Aging-related alterations in the distribution of Ca²⁺-dependent PKC isoforms in rabbit hippocampus

The immunocytochemical and subcellular localization of the Ca2+-dependent protein kinase C (cPKC) isoforms (PKCalpha, beta1, beta2, and gamma) was examined in rabbit hippocampus of young (3 months of age; n = 11) and aging (36 months of age; n = 14) subjects. Detailed immunocytochemical analyses revealed a significant increase in PKCbeta1, beta2, and gamma immunoreactivity in principal cell bodies and associated dendrites, and interneurons of the hilar region in the aging rabbits. The number of PKCalpha- and gamma-positive interneurons in the aging stratum oriens declined significantly. PKCa was least affected in principal cells, showing an increase in immunostaining in granule cells only. Weakly PKC-positive principal cells intermingled between densely stained ones were seen in parts of the hippocampus in most of the aging rabbits, showing that the degree of aging-related alterations in PKC-immunoreactivity varies between neurons. Changes in PKC expression in the molecular and subgranular layer of the aging dentate gyrus suggested a reorganization of PKC-positive afferents to this region. Western blot analysis revealed a significant loss of PKC in the pellet fraction for all isoforms, and a tendency for increased levels of cytosolic PKC. However, no significant changes were found in total PKC content for any PKC isoform. A concurrent dramatic loss of the PKC anchoring protein receptor for activated C kinase (RACK1) in the pellet fraction was shown by Western blotting. These findings suggest that the loss of RACK1 contributes to the dysregulation of the PKC system in the aging rabbit hippocampus. The enhanced PKC-immunoreactivity might relate to reduced protein-protein interactions of PKC with the anchoring protein RACK1 leading to increased access of the antibodies to the antigenic site. In conclusion, the results suggest that memory deficits in aging rabbits are (in part) caused by dysregulation of subcellular PKC localization in hippocampal neurons. (C) 2004 Wiley-Liss, Inc.