John Martin Barrett, PhD - US grants

We are trying to unravel the mechanisms which control the functional properties of spinal cord neurons. We have found that muscle cells grown in culture produce a high molecular weight substance which can enhance the growth of the axons of spinal cord neurons. In this coming year we plan to make more accurate measurements of the chemical properties of this trophic substance. We will use gel filtration and ultracentrifuge sedimentation techniques to obtain measures of its molecular weight. Isoelectric focusing and ion exchange chromotography will be used in determining its isoelectric point. Using these techniques together with affinity chromotagraphy methods we will continue to improve purification methods for this trophic molecule. We will also try to elucidate the mechanism of activation of the slow potassium conductance system found in motoneurons and muscle cells. This year we hope to determine how membrane depolarization and internal calcium contribute to the activation of this conductance system.

Several properties of central cholinergic and dopaminergic neurons appear to be regulated in part by trophic factors originating from their target tissues. We propose to purify four of these factors and characterize their biological and chemical properties, using neuron-rich cultures of rat central neurons to assay trophic factor effects. In particular, we will study: (1) a factor made by muscle which enhances acetylcholine synthesis in spinal cord cultures, (2) a factor isolated from the neocortex which enhances acetylcholine synthesis and cholineacetyltransferase activity in cultures of rat basal forebrain cells. The activity of this factor is not blocked by antibodies against the classical nerve growth factor, and this factor does not affect spinal cord neurons. (3) a factor from the striatum which enhances dopamine synthesis in cultures of basal mesencephalon (includes substantia nigra) neurons, and (4) a serum-derived factor which greatly enhances the survival of both cholinergic and dopaminergic neurons. Bioassays will be used to follow the trophic activity through biochemical separation procedures. Immunological methods will be used to facilitate the final purification steps and obtain blocking antibodies to help in studying the biological functions of the factors. Purified trophic factor preparations will be tested to determine the range of their effects on target neurons (enhancement of transmitter synthesis, neurite outgrowth, ability to regenerate processes following trauma, and effects on cell survival, protein synthesis and electrophysiological membrane properties). We will also determine whether the trophic actions are reversible and modulatory, or irreversible and perhaps stage-specific. We plan also to extend these trophic factor studies to cultured primate neurons to determine whether the factors that are trophic for rat neurons also have similar actions on primate neurons, and whether primate tissues make similar trophic factors. Using fractions of serum from patients with neurodegenerative diseases affecting the brain regions we culture (amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease), we will test claims of specific neurotoxic serum factors, and determine whether these patients might have antibodies against the trophic factors and their receptors.

Several properties of central cholinergic and dopaminergic neurons appear to be regulated in part by trophic factors originating from their target tissues. We propose to purify four of these factors and characterize their biological and chemical properties, using neuron-rich cultures of rat central neurons to assay trophic factor effects. In particular, we will study: (1) a factor made by muscle which enhances acetylcholine synthesis in spinal cord cultures, (2) a factor isolated from the neocortex which enhances acetylcholine synthesis and cholineacetyltransferase activity in cultures of rat basal forebrain cells. The activity of this factor is not blocked by antibodies against the classical nerve growth factor, and this factor does not affect spinal cord neurons. (3) a factor from the striatum which enhances dopamine synthesis in cultures of basal mesencephalon (includes substantia nigra) neurons, and (4) a serum-derived factor which greatly enhances the survival of both cholinergic and dopaminergic neurons. Bioassays will be used to follow the trophic activity through biochemical separation procedures. Immunological methods will be used to facilitate the final purification steps and obtain blocking antibodies to help in studying the biological functions of the factors. Purified trophic factor preparations will be tested to determine the range of their effects on target neurons (enhancement of transmitter synthesis, neurite outgrowth, ability to regenerate processes following trauma, and effects on cell survival, protein synthesis and electrophysiological membrane properties). We will also determine whether the trophic actions are reversible and modulatory, or irreversible and perhaps stage-specific. We plan also to extend these trophic factor studies to cultured primate neurons to determine whether the factors that are trophic for rat neurons also have similar actions on primate neurons, and whether primate tissues make similar trophic factors. Using fractions of serum from patients with neurodegenerative diseases affecting the brain regions we culture (amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease), we will test claims of specific neurotoxic serum factors, and determine whether these patients might have antibodies against the trophic factors and their receptors.

