During aging of the human brain and particularly in presenile dementia of the Alzheimer type (AD), there is an accumulation of abnormal fibrous organelles composed of paired, helically wound intermediate filaments (PHF). Recently, several laboratories have proposed that this fibrous material may be a post-translationally modified neurofilament (NF) protein. To date, the function of NF proteins is not well characterized, nor is there much information about the molecular structure of the NF 200K or NF 160K proteins. Most structural work has centered on the NF 70K subunit. Based on the new immunochemical and electron microscopic findings of PHF possibly being related to NF, we propose an integrated series of molecular studies which has as its goal the full antigenic characterization of the NF 200K protein from Alzheimer brain. Both polyclonal and monoclonal antibodies which cross-react with the NF 200K protein or PHF (as well as the monoclonals we produce) will be used to describe the determinants on the NF 200K protein at the primary structural level. Limited proteolytic fragments of the NF 200K protein will be used to identify the location of the various antigenic determinants and to investigate the still unknown domain structure of NF 200K. In the past, it has been very difficult to purify the NF subunits by conventional liquid chromatography, and most researchers have resorted to excising NF subunits from SDS-PAGE gels. We propose to try to improve these procedures by introducing HPLC techniques to this area of neurochemistry. Several different column methodologies will be tried in the isolation of the three NF subunits and also in the isolation of proteolytic subunits from the NF 200K subunit. This improvement in methodology is necessary for future sequencing and functional studies. The proteolytic NF 200K fragments will also be used as immunogens for the production of monoclonal antibodies, which will be tested for their cross-reactivity with PHF. We also propose to compare the relative amounts of asparagine, L-aspartic acid and D-aspartate between Alzheimer NF proteins and age-matched normal controls. The presence of D-aspartate in brain proteins has been recently proposed as both disrupting protein function and involved in aging. The NF 200K protein from affected areas of Alzheimer brain will be compared with NF 200K isolated from unaffected brain in an attempt to see if NF 200K protein is identical throughout the brain or if local modifications of NF 200K can occur.
| Kirschner, D A; Inouye, H; Duffy, L K et al. (1987) Synthetic peptide homologous to beta protein from Alzheimer disease forms amyloid-like fibrils in vitro. Proc Natl Acad Sci U S A 84:6953-7 |
