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

Viewpoint: spinocerebellar ataxias as diseases of Purkinje cell dysfunction rather than Purkinje cell loss

Spinocerebellar ataxias (SCAs) are a group of hereditary neurodegenerative diseases mostly affecting cerebellar Purkinje cells caused by a wide variety of different mutations. One subtype, SCA14, is caused by mutations of Protein Kinase C gamma (PKCγ), the dominant PKC isoform present in Purkinje cells. Mutations in the pathway in which PKCγ is active, i.e., in the regulation of calcium levels and calcium signaling in Purkinje cells, are the cause of several other variants of SCA. In SCA14, many of the observed mutations in the PKCγ gene were shown to increase the basal activity of PKCγ, raising the possibility that increased activity of PKCγ might be the cause of most forms of SCA14 and might also be involved in the pathogenesis of SCA in related subtypes. In this viewpoint and review article we will discuss the evidence for and against such a major role of PKCγ basal activity and will suggest a hypothesis of how PKCγ activity and the calcium signaling pathway may be involved in the pathogenesis of SCAs despite the different and sometimes opposing effects of mutations affecting these pathways. We will then widen the scope and propose a concept of SCA pathogenesis which is not primarily driven by cell death and loss of Purkinje cells but rather by dysfunction of Purkinje cells which are still present and alive in the cerebellum

Carbonic Anhydrase 8 Expression in Purkinje Cells Is Controlled by PKCγ Activity and Regulates Purkinje Cell Dendritic Growth

Purkinje cell dendritic development is severely compromised after chronic activation of protein kinase C (PKC). In a recent transgenic mouse model of spinocerebellar ataxia 14, the ser361-to-gly (S361G) mutation of the protein kinase C gamma (PKCγ) gene was expressed in Purkinje cells. Purkinje cells from these mutant mice in organotypic slice cultures have the same stunted dendritic tree as Purkinje cells after pharmacological activation of PKC. Because the transgene is exclusively present in Purkinje cells, cerebellar tissue from these mice is an attractive starting material for searching genes which might be interacting with PKCγ in Purkinje cells for inducing the stunted dendritic growth. We have performed a microarray analysis and identified several candidate genes with an increased messenger RNA (mRNA) expression in the PKCγ-S361G transgenic Purkinje cells. Out of these candidates, we have further studied carbonic anhydrase 8 (CA8). We show here that CA8 mRNA and protein expression is strongly induced in PKCγ-S361G transgenic Purkinje cells. Overexpression of CA8 in Purkinje cells in dissociated cultures strongly inhibited Purkinje cell dendritic development and produced a dendritic phenotype similar to PKCγ-S361G. There was no evidence for a direct binding of CA8 to either PKCγ or the type 1 IP3 receptor. Knockdown of CA8 with miRNA did not alter Purkinje cell dendritic development and did not protect Purkinje cells in dissociated cultures from the stunted dendritic growth induced by PKCγ-S361G or by PKC activation. Our results indicate that CA8 is a novel important regulator of Purkinje cell dendritic development and that its expression is controlled by PKCγ activity

Fpk1/2 kinases regulate cellular sphingoid long-chain base abundance and alter cellular resistance to LCB elevation or depletion.

Sphingolipids are a family of eukaryotic lipids biosynthesized from sphingoid long-chain bases (LCBs). Sphingolipids are an essential class of lipids, as their depletion results in cell death. However, acute LCB supplementation is also toxic; thus, proper cellular LCB levels should be maintained. To characterize the "sphingolipid-signaling intercross," we performed a kinome screening assay in which budding yeast protein kinase-knockout strains were screened for resistance to ISP-1, a potent inhibitor of LCB biosynthesis. Here, one pair of such DIR (deletion-mediated ISP-1 resistance) genes, FPK1 and FPK2, was further characterized. Cellular LCB levels increased in the fpk1/2∆ strain, which was hypersensitive to phytosphingosine (PHS), a major LCB species of yeast cells. Concomitantly, this strain acquired resistance to ISP-1. Fpk1 and Fpk2 were involved in two downstream events; that is, ISP-1 uptake due to aminophospholipid flippase and LCB degradation due to LCB4 expression. RSK3, which belongs to the p90-S6K subfamily, was identified as a functional counterpart of Fpk1/2 in mammalian cells as the RSK3 gene functionally complemented the ISP-1-resistant phenotype of fpk1/2∆ cells

Increased biological activity of protein Kinase C gamma is not required in Spinocerebellar ataxia 14

