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Establishment of Stably Transfected Rat Neuronal Cell Lines Expressing α-Synuclein GFP Fusion Proteins

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Abstract

Mutations in the α-synuclein gene have been linked to rare cases of familial Parkinson's disease (PD). α-Synuclein, a 140 amino acid polypeptide, is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice, Drosophila and marmosets (Challitrix jacchus) expressing either wild type (WT) or mutant human α-synuclein develop motor deficits, LB-like inclusions in some neurons and neuronal degeneration. The effects of human α-synuclein were investigated in a neuronal rat cell line (B103). Plasmids expressing WT and mutant human α-synuclein regulated by the cytomegalovirus (CMV) promoter were prepared and used for creating stably transfected neuronal rat cell lines. For localizing α-synuclein expression, stably transfected neuronal rat cell lines, expressing α-synuclein enhanced green fluorescent protein fusion proteins, regulated by either the CMV or the human platelet-derived growth factor ß promoter were generated. Over-expression of WT and A53T α-synuclein regulated by CMV promoter in stable transfectants resulted in formation of α-synuclein-immunopositive inclusion-like structures and mitochondrial alterations. Taken together, these results suggest that abnormal accumulation of α-synuclein could lead to mitochondrial alterations that might result in oxidative stress and eventually, cell death.

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Acknowledgments

All plasmids, but also the B103 cell line which were used in this work, were a gift of Elizer Masliah (Department of Neuroscience, University of California San Diego, La Jolla, USA). We would like to thank him for that and his laboratory for doing the Ribonuclease Protection Assay.

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Authors and Affiliations

  1. JSW Life Sciences, Grambach, Austria

    Gert Schwach, Michaela Tschemmernegg, Edith Schreiner & Manfred Windisch

  2. Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Graz, Austria

    Roswitha Pfragner

  3. Institute for Electron Microscopy, University of Technology, Graz, Austria

    Elisabeth Ingolic

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  1. Gert Schwach
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  2. Michaela Tschemmernegg
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  3. Roswitha Pfragner
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  4. Elisabeth Ingolic
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  5. Edith Schreiner
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  6. Manfred Windisch
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Correspondence to Roswitha Pfragner.

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Schwach, G., Tschemmernegg, M., Pfragner, R. et al. Establishment of Stably Transfected Rat Neuronal Cell Lines Expressing α-Synuclein GFP Fusion Proteins. J Mol Neurosci 41, 80–88 (2010). https://doi.org/10.1007/s12031-009-9289-1

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  • DOI: https://doi.org/10.1007/s12031-009-9289-1

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