Michael W. Miller

Growth factor-mediated neural proliferation: target of ethanol toxicity

Ethanol exposure during development is teratogenic. The central nervous system (CNS) is particularly susceptible to ethanol toxicity. In fact, heavy gestational ethanol consumption is one of the leading known causes of mental retardation in the Western world. Ethanol exposure disrupts the proliferation of glia and neuronal precursors in the developing CNS. Depending upon cell population and blood ethanol concentration, ethanol can either inhibit or stimulate cell proliferation. Two features of cell proliferation that are affected by ethanol exposure are the growth fraction (the proportion of cells that is actively cycling) and the cell cycle kinetics, particularly in the length of the G1 phase of the cell cycle. Cell proliferation in the developing CNS reflects the action of positive (mitogenic growth factors) and negative (anti-proliferative factors) regulators. Increasing evidence shows that ethanol interferes with the action of growth factors. In vitro systems are a good model to investigate ethanol neurotoxicity, since the effects of ethanol on cultured cells parallel the effects of ethanol in the developing CNS. The inhibitory effects of ethanol on cell proliferation may result from interference with mitogenic growth factors (e.g., bFGF, EGF, PDGF, IGF-I). Conversely, the stimulatory effects of ethanol may result from the interference with growth inhibiting factors (e.g., TGFbeta1). Interestingly, both in vivo and in vitro studies show that proliferating neural cells display differential sensitivity to ethanol. This differential sensitivity correlates with their response to mitogenic growth factors; that is, cells that are actively regulated by mitogenic growth factors are much more susceptible to ethanol than cells that are less or unresponsive to such factors. Ethanol interference with growth factor action could occur at three levels: ligand production, receptor expression, and/or signal transduction. Thus, ethanol-induced alterations in the developing CNS that characterize fetal alcohol syndrome apparently result from alterations in the regulatory action of growth factors.

Effects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus.

Prenatal exposure to ethanol causes neuronal death in somatosensory cortex, but apparently not in the ventrobasal nucleus of the thalamus. Effectors such as bcl-2, bax, and caspase 3 can determine whether a neuron survives or dies. We hypothesize that ethanol differentially affects the expression of these proteins in the cortex and thalamus during the periods of naturally occurring and ethanol-induced neuronal death. Pregnant rats were fed ad libitum with an ethanol-containing liquid diet (Et) or pair-fed an isocaloric non-alcoholic diet (Ct). Samples were collected from fetuses (gestational day (G) 16 and G19) and pups (postnatal day (P) 0 through P30) and examined for bcl-2, bax, or caspase 3 expression using a quantitative immunoblotting procedure. Prenatal exposure to ethanol reduced cortical bcl-2 expression, but not bax expression on P6. Hence, the bcl-2/bax ratio was lower in Et-treated rats than in controls. In contrast, thalamic expression of neither bcl-2 nor bax was significantly different in the two groups of rats. Thus, the thalamic bcl-2/bax ratio was unaffected by exposure to ethanol. During the period of naturally occurring neuronal death, the expression of the active (20 kDa) and inactive isoforms (32 kDa) of caspase 3 was altered in the cortices of Et-treated rats, but not in their thalami. Thus, prenatal exposure to ethanol affected the early postnatal expression of death-related proteins in the cortex, but not in the thalamus. These biochemical changes concur with anatomical data on the spatial and temporal selectivity of ethanol toxicity in the developing CNS.

