Ephraim Yavin   Oxidative stress, bioactive lipids and cell death in cell cultures and in fetal brain during intrauterine life

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e-mail: ephraim.yavin@weizmann.ac.il

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Objectives

The disruption of the balance between free radical production and cellular defense mechanisms as a consequence of ischemia, anoxia and reoxidation injury have been implicated in a wide variety of acute and chronic neurologic disorders. During neuronogenesis, a process which almost exclusively occurs in utero, a large number of cells die through a process called programmed neuronal death (PND) or apoptosis. PND is a physiological phenomenon of cardinal importance for the development and ultimate shaping of neuronal assemblies in the mature nervous system. In addition to programmed death, loss of cells in the nervous system can occur by necrosis after an acute injury. It is usually the result of a precipitous environmental stress such as release of excitotoxins, mechanical injury, abrupt changes in pH and cellular osmolarity or hypoxia/ischemia. Stress-triggered cell death may assume either a necrotic or apoptotic form by yet to be clarified mechanisms.

Although morphologically and molecularly distinct, death by apoptosis and necrosis share several common features including generation of free radicals and typical DNA fragmentation. Lipids are essential membrane constituents but in addition, some participate in signalling cascades during normal cell function while others are targets for lipid peroxides during oxidative stress. Understanding the exact role of lipids in signalling cascades and in cell damage and death is the main focus of our studies.

Recent Findings

Cell culture models: A new permanent, actively proliferating cell line, OLN-93, expressing limited properties of oligodendroglia cell precursors was used to examine the effects of toxic doses of H₂O₂ on activation of cell cycle associated proteins and cell death. Exposure of G1-arrested cells to H₂O₂, resulted in a transient halt in cell proliferation. Cell death was assessed by a reduction in total cell DNA, accumulation of propidium iodide-stained cells in the sG1 phase, DNA ladder and numerous DAPI-stained nuclear fragments, all characteristic of apoptotic death. The expression of p21^Waf1 and p27^Kip1 growth regulatory proteins has been examined. There was a significant increase in p21^Waf1 protein and mRNA levels. p27^Kip1 protein levels were diminished within a short time following stress. The data suggest that G1-arrested OLN-93 cells are sensitive to H₂O₂ stress and undergo apoptosis. In contrast, cells which are in the non arrested G1 phase escape death and in response to stress, elevate p21^Waf1 levels which may then act in the rescue process (Kameshwar et al.).

The effect of cell density on survival of primary cerebral cortex cells after anoxia/hypoglycemia episodes is investigated (Yavin and Marie-Billia, 1997). Incubation of cells for up to several hours under argon in conjunction with glucose deprivation, enhanced apoptotic cell death in neuronal cells as assessed by DAPI staining and reduced neutral red dye uptake. Time lapse cinematography and DAPI staining revealed a form of cell death involving exclusion of the nucleus. Neutral red dye uptake was enhanced with increasing density and in vitro maturation but was reduced in dense cultures exposed to anoxic/hypoglycemic conditions. The data suggest that cell density may play a critical role in brain organogenesis and that anoxia/hypoglycemia is more deleterious in dense than sparse cells. Further to these studies, phospholipid polar head groups and polyunsaturated fatty acid composition in cells are modified by long term feeding and the impact of the changes on stress-induced cell death is investigated.

In vivo studies: The developing brain accumulates docosahexaenoic acid (DHA) in relatively large quantities already prior to birth (Green and Yavin, 1996). The objective of our work is to unravel the role of DHA in the fetal brain during intrauterine life and elucidate its possible role during oxidative stress in utero and at birth (Green and Yavin, in press). Our working hypothesis is that DHA is a necessary component for the normal brain development and its selective accumulation during the last days of gestation in the fetus is crucial for increasing the scavenging capacity of brain tissue towards birth. In addition DHA is an important membrane component which may accentuate the expression of various signalling cascades including that of PKC.

Because of its ubiquity in neuronal membranes and possible role in synaptogenesis, we recently devised a method for selectively and rapidly enriching fetal brain DHA without the confounding effects of other n-3 PUFA (Green and Yavin, 1995). This was accomplished by intraamniotic administration of ethyl-DHA (Et-DHA). Analysis of brain and liver phospholipids showed significant changes in both polar head group and FA composition. Phosphatidyl serine levels for example, were nearly doubled.

Excessive DHA accretion in utero may also be detrimental during oxidative stress around birth. To study these aspects, optimal conditions for TBARS production by brain slices after intraamniotic administration of Et-DHA or after depriving the maternal diet of n-3 precursors during pregnancy were established (Glozman and Yavin, 1997). Studies were recently conducted on prenatal rats given a single intraamniotic injection of Et-DHA to evaluate the role of DHA on oxidative stress during episodes of ischemia. Fetal global ischemia was induced by obstruction of the maternal-fetal blood circulation (Glozman et al., in press). A time-dependent decrease in the ability of brain slices from animals treated with Et-DHA to produce thiobarbituric acid reactive substance (TBARS), most pronounced after one day was noticed. The protective effect of a single dose of Et-DHA in utero remained evident up to 3 days after injection and was still notable after birth. In agreement with a reduction in TBARS production by slices, the endogenous levels of TBARS in brains of Et-DHA-treated animals was lower than the controls. Et-DHA injected fetuses exhibited significantly higher levels of esterified DHA than the non-injected controls.

