MOLECULAR PROBES TO STUDY ALZHEIMER'S DISEASE

The development and maintenance of cell shape depends on the assembly of microtubules from tubulin monomers. This process is facilitated by microtubule-associated protein-2 (MAP-2) which is expressed throughout neuronal development. MAP-2 binds to tubulin and reduces the critical concentration of tubulin required to polymerize into microtubules. Multiple MAP-2 isoforms regulate process outgrowth and the spacing of microtubules. The goal of this proposal is to understand the regulation of MAP-2 function and to determine the temporal and spatial expression of newly isolated MAP-2 isoforms in brain and spinal cord. We have cloned and characterized six novel MAP-2 exons. By in situ hybridization and antisense strategies, we will test the hypothesis that unique regions of the 5/UTR and/or CA repeats located in the 3/ UTR play a role in intracellular localization of MAP-2 transcripts or targeting to subpopulations of neurons. Using the rat as a model system, we will test the hypothesis that MAP_2 isoforms containing newly identified exons 8 and/or 13 are developmentally regulated in subpopulations of rat spinal cord and brain neurons. Preliminary data support this hypothesis. RT- PCR, and Northern and Western blot analyses will determine the developmental expression of the transcripts and the respective proteins. The neuronal subpopulations will be examined by immunocytochemistry, and the association of these isoforms with microtubules will be examined by electron microscopy. Western blots and immunocytochemistry will be carried out using MAP-2-specific polyclonal antibodies generated in our laboratory from exons 8 and 13 translated sequences. In human brain and spinal cord, we will determine the sub-populations of neurons expressing these MAP-2 isoforms. Also, brain sections from normal, Alzheimer disease and Lewy body disease will be compared. This study will permit us to assess for the first time the developmental expression of novel MAP-2 isoforms expressing exons 8 and 13 and to understand their role in the development and maintenance of the differentiated state. Further, the ability to distinguish between the multiple MAP-2 isoforms will permit analysis of neurodegenerative diseases as assessed by aberrant MAP-2 expression.