Ephraim Yavin

Docosahexaenoic acid abundance in the brain: a biodevice to combat oxidative stress.

Abstract Docosahexaenoic acid (DHA) (22:6) is a polyunsaturated fatty acid of the n−3 series which is believed to be a molecular target for lipid peroxides (LPO) formation. Its ubiquitous nature in the nervous tissue renders it particularly vulnerable to oxidative stress, which is high in brain during normal activity because of high oxygen consumption and generation of reactive oxygen species (ROS). Under steady state conditions potentially harmful ROS and LPO are maintained at low levels due to a strong antioxidant defense mechanism, which involves several enzymes and low molecular weight reducing compounds. The present review emphasizes a paradox: a discrepancy between the expected high oxidability of the DHA molecule due to its high degree of unsaturation and certain experimental results which would indicate no change or even decreased lipid peroxidation when brain tissue is supplied or enriched with DHA. The following is a critical review of the experimental data relating DHA levels in the brain to lipid peroxidation and oxidative damage there. A neuroprotective role for DHA, possibly in association with the vinyl ether (VE) linkage of plasmalogens (pPLs) in combating free radicals is proposed.

MECHANISMS OF DOCOSAHEXAENOIC ACID ACCRETION IN THE FETAL BRAIN

Docosahexaenoic acid (DHA, 22:6 n‐3) is the major polyunsaturated fatty acid (PUFA) in the adult mammalian brain. DHA is an essential fatty acid (FA) since it, or its short chain precursor, α‐linolenic acid (LnA, 18:3 n‐3), have to be obtained in the diet. Moreover, dietary n‐3 FA deficiency is associated with biochemical changes in the brain and with disturbances in vision and other neurological parameters. Under normal nutritional conditions, fetal brain DHA accumulation is substantial, with a “DHA accretion spurt” being demonstrated in the last period of gestation. This accumulation is supported by the maternal supply of DHA or LnA, but selectivity of DHA accumulation is probably a placental function whose mechanism is lately being clarified. The fetal gastrointestinal (G‐I) tract may be instrumental in supplying DHA to the fetal brain under certain conditions, such as following intra‐amniotic administration of ethyl‐docosahexaenoate (Et‐DHA). In this pathway, DHA is supplied independently of the maternal metabolism, and the fetal liver is apparently involved. The fetal G‐I tract may be advantageous for DHA supply in cases of maternal–placental insufficiency resulting in intrauterine growth retardation. The fetal brain itself is capable of metabolizing LnA to DHA, without the participation of the fetal liver, thus contributing to the accumulation of its own DHA during one of the most crucial periods of its development. J. Neurosci. Res. 52:129–136, 1998. © 1998 Wiley‐Liss, Inc.

Hydrogen peroxide induces nuclear translocation of p53 and apoptosis in cells of oligodendroglia origin

The observation that apoptosis is an inherent pathway in oligodendrocytes development coupled with the notion that wild-type p53 is expressed in these cells, prompted us to investigate the interrelationship between the two phenomena. Using a permanent oligodendroglia-like cell line (OLN 93), we examined the role of p53 protein in apoptosis following a DNA insult induced by a brief exposure to H2O2. A marked translocation of p53 from the cytosolic to the nuclear compartment was notable by 20 min, following a 5 min treatment with 1 mM H2O2 as identified by cell immunostaining. By 48 h following H2O2 addition, nearly 60% of the cells exhibited p53 in the nuclei. At this time, a large proportion of the cells underwent apoptosis as identified by DAPI nuclear staining. The genotoxic-induced p53 relocalization appeared to be cell cycle phase specific; thus OLN 93 cultures enriched for cells in the G0/G1 stage by serum starvation, and abundant in nuclear-associated p53, were more susceptible to H2O2-induced apoptosis than their untreated counterparts and than double thymidine block, G1/S enriched, cultures. Analysis of the expression of p53 downstream genes indicated that p21 and mdm2 were upregulated following p53 nuclear translocation. From the kinetics of protein accumulation, it appears that mdm2 enhancement accelerated the exit of p53 from the nucleus to the cytosol. Our results suggest that following stress, oligodendroglia-like cells are induced to undergo p53-dependent apoptosis, an event that coincides with p53 nuclear translocation and is cell-cycle related.

Survival and maturation of cerebral neurons on poly(l-lysine) surfaces in the absence of serum

Abstract Several phenotypic properties of dissociated rat embryo cerebral cells cultivated in the absence of serum on poly( l -lysine) coated surfaces were investigated. A great enrichment of neuronal cells is attained after 3–4 weeks in culture as a result of cessation of nonneuronal cell proliferation. The monolayer pattern of cell-cell reassociation and neuritic sprouting is promoted by poly( l -lysine) and it is independent of serum presence or the existence of a layer of nonneuronal cells. Synaptic profiles and typical regions of neuropil are prominent. The neuronal plasma membrane is often characterized by irregular foldings at points of cell-cell contacts. A few biochemical parameters investigated lend support to the suggestion that the cerebral neurons acquire a certain state of functional maturation. It is proposed that substances secreted by the cells or released as a result of cell death may be conspicuous components for long-term survival of the cerebral neurons.

