Mechanisms of PKC gamma-mediated inhibition of dendritic growth in cerebellar purkinje cells

Spinocerebellar ataxias (SCA) are a group of cerebellar diseases characterized by progressive ataxia and cerebellar atrophy accompanied by a loss of Purkinje cells. Within SCA, Spinocerebellar ataxia type 14 (SCA14) is a subtype inherited in an autosomal dominant fashion and caused by missense, deletion or splice site mutations in the PRKCG gene, which is coding for protein kinase C (PKC) gamma (γ) (Yabe et al., 2003). Previous studies in our lab have shown that chronic activation of PKC with the PKC activator phorbol-12-myristate-13-acetate (PMA) in organotypic cerebellar slice cultures drastically inhibits the growth and development of the Purkinje cell dendritic tree (Metzger et al., 2000). This result is intriguing and could mean that the degeneration of the Purkinje cell dendritic tree in SCA14 may be caused by the increased activity of PKC. Another study has shown most SCA14 mutations in PKCγ showed an increased activity of PKCγ in transfected cells (Adachi et al., 2008). These findings raise the possibility that SCA14 might be related to increased PKC activity. The mechanisms by which increased PKC activity may lead to inhibition of Purkinje cell dendritic growth and perhaps even to degeneration are not known and it is not clear which proteins are involved in PKC signalling related to dendritic growth in Purkinje cells. In this project, we took advantage of a mouse model for SCA14, which was developed in our lab. In S361G mutated PKC gamma (mPKCγ) transgenic mice carrying a PKCγ transgene with a mutation from a human SCA14 allele we have shown that PKC activity is increased and Purkinje cell dendritic growth is strongly inhibited in slice cultures. We then tested whether other SCA14 mutations, in particular located in the C1 domain, would show similar effects on Purkinje cell dendritic development as the S361G mutation. We constructed several PKCγ mutants carrying mutations from human SCA14 patients and transfected them to Purkinje cells. We found that mutations in the catalytic domain caused severe inhibition of Purkinje cell dendritic development. In contrast, mutations in the C1 domain didn’t show this effect. Our findings suggest that mutations in the PKCγ gene causing SCA14 can have different effects on PKC biological activity in Purkinje cells and that multiple mechanism may be involved in the pathogenesis of SCA14. In order to search for molecules involved in signal transduction of mPKCγ, we performed a gene chip microarray analysis using mPKCγ transgenic mice and identified Carbonic anhydrase 8 (Car8) and type 1 inositol 1, 4, 5-trisphosphate receptor (IP3R1) as mRNAs and proteins being upregulated in mPKCγ transgenic mice. Furthermore, Car8 over-expression in Purkinje cells resulted in the formation of small, stunted dendritic trees in Purkinje cell similar to those after PKCγ activation implying that Car8 negatively regulates dendritic development. On the other hand, Car8 knocked down failed to rescue the morphology of the dendritic tree in Purkinje cells from mPKCγ transgenic mice or after pharmacological PKC activation. This indicates that Car8 is not directly downstream of mPKCγ signalling for Purkinje cell dendritic development but is likely to be part of a larger signalling network including PKCγ and IP3R1 which controls dendritic growth of Purkinje cells.