Enhanced survival of wild-type and Lurcher Purkinje cells in vitro following inhibition of conventional PKCs or stress-activated MAP kinase pathways

Cerebellum. 2013 Jun;12(3):377-89. doi: 10.1007/s12311-012-0427-x.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analysis of Variance
  • Animals
  • Animals, Newborn
  • Calbindins / metabolism
  • Calcium / metabolism
  • Caspase 3 / metabolism
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Cerebellum / cytology*
  • Enzyme Inhibitors / pharmacology
  • Female
  • Gene Expression Regulation, Enzymologic / drug effects
  • Gene Expression Regulation, Enzymologic / genetics
  • Male
  • Mice
  • Mice, Inbred CBA
  • Mice, Transgenic
  • Mitogen-Activated Protein Kinases / metabolism*
  • Organ Culture Techniques
  • Protein Kinase C / metabolism*
  • Purkinje Cells / drug effects*
  • Receptors, Glutamate / genetics*
  • Signal Transduction / drug effects*
  • Signal Transduction / genetics

Substances

  • Calbindins
  • Enzyme Inhibitors
  • Receptors, Glutamate
  • glutamate receptor delta 2
  • Protein Kinase C
  • Mitogen-Activated Protein Kinases
  • Caspase 3
  • Calcium