Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background (protein kinase CyhippocampusyMacs)

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an '50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKC« expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BLy6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BLy6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKC« expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes. Protein kinase C (PKC) is a family of serineythreonine kinases implicated in synaptic plasticity and information storage processes (24). The myristoylated alanine-rich C kinase substrate (MARCKS) is a primary PKC substrate that binds plasma membrane via N-terminal myristoylation and electrostatic interactions with the phosphorylation site domain, binds calmodulin in a calcium-dependent manner, and cross-links filamentous (F) actin, all in a PKC phosphorylation-reversible manner (1, 3). Accordingly, MARCKS is proposed to regulate membrane– cytoskeleton plasticity in response to PKCand calcium-mediated signaling (1, 3). Moreover, MARCKS is developmentally regulated (13), is enriched in neuronal growth cones (R.K.M., E. A. Wees, P. J. Meberg, T. B. Kuhn, and R.H.L., unpublished data), and is highly expressed in select regions of the developing and adult rat brain (12, 13). In particular, the granule cell layer of the hippocampus exhibits a high level of MARCKS gene expression (12, 13). In addition, the granule cell axons (mossy fibers), which have been implicated in the spatial learning ability of inbred mice (reviewed in ref. 17), express MARCKS protein at high levels in the adult brain (14). Because granule cell neogenesis occurs well into adulthood in a manner regulated by environmental demands (9), MARCKS may contribute to mossy fiber maturation and synaptic plasticity throughout the life of the animal. The creation of a mutant mouse strain that exhibits reductions in MARCKS expression has permitted the assessment of the contribution of MARCKS to hippocampal mossy fiber development and associated spatial learning processes. Homozygous mutant mice that do not express MARCKS exhibit abnormal brain development characterized by decreased brain size, collosal and commissural agenesis, cortical lamination abnormalities, particularly in the neocortex and hippocampus, exencephaly, and perinatal lethality (20). This phenotype is ‘‘rescued’’ (complemented) by transgenic expression of epitope-tagged human MARCKS driven by 3.4 kilobases of human MACS promoter (21), indicating that the phenotype is caused by the MARCKS deficiency alone. In the present study, we used heterozygous Macs mutant mice, which do not exhibit any gross neuroanatomical abnormalities, and MACS transgenic mice to assess the role of MARCKS in hippocampal mossy fiber development and spatial learning in the Morris water maze.