Caspase-independent component of retinal ganglion cell death, in vitro

Invest Ophthalmol Vis Sci. 2004 Nov;45(11):4049-59. doi: 10.1167/iovs.04-0490.


Purpose: Although in vitro and in vivo models demonstrate caspase activation in retinal ganglion cells (RGCs) undergoing apoptosis, the caspase-independent component of RGC death is unclear. Identification of the precise mechanisms of cell death in these distinct neurons is essential for the development of effective neuroprotective strategies in glaucoma. Because TNF-alpha and hypoxia have been implicated in RGC death during glaucomatous optic nerve degeneration, this study was conducted to determine whether RGCs survive exposure to TNF-alpha or hypoxia in the presence of caspase inhibitor treatment, and whether mitochondrial dysfunction is involved in RGC death induced by these glaucomatous stimuli.

Methods: Primary cultures of rat RGCs were exposed to TNF-alpha or hypoxia for up to 48 hours. The temporal relationship of RGC death with the loss of mitochondrial membrane potential and the release of cell death mediators, including cytochrome c and apoptosis-inducing factor (AIF), was studied in the absence and presence of specific inhibitors of caspases. In addition, treatment with a free-radical scavenger, 4-hydroxytetramethylpiperidine-1-oxyl (tempol; 5 mM), was used in some experiments. Cell viability was assessed using calcein assay, and annexin V binding combined with propidium iodide staining was used for the distinction of apoptotic and necrotic cells. Caspase-3-like protease activity was measured using a fluorometric assay, and for the in situ detection of caspase activity, immunocytochemistry was performed with a cleavage-site-specific antibody. The time course of alterations in the mitochondrial membrane potential and the release of cell death mediators in individual cells undergoing cell death were assessed with a fluorescent tracer and subsequent immunocytochemistry. In addition, a fluorescent dye, dihydroethidium was used to assess the generation of reactive oxygen species (ROS).

Results: Findings of this study revealed that the loss of mitochondrial membrane potential and the release of cell death mediators accompanied RGC death induced by TNF-alpha or hypoxia. Although caspase inhibitor treatment temporarily decreased the rate of apoptosis, caspase inhibition was not adequate to block RGC death if the mitochondrial membrane potential was lost and mitochondrial mediators were released. Despite the inhibited caspase activity, survival rate was less than 70% after a 48-hour incubation with death stimuli, and both apoptotic and necrotic cells were detectable in these cultures. When combined with caspase inhibition, tempol reduced the production of ROS and provided an additional 20% increase in RGC survival.

Conclusions: Based on these novel findings, RGC death induced by TNF-alpha or hypoxia involves a caspase-independent component, and reducing the free-radical generation provides additional protection of RGCs temporarily saved by caspase inhibition. Therefore, neuroprotective strategies in glaucoma should include tools to improve the ability of these neurons to survive the cytotoxic consequences of mitochondrial dysfunction.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Animals, Newborn
  • Apoptosis Inducing Factor
  • Apoptosis*
  • Caspase Inhibitors
  • Caspases / physiology*
  • Cell Hypoxia
  • Cell Survival
  • Cells, Cultured
  • Cyclic N-Oxides / pharmacology
  • Cytochromes c / metabolism
  • Enzyme Inhibitors / pharmacology
  • Flavoproteins / metabolism
  • Fluorometry
  • Free Radical Scavengers / pharmacology
  • Membrane Potentials
  • Membrane Proteins / metabolism
  • Mitochondria / metabolism
  • Mitochondria / pathology
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism
  • Retinal Ganglion Cells / drug effects
  • Retinal Ganglion Cells / enzymology
  • Retinal Ganglion Cells / pathology*
  • Spin Labels
  • Tumor Necrosis Factor-alpha / pharmacology


  • Aifm1 protein, rat
  • Apoptosis Inducing Factor
  • Caspase Inhibitors
  • Cyclic N-Oxides
  • Enzyme Inhibitors
  • Flavoproteins
  • Free Radical Scavengers
  • Membrane Proteins
  • Reactive Oxygen Species
  • Spin Labels
  • Tumor Necrosis Factor-alpha
  • Cytochromes c
  • Caspases
  • tempol