Sharon Goodenough

Increased TUNEL positive cells in human alcoholic brains

Alcohol-sensitive neuronal cell loss, which has been reported in the superior frontal cortex and hippocampus, may underlie the pathogenesis of subsequent cognitive deficits. In the present study, we have used the TUNEL labeling to detect the DNA damage in human alcoholic brains. Seven out of eleven alcoholics exhibited TUNEL-positive cells in both superior frontal cortex and hippocampus, which were co-localized with GFAP immunoreactivity. In contrast, almost no positive cells were detected in the non-alcoholic controls. None of the TUNEL-positive cells showed any typical morphological features of apoptosis or necrosis. TUNEL-positive cells observed in the present study may indicate DNA damage induced by ethanol-related overproduction of reactive oxygen species.

Immediate Early Gene Expression and Delayed Cell Death in Limbic Areas of the Rat Brain after Kainic Acid Treatment and Recovery in the Cold

Systemic injection of kainic acid (KA) results in characteristic behaviors and programmed cell death in some regions of the rat brain. We used KA followed by recovery at 4 degrees C to restrict damage to limbic structures and compared patterns of immediate early gene (IEG) expression and associated DNA binding activity in these damaged areas with that in spared brain regions. Male Wistar rats were injected with KA (12 mg/kg, i.p.) and kept at 4 degrees C for 5 h. This treatment reduced the severity of behaviors and restricted damage (observed by Nissl staining) to the CA1 and CA3 regions of the hippocampus and an area including the entorhinal cortex. DNA laddering, characteristic of apoptosis, was first evident in the hippocampus and the entorhinal cortex 18 and 22 h after KA, respectively. The pattern of IEG mRNA induction fell into three classes: IEGs that were induced in both damaged and spared areas (c-fos, fos B, jun B, and egr-1), IEGs that were induced specifically in the damaged areas (fra-2 and c-jun), and an IEG that was significantly induced by saline injection and/or the cold treatment (jun D). The pattern of immunoreactivity closely followed that of mRNA expression. Binding to the AP-1 and EGR DNA consensus sequences increased in all three regions studied. This study describes a unique modification of the animal model of KA-induced neurotoxicity which may prove a useful tool for dissecting the molecular cascade that ultimately results in programmed cell death.

In vivo pharmacological study of spermine-induced neurotoxicity.

Spermine-induced neurotoxicity and its pharmacological manipulation was studied in the rat striatum in vivo. Spermine (50, 100, 250 nmol) was injected into the striatum and the volume of damage quantified by computer-based image analysis. Spermine produced a dose-dependent increase in the volume of damage. Co-administration of MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; dizocilpine, 60 nmol), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (25, 40 nmol) and pretreatment with pentobarbital (40 mg/kg, i.p.) significantly reduced the volume of damage induced by 100 nmol spermine. MK-801 (30 nmol) was also effective in reducing the damage induced by 50 nmol spermine. Treatment with a specific inhibitor of nitric oxide synthase, N omega-nitro-L-arginine methyl ester (50 mg/kg, i.p., twice daily for 10 days) was ineffective. These results suggest an involvement of both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the cascade of spermine-induced neurotoxicity.

Differential expression of Egr-1-like DNA-binding activities in the naive rat brain and after excitatory stimulation

Abstract: Egr‐1 and related proteins are inducible transcription factors within the brain recognizing the same consensus DNA sequence. Three Egr DNA‐binding activities were observed in regions of the naive rat brain. Egr‐1 was present in all brain regions examined. Bands composed, at least in part, of Egr‐2 and Egr‐3 were present in different relative amounts in the cerebral cortex, striatum, hippocampus, thalamus, and midbrain. All had similar affinity and specificity for the Egr consensus DNA recognition sequence. Administration of the convulsants NMDA, kainate, and pentylenetetrazole differentially induced Egr‐1 and Egr‐2/3 DNA‐binding activities in the cerebral cortex, hippocampus, and cerebellum. All convulsants induced Egr‐1 and Egr‐2 immunoreactivity in the cerebral cortex and hippocampus. These data indicate that the members of the Egr family are regulated at different levels and may interact at promoters containing the Egr consensus sequence to fine tune a program of gene expression resulting from excitatory stimuli.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain.

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Sp1. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity.

Kainic acid induces 14-3-3 ζ expression in distinct regions of rat brain

Areas of the limbic system of adult male Wistar rats were screened for kainic-acid-induced gene expression. Polymerase-chain-reactionbased differential display identified a 147-bp cDNA fragment, which represented an mRNA that was upregulated in the entorhinal cortex and hippocampus in the kainic-acid-treated animals. The sequence was 97.8% homologous to rat 14-3-3 zeta isoform mRNA. Detailed Northern analysis revealed increased mRNA levels in the entorhinal cortex I h after kainic acid exposure and continued elevation 24 h post-injection in both the entorhinal cortex and hippocampus. Western blot analyses confirmed that the protein product of this gene was also present in increased amounts over the same time period. Immunohistochemistry and terminal transferase-mediated dUTP nick end labelling (TUNEL) detected expression of 14-3-3 protein exclusively in the entorhinal cortex and hippocampus, and only in TUNEL-positive neuronal cells. Expression of the tumor suppressor protein, p53 was also induced by kainate injection, and was co-localized with 14-3-3 zeta protein in selected cells only in the affected brain regions. The increase gene expression of 14-3-3 represents a transcription-mediated response associated with region selective neuronal damage induced by kainic acid