Intraventicular perfusion of leupeptin, a thiol proteinase inhibitor, or of chloroquine, an inhibitor of lysosomal enzymes in general, causes a massive increase in ceroid-lipofuscin (CL) in neurons throughout the central nervous system. This This simple, rapid and reliable technique thus provides a means for inducing in young animals one of the hallmarks of brain aging. It may prove possible to use this experimental method to induce other concomitants of aging, as well, thus generating a model for the study of biochemical mechanisms underlying aging. The goals of the proposed experiments are 1) to determine if other morphological manifestations of aging are induced by proteinase or lysosomal enzyme inhibitors, 2) to determine if a new and specific inhibitor of the lysosomal thiol proteinase, cathepsin B, produces the morphological anomalies previously seen with leupeptin treatment and 3) to determine if lipofuscin accumulation is proportional to lifespan in super-aged mice. The three sets of experiments will each use a combination of light and electron microscopic techniques. The first set will determine quantitatively if leupeptin treatment causes astroglial hypertrophy or decreases in synapse density in hippocampus, as well as cell death in hippocampus or cerebellum. Further, several inhibitors will be tested for their ability to cause the accumulation of amyloid plaques or neurofibrillary tangles in the brains of young rats. The second set of experiments will ascertain the nature of the morphological changes (if any) caused by specific inhibition of cathepsin B. The third set will utilize mice raised on a restricted diet which greatly increases lifespan. Lipofuscin content in dentate gyrus granule cells will be quantified in super-aged mice which are 50% older than control mice raised on a smiliar diet. The brains of such mice will also be examined for other morphological manifestations of aging. Together, these experiments could produce a powerful probe for studying aging at the cellular and molecular level, and shed new light on the relationship between mechanisms of aging and lipofuscin accumulation.