Abstract Spinocerebellar ataxia (SCA) is an autosomal dominant neurodegenerative disorder characterized by slowly progressive cerebellar dysfunction. Currently, 42 SCA types are known, some of which are caused by CAG repeat expansions, but others are caused by point mutations or deletions. Spinocerebellar ataxia type 14 (SCA14) is caused by missense mutations or deletions in the PRKCG gene, coding for protein kinase C gamma (PKCγ). It is still not well understood how these mutations eventually cause Purkinje cell dysfunction and death. Because PKCγ is a well characterized signaling protein highly expressed in Purkinje cells SCA14 offers the chance to better understand the pathogenesis of Purkinje cell dysfunction and death. Altered biological activity of PKCγ would be the simplest explanation for the disease phenotype. There are indeed indications that the enzymatic activity of mutated PKCγ proteins could be changed. Many mutations found in SCA14 families are located in the regulatory C1B and C1A domain, while a few mutations are also found in the C2 and in the catalytic C3 and C4 domains. For many of these mutations an increased enzymatic activity could be demonstrated in cell-based assays, but it remains unclear whether there is indeed an altered biological activity of the mutated PKCγ proteins within living Purkinje cells. In this study we used the dendritic morphology of developing Purkinje cells to detect increased biological activity of PKCγ after expression of different mutated PKCγ proteins. Our results indicate that two out of three known mutations in the catalytic domain of PKCγ did indeed show increased biological activity. On the other hand, none of the five tested mutations located in the regulatory C1 or the C2 domain showed an increased biological activity. Our findings indicate that SCA14 mutations located in different domains of the PRKCG gene cause SCA14 by different mechanisms and that an increased constitutive activity of PKCγ may be one, but cannot be the only mechanism to cause disease in SCA14

Transcriptome Profile of a New Mouse Model of Spinocerebellar Ataxia Type 14 Implies Changes in Cerebellar Development

The autosomal dominant inherited spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders characterized by cerebellar atrophy and loss of Purkinje neurons. Spinocerebellar ataxia type 14 (SCA14) is a rare variant of SCAs caused by missense mutations or deletions in the PRKCG gene encoding the protein kinase C γ (PKCγ). Although mutated PKCγs are responsible for SCA14, it is still unclear exactly how mutated PKCγs are involved in SCA14 pathogenesis. Therefore, it is important to study how PKCγ signaling is altered in the cerebellum, which genes or signaling pathways are affected, and how this leads to neurological disease. In this study, we used a mouse line carrying a knock-in pseudo-substrate domain mutation in PKCγ (PKCγ-A24E) as an SCA14 model and performed RNA sequencing (RNA-seq) analysis at an early developmental timepoint (postnatal day 15) to investigate changes in the gene profile compared to wildtype mice. We analyzed both heterozygous (Het) PKCγ-A24E mice and homozygous (Homo) PKCγ-A24E mice for transcriptomic changes. The Het PKCγ-A24E mice reflects the situation observed in human SCA14 patient, while Homo PKCγ-A24E mice display stronger phenotypes with respect to Purkinje cell development and behavior. Our findings highlight an abundance of modifications affecting genes involved in developmental processes, suggesting that at least a part of the final phenotype is shaped by altered cerebellar development and is not only caused by changes in mature animals

Increased protein kinase C gamma activity induces Purkinje cell pathology in a mouse model of spinocerebellar ataxia 14

Spinocerebellar ataxias (SCAs) are hereditary diseases leading to Purkinje cell degeneration and cerebellar dysfunction. Most forms of SCA are caused by expansion of CAG repeats similar to other polyglutamine disorders such as Huntington's disease. In contrast, in the autosomal dominant SCA-14 the disease is caused by mutations in the protein kinase C gamma (PKCgamma) gene which is a well characterized signaling molecule in cerebellar Purkinje cells. The study of SCA-14, therefore, offers the unique opportunity to reveal the molecular and pathological mechanism eventually leading to Purkinje cell dysfunction and degeneration. We have created a mouse model of SCA-14 in which PKCgamma protein with a mutation found in SCA-14 is specifically expressed in cerebellar Purkinje cells. We find that in mice expressing the mutated PKCgamma protein the morphology of Purkinje cells in cerebellar slice cultures is drastically altered and mimics closely the morphology seen after pharmacological PKC activation. Similar morphological abnormalities were seen in localized areas of the cerebellum of juvenile transgenic mice in vivo. In adult transgenic mice there is evidence for some localized loss of Purkinje cells but there is no overall cerebellar atrophy. Transgenic mice show a mild cerebellar ataxia revealed by testing on the rotarod and on the walking beam. Our findings provide evidence for both an increased PKCgamma activity in Purkinje cells in vivo and for pathological changes typical for cerebellar disease thus linking the increased and dysregulated activity of PKCgamma tightly to the development of cerebellar disease in SCA-14 and possibly also in other forms of SCA