Allan J. Tobin - US grants

The experiments in Part I of this proposal will test the hypothesis that alterations in the structure and regulation of the GABA synthetic enzyme, glutamate decarboxylase (GAD), or of GABAA receptor polypeptides underlie the seizure disorders in animal models of epilepsy. Part I encompasses two specific aims: (1) to determine whether one of the genes encoding GAD or GABA receptor polypeptides is altered in one of the existing genetic mouse models of epilepsy, and (2) to determine whether the expression of GAD or GABA receptor genes is altered in several genetic and experimental models or epilepsy. The experiments proposed in Part II of this proposal will attempt to program the altered expression of GAD and GABA receptor polypeptides in cell lines and in transgenic mice. Part II encompasses three specific aims: (3) to program high level expression of GAD in subclones of existing cell lines, (4) to determine the mechanism of GABA release in these engineered cell lines, and (5) to determine whether seizure susceptibility is altered in transgenic mice with altered expression of GAD or GABA receptors. Direct injection of GABA agonists into the brain in some cases prevents seizure propagation and cell death. A long term goal of the experiments proposed in specific aims #3 and 4 is the production of engineered cell lines that could provide a continuing and regulatable source of GABA. Such "designer neurons" may be ultimately useful in transplantation therapies for intractable seizures or for such neurodegenerative diseases as Huntington's disease. If we succeed in producing cells that release GABA, either constitutively or in response to depolarization, we will then, in collaboration with other investigators, address the problems of transplantation into experimental animals and testing for seizure suppression.

This project will use recombinant DNA probes for individual species of mRNA to study morphogenesis and cytodifferentiation in the developing mouse cerebellum. By examining the cellular distribution and the temporal pattern of individual gene expression in normal and mutant mice, we will evaluate four hypotheses: (1) The five types of neuron of the mature cerebellum express sets of genes that only partially overlap, both during cytodifferentiation and in the fully differentiated state; (2) individual neuron types are most likely to differ in the particular species of messenger RNAs that accumulate to high or moderate concentrations; (3) genes for glutamic acid decarboxylase are expressed at different times in four of the five neuron types; and (4) neuroblasts in the two germinal layers that give rise to the five cerebellar neuron types do not begin to express cell type specific genes until they start their characteristic migrations into new microenvironments. The preparation of probes for mRNAs preferentially expressed in individual neuron types will also allow the examination of a further hypothesis concerning hereditary ataxias both in mice and humans - that the molecular lesions in these conditions are located in genes that are preferentially expressed in the affected cell types. This hypothesis will be tested for four cerebellar mouse mutants whose primary defects appear to be in Purkinje cell development. We will also search for changes in the structure of cerebellum-specific genes in humans with hereditary ataxias. This project will provide basic information about the expression of specific genes during the development of a region of the brain that has been extremely well studied from morphological, physiological and genetic perspectives. If the proposed hypotheses about the molecular lesions in murine and human hereditary ataxias are correct, this work will also lead to a direct approach to diagnosis, and future therapies, for human neurogenetic disorders.

The experiments in Part I of this proposal will test the hypothesis that alterations in the structure and regulation of the GABA synthetic enzyme, glutamate decarboxylase (GAD), or of GABAA receptor polypeptides underlie the seizure disorders in animal models of epilepsy. Part I encompasses two specific aims: (1) to determine whether one of the genes encoding GAD or GABA receptor polypeptides is altered in one of the existing genetic mouse models of epilepsy, and (2) to determine whether the expression of GAD or GABA receptor genes is altered in several genetic and experimental models or epilepsy. The experiments proposed in Part II of this proposal will attempt to program the altered expression of GAD and GABA receptor polypeptides in cell lines and in transgenic mice. Part II encompasses three specific aims: (3) to program high level expression of GAD in subclones of existing cell lines, (4) to determine the mechanism of GABA release in these engineered cell lines, and (5) to determine whether seizure susceptibility is altered in transgenic mice with altered expression of GAD or GABA receptors. Direct injection of GABA agonists into the brain in some cases prevents seizure propagation and cell death. A long term goal of the experiments proposed in specific aims #3 and 4 is the production of engineered cell lines that could provide a continuing and regulatable source of GABA. Such "designer neurons" may be ultimately useful in transplantation therapies for intractable seizures or for such neurodegenerative diseases as Huntington's disease. If we succeed in producing cells that release GABA, either constitutively or in response to depolarization, we will then, in collaboration with other investigators, address the problems of transplantation into experimental animals and testing for seizure suppression.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The Molecular Biology Core will be responsible for the production and in vitro characterization and maintenance o GABA-producing cells that will be used in all three projects of the Center. In addition, the core will develop and use DNA microarray technology to identify candidate genes that are altered in the subthalamic nucleus and its target areas in the animal models of Parkinson's disease used in the Center.