Ganglioside Profiles during Neural Tissue Development

The accumulation and species distribution of gangliosides in cultured cerebral cells and in the late prenatal stage of the developing rat brain were examined as a function of their growth state. The concentration of lipid-bound sialic acid in cerebral cells rises from a value of about 5 nmol/mg protein at day 1 to a peak level of 16.2 nmol/mg protein by 12 days, followed by a substantial decrease after 25 days in culture. When expressed per cell DNA, a linear increase of about 10-fold, notably after 10 days in culture, in the amount of sialic acid is seen. The most significant change in the species distribution is an apparent accumulation of disialo ganglioside (GD1a) trisialo ganglioside (GT) and tetrasialo ganglioside (GQ), whereas a marked reduction of GD3 is evident. The accumulation of sialic acid and the developmental patterns of the ganglioside species in vitro reasonably agree with the developing prenatal brain. Thus, by birth, both GT and GQ account for about 36% of the total brain gangliosides as compared to 40% in the mature cerebral cultures. Since in the newborn rat brain synaptogenesis is not prominent the increase in the ganglioside complexity could be associated with neuritic sprouting and establishment of nonsynaptic interneuronal connections. Rabbit antiserum raised against pure GD1a reacted preferentially with the neuronal cell bodies and neurite extensions as observed by an indirect immunofluorescence technique. A progressive increase in immunofluorescence labeling paralleled accumulation of polysialo-gangliosides and culture maturation. In this context, the dissociated cerebral cells provide a powerful tool for investigating aspects of neuronal development because of the possibility of correlating neurochemical data with ultrastructure and cell-surface-related events.

ERK activation and nuclear translocation in amyloid-β peptide- and iron-stressed neuronal cell cultures

Oxidative stress in the human brain has been strongly implicated as the cause of neuronal cell losses in Alzheimers disease patients, but the exact mechanism still remains unknown. In this report several oxidative stress parameters and an associated signalling transduction cascade predating neuronal cell death in cultures treated with the oxidative stressors Fe2+ (5 µm) and the amyloid beta (Aβ1−40) peptide (5 µm) were studied. Production of reactive oxygen species as detected by dichlorofluorescein staining was apparent within 5 min in the presence of both agents. Lipid peroxide content increased by approximately 10‐fold after 2 h, while mitochondrial activity was impaired by 40% after 6 h. Caspase‐3 activity was elevated 5–6 fold, all indicative of oxidative cell stress. The combined presence of Aβ1−40 and Fe2+ resulted in a rapid (5 min) ERK activation followed by a decline by 30 min and a second activation that continued up to 24 h when nuclear translocation was noticed. Neither treatment with Fe2+ nor that with Aβ1−40 alone caused similar changes. Addition of either deferroxamine (DFe, 25 µm), catalase (0.4 mg/mL) or N‐acetyl cysteine (0.5 mm) – the last two known as suppressants of oxidative stress – attenuated ERK activation and nuclear translocation. The mitogen‐activated protein/ERK kinase (MEK) inhibitor U0126 blocked ERK and caspase 3 activation, suppressed ERK translocation and reduced the number of apoptotic cells, suggesting a central role for the ERK signalling cascade in Aβ1−40 plus Fe2+ (Aβ1−40/Fe2+) ‐induced apoptotic death. The full peptide Aβ1−42 was very effective at 0.5 µm while the inverse peptide Aβ40−1 at 5 µm was ineffective. The acetyl‐amyloid‐β protein amide fragment 15–20 (V‐pep) known to be an Aβ aggregation inhibitor, prevented Aβ1−40/Fe2+‐induced toxicity. These findings indicate that metal ions chelators and antioxidants suppress the Aβ1−40/Fe2+‐induced oxidative stress cascade and may be beneficial in reducing the severity of Alzheimers disease.

Nervous System‐Specific Proteins in Developing Rat Cerebral Cells in Culture

Abstract: The nervous system‐specific proteins: synaptin, D1, D2, D3, glial fibrillary acidic protein (GFA) and 14‐3‐2, were quantified in dissociated cerebral cells from the foetal rat brain at various times of growth in culture. By approximately 1 week in culture, the neuronal membrane markers synaptin, D1, D2, and D3 could all be demonstrated. A maximum concentration of 10–20% for synaptin, D1, and D3 and 160% for D2, in comparison with the levels in adult forebrain, was attained during the 2nd week in vitro. The astroglial gliofilament marker GFA increased continuously, reaching by 38 days of cultivation an 18‐fold higher level than the concentration in adult forebrain. The neuronal cytoplasm marker 14‐3‐2 could be demonstrated in trace amounts, and only after more than 1 week in vitro. Neuronal cell bodies and processes stained by indirect immunofluorescence using an anti‐D2 serum were strongly fluorescent after 1 week in vitro. Immunofluorescence staining for GFA revealed a cytoplasmatic filamentous network in perinuclear areas and processes of, presumably, astroblasts.

