Until the beginning of 1980s, nerve cell necrosis was thought to responsible for nerve cell death in human brain, stroke ischemia and other neurological diseases1. In the early 70’s and 80’s a new form of cell death, termed apoptosis, was discovered2,3. About the same time it was recognized that the massive death of neurons which occurred as part of vertebrate prenatal and postnatal brain development dependent on competition for trophic factors4. Death due to neurotrophic insufficiency was termed programmed cell death as it was thought to depend on the activation of an intrinsic program leading to activation of “death genes” causing self-destruction. Therefore this form of cell death required new protein and RNA synthesis5,6. The dependence on new protein synthesis was similar to that found in the cell death of cultured sympathoblasts deprived of nerve growth factor (NGF) which showed many of the characteristics of apoptosis7. Subsequently, it has become clear that a requirement for new protein synthesis is a hallmark of programmed neuronal death but not necessarily of neuronal apoptosis8.
Some forms of neuronal apoptosis can proceed without new protein synthesis despite displaying the typical characteristics of cell and nuclear shrinkage, chromatin condensation, DNA fragmentation, (see figure) neurite and growth cone blebbing and detachment from the matrix. For example, PC12 cells that have been exposed to serum but not NGF undergo apoptosis with serum withdrawal that is unaffected by treatment with transcriptional/translational blockers that greatly reduce protein synthesis9. In contrast, PC12 cell apoptosis caused by trophic withdrawal after 12 days of NGF exposure required new protein synthesis10. Therefore, neuronal apoptos can be dependent on new protein synthesis (programmed), independent of new protein synthesis (unprogrammed), or can be even facilitated by the inhibition of protein synthesis11. It has rapidly become apparent that neuronal apoptosis is not only the result of serum starvation and/or trophic factor withdrawal, but that a wide range of different insults such as chemicals, toxins, neurotransmitters, hypoxia, radical oxygen species and neurodegenerative diseases can induce this process. It may not be surprising that neuronal apoptosis is common in neurological disorders, if one takes the view that apoptosis is the product of low level insults which are insufficient to kill nerve cells but are sufficient to activate a “suicide” system.
Apoptosis has been divided into four stages:
- Cell cycle arrest
- Capacitation for apoptosis and proliferation
- Irreversible commitment to death or preapoptosis
- Nucleolysis, chromatolysis and proteolysis12
The molecular and cellular mechanisms leading to cell death in general, and apoptosis in particular, are largely unknown. However, emerging new concepts strongly suggest that under pathological conditions, an imbalance of intracellular signal transduction pathways may cause cell death. For example Ca2+ overload13 in neurons due to the loss of membrane potential or massive synaptic glutamate release leads to mitochondrial impairment14, rapid increase in the concentration of cytoplasmic reactive oxygen species with widespread peroxidation of membrane lipids and activation of Ca2+ -dependent proteolytic enzymes. Together, these changes caused neuronal cell death.
To study cell death and in order to develop a novel understanding of neuroprotection, there is a tremendous need for chemical tools to induce cell death models in vitro and in vivo, and requires the availability of reliable, potent apoptosis/cell death inducers to achieve death in primary and cloned neuronal cultures, brain slices, ganglia and other neuronal preparations.
Alomone Labs is pleased to introduce the first in a series of compounds which induce apoptosis/cell death by four defined cellular mechanisms.
- Increase in intracellular Ca2+ to achieve Ca2+ overload
- Inhibition of protein kinase C (PKC)
- Inhibition of protein phosphatases
- Inhibition of macromolecules, (DNA, RNA and protein) synthesis
- The Ca2+ ionophore A23187 induces cell death in cultures of embryonic striatal neurons, causing both apoptos is and necrosis15. Oxidative stress, mitochondrial impairment and activation of caspases and phospholipases are important steps in A23187-induced cell death15,16,17. Thapsigargin, an inhibitor of the ER Ca2+-ATP-ase, by increasing intracellular Ca2+ induces apoptosis of young cerebellar granule neurons18, PC12 cells19, retinal cells20 by activation of calmodulin/calcineur in, BAD dephosphorylation and activation of caspase 9 and DNA fragmentation2.