Proliferation and death of cultured fetal neocortical neurons: effects of ethanol on the dynamics of cell growth

Neuronal number in the mature CNS is determined by the balance of cell proliferation and death. The effects of ethanol on cell proliferation and death were examined in primary cultures of neocortical neurons derived from 16-day-old rat fetuses. The cells were treated with ethanol (0 or 400 mg/dl) and examined for (1) immunohistochemical identity, (2) cell cycle kinetics using a cumulative bromodeoxyuridine labeling technique, (3) viable cell number via a trypan blue assay, and (4) the incidence of cell death with terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 immunhistochemistry. After two days in culture, most (>85%) cells expressed a neuron-specific antigen(s) whether or not ethanol was added to the culture medium. Ethanol affected the proliferation of the cultured cells, e.g., the length of the cell cycle was greater in the ethanol-treated cells than in controls. The number of trypan blue-negative (viable) cells was profoundly decreased by ethanol exposure. This decrease was accompanied by increases in the frequencies of TUNEL- and caspase 3-positive cells and of cells exhibiting nuclear condensations. Thus, ethanol decreases the number of viable cells in vitro by slowing cell proliferation and increasing the incidence of cell death. The expression of the death indices in untreated cultures is most consistent with a single (apoptotic) pathway of cell death, rather than simultaneous apoptotic and necrotic modes of death. Furthermore, it appears that ethanol initiates an apoptotic death among cultured cortical neurons.

Effects of prenatal exposure to ethanol on callosal projection neurons in rat somatosensory cortex.

The distribution and density of callosal projection neurons in the somatosensory cortex of mature rats was altered by prenatal exposure to ethanol. The density of callosal neurons was significantly greater in ethanol-treated rats than in controls. Ethanol exposure also altered the laminar distribution of callosal projection neurons. Whereas in control rats the cell bodies of callosal projection neurons were in layers II/III and V, in ethanol-treated rats most of these neurons were distributed in layers V and VI. Many of the ectopic neurons were generated toward the end of cortical neuronogenesis (i.e., on gestational day 20). This contrasts with controls wherein co-generated cohorts were distributed in layer II/III. Thus, the connectional phenotype of the callosal projection neurons is retained regardless of its laminar residence. These ethanol-induced abnormalities apparently result from defects in neuronal migration and axonal pruning.

Expression of bcl-2, bax, and caspase-3 in the brain of the developing rat.

Naturally occurring neuronal death (NOND) is generally considered to be apoptotic. Apoptosis is an active form of cell death in which the regulation of specific proteins produces anti- or pro-apoptotic signals. Two of the protein families involved in this regulation are the bcl proteins and caspases. A quantitative immunoblotting technique was used to examine the temporal expression of bcl-2, bax, and two isoforms of caspase 3 (an active 20 kDa isoform and the inactive 32 kDa precursor) throughout the developing neuraxis. Long-Evans rat fetuses were collected on gestational day (G) 16 and G19, and pups were harvested on postnatal day (P) 0, P3, P6, P12, P21, and P30. Brains were divided into five segments: cortex, thalamus, midbrain, medulla/pons, and cerebellum. In general, the expression of bax increased and the ratio of bcl-2 expression to bax expression decreased concurrent with published data on the onset of NOND in a given area. The timing of these events was paralleled by an increase in the expression of active caspase 3. Unlike the bcl proteins, caspase 3 expression returned toward fetal levels as the brain matured. The timing of the changes in bcl protein and caspase expression show that both protein families are involved in promoting neuronal death. Reductions in caspase expression (and not bcl-2 and bax expression) are key to ending the period of NOND.

Effect of ethanol on neurotrophin-mediated cell survival and receptor expression in cultures of cortical neurons

The interaction of ethanol and neurotrophin-mediated cell survival was examined in primary cultures of cortical neurons. Cells were obtained from rat fetuses on gestational day 16 and maintained in a medium supplemented with either 10% or 1.0% fetal calf serum (FCS). Exogenous nerve growth factor (NGF; 20 ng/ml), brain-derived neurotrophic factor (BDNF; 20 ng/ml) or neurotrophin 3 (NT-3; 20 ng/ml) was added to the cultures alone, or in combination with ethanol (400 mg/dl). The number of viable neurons was determined after a 48 h treatment with a growth factor and/or ethanol. The effects of ethanol on the expression of high affinity neurotrophin receptors (trkA, trkB, and trkC) and the low-affinity receptor (p75), were analyzed using Western immunoblots. In untreated cultures, 22.7% and 26.3% of the cells raised in a medium containing 10% and 1.0% FCS, respectively, were lost. Only NGF prevented the death of the cultured cortical neurons. Ethanol was toxic; it caused a 23.5% and 16.7% loss of cells (for cells grown in a medium containing 10% and 1.0% FCS, respectively) beyond that occurring naturally in an untreated culture. Ethanol completely blocked the NGF-mediated cell survival. In general, BDNF and NT-3 did not offset the toxic effect of ethanol. Immunoblotting studies showed that the expression of p75 was significantly (p < 0.05) lower (40%) in ethanol-treated cultures, but ethanol did not affect trk expression. Thus, ethanol has specific effects upon NGF-mediated cell survival and the effects on the low affinity receptor imply that p75 specifically plays an important role in NGF signaling.