Fatty acid composition of late embryonic and early postnatal rat brain.

The fatty acid (FA) composition and distribution in a variety of phospholipids (PL) and neutral lipids (NL) at two discrete stages during the embryonic rat brain development were investigated. Over 96% of the FA were acylated into fetal brain PL at embryonic day 17 after the peak of neuronal proliferation and at embryonic day 20, one day prior to delivery. Phosphatidylcholine constituted approximately 60% of the total PL pool, phosphatidylethanolamine (PE) 30%, phosphatidylserine (PS) 6%, and phosphatidylinositol (PI) 4%. The diacylglycerols and triacylglycerols constituted 1–2% of the fetal brain lipids. α-Linolenic acid (18∶3n−3) and linoleic acid (18∶2n−6) were found in very low amounts in all fetal brain PL and NL. The percentage of the n−6 polyunsaturated FA, consisting of arachidonic acid (AA), 22∶4n−6 and 22∶5n−6, remained unchanged in all the fractions, except in Pl, in which the proportion of AA increased. The concentration of docosahexaenoic acid (DHA) increased with age in all the fractions, with the bulk of accumulation accounted for by its increase in PE and, to a lesser extent, in PS. This finding suggests a “DHA accretion spurt” during the last three days of pregnancy.

Lipid constituents in oligodendroglial cells alter susceptibility to H2O2‐induced apoptotic cell death via ERK activation

The present work examines the effect of membrane lipid composition on activation of extracellular signal‐regulated protein kinases (ERK) and cell death following oxidative stress. When subjected to 50 µm docosahexaenoic acid (DHA, 22 : 6 n‐3), cellular phospholipids of OLN 93 cells, a clonal line of oligodendroglia origin low in DHA, were enriched with this polyunsaturated fatty acid. In the presence of 1 mmN,N‐dimethylethanolamine (dEa) a new phospholipid species analog was formed in lieu of phosphatidylcholine. Exposure of DHA‐enriched cells to 0.5 mm H2O2, caused sustained activation of ERK up to 24 h. At this time massive apoptotic cell death was demonstrated by ladder and TUNEL techniques. H2O2‐induced stress applied to dEa or DHA/dEa co‐supplemented cells showed only a transient ERK activation and no cell death after 24 h. Moreover, while ERK was rapidly translocated into the nucleus in DHA‐enriched cells, dEa supplements completely blocked ERK nuclear translocation. This study suggests that H2O2‐induced apoptotic cell death is associated with prolonged ERK activation and nuclear translocation in DHA‐enriched OLN 93 cells, while both phenomena are prevented by dEa supplements. Thus, the membrane lipid composition ultimately modulates ERK activation and translocation and therefore can promote or prevent apoptotic cell death.

Intraamniotic Ethyl Docosahexaenoate Administration Protects Fetal Rat Brain from Ischemic Stress

Abstract: Studies were conducted on the prenatal rat given a single intraamniotic injection of ethyl docosahexaenoate (Et‐DHA; 9.6–12 mmol per fetus) or subjected to an n‐3 fatty acid‐deficient diet to assess the role of docosahexaenoate on oxidative stress during episodes of ischemia. 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 1 day (from 58.1 ± 4.22 to 15.9 ± 1.6 nmol/mg of DNA), was noticed on stimulation with Fe2+. Brain slices from fetuses treated for 1 day with Et‐DHA and those from untreated fetuses produced TBARS levels of 46.7 ± 6.5 and 114.8 ± 10.8 nmol/mg of DNA, respectively, after a 20‐min occlusion of the fetal‐maternal circulation at embryonic day 20, suggesting a protective effect of Et‐DHA. The protective effect of a single dose of Et‐DHA in utero remained high up to 3 days after injection (p < 0.001) and was long‐lasting, yet not significant, up to 3 days following birth. In agreement with a reduction in TBARS production by slices, the endogenous levels of TBARS in brains of Et‐DHA‐treated animals were lower than in the controls. Et‐DHA‐injected fetuses exhibited significantly higher levels of esterified DHA than the non‐injected controls. n‐3‐deficient diet given to dams for 2 weeks before birth did not affect the levels of TBARS production in control fetal brain slices but abolished the increase caused by ischemia. Et‐DHA administration for 24 h to n‐3‐deficient fetuses reduced the amount of TBARS produced by the fetal brain slices from 49.1 ± 8.5 to 31.7 ± 4.1 nmol/mg of DNA. A protective effect from oxidative damage after postischemic oxidative stress in fetal brain following DHA supplements is suggested, whereas the effect of n‐3 fatty acid deficiency in this regard is more ambiguous.