Singlet oxygen (O2[1 delta g]) is a very reactive molecule that can be produced by living cells and may contribute to cytotoxicity. The pineal hormone melatonin has been reported to possess potent antioxidant activity, and to be capable of scavenging O2(1 delta g). We investigated whether melatonin might reduce the neurotoxic action of O2(1 delta g). The cytotoxic effect of singlet oxygen was studied in primary cultures of cerebellar granule neurons pretreated with a photosensitive dye, rose bengal, and exposed to light--a procedure that generates O2(1 delta g). We found that this procedure triggers neuronal death, which is preceded by mitochondrial impairment (assayed by the rate of the reduction of MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, into formazan), and by DNA fragmentation--a marker of apoptosis. DNA fragmentation was determined in situ by terminal deoxynucleotidyl transferase assay; cell death was assayed with 0.4% trypan blue solution--viable cells with an intact membrane are not permeable to trypan blue; dead cells are, and thus, they are stained blue. Neuroprotection was obtained with the pineal hormone melatonin. In a cell-free system, melatonin also protected the enzyme creatine kinase (EC 2.7.3.2) from the rose bengal-induced injury. The results suggest that melatonin might counteract the cytotoxic action of singlet oxygen. Further studies are needed to clarify the exact role singlet oxygen and melatonin might play in neurodegenerative 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 report that serum removal triggers cell injury, which is characterized by signs of apoptosis. Actual cell death (trypan blue permeability) occurred 24 and 48 hr after serum removal. At earlier times (6 and 8 hr 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 μM ascorbic acid]. 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‐10,11‐dihydroxy‐5H‐dibenzo(a,d)cyclohepten‐5,10‐imine (MK‐801) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione also provided neuroprotection. However, oxidative stress appears to precede glutamate receptor activation: within the 8 hr period of serum deprivation, mannitol was protective when present either during only the first or last 4 hr; MK‐801 was protective only when present for the entire 8 hr period or in the last, but not first 4 hr of serum deprivation. Serum deprivation of mature cerebellar granule neurons can be used to study mechanisms of oxidative stress‐induced apoptosis. © 1996 Wiley‐Liss, Inc.

No abstract

Typically, primary cultures of rat cerebellar granule neurons are grown in the presence of 25 mM KCl and are considered to mature by ∼7 days in vitro. Potassium deficiency was created by growing the neurons from days 1 to 4 in the presence of 12.5 mM KCl (immature cultures) or by switching the mature neurons grown with 25 mM KCl to 12.5 mM KCl. In both conditions we observed neuronal death that bears the signs of apoptosis, i.e., DNA fragmentation determined qualitatively by agarose gel electrophoresis of DNA and quantitatively by in situ terminal deoxynucleotidyl transferase assay. The protein synthesis inhibitors cycloheximide and anisomycin provided neuroprotection in the mature cultures but potentiated the toxic effect of KCl deprivation in the immature neurons. The results suggest that a prudent use of protein synthesis inhibitors is critical in experiments with primary neuronal cultures.

Protein phosphorylation is kept in balance by an orchestrated action of kinases and phosphatases; when this balance is lost, neuronal apoptosis may occur. Okadaic acid (OKA), a marine toxin that inhibits specifically protein phosphatases 1 and 2A (EC 3.1.3.16), and staurosporine, an inhibitor of protein kinase C (PKC; EC 2.7.1.37), induced apoptosis in primary cultures of rat cerebellar granule neurons. We assayed apoptosis by the DNA gel electrophoresis, by the in situ TUNEL assay, and by morphological appearance following propidium iodide staining. Cell viability was assessed by the Trypan blue assay. Both OKA- and staurosporine-induced neuronal apoptosis were prevented by a macromolecular synthesis inhibitor actinomycin D and by a group of isoquinolinesulfonamide kinase inhibitors (H-7, 1-[5-isoquinolinesulfonyl]-2-methylpiperazine; H-8, N-¿2-[methylamino]ethyl¿-5-isoquinolinesulfonamide; H-9, N-(2-aminoethyl)-5-isoquinolinesulfonamide, but not by inhibitors of PKC, cyclic-GMP- and cyclic-AMP-dependent kinases, calcium/calmodulin-dependent kinases, tyrosine kinases, or by antioxidants. We postulate that a common mechanism, possibly an increased protein phosphorylation, is responsible for apoptosis triggered by an inhibition of phosphatases 1 and 2A and PKC. Elucidating the isoquinolinesulfonamide-sensitive mechanism may help us find new therapies for neurodegenerative diseases that involve apoptosis.