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Alcohol induces Golgi fragmentation in differentiated PC12 cells by deregulating Rab1-dependent ER-to-Golgi transport

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Abstract

In the present study, we analyze the effects of ethanol on the Golgi structure and membrane transport in differentiated PC12 cells, which are used as a model of neurons. Chronic exposure to moderate doses of ethanol induces Golgi fragmentation, a common characteristic of many neurodegenerative diseases. Alcohol impaired the lateral linking of stacks without causing microtubule damage. Extensive immunocytochemical and western blot analyses of representative Golgi proteins showed that few, but important, proteins are significantly affected. Thus, alcohol exposure induced a significant ER-to-Golgi transport delay, the retention of the GTPase Rab1 in the Golgi membranes and the accumulation of tethering factor p115 in the cytosol. These modifications would explain the observed fragmentation. The amount of p115 and the stacking protein GRASP65 increased in alcohol-treated cells, which might be a mechanism to reverse Golgi damage. Importantly, the overexpression of GTP-tagged Rab1 but not of a dominant-negative Rab1 mutant, restored the Golgi morphology, suggesting that this protein is the main target of alcohol. Taken together, our results support the view that alcohol and neurodegenerative diseases such as Parkinson have similar effects on intracellular trafficking and provide new clues on the neuropathology of alcoholism.

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Acknowledgments

We are grateful to the Image Analysis Service of the University of Murcia for help with the 3D reconstruction. This work was supported by grants from the Ministerio de Ciencia e Innovación (Spain) (BFU2008-00477/BMC and Consolider COAT CSD2009-00016) and Fundación Séneca de la Comunidad Autónoma de la Región de Murcia (04542/GERM/06) to JAM-M, from the Ministerio de Ciencia e Innovación (SAF2008-00305) and Red de Trastornos Adictivos (RTA, RETICS, ISCIII, grant RD06/0001/0009) to JR-P and from Ministerio de Ciencia e Innovación (BFU2010-17116) to MMM. MPM and GE-P are recipients of RTA fellowships.

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  1. Mónica Tomás

    Present address: Department of Anatomy, Medical School, University of Valencia, Valencia, Spain

Authors and Affiliations

  1. Department of Cell Biology and Histology, School of Medicine, University of Murcia, 30100, Murcia, Spain

    Mónica Tomás, Emma Martínez-Alonso & José A. Martínez-Menárguez

  2. Centro de Investigación, Hospital La Fe, 46009, Valencia, Spain

    María Pilar Marín, Guillermo Esteban-Pretel & Jaime Renau-Piqueras

  3. Department of Cell Biology, Physiology and Immunology, IMIBIC, CIBERobn, University of Cordoba, 14014, Cordoba, Spain

    Alberto Díaz-Ruiz, Rafael Vázquez-Martínez & María M. Malagón

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Correspondence to José A. Martínez-Menárguez.

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418_2012_970_MOESM1_ESM.pdf

Supplementary figure 1. Alcohol does not affect the microtubule cytoskeleton. Representative immunofluorescence images of distribution of α-tubulin and acetylated α-tubulin in control and ethanol-exposed cells. No differences concerning the distribution of these cytoskeletal proteins were found. Bar 25 μm. Supplementary figure 2. Alcohol does not affect the level of expression of proteins involved in ER-Golgi trafficking. Cell lysates obtained from control and ethanol-treated cell were separated by SDS-PAGE and immunolabelled with different antibodies. (PDF 1103 kb)

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Tomás, M., Marín, M.P., Martínez-Alonso, E. et al. Alcohol induces Golgi fragmentation in differentiated PC12 cells by deregulating Rab1-dependent ER-to-Golgi transport. Histochem Cell Biol 138, 489–501 (2012). https://doi.org/10.1007/s00418-012-0970-z

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