Research reportEffects of prenatal exposure to ethanol on the expression of bcl-2, bax and caspase 3 in the developing rat cerebral cortex and thalamus
Introduction
Prenatal ethanol exposure can alter the number of neurons in the mature central nervous system (CNS). Such damage varies among CNS structures; indeed, variations can even exist within the same motor or sensory system. For example, in the trigeminal-somatosensory system, the number of neurons in the primary somatosensory cortex [40] and in the principal sensory nucleus of the trigeminal nerve (PSN) [33], [35], [38] of ethanol-treated rats is one-third fewer than in controls. Neuronal number in the ventrobasal nucleus of the thalamus (VB), however, is unaffected by ethanol [44].
The number of neurons within a structure is the sum of two opposing processes: the additive effect of cell acquisition (proliferation and neuronal migration) and the subtractive effect of neuronal death. Ethanol affects both processes in vivo. Studies show that exposure to ethanol during early development can reduce the proliferation of neuronal precursors [15], [35], [36], [39] and alter the timing of neuronal migration [28], [29], [31], [34]. A recent longitudinal study of neuronal number in the developing PSN shows that ethanol reduces neuronal acquisition and increases neuronal death [35]. This dual effect of ethanol on cell proliferation and neuronal death is also evident in vitro. Ethanol causes the death of cultured cortical neurons and inhibits the neuronal proliferation seen in primary neuronal cultures [13], [55]. It is noteworthy that the balance of the effects of ethanol on cell proliferation and death in vitro is similar to the effects seen in the PSN in vivo.
Various features of ethanol-induced cell death echo those associated with naturally occurring apoptosis [5], [7], [53]. These include morphological changes (e.g., in the cytoskeleton and nucleus) and biochemical changes [16]. For example, chronic exposure to ethanol can increase the incidence of DNA damage in vitro [2], [13], [21], [27] and in vivo [5], [53]. Furthermore, prenatal exposure to ethanol causes transient increases in the cortical expression of a protein recognized by the antibody ALZ-50 [19], a protein associated with naturally occurring neuronal death (NOND) [1], [37], [58]. Likewise, p53 is upregulated during NOND [19], [37], [52], [59]. p53 can act as a transcription factor for bax [42] and directly affects the relative expression of pro- and anti-apoptotic bcl proteins. The balanced expression of bcl proteins is considered to be a critical determinant in cell survival [18], [50].
Effectors that are downstream of the bcl proteins in the apoptotic cascade include the caspases [6], [41]. Activation of caspases results in the cleavage of crucial cytoskeletal and regulatory proteins and, ultimately, in cell death [24]. Ethanol causes the death of primary cultured cerebellar granule neurons [23], [51] and neocortical neurons [55]. This is associated with the upregulation of caspase activity [48] which can be offset by treatment with the anti-apoptotic agent N-methyl-d-aspartate [3] or with ganglioside treatment [54].
The temporal expression of bcl-2, bax and caspase 3 [45], [56], [57] shows changes during normal development. These changes coincide with the period of NOND in the cerebral cortex [45], [57], thalamus and midbrain/pons [45]. For example, the rise in expression of an active (20 kDa) isoform of caspase 3 temporally correlates with an increase in bax expression, and with a decrease in the ratio of bcl-2 to bax expression [45], suggesting an interaction between these proteins.
Ethanol-induced neuronal death is evident in select regions of the CNS, e.g. in the somatosensory cortex, but not in the VB. This death may be apoptotic, and may result from a direct toxic effect of ethanol on the cell, or it may occur indirectly by the ability of ethanol to exacerbate processes associated with NOND. We hypothesize that prenatal exposure to ethanol differentially alters the relative expression of proteins involved in naturally occurring neuronal apoptosis, e.g. bcl-2, bax and caspase 3. These effects should be evident in cortex where ethanol causes neuronal death, but not in the thalamus. Therefore, a quantitative immunoblotting technique was used to examine the temporal expression of bcl-2, bax and caspase 3 proteins in the cerebral cortex and the thalamus in ethanol-treated rats.
Section snippets
Animals, feeding, and care
Pregnant Long-Evans rats were obtained from Harlan-Sprague-Dawley (Indianapolis, IN, USA) on gestational day (G) 4. The day on which a dam was first identified as having a sperm-positive vaginal plug was designated as G1. Animals were maintained in a temperature/humidity controlled facility in which the light/dark cycle was 12 h/12 h.