DESCRIPTION: (Adapted from the Investigator's Abstract): The central hypothesis of this proposal is that GAD65 and GAD67 catalyze the synthesis of distinct pools of GABA-one vesicular and the other cytosolic. According to our working model, membrane-associated GAD65 produces vesicular GABA and cytosolic GAD67 produces cytosolic GABA. Whereas vesicular GABA, released by exocytosis, is likely to be particularly important in point to point signaling via synapses, cytosolic GABA, released via plasma-membrane transporters, may serve a longer-range paracrine role-inhibiting neuronal activity in a limited region of the nervous system. To address this hypothesis, we will undertake three sets of studies of genetically modified cells in culture, as well as of primary cell cultures from wild-type, GAD65- and GAD67 deficient mice. These experiments are organized around three Specific Aims: (1) to identify the determinants of the distinctive intracellular distributions of GAD65 and GAD67 (2) to test the distinct roles of the two GADs in vesicular and nonvesicular GABA release; and (3) to examine the functional significance of changes in the ratio of GAD65 to GAD67 and of apoGAD65 to -holoGAD65. An important long-term goal of this project period is the development of presynaptic pharmacological interventions in GABA signaling. Specifically, the establishment of cell culture model systems to study GABA dynamics will also enable us to use these systems as a rapid and high-throughput screen for drugs that selectively affect one or the other branch of the GABA release system. Drugs identified in such screens will have major importance as candidates for the treatment of epilepsy, movement disorders such as Parkinson's Disease, and ischemic or traumatic injury to the nervous system.

DESCRIPTION: (Adapted from the Investigator's Abstract): The central hypothesis of this proposal is that GAD65 and GAD67 catalyze the synthesis of distinct pools of GABA-one vesicular and the other cytosolic. According to our working model, membrane-associated GAD65 produces vesicular GABA and cytosolic GAD67 produces cytosolic GABA. Whereas vesicular GABA, released by exocytosis, is likely to be particularly important in point to point signaling via synapses, cytosolic GABA, released via plasma-membrane transporters, may serve a longer-range paracrine role-inhibiting neuronal activity in a limited region of the nervous system. To address this hypothesis, we will undertake three sets of studies of genetically modified cells in culture, as well as of primary cell cultures from wild-type, GAD65- and GAD67 deficient mice. These experiments are organized around three Specific Aims: (1) to identify the determinants of the distinctive intracellular distributions of GAD65 and GAD67 (2) to test the distinct roles of the two GADs in vesicular and nonvesicular GABA release; and (3) to examine the functional significance of changes in the ratio of GAD65 to GAD67 and of apoGAD65 to -holoGAD65. An important long-term goal of this project period is the development of presynaptic pharmacological interventions in GABA signaling. Specifically, the establishment of cell culture model systems to study GABA dynamics will also enable us to use these systems as a rapid and high-throughput screen for drugs that selectively affect one or the other branch of the GABA release system. Drugs identified in such screens will have major importance as candidates for the treatment of epilepsy, movement disorders such as Parkinson's Disease, and ischemic or traumatic injury to the nervous system.

