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

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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 GluRδ2Lc mutation.

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Acknowledgments

This work was supported by a NIH grant NS 34309 to M.W.V. and J.M. and by funds from “Centre National de la Recherche Scientifique” and Université Pierre et Marie Curie; Laboratoire Internationaux Associé grant from UPMC. MV was recipient of a visiting professorship grant from UPMC. We thank Fabrice Machulka for his assistance in animal breeding. We gratefully acknowledge the considerable contribution of Dr. Pauline Cavelier in data acquisition. We thank the Cell Imaging and Flow Cytometry facility of the IFR83 (Paris, France) for access and technical support in microscopy.

Conflicts of Interest

The authors certify that there is no conflict of interest concerning the work presented in this manuscript.

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Author notes

  1. Michael W. Vogel

    Present address: Division of Neuroscience and Basic Behavioral Science, National Institutes of Mental Health, 6001 Executive Blvd, Bethesda, MD, 20892-9645, USA

  2. Rebecca McFarland

    Present address: Department of Biology, University of Maryland Baltimore County, Baltimore, MD, 21201, USA

Authors and Affiliations

  1. CNRS, UMR7102, Paris, 75005, France

    Hadi S. Zanjani, Ann M. Lohof & Jean Mariani

  2. Université Pierre et Marie Curie-P6, UMR7102, Paris, 75005, France

    Hadi S. Zanjani, Ann M. Lohof & Jean Mariani

  3. Institut de la Longévité, Hôpital Charles Foix, 94205, Ivry-Sur-Seine, France

    Jean Mariani

  4. Maryland Psychiatric Research Center, Department of Psychiatry, University of Maryland School of Medicine, P.O. Box 21247, Baltimore, MD, 21228, USA

    Rebecca McFarland & Michael W. Vogel

  5. Equipe Développement et Vieillissement du Système Nerveux, UMR NPA 7102 CNRS et Université Pierre et Marie Curie, Case 14, 9, Quai St. Bernard, 75005, Paris, France

    Hadi S. Zanjani

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  1. Hadi S. Zanjani
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Correspondence to Hadi S. Zanjani.

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Zanjani, H.S., Lohof, A.M., McFarland, R. et al. Enhanced Survival of Wild-Type and Lurcher Purkinje Cells In Vitro Following Inhibition of Conventional PKCs or Stress-Activated MAP Kinase Pathways. Cerebellum 12, 377–389 (2013). https://doi.org/10.1007/s12311-012-0427-x

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