AXONOGENESIS AND NEURODEGENERATION--ROLE OF NEUROTUBULIN

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Neurons are asymmetric cells that extend dendrites and axons to communicate with ne another and with non-neuronal cells. The growth of these processes during development and regeneration depends on the elongation of microtubules (MTs) which are a principal component of the neuronal cytoskeleton. MTs within the dendrites and the soma differ from the axonal-MTs with respect to their biochemical properties. For example the MTs of the soma and the dendrites are associated with MAP2 but not tau whereas MTs in the axon are associated exclusively with tau. These biochemical distinctions among subcellular MTs are disrupted in various age-related degenerative pathologies, such as Alzheimer's disease. We recently demonstrated that neurons express five distinct beta-tubulin isotypes, encoded by different genes, that differ primarily in their carboxyterminal MAP-binding domain (Joshi and Cleveland, 1989). Furthermore, our preliminary results strongly suggest that certain MTs of the soma are distinct in tubulin isotype composition compared to those of the axon. Thus, we propose to test the hypothesis that during the normal development of neurons different tubulin isotypes are recruited into the somatodendritic and axonal compartments where they differentially associate with MAPs. Using our beta-tubulin isotype specific antibodies and cultured rat hippocampal neurons, which exhibit in vivo morphology, we are in a excellent position to test this hypothesis. Specifically, we will a) use immunofluorescence microscopy and quantitative imaging to determine if the dendrites, soma, and axon contain different relative amounts of each beta-tubulin isotype; b) determine when differences in the specific tubulin-isotype localization arise during development; c) determine if the composition of microtubule subsets within each subcellular compartment is different by immunogold electron microscopy and serial section reconstruction analysis; d) evaluate the utility of antisense oligonucleotides to specifically inhibit synthesis of neuronal Type-II tubulin; and if successful, e) determine the phenotypic consequences for the development of axons and dendrites by morphological, immunological and ultrastructural approaches; and f) determine whether the somatodendritic neurofibrillary tangles in Alzheimer's disease contain type-II tubulin isotypes. In addition to revealing the fundamental determinants of neuronal MT properties, these studies will form a new basis for investigating a hypothesis that certain age-related diseases result from disruption of mechanisms which regulate tubulin isoform expression.