A grant has been awarded to Dr. Arthur W. Toga at the Laboratory of Neuro-Imaging (LONI) of the University of California, Los Angeles (UCLA) to enhance the graphic and compute capabilities of an existing supercomputer for distributed use in neuroinformatics. Unabated advancements in imaging technology today has provided researchers with the ability to produce very high-resolution, time-varying, multidimensional data sets of the structural and functional development of the dynamic human brain. The complexity of the new data, however, require immense computing power for effective analyzation and study.
LONI and its collaborators will upgrade the facility's SGI Onyx2 supercomputer with additional graphics pipelines, computational nodes, and the incorporation of high speed networking equipment. The graphics pipelines will allow the visual interpretation of brain data and, in addition, drive a Data Immersive Visualization Environment (DIVE). The premise behind the DIVE is to provide investigators with the unique ability to visually step inside their data and analyze it in new, unexpected ways. The additional computational nodes will accelerate the speed of both interactive manipulation of multidimensional brain data sets and complex offline computations. The incorporation of high speed networking equipment will facilitate local and offsite network access to these computer resources and improve overall communication between the local, national, and international communities of neuroscientists.
This instrumentation not only benefits ongoing research in brain development, heritability and function but because analysis and interpretation of multidimensional brain data is elevated to the next level, it will open the doors to new insights and better understanding of brain structure and function in health and disease.

Huntington's disease is a devastating neurodegenerative disease that is inherited as an autosomal dominant. Disease-causing alleles encode expanded polyglutamine tracts in the aminoterminal region of huntingtin (htt), a large protein whose normal function is unknown. The unifying hypothesis in this proposal is that htt containing an expanded polyglutamine tract (htt-ex) causes proteasome poisoning by irreversibly blocking proteasomes. We argue that poisoned proteasomes in postmitotic neurons results in a failure of normal protein turnover and in cell dysfunction and death. Wild-type htt (htt-wt should not produce such effect. The proposed studies will consider not only the poisoned- proteasome hypothesis but also alternative hypotheses, in which the pathogenic action of htt-ex does not depend on proteasomes. We have organized these studies around three Specific Aims: 1. To determine whether the intracellular distribution and the pathogenic effects of htt-ex expression vary with cell type and proliferative state. 2. To examine the effect of htt-ex expression on proteasome function and on the turnover of specific proteins, including htt itself. 3. To examine the effects of htt-ex expression on neuronal function and to determine whether known proteasome inhibitors can mimic these effects. At the conclusion of these three sets of proposed experiments we will know 1) whether proteasome location and function change in response to cell cycle and htt expression; (2) whether htt affects proteasome function and protein turnover either directly or indirectly; (3) whether htt-ex alters cellular function in dividing and stationary, differentiated cells; and (4) whether proteasome inhibitors mimic htt-ex-induced changes in neuronal function.

Both experimental animals and humans can regain the ability to stand or to step after a complete spinal cord transection. The ability to execute these tasks depends on specific training regimens, illustrating the importance of motor leading in the spinal cord. Low-thoracic transection and subsequent training leads to a persistent increase in the total inhibitory capacity of the lumbar spinal cord, exhibited by increases in GAD67, a GABA-synthesizing enzyme, and its mRNA, as well as in the alpha-1 subunit of glycine receptor and in gephyrin, a protein associated with glycine receptors. However, repetitive hindlimb training, such as stepping, returns the levels of GAD67 and glycine receptors towards normal. The central hypothesis of this proposal is that task-specific, repetitive training selectively modulates the inhibition within sensorimotor pathways associated with the execution of that task. Using a robotic device, we will test this hypothesis with a well-defined standing task in neonatally transected (T12-13) rats. Stand training allows us to compare training-induced changes in neurons associated with plantarflexion (facilitation of the soleus motor pool) and neurons associated with dorsiflexion (inhibition of the tibialis anterior motor pool). We will test three hypotheses: (1) that stand training decreases the inhibitory capacity of specific neurons associated with the ankle dorsiflexor (tibialis anterior); (2) that training selectively alters the ratio of inhibitory and excitatory synapses on the somata of individual motoneurons in motor pools associated with soleus, and (3) that pharmacologically induced changes in motor performance of spinally transected rats reflect these alterations in GABAergic and glycinergic inhibition in plantarflexion- and dorsiflexor-associated neurons as noted in the first two hypotheses. A major innovation in this work is the ability to train motor tasks, and to quantify the kinematics of standing and stepping using a newly developed robotic device. This device will allow us to impose strictly repetitive training and to assess the progress of individual animals with great precision. The proposed studies address the anatomical and molecular bases of the plasticity that may underlie rehabilitative training after spinal injury. This work will lead to better ways of testing the effectiveness of alternative training strategies and associated pharmacological interventions.