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. 2017 Jan;22(1):89-101.
doi: 10.1038/mp.2016.12. Epub 2016 Mar 8.

mTORC1-dependent Translation of Collapsin Response Mediator protein-2 Drives Neuroadaptations Underlying Excessive Alcohol-Drinking Behaviors

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Free PMC article

mTORC1-dependent Translation of Collapsin Response Mediator protein-2 Drives Neuroadaptations Underlying Excessive Alcohol-Drinking Behaviors

F Liu et al. Mol Psychiatry. .
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Abstract

Mammalian target of rapamycin complex 1 (mTORC1) has an essential role in dendritic mRNA translation and participates in mechanisms underlying alcohol-drinking and reconsolidation of alcohol-related memories. Here, we report that excessive alcohol consumption increases the translation of downstream targets of mTORC1, including collapsin response mediator protein-2 (CRMP-2), in the nucleus accumbens (NAc) of rodents. We show that alcohol-mediated induction of CRMP-2 translation is mTORC1-dependent, leading to increased CRMP-2 protein levels. Furthermore, we demonstrate that alcohol intake also blocks glycogen synthase kinase-3β (GSK-3β)-phosphorylation of CRMP-2, which results in elevated binding of CRMP-2 to microtubules and a concomitant increase in microtubule content. Finally, we show that systemic administration of the CRMP-2 inhibitor lacosamide, or knockdown of CRMP-2 in the NAc decreases excessive alcohol intake. These results suggest that CRMP-2 in the NAc is a convergent point that receives inputs from two signaling pathways, mTORC1 and GSK-3β, that in turn drives excessive alcohol-drinking behaviors.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alcohol intake promotes the translation of CRMP-2 mRNA via mTORC1. (a) Timeline of experiment depicted in (b). (b) Rats were subjected to IA20%-2BC-drinking procedure for 2 months. The NAc was removed at the end of the last 30 min binge alcohol-drinking session. Control animals underwent the same paradigm but had access to water only (W). Polysomes were isolated and mRNA levels were determined by RT-PCR analysis. The PCR products were separated on 1.5% agarose gels, photographed by Image Lab and quantified by using ImageJ. The vertical lines indicate that the samples were from the same gel but were not run in adjacent lanes. Data are expressed as the average ratio of each gene to GAPDH±s.e.m., and are expressed as percentage of water control. Significance was determined using two-tailed unpaired t-test. Arc, t(6)=2.805, P=0.031; CaMKIIα, t(6)=2.475, P=0.048; CRMP-2, t(6)=2.914, P=0.027; GluA1, t(6)=2.569, P=0.042; Homer2, t(6)=3.276, P=0.017; GluN1, t(6)=1.283, P=0.247; PSD-95, t(6)=3.209, P=0.018; RACK1, t(6)=0.902, P=0.401; TrkB, t(6)=0.903, P=0.401. (c) Timeline of experiment depicted in (d). Three hours before the end of the last 24 h of alcohol withdrawal, rats were systemically administered with 10 mg kg−1 of rapamycin or vehicle. (d) The NAc was removed 3 h after rapamycin or vehicle treatment and was subjected to polysomal RNA fractionation. mRNA levels were measured by RT-qPCR analysis. Data are expressed as the average ratio of CRMP-2 to GAPDH±s.e.m., and expressed as percentage of water plus vehicle. Significance was determined using two-way ANOVA and the method of contrasts. Two-way ANOVA showed a significant main effect of alcohol (F(1,16)=5.07, P=0.039) and rapamycin (F(1,16)=9.087, P=0.008) but no interaction (F(1,16)=1.123, P=0.305); and the method of contrasts detected a significant difference between water and alcohol within the vehicle group (P=0.048) and a significant difference between vehicle and rapamycin within the alcohol group (P=0.029). (b) n=4, (d) n=5 for each group. *P<0.05.
