Enhanced Survival of Wild-Type and Lurcher Purkinje Cells In Vitro Following Inhibition of Conventional PKCs or Stress-Activated MAP Kinase Pathways

• DOI: 10.1007/s12311-012-0427-x
• Publication Date: 2012-11-07T07:29:12Z
• Authors: Hadi S. Zanjani; Ann M. Lohof; Rebecca McFarland; Michael W. Vogel; Jean Mariani
• Keywords: Cell death; Male; 0301 basic medicine; Calbindins; Cell Survival; [SDV]Life Sciences [q-bio]; Lurcher mutant mouse; Mice, Transgenic; Gene Expression Regulation, Enzymologic; Mice; Purkinje Cells; 03 medical and health sciences; Organ Culture Techniques; Cerebellum; Animals; PKC; Enzyme Inhibitors; Organotypic culture; Protein Kinase C; Analysis of Variance; Caspase 3; [SDV] Life Sciences [q-bio]; Animals, Newborn; Receptors, Glutamate; Purkinje cells; Mice, Inbred CBA; MAP kinase; Calcium; Female; JNK; Mitogen-Activated Protein Kinases

Recent studies using both dissociated and organotypic cell cultures have shown that heterozygous Lurcher (Lc/+) Purkinje cells (PCs) grown in vitro share many of the same survival and morphological characteristics as Lc/+ PCs in vivo. We have used this established tissue culture system as a valuable model for studying cell death mechanisms in a relatively simple system where neurodegeneration is induced by a constitutive cation leak mediated by the Lurcher mutation in the δ2 glutamate receptor (GluRδ2). In this study, Ca(++) imaging and immunocytochemistry studies indicate that intracellular levels of Ca(++) are chronically increased in Lc/+ PCs and the concentration and/or distribution of the conventional PKCγ isoform is altered in degenerating Lc/+ PCs. To begin to characterize the molecular mechanisms that regulate Lc/+ PC death, the contributions of conventional PKC pathways and of two MAP kinase family members, JNK and p38, were examined in slice cultures from wild-type and Lc/+ mutant mouse cerebellum. Cerebellar slice cultures from P0 pups were treated with either a conventional PKC inhibitor, a JNK inhibitor, or a p38 inhibitor either from 0 to 14 or 7 to 14 DIV. Treatment with either of the three inhibitors from 0 DIV significantly increased wild type and Lc/+ PC survival through 14 DIV, but only Lc/+ PC survival was significantly increased following treatments from 7 to 14 DIV. The results suggest that multiple PC death pathways are induced by the physical trauma of making organotypic slice cultures, naturally-occurring postnatal cell death, and the GluRδ2 (Lc) mutation.