Immunohistochemical examination of the INK4 and Cip inhibitors in the rat neonatal cerebellum: cellular localization and the impact of protein calorie malnutrition

Expression of the cyclin-dependent kinase inhibitors (CKIs) has been linked to the inhibition of cellular proliferation and the induction of different iation. Based on structure function analysis, two distinct families of CDKI s, the INK4 and the Cip/Kip family have been identified. The INK4 family me mber p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in n ormal development of the CNS and cerebellum, Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D 1 in orchestrating growth factor-induced cellular proliferation. The neonat al rat cerebellum undergoes proliferative growth and differentiation, local ized to distinct topographical regions and cell types. The cell type and th e temporal profile of CKI expression during postnatal cerebellar developmen t had not been described. The current studies determined the specific cereb ellar cell types in which the CKIs were expressed during post natal develop ment by co-staining for cell-type specific markers. p16(Ink4a) and p27(Kip1 ) immunostaining was identified in both neurons and glial cells, increasing progressively between postnatal days 6 to 13 into adulthood. By contrast, neuronal and glial cell p21(Cip1) Staining was prominent at days 6-11 and d ecreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal gr anular layers. Dual immunostaining demonstrated cyclin D1 within cells expr essing CKI (p16(Ink4a), p21(Cip1), p27(Kip1)). Cerebellar cellular growth a rrest, induced by protein-calorie malnutrition, inhibited cyclin D1 protein levels without affecting CKI immunostaining suggesting CKI do not mediate the developmental arrest. These results demonstrate that the CKIs are induc ed by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo. (C) 2000 Elsevier Science B.V. All righ ts reserved.