We have chosen as auxiliary models the murine neuroblastoma cell (clone N1E-115) and the avian salt gland (duck), since like the nerve ending preparation, the cells do not engage in exocytotic secretion. In addition to the advantages of studying intact cells which seemingly share a common mechanism, there is the useful technical property that in these cells lipid changes associated with the receptor-ligand response will easily be distinguished from the more gross effects seen in lipid metabolism following exocytotic secretion seen in most other models. Parameters of interest associated with the intracellular response include changes in cyclin GMP and Ca2+. In addition, we hope to reconstruct a broken cell preparation in which the receptor-ligand interaction will directly affect the action of a phospholipase C-type phosphodiesterase which breaks down PIP2 to IP3 and diacylglycerol. A prior suggestion that PIP2 cleavage yields a 1,2-cyclic IP3 derivative (cIP3) will be reinvestigated in view of its possible messenger function. These experiments have relevance, not only in that they may constitute a novel second messenger system, but also that they may explain the therapeutic role of Li+ in affective disorders, since low concentrations of Li+ are known to affect the breakdown of intracellular inositol monophosphate produced from the degradation of the inositol lipids.

This program project merges two long-term and actively ongoing interests of this laboratory: the quantitative determination of muscarinic receptors in the brain, and the biochemistry of receptor activation via the phosphoinositide cleavage pathway. These approaches to understanding cholinergic signal transduction in the CNS, the first extracellular and the second intracellular, and here combined to explore experimental models of cholinergic dysfunction. Much of our biochemical knowledge regarding receptor function up to the present has been restricted to the receptor binding of labeled ligands in vitro to membranes, synaptoneurosomes or brain sections following acute or chronic exposure to a neuroactive agent. We here examine the effects of chronic in vivo conditions, such as deafferentation or exposure to drugs, not only upon the regulation of receptor number and affinity, but also on the regulation of the function of PPI-related second messengers, a consequence of receptor activation. These include: the diacylglycerol-activated protein kinase C and the mobilization of Ca2+ following inositol trisphosphate release. In vitro models of chronicity involve long-term exposure of neuroblastoma and primary neuronal or glial cultures to various anticholinergic agents, antidepressants and to Li+. A proposed developmental model is based on our observed enhanced coupling of muscarinic receptors to the cell response in fetal and neonatal brain. Possible interaction of muscarinic receptor regulation with other neurotransmitter-effector systems will also be explored. We will also investigate the role of phosphoinositides in presynaptic function, as well as the effects of the various drug treatments on acetylcholine release. Techniques that will be brought to bear upon this problem include 32P-labeled phosphoinositide turnover, (3H)inositol phosphate accumulation, and chemical determination of diacylglycerol and of the phosphoinositides. Inositol phosphate metabolism will be investigated, including the formation and breakdown of inositol trisphosphate and inositol tetraphosphate. Protein phosphorylation under control and receptor-regulated conditions will be examined with particular emphasis on protein kinase C- mediated reactions in synaptic vesicles and growth cones. Changes in receptor number and affinity will be localized via quantitative receptor autoradiography.

Biochemical, pharmacological and molecular biological approaches are integrated in an investigation of phosphoinositide (PPI)-mediated signal transduction in the CNS, with emphasis on PPI-linked muscarinic receptors (mAChR)s, toward the eventual goal of establishing the cellular basis of their regulation in normal and dysfunctional brain states. In vivo studies offer the advantage that physiological and anatomical relationships in the experimental preparation remain largely intact. They are complemented by studies in cultured cell lines of neural origin preparations that are vastly simpler and experimentally accessible. Together, the two approaches permit one to pose straightforward yet cogent experimental questions. Based on the known distribution of mRNAs for AChR isoforms in brain, we will explore their regulation using the combined approaches of receptor autoradiography, immunoprecipitation and mRNA hybridization methods. Diisopropylfluorophosphate- and trihexyphenidyl-treated rats will be used to study effects of chronic cholinergic stimulation. Cultured cell studies will explore distribution of mAChRs and the mechanism of homologous agonist-induced receptor sequestration, including the possible role for G-proteins in this process. A cell line expressing multiple PPI-linked receptors (in addition to adenyl cyclase-linked receptors) will be used to determine the extent of cross-talk among different receptors and intracellular signalling systems. We will explore to what degree IP3 receptor function may be regulated by cyclic AMP-dependent protein kinase. Effects of overexpression of key enzymes and receptors on signal transduction events will be investigated. Evidence in cultured cells will be brought to bear for or against the hypothesis that Li+ produces its therapeutic effect in manic depressive psychoses as a consequence of its inhibition of inositol monophosphate phosphatase (IP-Pase), which has been presumed to result in depletion of intracellular inositol available to phosphatidylinositol (PI) synthase. We will measure inositol and the inositol phosphates, the inositol lipids and their precursors: phosphatidate, DAG, and CDP-DAG, under conditions of rest and muscarinic stimulation in the presence and absence of Li+. Analytical and labeling studies on the PPI cycle lipids will take advantage of their known enrichment in the stearoyl arachidonoyl DAG species. We will also further explore in vivo and in vitro the regulation and properties of the key lipid enzyme, PI synthase. The gene for the latter will be cloned, antibodies will be prepared for immunohistochemistry studies, and the possibility of multiple genes encoding PI synthase will be explored. The enhancement or depletion of mRNA levels for this enzyme, as well as inositol 3P-synthase and IP-Pase, will be measured by northern blots, and where appropriate in sections of whole brain, by in situ hybridization. Together, the individual subprojects will contribute to the common goal of understanding regulatory mechanisms of signal transduction pathways in the CNS that underlie abnormal behavioral states.