Figure 2
Figure 2
Binge drinking of alcohol and withdrawal increase CRMP-2 protein levels. (a) Timeline of experiment depicted in (be). The NAc was removed 30 min after the beginning (b and c, binge, B) or 24 h after the end of the last drinking session (d–e, withdrawal). Protein levels were determined by western blot analysis. ImageJ was used for optical density quantification. Data are expressed as the average ratio±s.e.m. of CRMP-2 or RACK1 to GAPDH, and are expressed as percentage of water (W) control. Significance was determined using two-tailed unpaired t-test. (b) Protein levels in the total homogenate of binge-drinking rats. CRMP-2, t(18)=2.537, P=0.02. (c) Protein levels in the synaptic fraction of binge drinking rats. CRMP-2, t(18)=3.596, P=0.002. (d) Protein levels in the total homogenate 24 h after withdrawal. CRMP-2, t(18)=4.182, P<0.001. (e) Protein levels in the synaptic fraction 24 h after withdrawal. CRMP-2, t(18)=4.887, P<0.001. n=10 for each group. *P<0.05, **P<0.01, ***P<0.001.
Figure 3
Figure 3
Binge drinking of alcohol blocks the phosphorylation of CRMP-2 and increases microtubule content. (ai) Rats were trained to drink alcohol as described in Figures 1a. (ad) The NAc was removed 30 min after the beginning of the last drinking session (binge, B). Phosphorylation of GSK-3β and CRMP-2 was determined by western blot analysis. Quantification was conducted as in Figure 2. The vertical lines indicate that separates groups were from the same gel but were not run in adjacent lines. Data are expressed as the average ratio of phospho-GSK-3β to total GSK-3β or phospho-CRMP-2 to total CRMP-2±s.e.m., and are expressed as percentage of water (W) control. Significance was determined using two-tailed unpaired t-test. (a) [S9]GSK-3β phosphorylation in total homogenate, t(10)=3.141, P=0.011. (b) [S9]GSK-3β phosphorylation in the synaptic fraction, t(10)=3.574, P=0.005. (c) [T514]CRMP-2 phosphorylation in total homogenate, t(14)=3.119, P=0.008. (d) [T514]CRMP-2 phosphorylation in the synaptic fraction, t(14)=3.18, P=0.007. (ei) The NAc were removed 24 h after the end of the last drinking session (withdrawal). (e and f) Microtubule-binding assay. (e) CRMP-2 and tubulin levels in the pellet fraction were determined by western blot analysis. (f) Optical density quantification of Microtubules-bound CRMP-2 is expressed as the ratio of CRMP-2 (pellet) to tubulin (pellet)±s.e.m. t(11)=2.271, P=0.044. (gi) Microtubule content assay. Tubulin and GAPDH levels in fraction were detected by western blot analysis. Optical density quantification of the microtubule content (h) and total tubulin (i) are expressed as the ratio of tubulin (pellet) to tubulin (supernatant)±s.e.m. t(14)=2.42, P=0.029, and tubulin (total) to GAPDH (total), t(14)=1.207, P=0.248, respectively. (j) Diagram of the proposed signaling cascade. Excessive drinking of alcohol leads to newly synthesized CRMP-2 in a hypophosphorylated state through the activation of mTORC1 and the inhibition of GSK-3β that in turn promotes microtubule assembly. (a, b) n=6, (cd, hi) n=8 for each group; (f) n=7 water, n=6 withdrawal. *P<0.05, **P<0.01.
Figure 4
Figure 4