Transforming growth factor β1 regulates the expression of cyclooxygenase in cultured cortical astrocytes and neurons

Abstract: The hypothesis that transforming growth factor β1 (TGFβ1) regulates the synthesis of prostaglandins by CNS tissue was tested by using purified cultures of cortical astrocytes or neurons that were obtained from rat pups on postnatal day 4 or 5 or fetuses on gestational day 16, respectively. The cells were exposed to TGFβ1 for 2 days. The synthesis of prostaglandins depends upon the production and conversion of arachidonic acid, steps that are catalyzed by phospholipase A2 (PLA2) and cyclooxygenase (COX), respectively. Prostaglandin E2 (PGE2) concentration was determined by radioimmunoassay. The expression of cytosolic PLA2 and COX (the constitutive COX1 and the inducible COX2) was assessed by using immunohistochemical and quantitative immunoblotting procedures. Astrocytes produced much more PGE2 than neurons, suggesting that glial cells are an important source of PGE2 in the CNS. TGFβ1 increased the production of PGE2 by astrocytes and neurons in a concentration‐dependent manner. Furthermore, TGFβ1 enhanced COX activity; the inhibitor indomethacin completely blocked TGFβ1‐mediated PGE2 synthesis. Cultured astrocytes and neurons expressed the three enzymes: cytosolic PLA2, COX1, and COX2. Cytosolic PLA2 expression was unaffected by TGFβ1 treatment. In contrast, COX expression was altered by TGFβ1 treatment in a concentration‐dependent fashion. COX1 was increased by TGFβ1, but only in astrocytes. TGFβ1 increased COX2 expression in astrocytes and neurons. Thus, TGFβ1‐induced increases in PGE2 concentration are regulated by COX. This study suggests that TGFβ1 is an important regulator of immune and inflammatory processes in the CNS.

Effect of early exposure to ethanol on the protein and DNA contents of specific brain regions in the rat

A hallmark of fetal alcohol syndrome is microencephaly. Microencephaly can result from either a reduction in the number of cells, in the amount of neuropil, or both. Inasmuch as CNS structures develop along different time lines, the window of vulnerability to ethanol for each structure may differ. Therefore, the growth of the brain after prenatal exposure to ethanol was compared with that following postnatal ethanol exposure. Rats were exposed to ethanol prenatally (between gestational days 6 and 21 via oral consumption by the pregnant dam) or postnatally (between postnatal days 4 and 12 via intubation). The mean of the daily peak blood ethanol concentration was 146 +/- 16 and 127 +/- 18 mg/dl for the rats exposed to ethanol pre- or postnatally, respectively. Pair-fed control rats were generated for both the pre- and postnatal conditions. Wet weights, DNA content, and protein content were quantified for 6 brain segments (right and left neocortices, right and left hippocampi, the cerebella, and brainstems) of 21-day-old rats. The total weight of the brain was reduced 12% by pre- or postnatal exposure to ethanol. Only the weights of the neocortex and the cerebellum were significantly reduced by pre- or postnatal treatment. Interestingly, postnatal ethanol exposure increased the weight of the hippocampi. The DNA and protein contents of specific brain segments were affected by ethanol exposure, but all structures were not affected similarly. The greatest effect was on the neocortex; both measures decreased following pre- or postnatal ethanol exposure. The DNA and protein contents of the cerebellum were also affected by pre- or postnatal ethanol exposure. In contrast, while DNA content in the hippocampus was unaffected by prenatal exposure, it was significantly increased by postnatal ethanol exposure. Thus, ethanol-induced microcephaly and differences in DNA and protein content result from changes in cell number and in neuropil volume. These effects, however, differ among CNS structures in a time-dependent manner suggesting that the primary, but not sole target of ethanol is the proliferating cell.

