Andrej Rotter - US grants

Neurotransmitter receptors for acetylcholine, gamma-aminobutyric acid, noradrenaline, serotonin and histamine in the cerebellar cortex of the mouse will be visualized by light-microscopic autoradiography of receptor-bound ligands. The localization of the receptors to regions and/or layers of the cerebellar cortex during postnatal development will be studied with special emphasis on the developmental stage at which receptors first appear. We will attempt to correlate the appearance of receptors with other known ontogenetic events, such as cell migration and synapse formation. MIce with genetic mutations affecting the structure, organization and development of the crebellar cortex will be used in studies aimed at elucidating the cellular localization of neurotransmitter receptors and/or any abnormalities in their normal pattern of development. to determine to what extent receptor appearance and localization is governed by the orderly structure and connections of the cerebellar cortex, cells from the rat cerebellum will be grown in dispersed cell culture and receptors visualized by autoradiography of receptor-bound ligands at various developmental stages. The stage of cerebellar development in culture at which receptors become "functional" will be studied by the autoradiographic localization of changes in 2-deoxy-D-glucose utilization following administration of receptor agonists. Monoclonal antibodies, directed against cell surfaces of rat cerebellar neurons will be raised in mice to obtain reagents necessary for the identification of neuronal cell types in culture. These neurochemical studies could lead to a better understanding of malfunctions affecting the nervous system.

There are several drugs currently used to treat hypertension which appear to act on the cardiovascular control centers of the central nervous system. Such drugs include clonidine and Alpha-methyldopa, both of which are Alpha2-adrenergic receptor agonists. Other CNS receptors may also be involved in hypertensive states and their identity might lead to the development of new anti-hypertensive drugs. We wish to study the distribution of five neurotransmitter and their corresponding receptors in central nervous system cardiovascular control centers (for example, the nucleus ambiguus, nucleus of the Xth nerve, nucleus of the solitary tract and the intermediolateral column of the spinal cord), in order to determine which drugs may potentially act at these sites. Markers for adrenergic, cholinergic, gamma-aminobutyric acid, glutamate, and serotonin transmission and their receptors will be detected by light microscope histochemistry and autoradiography. Receptor concentrations of cardiac and vasomotor control centers in adult rats will also be determined after deafferentation and axotomy. The changes observed will indicate whether the receptors tested are localized pre- or postsynaptically. In addition, the concentration of neurotransmitter and drug receptors in the cardiovascular control centers of neonatal and adult normotensive and spontaneously hypertensive rats will be compared. This comparison might reveal the chemical nature of central transmitter system malfunction underlying hypertension.

Recent experimental studies have provided strong evidence that the gamma-aminobutyric acid/benzodiazepine (GABA/BZ) receptor mediates some of the effects of alcohol intoxication. The aim of this proposal is to study the effect of alcohol ingestion on the expression of GABA/BZ receptor subunit mRNAs in the mouse central nervous system. GABA/BZ receptor subunit mRNAs will be autoradiographically localized following Northern blot and in situ hybridization with subunit specific complementary [32p] and [35S]-oligonucleotide probes. These techniques will also be used to study the expression of GABA/BZ receptor subunit mRNA in granule cell cultures from developing normal mouse cerebella in the presence and absence of alcohol. The following experimental questions will be asked: What is the anatomical localization of subunit mRNAs coding for the alpha, beta and gamma subunits of the GABA/BZ receptor complex in adult murine central nervous system? How does the expression of subunit mRNAs coding for the alpha, beta and gamma subunits of the GABA/BZ receptor complex in the adult mouse brain change following chronic alcohol intoxication? Are there changes in the expression of MRNA coding for GABA/BZ receptor subunits in developing mouse brain in response to chronic maternal alcohol consumption? What is the difference in expression of GABA/BZ receptor subunit mRNAs in the CNS of ethanol-sensitive (LS) and ethanol-insensitive (SS) mice? How is the expression of GABA/BZ receptor subunit mRNAs in neuronal cultures of cerebellar granule cells affected by exposure to alcohol? The above studies will provide preliminary data of the effects of alcohol at the molecular genetic level in a specific neurotransmitter receptor system.

