Ethanol impairs activation of retinoic acid receptors in cerebellar granule cells in a rodent model of fetal alcohol spectrum disorders

Abstract

Background: Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to ethanol toxic effects. Retinoic acid (RA) is a physiologically active metabolite of vitamin A that is locally synthesized in the cerebellum. Studies have shown that RA is required for neuronal development, but it remains unknown if ethanol impairs RA signaling and thus induces neuronal malformations. In this study, we tested the hypothesis that ethanol impairs the expression and activation of RA receptors in cerebellum and in cerebellar granule cells.

Methods: The cerebellum of ethanol unexposed and exposed pups was used to study the expression of retinoic acid receptors (RARs or RXRs) by immunohistochemistry and by Western blot analysis. We also studied the effect of ethanol on expression of RA receptors in the cerebellar granule cells. Activation of RA receptors (DNA-binding activities) in response to high-dose ethanol was determined by electrophoretic mobility shift and supershift assays.

Results: Findings from these studies demonstrated that ethanol exposure reduced the expression of RARalpha/gamma while it increased the expression of RXRalpha/gamma in the cerebellum and in cerebellar granule neurons. Immuno-histological studies further strengthened the expression pattern of RA receptors in response to ethanol. The DNA-binding activity of RARs was reduced, while DNA-binding activity of RXRs was increased in response to ethanol exposure.

Conclusion: For the first time, our studies have demonstrated that high-dose ethanol affects the expression and activation of RA receptors, which could impair the signaling events and induce harmful effects on the survival and differentiation of cerebellar granule cells. Taken together, these findings could provide insight into the treatment options for brain defects caused by excessive ethanol exposure, such as in Fetal Alcohol Spectrum Disorders.

Fig. 1

Immunofluorescence staining of RA receptors in unexposed and ethanol-exposed rats shows the effects of ethanol on the expression of RA receptors in the cerebellum. (A) Reduced expression of RARα and RARγ in the external granule layer (EGL) of ethanol-exposed rats (arrow). (B) Increased expression of RXRγ in the EGL of ethanol-exposed rats (arrow). For nonspecific staining, sections were incubated with IgG instead of primary antibodies.

Fig. 2

Ethanol changes the expression of RA receptors in the cerebellum, as shown by Western blot analysis. Cerebella of unexposed and ethanol-exposed pups were homogenized in RIPA buffer for protein extraction. Equal amount of extracted proteins from the unexposed and ethanol-exposed samples was separated by SDS–PAGE and transferred to a nitrocellulose membrane for Western blotting using anti-RA receptor antibodies. (A) Western blot analysis of cerebellar proteins using anti-RARα, β, and γ antibodies. (B). Western blot analysis of cerebellar proteins using anti-RXRα, β, and γ antibodies. Membranes were re-probed with an anti-actin antibody to examine loading differences. The expression of RARs and RXRs was quantified by scanning densitometry of their respective bands. The ratio of the scanned density of the band belonging to the individual receptor with that of the actin is represented as fold increase or decrease. Results are mean ± SD of three separate experiments.

Fig. 3

Ethanol alters the cytoplasmic and nuclear distribution of RA receptors in the cerebellum. Cytoplasmic and nuclear proteins were isolated from cerebella of unexposed and ethanol-exposed pups. Equal amount of proteins were separated by SDS–PAGE and transferred to a nitrocellulose membrane for Western blotting using anti-RA receptor antibodies. (A) Western blot analysis of cytoplasmic and nuclear proteins using anti-RARα, β, and γ antibodies. Membranes were blotted with a TFIID antibody to check the purity of nuclear proteins. Total cerebellar lysates were run in a parallel set to monitor the level of actin in each sample (data not presented). The amounts of RARα, β, and γ in the cytoplasmic and nuclear fractions were calculated by scanning densitometric analysis of respective bands. The ratio of the scanned density of the band belonging to the individual receptor with that of the actin is represented as fold increase or decrease. Results are mean of ± SD of three separate experiments. (B) Similar methods were used to analyze ethanol-induced changes in the cytoplasmic and nuclear levels of RXRs.