This revised application proposes studies using cultured rat basal forebrain neurons and ion imaging techniques to investigate mechanisms underlying increases in intracellular [Ca2+] and choline acetyltransferase (ChAT) activity produced by brief exposure to neurotrophins, and neurotrophin-mediated protection from hypoglycemic stress. By imaging neurons filled with the ratiometric Ca-sensitive dye fura2, we have found that certain members of the neurotrophin family, which includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4/5 (NT3, NT4/5), induce transient increases in [Ca2+]i in subsets of basal forebrain neurons. We have also found that brief (15 min) exposure to certain neurotrophins leads to a significant increase in the activity of the acetylcholine-synthesizing enzyme, ChAT, measured 24 hr later. Mechanisms underlying the [Ca2+]i transients will be investigated by determining which neuronal types and neurotrophin receptors are involved, and whether the changes in [Ca2+]i are accompanied by release of internal [Ca2+] stores and/or changes in intracellular [Na+] and/or [H+]. For both the (Ca2+]i and the ChAT responses to brief neurotrophin exposure, we will investigate whether tyrosine (or other) kinase activity is required, whether synaptic or action potential activity is required, and whether the phenomena change during neuronal maturation. We will also determine whether the enhancement of ChAT activity by brief neurotrophin exposure requires changes in [Ca2+]i, transcription, and/or protein synthesis. Our studies have indicated that certain neurotrophins significantly increase basal forebrain neuronal survival and ChAT activity after a hypoglycemic stress. We propose to determine how the degree of protection varies with the identity, concentration and time of application of the neurotrophin. Mechanisms underlying stress protection will be investigated by determining which types of neurons are protected, and whether the stress protection requires tyrosine (or other) kinase activity, transcription and/or protein synthesis, and varies with synaptic activity. These studies will provide important new information concerning how various members of the neurotrophin family exert their trophic effects on basal forebrain neurons, including the central cholinergic neurons implicated in memory mechanisms.

DESCRIPTION (From the applicant's abstract): I have demonstrated that neurotrophins (NTs) protect cultured embryonic rat basal forebrain cholinergic neurons during hypoglycemic stress. The goal of the proposed study is to identify important second messenger and early effector pathways involved in this protection. Many of the experiments will utilize cholinergic neurons purified by immunopanning. I have preliminary evidence that the following contribute importantly to NT-mediated protection of cholinergic neurons during hypoglycemic stress: (1) activation of phosphotidyl inositol (PI) 3-kinase, and (2) activation of NF-kB (perhaps) via the p75 NT receptor (p75NTR). Receptor-blocking antibodies, neurons from receptor-deficient mice, and mutant forms of nerve growth factor (NGF) will be used to test the involvement of the p75 NTR and TrkA NGF receptors. Assays of kinase activity and immunohistochemical staining for activated forms of signalling molecules will be used to clarify the stress-protective pathways activated by NTs under hypoglycemic conditions. Inhibitors and activators of specific kinases and introduction (by transfection) of mutant kinases will be tested to determine whether they block, mimic or occlude NT-mediated protection. To investigate early effector pathways by which NTs might produce their stress-protection, imaging techniques will be used to measure NT effects on cytosolic [Na+] and [Ca2+], mitochondrial [Ca2+] and mitochondrial membrane potential. Alternative energy substrates, respiration measurements and inhibitors of specific mitochondrial complexes will be tested to determine whether NT-mediated protection requires up-regulation of plasma membrane glucose transporters and/or alterations in mitochondrial energy and/or Ca2+ metabolism. I have recently discovered that certain bone morphogenetic proteins (BMPs) robustly enhance NT-mediated protection during hypoglycemic stress, and the proposed experimental plan integrates studies to uncover basic mechanisms underlying this synergistic effect.

An interdepartmental group of NIH-supported investigators seeks funds for a Perkin-Elmer Wallac Ultraview confocal microscope system, to replace and upgrade an existing Noran system that has several major deficiencies and is starting to fail. The requested system has several important advantages that will benefit many NIH-sponsored research programs at this institution. The noel design of this confocal system allows use of very low intensities of excitation light, thus minimizing phototoxicity and bleaching. The high sensitivity of the light-detection systems permits measurements of weaker signals than can be detected by our current confocal system. The requested system also has a much better data-collecting ability (14 bit sampling) than the existing system (8 bits). The application details why the requested confocal system is essential and/or important for optimal progress of NIH-sponsored projects of major users from five laboratories. Four of the laboratories will be imaging living tissues, and share research interests in mitochondrial function. All major users have a history of cooperative interactions, and one has access to any other suitable confocal facility. The confocal needs of five additional laboratories are briefly summarized. All major users attended a demonstration of the requested confocal system at the University of Miami, during which some of the tissues and fluorophores to be imaged were examined. In all cases the requested confocal system, facilitating experiments that are now difficult or impossible to perform.