DESCRIPTION (Adapted from applicant's abstract): Affective disorders, psychosis and mental retardation are a group of mental health diseases thought to result, in part, from the disruption of normal developmental processes within the nervous system. Fundamental to the pathophysiology of mental and behavioral disorders are alterations in basic molecular properties produced by genetic abnormalities or by unfavorable environments such as disease or substance abuse. Such molecular disruptions could result in changes in cell-cell interactions leading to a variety of defects ranging from malformation of neural tube compartments to inappropriate synapse formation. We have cloned a novel receptor-like protein tyrosine phosphatase (RPTP-rho) whose expression is entirely restricted to the central nervous system. It is a developmentally regulated molecule which defines a sharp anterior-posterior boundary in the murine cerebellar cortex between regions derived from the embryonic mes- and metencephalon. In addition to their generally accepted role in intracellular signal transduction through the regulation of protein tyrosine phosphorylation, RPTP molecules have been implicated in various cell adhesion mechanisms, including cell-cell or cell-extracellular matrix recognition and axon guidance. The sequence of RPTP-rho suggests membership in a molecular family which acts as direct signal transducers of cell contact phenomena. We propose to extend our preliminary data by performing a series of experiments grouped into four specific aims. The first aim is to generate anti-RPTP-rho antibodies for the immunocytochemical localization of the RPTP-rho protein at the light and electron microscopic levels. The second aim is to investigate the role of the extracellular domain in cell aggregation and cell adhesion. In the third aim, the role of the RPTP-rho intracellular domain in signal transduction via association with cadherins/catenins will be studied. Finally, in the fourth specific aim, the RPTP-rho promotor will be cloned, sequenced and characterized with a view to providing the basis for the identification of sequences responsible for the distinct rostrocaudal distribution of the RPTP-rho transcript in the cerebellar cortex and for future gene inactivation studies. The proposed studies will clarify the developmental role of RPTP-rho in the brain and may indicate new molecular mechanisms underlying developmental neuropsychiatric disorders.

The major function of the cerebellum is to integrate sensory input and motor output, thus modulating movement and balance. Senescent changes in the cerebellum are thought to contribute to the impairment in balance and motor coordination frequently observed in aged individuals. Aging-related changes in neuronal properties and resulting neuronal loss have been well documented in the cerebellum, and alterations in the function, or levels, of specific molecular components have been described. It is plausible that cerebellar senescence is the result of cumulative changes in the expression of many genes. To date only a fraction of the genes comprising the human genome have been assayed by DNA microarrays for senescent changes. Because DNA microarray technology is a "closed" system capable of detecting only known genes, we propose to conduct a global analysis of gene expression in the adult and aged mouse cerebellum using SAGE (Serial Analysis of Gene Expression). SAGE is an "open" system capable of discovering and digitally quantifying both known and, as yet, unknown genes. The proposed experiments involve the construction, sequencing and bioinformatic analysis of cerebellar SAGE libraries from a 5-month and 30-month old mouse. The resulting comprehensive and quantitative SAGE database of gene expression, which includes that of novel genes in the adult and aged mouse cerebellum, will be disseminated via the NCBI website. Future experiments will examine the effect of caloric restriction on gene expression in the aged mouse cerebellum, and a full characterization of novel genes whose expression is substantially altered during aging.

DESCRIPTION (provided by applicant): Excessive consumption of alcohol is known to have toxic effects on the fetal, adult and aging nervous system. It is well established that cerebellar neurons are subject to the damaging effects of alcohol administration throughout development. Because the major function of the cerebellum is to modulate movement and balance by integrating sensory input and motor output, any injury to component neurons is likely to lead to the impairment of motor co-ordination. It is plausible that alcohol-induced cerebellar damage is the result of cumulative changes in the expression of many genes. To date, only a fraction of the genes comprising the human and mouse genomes have been assayed for alcohol-induced changes, either individually or by DNA microarrays. Because DNA microarray technology is a 'closed' system, capable of detecting only known genes, we propose to conduct a global analysis of gene expression in the alcohol-exposed mouse cerebellum using Serial Analysis of Gene Expression (SAGE). SAGE is an 'open' system capable of detecting and digitally quantifying both known and, as yet, unknown genes. The proposed experiments involve the construction, sequencing and bioinformatic analysis of routine cerebellar SAGE libraries during the early postnatal period, adulthood and senescence, shortly after exposure to "binge" amounts of alcohol. The resulting comprehensive and quantitative SAGE database of alcohol-induced changes in gene expression, which includes that of novel genes, will be disseminated via the NCBI website. Future long-term experiments, beyond the scope of this R03 and proposed as part of a planned R01 submission, will be aimed at (1) construction of "timed" cerebellar SAGE libraries at several time intervals after alcohol exposure; (2) preparation of cerebellar SAGE libraries from mice chronically exposed to alcohol; (3) the full characterization of novel cerebellar genes whose expression is substantially altered as a result of alcohol administration, and (4) the examination of experimental strategies aimed at reversing the toxic effects of alcohol ingestion on cerebellar neurons.