Differential effects of ethanol on the expression of cyclo-oxygenase in cultured cortical astrocytes and neurons

The developing central nervous system is a primary target of ethanol toxicity. The teratogenic effect of ethanol may result from its action on prostaglandins. Prostaglandins are generated through the release of arachidonic acid (AA) by the action of cytosolic phospholipase A2 (cPLA2) on membrane‐bound phospholipids and the catalytic conversion of AA to prostaglandin E2 (PGE2) by cyclo‐oxygenase (COX). COX is expressed in two isoforms, constitutive COX1 and inducible COX2. Cultured astrocytes and neurons from immature cerebral cortex were used as in vitro models to investigate the effect of ethanol on PGE2 synthesis. In both cell types, neither the activity nor the expression of cPLA2 was affected by ethanol. PGE2 was synthesized by astrocytes and neurons. Ethanol (200–400u2003mg/dL for 24u2003h) significantly increased PGE2 production in both cell types and the ethanol‐induced increase in PGE2 accumulation in astrocytes was significantly greater than in neurons. These increases resulted from the effects of ethanol on COX. Overall COX activity was up‐regulated by ethanol in astrocytes and neurons, and indomethacin, a nonselective blocker for COX, eliminated the ethanol‐induced increases of COX activity in both cell types. Increased COX activity in astrocytes resulted from an increase in COX2 expression. NS‐398, a selective COX2 blocker, completely inhibited ethanol‐induced alterations in COX activity. In neurons, however, ethanol had a direct effect on COX activity in the absence of a change in COX expression. NS‐398 only partially blocked ethanol‐induced increases in neuronal COX activity. Thus, astrocytes are a primary target of ethanol and ethanol‐induced increases in glial PGE2 synthesis are mediated by COX, principally COX2. Ethanol toxicity may be mediated through PGE2 in immature cortical cells.

Ethanol Inhibits Basic Fibroblast Growth Factor‐Mediated Proliferation of C6 Astrocytoma Cells

Abstract: Early ethanol exposure alters the proliferative activity of glial and neuronal precursors in the developing CNS. The present study tests the hypothesis that ethanol‐induced alterations in cell proliferation result from interference with growth factors. An in vitro model of astroglia (C6 astrocytoma cells) was used to study the effects of ethanol on proliferation mediated by basic fibroblast growth factor (bFGF). bFGF stimulated the proliferation of C6 cells. This bFGF‐enhanced proliferation was evident by increases in total cell number, DNA synthesis (as measured by [3H]thymidine incorporation), and the number of cells that took up bromodeoxyuridine. A synthetic peptide that specifically blocked the binding of bFGF to its high‐affinity receptor completely abolished the proliferation‐promoting effect of bFGF. The action of another mitogen for C6 cells, insulin‐like growth factor‐1, was not affected by this peptide. Therefore, the bFGF‐stimulated proliferation was mediated through a specific bFGF receptor. Ethanol inhibited bFGF‐mediated proliferation in a concentration‐dependent manner. Ethanol concentrations of 100 and 200 mg/dl partially inhibited bFGF‐mediated proliferation (by 58 and 74%, respectively), whereas concentrations of ≥400 mg/dl completely abolished the growth‐stimulating effect of bFGF. Our data show that ethanol alters proliferative activity of C6 cells by disrupting the action of bFGF. The target of ethanol neurotoxicity is a receptor‐mediated activity. bFGF can affect cell proliferation by a non‐receptor‐mediated intracellular pathway, but ethanol does not have an impact on this pathway.