Efrain C. Azmitia - US grants

The proponents of this New York Academy of Science symposium met before and after the International Symposium in Lund to select those aspects of brain transplantation which deserved the greatest attention. Two topics, seemingly at either end of a continum, were chosen because of the (1) emergence of new data; (2) need for exchange of techniques and ideas among scientist engaged in basic and applied research; and (3) importance to the progress of neuronal transplantation procedures in clinical medicine. The specific topics selected by us can be grouped into two general areas: molecular and cellular responses in adult brain to donor neural cells and tissues; and models of aging, dementia, and neurodegenerative diseases in primate and subprimate brain. The present proposal begins with three topics central to cellular mechanisms involved in the integration of donor cells with host brain: (1) Interaction of neurons and glial cells, (2) production of trophic substances following transplantation, and (3) transplantation of specific cells. It is expected that the cellular and molecular events underlying the interactions between neurons, glial, and endothelial cells and important insights into procedures to best integrate adult host and donor CNS cells will be thoroughly discussed. The second half of the proposed symposium is devoted to models of aging, dementia, Alzheimer's Disease, Parkinsonism and Huntington's Chorea. The first session will deal with extrapyramidal disorders produced by localized lesions with 6-OH-dopamine and ibotenic acid. The second session deals with aging and dementia. This session presents exciting findings on reversal of age-related deficits by neuronal transplantation and also on the effects of cortical transplants on improving cognitive deficits produced by brain damage. The final session will deal directly with the important topic of brain transplantation into humans and primates. Very recent studies will be presented on MPTP lesioned monkeys and a progress report on the human studies performed in Sweden.

This proposal is a request for funds to obtain a transmission electron microscope. The major users of the instrument have proposed four main projects which require the use of the instrument. First, the morphology of the neuromuscular junction and synaptosomes during age-associated changes will be quantified. Second, the frequency of myelinated monoaminergic nerve fibers in rat and monkey central nervous systems will be determined to serve as a baseline for comparisons between species, over time and after experimental manipulations. Third, quantitative determination of cholinergic axons in several regions of the brain, together with quantitation of the innervation of several connections between areas within the brain will be determined. Lastly, a quantitative study of the morphology of regenerating peripheral nerve and nerve muscle development during ontogeny has been proposed.

Serotonin plays an important role in neurodevelopment by causing changes in forebrain naturation, neurite outgrowth, expression of postsynaptic receptors, and on the apprearance of inhibitory behaviors. This research will focus on the proposition that a component of the action of 5-HT on serotonergic neurons (autoregulation) may be its intracellular metabolism by monoamine oxidase (MAO). The hypothesis is based on the fact that metabolism of 5-HT by MAO generates H202, which is highly toxic to neurons because it can form hydroxy-free radicals. The enzyme MAO exists in two forms, A and B. MAO-A has a higher affinity for 5-HT than MAO-B, but this latter form is localized to serotonergic neurons and astrocytes. Preliminary studies indicate that inhibition of MAO-B is effective in augmenting fetal 5-HT development. The studies are clinically relevant because of the widespread use of monoamine oxidase inhibitors in depressed patients. They may also help understand age-related degenerative processes underlying dementia since MAO-B is increased during aging while serotonergic neurons, important in learning and memory, are reduced.

A central issue in developmental neurobiology is the role of cell-cell interactions in regulating neuronal differentiation. Astrocytes secrete factors that influence neuronal differentiation, including S-100 beta, a factor that affects serotonergic neurons. In turn, the secretion of S-100 beta from astrocytes is regulated by activation of serotonergic neurons. The mechanisms of these cellular interactions will be investigated at the molecular level by studying the transcription, translation and release of S-100 beta. In addition, the regulatory role of steroid hormones, specifically the glucocorticoids, in these processes will be studied. The PI will undertake these studies at Cold Spring Harbor during a one year Sabbatical leave where he will be trained in molecular and protein techniques in the laboratory of Dr. Daniel Marshak.