- Staurosporine is a very potent, selective inhibitor of PKCs, and induces apoptosis in many cell types: glioblastoma22, cochlear neurons23, dopaminergic cells24, cortical neurons25 by interfering with Bcl2 phosphorylation, increasing caspase 3 activity and causing DNA fragmentation.
- Okadiac acid is a potent inhibitor of serine/threonine phosphatases 1 and 2A and induce apoptosis, by induction of new cell death genes, in rat pituitary cells26, human neuroblastoma27, hypothalamic neurons28, motor neurons29, cortical neurons30, glyoma cells31, cerebellar granule cells32,33, retinal ganglion cells34 and many other neuronal and non-neuronal cells.
- Protein synthesis inhibitors such as Puromycin are well known universal inhibitors of apoptosis35-37. Also, inhibitors of RNA synthesis such as actinomycin D act as very potent inducers of apoptosis by activation of stress-kinase JANK/SAPK and proapoptotic protein Bax38-40.
- Degirolami, U. et al. (1989) J.Neuropathol.Exp. Neurol. 43, 57.
- Kerr, J.F.R. et al. (1972) Curr.Biol. 4, 662.
- Willie, A.H. (1987) Int.Rev.Cytol. 17, 755.
- Oppenheim, R.W. (1989) Trends.Neurosci. 12, 252.
- Oppenheim, R.W. (1989) Ann. Rev. Neurosci. 14, 1356.
- Oppenheim, R.W. et al. (1990) Dev.Biol. 138, 104.
- Martin, D.P. et al. (1998) J.Cell.Biol. 119, 1669.
- Johnson, E.M. et al. (1995) J. Neurotrauma. 12, 843.
- Rukenstein, A. et al. (1991) J.Neurosci. 11, 2552.
- Mesner, P.W. et al. (1992) J.Cell.Biol. 119, 1669.
- Koh, J.Y. and Cotman, C.W. (1992) Brain Res. 587, 233.
- Kroemer, G. et al. (1995) FASEB J. 9, 1277.
- Choi, D.W. et al. (1995) Trends Neurosci. 18, 58.
- Richter, C. et al. (1995) BBA. 1271, 67.
- Peterson, A. et al. (2000) Brain.Res. 857, 20.
- Ray, S.K. et al. (2000) Brain Res. 852, 326.
- Atsumi, G. et al. (2000) J.Biol.Chem. 275, 18248.
- Levick, V. et al. (1995) Brain Res. 676, 325.
- Guo, Q. et al. (1997) J.Neurosci. 17, 4212.
- Chiarini, L.B. et al. (2000) Cell.Death Differ. 7, 272.
- Tombal, B. et al. (2000) Prostate. 43, 303.
- Begemann, M. et al. (1998) Anticancer Res. 18, 3139.
- Zirpel, L. et al. (1998) J.Neurophysiol. 79, 2288.
- Oh, Y.J. et al. (1997) Brain Res.Mol.Brain Res. 51, 133.
- Hou, S.T. et al. (2000) J.Neurochem. 75, 91.
- Ritz, V. et al. (1999) Naunyn.Schmiedebergs. Arch. Pharmacol. 360, 116.
- Francois, F. and Grimes, M.L. (1999) J.Neurochem. 73, 1773.
- Sortino, M.A. et al. (1999) Endocrinol. 140, 4841.
- Zhou, H. et al. (1999) J.Biol.Chem. 273, 16568.
- Kim, D.H. et al. (2000) Mol.Cells. 10, 83.
- Chin, L.S. et al. (2000) J.Biomed.Sci. 7, 152.
- Chalecka-Franaszek, E. and Chuang, D.M. (1999) PNAS. USA 96, 8745.
- Cagnoli, C.M. et al. (1996) Life.Sci. 58, 295.
- dosSantos, A.A. and deAraujo, E.G. (2000) Brain.Res. 853, 338.
- Ghibelli, L. et al. (1999) FASEB J. 13, 2031.
- Schlapbach, R. and Fontana, A. (1997) BBA. 1359, 174.
- Meijerman, I. et al. (1999) Toxicol.Appl.Pharmacol. 156, 46.
- Kleff, J. et al. (2000) Int.J.Cancer. 86, 399.
- Piva, T.J. et al. (2000) J.Cell.Biochem. 76, 625.
- Norita, Y. et al. (2000) Cancer Chemother. Pharmacol. 45, 149.