Ethanol-induced increase in Fyn kinase activity in the dorsomedial striatum is associated with subcellular redistribution of protein tyrosine phosphatase α

Abstract

In vivo exposure of rodents to ethanol leads to a long-lasting increase in Fyn kinase activity in the dorsomedial striatum (DMS). In this study, we set out to identify a molecular mechanism that contributes to the enhancement of Fyn activity in response to ethanol in the DMS. Protein tyrosine phosphatase α (PTPα) positively regulates the activity of Fyn, and we found that repeated systemic administration or binge drinking of ethanol results in an increase in the synaptic localization of PTPα in the DMS, the same site where Fyn resides. We also demonstrate that binge drinking of ethanol leads to an increase in Fyn activity and to the co-localization of Fyn and PTPα in lipid rafts in the DMS. Finally, we show that the level of tyrosine phosphorylated (and thus active) PTPα in the synaptic fractions is increased in response to contingent or non-contingent exposure of rats to ethanol. Together, our results suggest that the redistribution of PTPα in the DMS into compartments where Fyn resides is a potential mechanism by which the activity of the kinase is increased upon ethanol exposure. Such neuroadaptations could be part of a mechanism that leads to the development of excessive ethanol consumption.

Figure 1. Repeated systemic administration of ethanol does not alter total protein levels of PTPα in the DMS of Sprague Dawley Rats

Rats were treated with saline or ethanol (2 g/kg, i.p.) once a day for 7 days. DMS was dissected 16 hrs after the last injection. (a) PTPα in total homogenates was detected with anti-PTPα antibodies (1:1000). Images are representative of n=6 (saline), n=6 (ethanol). (b) Schematic representation of the synaptosomal fractionation procedure. Anti-PSD95 (1:10,000) and anti-Synapsin 1a/b antibodies (1:2000) were used as synaptosomal membrane markers; anti-CREB antibody (1:1000) was used as a non-synaptic membrane marker; and anti-Actin antibody (1:2000) was used as loading control. H: Total homogenate, P1: Nuclei and large debris, P2: Crude synaptosomal membrane, S2: Cytosol and light membranes. (c) Repeated systemic administration of ethanol alters the protein levels of PTPα in the synaptosomal fraction. Images are representatives of n=8 (saline), n=8 (ethanol). (d) Repeated systemic administration of ethanol does not alter the protein levels of Fyn (polyclonal anti-Fyn antibodies, 1:500) in the synaptosomal fraction. Image is representative of n=3 (saline), n=3 (ethanol). Bar graphs summarize the averaged change in protein levels of PTPα in total homogenates (a), PTPα in synaptosomal fractions (c), and Fyn in synaptosomal fractions (d). Band intensity of PTPα or Fyn were normalized to the level of GAPDH and plotted as percentage of saline-treated rats. Results are expressed as mean ± SEM. ***P < 0.001, vs. saline (two-tailed Student’s t-test).

Figure 2. Repeated systemic administration of ethanol does not alter PTPα protein levels in synaptosomal membranes in the DLS and NAc of Sprague Dawley Rats

Rats were treated as in Fig. 1, the DLS and NAc were dissected 16 hrs after the last injection and the level of PTPα in synaptosomal membranes was determined. Repeated systemic administration of ethanol did not alter the protein levels of synaptic PTPα in the DLS (a) and in the NAc (b). Images are representative of n=6 (saline), n=6 (ethanol). Bar graphs summarize the averaged change in protein levels of PTPα in synaptic membranes. Band intensity of PTPα was normalized to the level of GAPDH and plotted as percentage of saline-treated rats. Results are expressed as mean ± SEM. P > 0.05 vs. saline (two-tailed Student’s t-test).

Figure 3. Binge drinking of ethanol increases protein levels of PTPα in synaptic membranes in the DMS of Long Evans rats

Rats underwent an intermittent access to 20% ethanol in a 2-bottle choice paradigm for 7–8 weeks. Thirty minutes after the initiation of the last drinking session, DMS tissue was dissected and protein levels of PTPα in homogenates (a), and synaptic membranes (b) as well as the level of Fyn in the synaptosomal fraction were measured. (a) Image is representative of n=9 (water), n=9 (ethanol). (b) Images are representative of n=8 (water), n=6 (ethanol). (c) Image is representative of n=3 (water), n=3 (ethanol). Bar graphs summarize the averaged changes in protein levels of PTPα in total homogenates (a), as well as PTPα (b) and Fyn (c) in the synaptosomal fraction. Band intensity of PTPα or Fyn was normalized to the level of GAPDH and plotted as percentage of water-only drinking rats. Results are expressed as mean ± SEM. **P < 0.01 vs. water (two-tailed Student’s t-test).

Figure 4. Binge drinking of ethanol increases Fyn activation in the DMS of Long Evans rats

Rats underwent ethanol drinking procedue as described in Fig. 3, thirty minutes after the initiation of the last drinking session, DMS tissue was dissected and Fyn total was IP-ed from homogenates with mouse monoclonal anti-Fyn antibodies, and anti-mouse IgG antibodies as a control. Active Fyn was detected by the anti-pY420/418[Fyn/Src] antibodies (1:500) (a). In a separate membrane, the anti-pY531/529 [Fyn/Src] antibodies (1:1000) detected inactive Fyn kinase (b). Both membranes were stripped and re-probed with polyclonal anti-Fyn antibodies to measure the level of IP-ed Fyn. Images are representative of n=3 (water), n=3 (ethanol). Bar graphs summarize the averaged effect of ethanol treatment on the levels of active and inactive Fyn by quantification of the level of pY420/418 or pY531/529 to total IP-ed Fyn. Data are expressed as percentage of water control, and are expressed as mean ± SEM. **P < 0.01, *P < 0.05 vs. water-drinking rats (two-tailed Student’s t-test).

Figure 5. Binge drinking of ethanol results in redistribution of PTPα, but not Fyn to lipid rafts in the DMS of Long Evans rats

(a) Characterization of the distribution of PTPα and Fyn in raft and non-raft fractions under non-stimulated conditions. Anti-Flotillin-1 antibody (1:1000), was used as a lipid rafts marker; and anti-TrfR antibody (1:500) was used as a non-lipid rafts marker. (b) Ethanol-binge drinking increases PTPα protein levels in lipid rafts in the DMS. Anti-Flotillin-1 and anti-TrfR antibodies were used as fractionation markers. The image is a representative of 2 independent experiments.

Figure 6. Repeated systemic injections and binge drinking of ethanol increase the level of tyrosine phosphorylation of PTPα in DMS synaptic membranes of Sprague Dawley and Long Evans rats

Sprague Dawley Rats (a) or Long Evans rats (b,c) were systemically treated with saline or ethanol (a,b) or underwent an ethanol binge-drinking session (c) as described in Figs. 1 and 3, respectively. Phosphorylated PTPα was detected using anti-pY789[PTPα] antibodies (1:250). Protein levels of PTPα (1:500), Fyn (1:500) and GAPDH (1:25,000) were also measured. Images are representative of n=3 (saline), n=3 (ethanol) (a,b), n=2 (water), n=2 (ethanol) (c). Bar graphs summarize the averaged change in phosphorylated levels of PTPα in synaptosomal membranes. Band intensities of pY789[PTPα] were normalized to the level of GAPDH and plotted as percentage of saline (a,b) or water (c) controls. Results are expressed as mean ± SEM. *P < 0.05 vs. control (one-tailed Student’s t-test).