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Adenylyl cyclases 1 and 8 initiate a presynaptic homeostatic response to ethanol treatment.

Conti AC, Maas JW, Moulder KL, Jiang X, Dave BA, Mennerick S, Muglia LJ - PLoS ONE (2009)

Bottom Line: While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition.DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment.These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Washington University in St. Louis, St. Louis, Missouri, United States of America. contia@psychiatry.wustl.edu

ABSTRACT

Background: Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition.

Methodology/principal findings: Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment.

Conclusions/significance: These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.

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Ethanol-induced phosphorylation of synapsin I and II and eEF-2 is not compromised in AC8KO hippocampus.(A) Immunoblot analysis of whole cell lysates from WT and AC8KO mice demonstrates increased expression of phosphorylated synapsin I and II (psyn I, II) and eEF-2 (peEF-2) in WT and AC8KO hippocampus following ethanol treatment compared to saline controls. (B) Quantification of phospho-synapsin I, II and phospho-eEF-2 expression normalized to total synapsin I, II (syn I, II) or eEF-2 expression, respectively. Ethanol significantly induced phosphorylation of synapsin I, II and eEF-2 in WT and AC8KO mice. (**, p<0.05 vs. respective saline control).
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pone-0005697-g004: Ethanol-induced phosphorylation of synapsin I and II and eEF-2 is not compromised in AC8KO hippocampus.(A) Immunoblot analysis of whole cell lysates from WT and AC8KO mice demonstrates increased expression of phosphorylated synapsin I and II (psyn I, II) and eEF-2 (peEF-2) in WT and AC8KO hippocampus following ethanol treatment compared to saline controls. (B) Quantification of phospho-synapsin I, II and phospho-eEF-2 expression normalized to total synapsin I, II (syn I, II) or eEF-2 expression, respectively. Ethanol significantly induced phosphorylation of synapsin I, II and eEF-2 in WT and AC8KO mice. (**, p<0.05 vs. respective saline control).

Mentions: Using immunoblot and immunohistochemistry techniques, impairments in synapsin phosphorylation were evaluated, as a representative presynaptic phospho-protein regulated by AC/PKA activity. Also, eEF-2 and dynamin phosphorylation was examined as representatives of the non-PKA substrate proteins identified. Whole cell hippocampal and cortical lysates were collected from WT, DKO, AC1KO and AC8KO mice 45 min following ethanol exposure. Phosphorylation of synapsins I and II was significantly increased in the hippocampus (Figure 2) and cortex (data not shown) of WT mice after ethanol treatment. These robust increases in phosphorylation of synapsin I and II were not observed in either brain region of DKO or AC1KO mice (Figures 2 and 3). Levels of phospho-synapsin I and II in ethanol-treated AC1KO and DKO mice were unchanged from those of saline-treated WT and DKO controls (Figures 2 and 3). The phosphorylation of synapsin in AC8KO mice resembled that of WT mice in both the hippocampus and cortex following ethanol exposure (Figure 4, data not shown). Similarly, while WT and AC8KO mice demonstrated significant increases in eEF-2 phosphorylation in the hippocampus (Figure 4) and cortex (data not shown) following ethanol treatment compared to controls, levels of phospho-eEF-2 in DKO and AC1KO mice were unchanged from saline-treated controls (Figures 2 and 3). Likewise, dynamin phosphorylation in WT and DKO hippocampal extracts was examined following acute ethanol treatment (Figure 5). Phosphorylation of dynamin was significantly increased in WT mice compared to saline-treated controls, while DKO mice demonstrated no change from controls in levels of phospho-dynamin following ethanol exposure (Figure 5).


Adenylyl cyclases 1 and 8 initiate a presynaptic homeostatic response to ethanol treatment.

Conti AC, Maas JW, Moulder KL, Jiang X, Dave BA, Mennerick S, Muglia LJ - PLoS ONE (2009)

Ethanol-induced phosphorylation of synapsin I and II and eEF-2 is not compromised in AC8KO hippocampus.(A) Immunoblot analysis of whole cell lysates from WT and AC8KO mice demonstrates increased expression of phosphorylated synapsin I and II (psyn I, II) and eEF-2 (peEF-2) in WT and AC8KO hippocampus following ethanol treatment compared to saline controls. (B) Quantification of phospho-synapsin I, II and phospho-eEF-2 expression normalized to total synapsin I, II (syn I, II) or eEF-2 expression, respectively. Ethanol significantly induced phosphorylation of synapsin I, II and eEF-2 in WT and AC8KO mice. (**, p<0.05 vs. respective saline control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682654&req=5

pone-0005697-g004: Ethanol-induced phosphorylation of synapsin I and II and eEF-2 is not compromised in AC8KO hippocampus.(A) Immunoblot analysis of whole cell lysates from WT and AC8KO mice demonstrates increased expression of phosphorylated synapsin I and II (psyn I, II) and eEF-2 (peEF-2) in WT and AC8KO hippocampus following ethanol treatment compared to saline controls. (B) Quantification of phospho-synapsin I, II and phospho-eEF-2 expression normalized to total synapsin I, II (syn I, II) or eEF-2 expression, respectively. Ethanol significantly induced phosphorylation of synapsin I, II and eEF-2 in WT and AC8KO mice. (**, p<0.05 vs. respective saline control).
Mentions: Using immunoblot and immunohistochemistry techniques, impairments in synapsin phosphorylation were evaluated, as a representative presynaptic phospho-protein regulated by AC/PKA activity. Also, eEF-2 and dynamin phosphorylation was examined as representatives of the non-PKA substrate proteins identified. Whole cell hippocampal and cortical lysates were collected from WT, DKO, AC1KO and AC8KO mice 45 min following ethanol exposure. Phosphorylation of synapsins I and II was significantly increased in the hippocampus (Figure 2) and cortex (data not shown) of WT mice after ethanol treatment. These robust increases in phosphorylation of synapsin I and II were not observed in either brain region of DKO or AC1KO mice (Figures 2 and 3). Levels of phospho-synapsin I and II in ethanol-treated AC1KO and DKO mice were unchanged from those of saline-treated WT and DKO controls (Figures 2 and 3). The phosphorylation of synapsin in AC8KO mice resembled that of WT mice in both the hippocampus and cortex following ethanol exposure (Figure 4, data not shown). Similarly, while WT and AC8KO mice demonstrated significant increases in eEF-2 phosphorylation in the hippocampus (Figure 4) and cortex (data not shown) following ethanol treatment compared to controls, levels of phospho-eEF-2 in DKO and AC1KO mice were unchanged from saline-treated controls (Figures 2 and 3). Likewise, dynamin phosphorylation in WT and DKO hippocampal extracts was examined following acute ethanol treatment (Figure 5). Phosphorylation of dynamin was significantly increased in WT mice compared to saline-treated controls, while DKO mice demonstrated no change from controls in levels of phospho-dynamin following ethanol exposure (Figure 5).

Bottom Line: While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition.DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment.These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Washington University in St. Louis, St. Louis, Missouri, United States of America. contia@psychiatry.wustl.edu

ABSTRACT

Background: Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition.

Methodology/principal findings: Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment.

Conclusions/significance: These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade.

Show MeSH
Related in: MedlinePlus