Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: Protection with antioxidants and glutamate receptor antagonists

Abstract

Apoptosis or programmed cell death is a morphologically distinct form of cell death that plays a major role during development. In apoptosis cells die through the activation of a cell-intrinsic suicide program which is regulated by many different signals that originate from both the intracellular and the extracellular milieu. This form of cell death appears to be a common feature in many biological processes where cell deletion is a mechanism for altering tissue structure and function. Over the last years compelling evidence has accumulated that apoptotic type of cell death may be the final common pathway in the neuropathology of a wide spectrum of neurological disease states, including ischemia, central nervous system trauma and some types of neurodegenerative diseases. Despite the identification of genes necessary for cell death the essential cellular events in apoptotic death remain largely unknown. A clear understanding of mechanisms that mediate apoptotic cell death will result in designing treatments that interfere with apoptotic mechanisms which could have important implications for developing therapeutic strategies for human diseases.Cerebellar granule neurons undergo apoptosis when deprived of chronic depolarization; serum deprivation has not been considered as a trigger of apoptosis in this culture. Here we found that serum removal triggers cell injury which is characterized by signs of apoptosis. Actual cell death (trypan blue permeability) occurred 24 and 48 h after serum removal. At earlier times (6 and 8 h after serum removal) we found significant impairment of mitochondrial functioning [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, assay) and an increase in the percentage of neurons showing signs of DNA fragmentation (in-situ terminal deoxynucleotidyl transferase assay, fluorescent assay). Protection was obtained by inhibiting RNA synthesis with actinomycin D and by antioxidants [1 mM: 1,4-diazobicyclo (2.2.2) octane, histidine, mannitol; 1% dimethyl sulfoxide; 0.01-1 uM ascorbic acid; 1, 10, 50 yM melatonin and 500 yM alpha tocopherol]. We also measured neuronal oxidation utilizing the oxidation-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate, and found a significant increase in the rate of neuronal oxidation as early as 15 min. after serum deprivation. The blockade of glutamate receptors by (+)-5-methyl-10,ll-dihydroxy-5H-dibenzo(a,d) cyclohepten-5,10- imine (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) also provided neuroprotection. However, oxidative stress appears to precede glutamate receptor activation: within the eight-hour period of serum deprivation, mannitol was protective when present either during only the first or last 4 h; MK-801 was protective only when present for the entire 8 h period or in the last, but not first 4 h of serum deprivation. These results are consistent with a role for oxidative stress in serum deprivation-induced apoptosis in cerebellar granule cell culture that can be manipulated with antioxidants and glutamate receptor antagonists.Key Words: apoptosis, programmed cell death, antioxidants, glutamate, free radicals