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 differentiation. Based on structure function analysis, two distinct families of CDKIs, the INK4 and the Cip/Kip family have been identified. The INK4 family member p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in normal development of the CNS and cerebellum. Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D1 in orchestrating growth factor-induced cellular proliferation. The neonatal rat cerebellum undergoes proliferative growth and differentiation, localized to distinct topographical regions and cell types. The cell type and the temporal profile of CKI expression during postnatal cerebellar development had not been described. The current studies determined the specific cerebellar cell types in which the CKIs were expressed during post natal development 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 decreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal granular layers. Dual immunostaining demonstrated cyclin D1 within cells expressing CKI (p16(Ink4a), p21(Cip1),p27(Kip1)). Cerebellar cellular growth arrest, 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 induced by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo.