It has become apparent in recent years that alcohol addiction involves a myriad of changes in neuronal cell function that appears to be restricted to specific brain regions. Two of the major neuronal systems affected by chronic alcohol exposure and withdrawal are the immediate-early gene transcription factors (ITFs) and the cytoskeleton, specifically microfilaments and microtubules.
In this research a rat model of ethanol dependence was used. The first part of this thesis focused on the Egr family of ITFs and the effect of chronic ethanol exposure and withdrawal on the protein expression and DNA-binding activity of Egr-1 and Egr- 3 in the cortex, hippocampus, striatum and cerebellum. Both Egr-1 and Egr-3 DNA-binding activity was reduced in the cortex and hippocampus following chronic ethanol exposure and was restored to the level of the pair-fed group at 16 hr of withdrawal. Cortical Egr-1 protein levels were not affected by
chronic ethanol exposure but increased at 16 hr of withdrawal. In contrast, Egr-3 protein levels did not undergo any change during withdrawal. There was no change in the level of either protein in the hippocampus. Egr DNA-binding activity in the striatum and cerebellum increased following 16 hr withdrawal. Egr-1 protein expression levels followed the changes observed in DNA-binding activity, whilst Egr-3 protein expression remained unchanged in the striatum. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. No change in the protein expression level or DNA-binding activity of the transcription factor Sp1 was observed. There was no significant change in expression of NGFI-A Binding protein 2 (NAB2) in the cortex and cerebellum. In the hippocampus, Egr inhibitor NAB2 expression increased following 16 hr withdrawal whilst in the striatum NAB2 expression decreased following chronic ethanol exposure and returned to pair-fed
levels at 16 hr of withdrawal. 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 ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependence and withdrawal. Furthermore, these effects are selective for certain transcription factors. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure affects the modulatory agents of Egr DNA-binding activity.
The second part of this thesis characterized a novel protein, neuronal protein 22 (NP22) which is exclusively expressed in neuronal tissue and is localized to the cytoplasm and processes of human and rat neurons. The rat protein (rNP22) is expressed constitutively throughout the rat brain. In contrast, in the human brain the human protein (hNP22)
had a restricted expression pattern with minimal levels in the hippocampal CA1 and cerebellum. rNP22 was expressed as early as prenatal day 15 with levels gradually increasing to adult levels by postnatal day 16. The rNP22 sequence shared significant homology with cytoskeleton-interacting proteins including SM22a, SM22P and calponin. rNP22 colocalized with cytoskeletal proteins including actin, tubulin, MAP2 and synaptophysin in normal rat brain tissue. hNP22 protein levels were elevated in human alcoholic frontal cortex. Increased hNP22 expression is also observed in the hippocampal CA3 and CA4 of human alcoholics compared to control subjects. There was no change in protein expression in the hippocampal CA1, CA2 and motor cortex. In the rat brain, rNP22 levels increased significantly following 16 hr withdrawal in the cortex, CA2 and dentate gyms. In contrast, a significant decrease in rNP22 expression following chronic ethanol exposure was observed in the striatum which returned to
pair-fed group levels after 16 hr withdrawal. No change was observed in the cerebellum. Protein associations of rNP22 were altered during alcohol withdrawal. rNP22 maintained its association with tubulin following chronic ethanol exposure and withdrawal. In contrast, colocalization between rNP22 and MAP2 was not observed in alcohol withdrawal samples. Colocalization of rNP22 and actin was not dramatically affected, although staining at the somatic periphery was observed in chronic ethanol and withdrawal rat samples. Taken together, these results suggest that NP22 has a significant role in the molecular changes affecting the neuronal cytoskeleton following chronic ethanol exposure and withdrawal. It is feasible that NP22 acts as part of a signalling cascade which transduces messages between the actin and tubulin cytoskeleton. Furthermore, these signals may affect the localization of excitatory and inhibitory receptors at the neuronal membrane and thus contribute to the development of
alcohol dependence and withdrawal phenomena.