Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells

International Journal of Molecular Sciences, vol. 23, pp. 2650

Abstract

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Authors 6

1. Wenting You

   Maastricht University · Maastricht University Medical Centre

   Affiliation as printed

   Department of Biochemistry, CARIM School for Cardiovascular Disease, Maastricht University, 6229 ER Maastricht, The Netherlands

   Department of Mental Health and Neuroscience, Maastricht University, 6229 ER Maastricht, The Netherlands

   University Eye Clinic Maastricht UMC+, Maastricht University Medical Center+, 6229 HX Maastricht, The Netherlands

2. Tos T. J. M. Berendschot

   Maastricht University · Maastricht University Medical Centre

   Affiliation as printed

   University Eye Clinic Maastricht UMC+, Maastricht University Medical Center+, 6229 HX Maastricht, The Netherlands

3. Kèvin Knoops

   Maastricht University

   Affiliation as printed

   The Maastricht Multimodal Molecular Imaging Institute, Maastricht University, 6229 ER Maastricht, The Netherlands

4. Marc A. M. J. van Zandvoort Aachen Institute of Molecular Cardiovascular Research

   RWTH Aachen University · Universitätsklinikum Aachen · Maastricht University

   Affiliation as printed

   Department of Molecular Cell Biology, CARIM School for Cardiovascular Disease, Maastricht University, 6229 ER Maastricht, The Netherlands

   Institute of Molecular Cardiovascular Research, Universitätsklinikum Aachen, 52074 Aachen, Germany

5. Carroll A.B. Webers

   Maastricht University · Maastricht University Medical Centre

   Affiliation as printed

   University Eye Clinic Maastricht UMC+, Maastricht University Medical Center+, 6229 HX Maastricht, The Netherlands

6. Chris P.M. Reutelingsperger corresponding

   Maastricht University

   Affiliation as printed

   Department of Biochemistry, CARIM School for Cardiovascular Disease, Maastricht University, 6229 ER Maastricht, The Netherlands