The broad objectives of this research program are to investigate the general characteristics of a class of brain extracellular glycoproteins (the ependymins) whose properties and turnover rates severely decrease with age. In previous studies these glycoproteins were found to have an important role in the molecular and cell biology of long-term memory (see Appendices I-III). In pilot experiments, double-labeling studies of the incorporation of valine into old mouse brain proteins of 0.5- to 2-year-old (C-14 labeled) as compared to 30-day-old (H-3 labeled) young adults indicated that the ependymins were the major proteins whose labeling showed an age-related decline. In additional experiments we have found that addition of the calcium chelating agent ethylenebis(oxyethylenenitrilo)tetraacetate (EGTA) to solutions of ependymins causes a rapid polymerization of the proteins into insoluble fibrous structures (see Figure 1). The capacity of these glycoproteins to form fibrous aggregates appears to severely decrease (5-fold) with aging in the mouse. Also, we have found that ependymin-like immunoreactivity is present in human cerebrospinal fluid. Analyses of 16 CSF samples from subjects (ages 0.44 to 83 years) showed a progressive decline in the polymerization properties of these proteins by about 5-fold as a function of aging. We will isolate, purify and characterize the ependymins of mouse brain (M.W. 64,000 and 32,000) in milligram quantities. Samples of the fibrous form will also be prepared. These will be used to raise monoclonal antibodies which selectively distinguish the unpolymerized from the polymerized proteins. Polyclonal antisera to these two forms will also be prepared. The antisera will be used to measure the level as well as the fiber-forming properties of the ependymins as a function of aging in mouse ECF samples and human CSF samples available in the McLean Hospital Brain Tissue Resource Center. Such studies will help establish the characteristics of these specific glycoproteins whose metabolism decreases with age and identify the mechanisms which lead to a deterioration of their properties. Eventually such results may provide a diagnostic assay method and leads for new approaches to treatments of certain types of memory dysfunctions that so often accompany the process of aging.