The Program Project consists of the cohesive efforts of established scientists from separate institutions. The CORES and projects are integrated to test the hypothesis that S-100beta is linked to the neuropathology associated with Alzheimer's Disease (AD), as well as to further our understanding of neuronal/glial interactions in the control and operation of CNS growth factors. An administrative core and five research cores are proposed to facilitate the interactions necessary to cost- effectively utilize the individuals' expertise and the excellent support facilities available at the respective research centers. Five research projects will address three overall research areas using tissue culture. Research Area 1 will be to study the detailed actions of factors acting on astrocytes to regulate the synthesis and release of S-100beta (eg. IL-1, 5- HT, Ca++, and glucocorticoids). The results of this research area will provide clues to the identity of key astrocytic regulatory-substances that may be altered in AD, and which can be analyzed in our extensive collection of Alzheimer's brains. Research Area 2 will examine the molecular and cellular events in astrocytes resulting from exposure to s-100beta. Changes in signal transduction pathways and in astrocytic morphology and proliferation will be studied. Research Area 3 will explore the mechanism of action of S-100beta on neurite extension. Does S-100beta enter the neurons to interact with specific microtubule-associated-proteins and/or does it bind to a cell surface receptor and produce a response via specific second messenger systems? The Results obtained from these three research areas can then be translated into in vivo experiments using whole animals. Procedures such as cell transplantation, in situ hybridization and immunocytochemistry can be applied to specific brain regions in the adult or aged animal. Predictions arising from these studies will be tested on human post mortem tissue which includes a large collection of AD brains. Our multidisciplinary efforts are not concerned solely with understanding astroglial/neuronal interactions but strives to answer basic questions of the role of S-100beta in Alzheimer's disease.

The Program Project consists of the cohesive efforts of established scientists from separate institutions. The CORES and projects are integrated to test the hypothesis that S-100beta is linked to the neuropathology associated with Alzheimer's Disease (AD), as well as to further our understanding of neuronal/glial interactions in the control and operation of CNS growth factors. An administrative core and five research cores are proposed to facilitate the interactions necessary to cost- effectively utilize the individuals' expertise and the excellent support facilities available at the respective research centers. Five research projects will address three overall research areas using tissue culture. Research Area 1 will be to study the detailed actions of factors acting on astrocytes to regulate the synthesis and release of S-100beta (eg. IL-1, 5- HT, Ca++, and glucocorticoids). The results of this research area will provide clues to the identity of key astrocytic regulatory-substances that may be altered in AD, and which can be analyzed in our extensive collection of Alzheimer's brains. Research Area 2 will examine the molecular and cellular events in astrocytes resulting from exposure to s-100beta. Changes in signal transduction pathways and in astrocytic morphology and proliferation will be studied. Research Area 3 will explore the mechanism of action of S-100beta on neurite extension. Does S-100beta enter the neurons to interact with specific microtubule-associated-proteins and/or does it bind to a cell surface receptor and produce a response via specific second messenger systems? The Results obtained from these three research areas can then be translated into in vivo experiments using whole animals. Procedures such as cell transplantation, in situ hybridization and immunocytochemistry can be applied to specific brain regions in the adult or aged animal. Predictions arising from these studies will be tested on human post mortem tissue which includes a large collection of AD brains. Our multidisciplinary efforts are not concerned solely with understanding astroglial/neuronal interactions but strives to answer basic questions of the role of S-100beta in Alzheimer's disease.

DESCRIPTION (Adapted from applicant's abstract): The serotonin system is known to be decreased in some depressed patients. Depression, in addition to its affective component, produces deficits in learning and memory which are normally associated with hippocampal synaptic function. These cognitive deficits are responsive to treatment with drugs that increase 5-HT. A loss of 5-HT in rats produces a decrease in synaptophysin, MAP-2 immunoreactivity and non-monoaminergic synapses in cortex and hippocampus. While these neuronal markers are decreased, we found an increase of trophic responses and of antipeptide labeling of the 5-HT-1A receptor protein. Likewise, in depressed humans, a fall of 5-HT in cortex is associated with an increase in 5-HT-1A receptor binding. In animals, activation of this receptor with a 5-HT-1A agonist rapidly reverses the changes induced by 5-HT loss. The long-term aim of this application is to test the hypothesis that the 5-HT-1A receptor regulates the fluctuation of adult hippocampal neurons between mature and immature states. Specifically, we propose that loss of 5-HT will induce a retraction of neuronal dendrites and an increase in 5-HT-1A receptor protein and gene expression. We predict 5-HT-1A receptor stimulation will restore the mature neuronal phenotype more effectively than either tricyclic antidepressants or specific serotonin reuptake inhibitors. 5-HT will be reduced by para-chlorophenylalanine (PCPA), para-chloroamphetamine (PCA) or 5,7-dihydroxytryptamine (5,7-DHT) and measured by HPLC or paroxetine binding. The neuronal morphology will be studied and compared to animals treated with a 5-HT-1A receptor antagonist. 5-HT-1A receptors labeled with our antipeptide antibodies will be studied morphometrically in vivo by immunocytochemistry and quantified in vitro by slot blots and immunoblots. Finally we will examine the 5-HT1A receptor mRNA using in situ hybridization. These results may help understand the etiology of depression and related disorders and provide insights for effective and novel treatments.