Rats were arbitrarily assigned to one of two treatment groups [28], [30]. One group was fed ad libitum a liquid diet containing 6.7% (v/v) ethanol (Et) from G11
Bcl proteins
The bcl-2-positive protein (25 kDa) was expressed in the offspring of Ct-fed dams throughout development (Fig. 1). Densitometric analyses showed that the relative amount of bcl-2 expression did not significantly change over development (Fig. 2). The temporal changes of bcl-2 expression in Et-treated rats followed the same pattern as that described for Ct-treated rats. Even so, there was a notable ethanol-induced difference. Prenatal exposure to ethanol significantly decreased bcl-2 expression
Protein expression in the developing prosencephalon
While interpreting the present data, it must be kept in mind that they represent the summed changes for a neuronal population(s). Neuronal development within a population is asynchronous. Thus, whereas the expression of death-related protein may be intense, but short-lived for an individual cell, alterations for a population may be dilute but evident over a broader period of time. This situation may be exacerbated by the removal of apoptotic cells that transiently altered bcl proteins or
Acknowledgements
We thank Dustin Hays for his technical assistance in the immunoblotting studies. This study was supported by grants from the Department of Veterans Affairs and from the National Institutes of Health (AA06916, AA07568, AA09611, and DE07734).
References (65)
- et al.
Transient expression of Alz-50 immunoreactivity in developing rat neocortex: a marker for naturally occurring neuronal death?
Brain Res.
(1989) - et al.
Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases
J. Biol. Chem.
(1996) - et al.
Cyclic AMP and ethanol interact to control apoptosis and differentiation in hypothalamic beta-endorphin neurons
J. Biol. Chem.
(1994) - et al.
Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of cyclin D1 during programmed cell death
Neuron
(1994) - et al.
Apoptosis and expression of p53 response proteins and cyclin D1 after cortical impact in rat brain
Brain Res.
(1999) - et al.
Cell proliferation in the embryonic mouse neocortex following maternal alcohol intoxication
Int. J. Dev. Neurosci.
(1985) - et al.
Expression of p53 and ALZ-50 immunoreactivity in rat cortex: effect of prenatal exposure to ethanol
Exp. Neurol.
(1998) - et al.
Basic fibroblast growth factor- and platelet-derived growth factor-mediated cell proliferation in B104 neuroblastoma cells: effect of ethanol on cell cycle kinetics
Brain Res.
(1997) - et al.
Recent advances on neuronal caspases in development and neurodegeneration
Neurochem. Int.
(1999) - et al.
Glial-derived neurotrophic factor (GDNF) prevents ethanol-induced apoptosis and JUN kinase phosphorylation
Dev. Brain Res.
(2000)
Circadian rhythm of cell proliferation in the telencephalic ventricular zone: effect of in utero ethanol exposure
Brain Res.
Effects of prenatal exposure to ethanol on callosal projection neurons in rat somatosensory cortex
Brain Res.
Neonatal transection of the infraorbital nerve increases the expression of proteins related to neuronal death in the principal sensory nucleus of the trigeminal nerve
Brain Res.
Effects of prenatal exposure to ethanol on systems matching: the number of neurons in the ventrobasal thalamic nucleus of the mature rat thalamus
Dev. Brain Res.
Expression of bcl-2, bax, and caspase-3 in the brain of the developing rat
Dev. Brain Res.
Enhanced caspase activity during ethanol-induced apoptosis in rat cerebellar granule cells
Eur. J. Pharmacol.
Bcl-2 prevents caspase-independent cell death
J. Biol. Chem.
Nuclear DNA strand breaks during ethanol-induced oxidative stress in rat brain
FEBS Lett.
Effect of ethanol on neurotrophin-mediated cell survival and receptor expression in cortical neuronal cultures
Dev. Brain Res.
Differential expression of rat brain bcl-2 family proteins in development and aging
Biochem. Biophys. Res. Commun.
Changes in caspase expression in Alzheimer’s disease: comparison with development and aging
Biochem. Biophys. Res. Commun.
Ethanol promotes apoptosis in cerebellar granule cells by inhibiting the trophic effect of NMDA
J. Neurochem.
Brain-derived neurotrophic factor mediates the anti-apoptotic effect of NMDA in cerebellar granule neurons: signal transduction cascades and site of ethanol action
J. Neurosci.
Bcl-2 and adenovirus E1B 19 kDa protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase
Oncogene
Ethanol-induced neural crest apoptosis is coincident with their endogenous death, but is mechanistically distinct
Alcohol. Clin. Exp. Res.
Local differences in the amount of early cell death in neocortex predict adult local specializations
Science
Down regulation of bcl-2 by p53 in breast cancer cells
Cancer Res.
Neuronal death in the development and aging of the cerebral cortex of the mouse
Neuropathol. Appl. Neurobiol.
Postnatal development of the mouse cerebral neocortex. IV. Evolution of the total cortical volume of the population of neurons and glial cells
J. Hirnforsch.
Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics
Br. J. Cancer
Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death
Semin. Cancer Biol.
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