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

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.

PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

“…Incubating neuronal PC12 cells with SR-18292 at a concentration of 100 µM after EtOH removal for 20 h resulted in 100% cell death. This is in stark contrast with our previous findings on cell viability that more than 90% neuronal PC12 cells survived and recovered from 3 h exposure to EtOH [ 17 ]. It indicates the critical role of PGC-1α-mediated mitochondrial biogenesis in the recovery of neuronal PC12 cells from EtOH-induced cellular degeneration.…”

“…A previous study in neuronal PC12 cells showed that, by removing the cell death stimulus EtOH, dying cells could survive and recover from diverse apoptotic events that are related to neuronal cell death, including neurite retraction, nuclear condensation, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, increased ROS generation and increased level of intracellular Ca 2+ [ 17 ]. In the present study, we analysed the mitochondrial ultrastructure in EtOH-treated neuronal PC12 cells and found fragmented mitochondria with swollen structure, disrupted cristae, and less electron-dense mitochondrial matrix (Fig.…”

A time window for rescuing dying retinal ganglion cells

“…Neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and ciliary neurotrophic factor (CNTF) have been demonstrated to increase neural repair and recovery, promoting neuroprotection and regeneration [ 42 ]. Our previous study in neuronal PC12 cells showed that recovery of mitochondrial impairment, including mitochondrial fragmentation and membrane potential loss, did not depend on exogenous NGF [ 17 ]. prRGCs are routinely cultured in media that contain the neurotrophic factors BDNF and CNTF.…”

“…Apoptosis is generally considered to be irreversible: once initiated, cells are committed to die. However, recent studies have shown that cells can reverse the process of cell death and recover to normal morphology when the cell death stimulus is removed, even at late stages of apoptosis [ 14 – 17 ]. Reversibility of apoptosis opens up a new, previously unexpected phenomenon in the field of regulated cell death, and may be helpful to rescue cells that are difficult to regenerate, like CNS neurons, such as RGCs.…”

“…Recently, we studied the reversibility of the cell death process in in vitro cell death models using various cell death triggers and neuronal cell lines [ 17 , 33 ]. Ethanol is a widely used cell death trigger [ 34 ], which can be easily removed by washing and changing the medium in order to study the reversibility of the cell death process [ 17 ]. In the current study, we used ethanol to study the reversibility of the cell death process in prRGCs.…”