Systemic administration of the CRMP-2 inhibitor lacosamide decreases binge drinking of alcohol in rodents. (a) Timeline of experiments depicted in (bf). (b) Rats were subjected to IA20%-2BC for 2 months. Vehicle (Veh) or lacosamide (LCM, 20 mg kg−1) was systemically administered 90 min before the beginning of a drinking session. Alcohol intake was measured 30 min after the beginning of a drinking session. Data are presented as mean±s.e.m. Significance was determined using two-tailed paired t-test, t(8)=4.176, P=0.003. (c) Rats were subjected to an intermittent access to sucrose solution two-bottle choice drinking procedure for 2 weeks. Vehicle (Veh) or lacosamide (LCM, 20 mg kg−1) was systemically administered 90 min before the beginning of a drinking session. Sucrose intake was measured 30 min after the beginning of a drinking session. Data are presented as mean±s.e.m. Significance was determined using two-tailed paired t-test, t(9)=−0.059, P=0.954. (df) Mice experienced 2 months of IA20%-2BC-drinking paradigm. Vehicle (Veh) or lacosamide (LCM, 20 or 50 mg kg−1) were systemically administered 90 min before the beginning of a drinking session. Alcohol intake (d), alcohol preference (e) and water intake (f) were measured at the end of a 4 h drinking session. Data are presented as mean±s.e.m. Significance was determined using one-way RM-ANOVA and post hoc Student–Newman–Keuls test. (d) Alcohol intake. One-way RM-ANOVA showed a significant main effect of lacosamide (F(2,20)=9.842, P=0.001), and post hoc Student–Newman–Keuls test, q=6.081, P=0.001. (e) Alcohol preference is expressed as the ratio of alcohol intake to total fluid intake. One-way RM-ANOVA showed a significant main effect of lacosamide (F(2,20)=8.715, P=0.002), and post hoc Student–Newman–Keuls test, q=5.453, P=0.003. (f) Water intake. One-way RM-ANOVA showed a significant main effect of lacosamide (F(2,20)=4.029, P=0.034), and post hoc Student–Newman–Keuls test, q=3.627, P=0.047. (b) n=9, (c) n=10, (d–f) n=11 for each group. *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5
Knockdown of CRMP-2 in the mouse NAc decreases excessive drinking of alcohol. (ae) A lentivirus expressing non-specific control shRNA (Ltv-shCT) (2 × 107 pg ml−1) or shRNA-targeting mouse CRMP-2 (Ltv-shCRMP-2) (2 × 107 pg ml−1) was infused bilaterally into the mouse NAc (1.2 μl per side). After 3 weeks of recovery, mice underwent IA20%-2BC drinking procedure for eight sessions. The NAc was dissected at the end of behavioral experiment and used for immunohistochemistry (a) and western blot analysis (b). (a) Ltv-shCRMP-2 infects NAc neurons. Slices were stained with anti-GFP antibodies. Left panel (× 5) depicts the specificity of the site of virus infection. Scale bar, 500 μm. Middle image ( × 40) depicts Ltv-shCRMP-2 infection of neurons. Scale bar, 25 μm. Right panel ( × 63) shows a representative neurite from an infected neuron. Scale bar, 5 μm. (b) Ltv-shCRMP-2 infection decreases CRMP-2 expression in the NAc. Left, the protein levels of CRMP-2, tubulin, RACK1 and GAPDH were determined by western blot analysis. Right, histogram depicts the ratio of CRMP-2 to GAPDH levels. Significance was determined using two-tailed unpaired t-test, t(6)=6.414, P=0.0007. (c) Timeline of experiments depicted in (d) and (e). Alcohol intake (d) and alcohol preference (e) were measured after each 24 h drinking session and expressed as an average of every two drinking sessions. (d) Alcohol intake. Two-way RM-ANOVA showed a significant main effect of virus infusion (F(1,20)=6.883, P=0.016), a significant effect of session (F(3,60)=51.502, P<0.001) and no interaction between virus infusion and session (F(3,60)=1.969, P=0.128). (e) Alcohol preference is expressed as the ratio of alcohol intake to total fluid intake. Two-way RM-ANOVA showed a significant main effect of virus infusion (F(1,20)=8.585, P=0.008), a significant effect of session (F(3,60)=29.666, P<0.001) and no interaction between virus infusion and session (F(3,60)=1.344, P=0.269). *P<0.05, **P<0.01, ***P<0.001. (b) n=4, (d and e) n=11 for each group.

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