DESCRIPTION: (Adapted from applicant's abstract) Depression is associated with affective and cognitive disorders. The applicant suggest some of these symptoms may be due to loss of hippocampal and cortical morphology (cytoskeletal collapse) induced by loss of serotonin. Loss of serotonin in the adult rat brain produces decreased dendritic length, dendritic spine number and size, synapse number and a reduction in immunoreactivity to antibodies against neuronal (Microtubule Associated Protein-2 and synaptophysin) and glial (S-lOOn) markers. Injection with a 5-HT1A antagonist produces similar loss of synapses and dendritic spines. The loss of neuronal and glial markers is reversed by treatment with 5-HTIA receptor agonists and S-100beta. This renewal application will test the hypothesis that the 5-HT1A receptor stabilize the neuronal cytoskeletal by targeting neurons and glial cells, and may protect neurons from death (apoptosis). The applicant would like to continue and expand our studies on the effects of 5-HT drugs on morphological reversal after 5-HT loss proposed in the onginal grant. In addition, the applicants now propose the 5-HT1A receptor may regulate the cytoskeleton of neurons, by acting both on glial (availability of S100f3) and neurons (receptor-induced changes in phosphorylation pathways (e.g. PKC, PKA and MAPK)). In addition, The applicant would like to test if 5-HT1A receptor stimulation or S lOObeta treatmnent will restore the cytoskeleton after exposure to coichicine. Coichicine promotes microtubule disassembly and promotes apoptosis in culture and in vivo. Rats will be injected with para-chloroamphetamine (PCA) to reduce 5-HT levels The applicant will treat these rats with either a 5-HT1A receptor antagonist, tricyclics, serotonin specific reuptake inhibitors (SSRJ) or MAO-A inhibitor. In addition, the applicants will study possible mechanisms of action after exposure of cultured neurons to 5-HT1A receptor agonist and S-lOObeta. The applicants hope to extend this work to primary hippocampal and cortical neuronal and glial cultures using wild type and knockout (S100beta and the 5-HT1A receptor) mice. Finally, the actions of S-HT1A agonist and S-lOObeta will be studied after microinjections of colchicine into the adult rat hippocampus and cortex. The applicant will focus on dendritic collapse and apoptosis of neurons. This work will continue our long-term research into the interactions between serotonin and adult neumplasticity.

DESCRIPTION: This is a request for an NIMH Senior Scientist Award (SSA). At the age of 52, with over 30 years of research experience, I request freedom from non-research duties in order to focus on an expanded research program and take advantage of a unique and timely opportunity to learn MRI in humans. The major aim of my present research program is to examine the basic cellular and pharmacological mechanism of serotonin-mediated neuroplasticity in the rodent brain and spinal cord. The research proposed for the next five years focuses my efforts on expanding my basic research objectives and extending them to include clinical research. In basic research, I will study the serotonin involvement with neurogenesis and apoptosis. My current hypothesis is that 5-HT1A receptor protein levels and expression are inversely correlated with neuronal maturation. Dr. Jacobs finds neurogenesis is stimulated by 5-HT1A receptor agonists. Dr. Banerjee reports 5-HT1A agonists activate MAP-kinase, which inhibits caspase-3 activity and apoptosis, in a neuronal cell line. I am positioned to move into these areas and expand on my current NIMH supported research that linked the 5-HT1A receptor to neuroplasticity and depression. I will expand my hypothesis to include events occurring during neurogenesis and apoptosis. The second major career plan involves acquiring the background and experience to perform and analyse MRI in the human brain. Dr. Mony DeLeon uses MRI to detect morphological changes in entorhinal cortex, amygdala and hippocampus at NYU Medical Center. My long-term aims are to use imaging techniques for longitudinal study of a psychiatric patient population where neuroplastic events are suspected. Therapeutic strategies using 5-HT drugs would enable direct testing of the clinical relevance of my basic research findings. Professional growth plans involve (a) supplemental training in human imaging technology; (b) explore the role of the 5-HT1A receptor in neurogenesis and apoptosis and (c) meet with national and international experts in the field of neuroplasticity as part of further development of therapeutic strategies for using pharmacology to reverse neuromorphological changes occurring in mental disorders.