Obstruction of the maternal fetal blood circulation is a serious stress which affects brain energy metabolism and may lead to impaired growth. Three distinct, maternal-independent routes (e.g. intraamniotic, intraperitoneal and intracerebral), for [1-¹³C]glucose utilization by fetal brain and liver tissues, before and after vascular occlusion of the maternal-fetal blood flow were examined by multinuclear magnetic resonance spectroscopy. Distinct kinetics of glucose and lactic acid accumulation were identified after ischemic episodes in both fetal brain and liver tissues. The data suggest that mobilization of glucose by fetal organs during oxygen deprivation and accumulation of lactate may act in concert to provide a protective mechanism for extending survival and for maintaining functional activities in the fetal brain.

Current Research Team

	Shosh Gil (Technician)

	Annette Brand, Ph.D. (Vis. Scientist)

	Pnina Green, MD, Ph.D. (Assoc. Vis. Scientist)

	Eduard Yakubov (Asst. Researcher)

	Faina Kuperstein (Graduate student)

Selected Publications

Green P, Kamensky B, and Yavin E. Replenishment of docosahexaenoic acid content in phospholipids of n-3 fatty acid-deficient fetuses by intraamniotic ethyl-docosahexaenoate administration. J. Neurosci. Res. 48, 264-272, 1997.

Yavin E and Marie Billia D. Death in early postmitotic cerebral cortex cells after transient oxygen and glucose deprivation is apoptotic. J. Neurosci. Res. 47, 471-478, 1997.

Glozman S, and Yavin E. Lipid peroxides are generated by fetal rat brain after episodes of global ischemia in utero. Neurochem. Res. 22,201-208, 1997.

Gil C , Ruiz-Meana M., Álava M., Yavin E, and Aguilera J. Tetanus toxin induces reversible protein kinase C translocation and increases polyphosphoinositide hydrolysis in rat cerebral cortex preparations J. Neurochem. 70, 1636-1643, 1998.

Glozman S, Green P and Yavin E. Intraamniotic ethyl-docosahexaenoate administration protects fetal rat brain from ischemic stress. J. Neurochem. 70, 2484-2491, 1998.

Brand A, Gil S, Leibfritz D, and Yavin E. Direct administration and utilization of [1-¹³C] glucose by fetal brain and liver tissues under normal and ischemic conditions: ¹H, ³¹P and ¹³C NMR studies. J. Neurosci. Res. 54, 97-108, 1998.

Green P and Yavin E. Mechanisms of docosahexaenoic acid accretion in the fetal brain. J. Neurosci. Res. 52, 129-136. 1998.

Kameshwar-Rao A.S.V.R., Richter-Landsberg C, Seger R, Givol D and Yavin E. H₂O₂-Induced apoptotic death in serum-deprived cultures of oligodendroglia origin is linked to cell differentiation. J. Neurosci. Res. 56, 447-556, 1999.

Uberti D, Yavin E, Gil S, Kameshwar-Rao A, Goldfinger N and Rotter V. Hydrogen peroxide induces nuclear translocation of p53 and apoptosis in cells of oligodendroglia origin. Mol. Brain Res. 65, 167-175, 1999.

Green P, Glozman S, Kamensky B and Yavin E. Developmental changes in rat brain membrane lipids and fatty acids - the preferential prenatal accumulation of docosahexaenoic acid J. Lipid Res. 40, 960-966, 1999.

Glozman S, Green P, Kamensky B, Weiner L, and Yavin E. Et-DHA administration during intrauterine life enhances prostanoid production and reduces free radicals generation in the fetal rat brain Lipids 34, s247-248, 1999.

Brand A, Gil S and Yavin E. N-Methyl bases of ethanolamine prevent apoptotic cell death induced by oxidative stress in cells of oligodendroglia origin. J. Neurochem. 74, 1596-1604, 2000.

Koudinova NV, Koudinov AR and Yavin E. Alzheimer's A[beta]_1-40 peptide modulates lipid synthesis in neuronal cultures and intact rat fetal brain under normoxic and oxidative stress conditions. Neurochem. Res. 25, 653-660, 2000.

Glozman S, Cerruti-Harris C, Groner Y and Yavin E. Docosahexaenoic acid-deficient phosphatidyl serine and high [alpha]-tocopherol in a fetal mouse brain over expressing Cu/Zn-superoxide dismutase. Biochim. Biophys. Acta. 1487, 135-144, 2000.

Kuperstein F, Reiss N, Koudinova NV, and Yavin E. Biphasic modulation of protein kinase C and enhanced cell death by amyloid beta peptide and anoxia in neuronal cultures. J. Neurochem. In press.

Brand A, Gil S, Seger R, and Yavin E. Lipid Constituents in Oligodendroglia Cells alter susceptibility to H2O2-induced apoptotic cell death via ERK activation. J. Neurochem. In press.

Yavin E, Glozman S, and Green P. Docosahexaenoic acid accumulation in the prenatal brain: prooxidant and antioxidant features. J. Mol. Neurosci. In press.

Rabinkov A, Miron T, Mirelman D, Wilchek M, Glozman S, Yavin E and Weiner L. S-allylmercaptoglutathione: the reaction product of allicin with glutathione possesses antioxidant and SH-modifying properties. Biochim. Biophys. Acta. In press.

Submitted

Green P, Glozman S, and Yavin E. Ethyl docosahexaenoate-associated decrease in fetal brain lipid peroxide production is mediated by activation of prostanoid and nitric oxide pathways.

Book chapters

Yavin E. Monosialogangliosides and nerve growth factor stimulate synthesis of glycoproteins in glioma cells, in Molecular signalling and regulation in glial cells: A key to remyelination and functional repair (G Jeserich, HH Althaus, C Richter-Landsberg and R Heumann, eds) Springer Verlag, Heidelberg pp 76-90, 1996.

Green P and Yavin E. Mechanisms of docosahexaenoic acid accretion in the fetal brain. J. Neurosci. Res. 52, 129-136, 1998.