Fig. 4

Ethanol inhibits DNA-binding activities of RARs and increases DNA-binding activities of RXRs. Cerebella of unexposed and ethanol-exposed pups were used for the isolation of nucleus and nuclear extracts. The EMSA for RAR (A) and RXR (B) was performed using 10 µg of nuclear extract. Lane 1: labeled probe. Lane 2: ethanol-unexposed sample. Lane 3: ethanol-exposed sample. Lane 4: ethanol-unexposed sample in the presence of cold competitor (unlabeled probe). Lane 5: ethanol-exposed sample in the presence of cold competitor (unlabeled probe).

Fig. 5

Ethanol changes the expression levels of RA receptors in CGNs. The cerebella of unexposed and ethanol-exposed pups were used for isolation of CGNs in the absence and presence of ethanol (80 mM) to avoid ethanol withdrawal effects. After primary culture for 24 hours in the absence and presence of ethanol (80 mM), CGNs were harvested and homogenized in RIPA buffer to extract proteins. Equal amounts of extracted proteins from unexposed and ethanol-exposed samples were run on an SDS–PAGE and transferred to nitrocellulose membrane for Western blotting using anti-RA receptor antibodies. (A) Western blot analysis of cerebellar proteins using anti-RARα, β, and γ antibodies. (B) Western blot analysis of cerebellar proteins using anti-RXRα, β, and γ antibodies. Membranes were re-probed with an anti-actin antibody to examine loading differences. The expression of RARs and RXRs was quantified by scanning densitometry of their respective bands. The ratio of the scanned density of the band belonging to the individual receptor with that of actin is represented as fold increase or decrease. Results are mean ± SD of three separate experiments.

Fig. 6

Ethanol alters the cytoplasmic and nuclear distribution of RA receptors in CGNs. The isolation and primary culture of CGNs from ethanol-exposed and unexposed pups was performed in the presence and absence of ethanol as noted in Fig. 5. Cytoplasmic and nuclear proteins were isolated from CGNs of unexposed and ethanol-exposed pups as noted in the Methods section. An equal amount of proteins was separated by SDS–PAGE and transferred to a nitrocellulose membrane for Western blotting using anti-RA receptor antibodies. (A) Western blot analysis of cytoplasmic and nuclear proteins using anti-RARα, β, and γ antibodies. Membranes were blotted with a TFIID antibody to check the purity of nuclear proteins. Total cell lysates were run in a parallel set to monitor the level of actin in each sample (data not presented). The amounts of RARα, β, and γ in the cytoplasmic and nuclear fractions were calculated by scanning densitometric analysis of respective bands. The ratio of the scanned density of the band belonging to the individual receptor with that of actin is represented as fold increase or decrease. Results are mean of ± SD of three separate experiments. (B) Similar methods were used to analyze the ethanol-induced changes in the cytoplasmic and nuclear levels of RXRs.

Fig. 7

Ethanol inhibits DNA-binding activities of RARα/γ and increases the DNA-binding activity of RXRγ. CGNs isolated and primary cultured from unexposed and ethanol-exposed pups (as noted in Fig. 5) were used for the isolation of nucleus and nuclear extract. (A) The EMSA for RAR (A-a) and RXR (A–b) was performed using 10 µg of nuclear extract. Lane 1: labeled probe. Lane 2: ethanol-unexposed sample. Lane 3: ethanol-exposed sample. Lane 4: ethanol-unexposed sample in the presence of cold competitor (unlabeled probe). Lane 5: ethanol-exposed sample in the presence of cold competitor (unlabeled probe). (B) Supershift assay for RAR (B-a) and RXR (B-b) performed in the presence of individual antibody against each receptor subtype. The antibodies were preincubated with the nuclear extract for 30 min before addition of the labeled probes. EMSA for the supershift assay was performed under similar conditions as noted in 7A.