Pharmacological inhibition of Receptor Protein Tyrosine Phosphatase β/ζ (PTPRZ1) modulates behavioral responses to ethanol

Abstract

Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that are upregulated in the prefrontal cortex after alcohol administration and have been shown to reduce ethanol drinking and reward. PTN and MK are the endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) β/ζ (a.k.a. PTPRZ1, RPTPβ, PTPζ), suggesting a potential role for this phosphatase in the regulation of alcohol effects. To determine if RPTPβ/ζ regulates ethanol consumption, we treated mice with recently developed small-molecule inhibitors of RPTPβ/ζ (MY10, MY33-3) before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with RPTPβ/ζ inhibitors, particularly with MY10, drank less ethanol than controls. MY10 treatment blocked ethanol conditioned place preference, showed limited effects on ethanol-induced ataxia, and potentiated the sedative effects of ethanol. We also tested whether RPTPβ/ζ is involved in ethanol signaling pathways. We found that ethanol treatment of neuroblastoma cells increased phosphorylation of anaplastic lymphoma kinase (ALK) and TrkA, known substrates of RPTPβ/ζ. Treatment of neuroblastoma cells with MY10 or MY33-3 also increased levels of phosphorylated ALK and TrkA. However, concomitant treatment of neuroblastoma cells with ethanol and MY10 or MY33-3 prevented the increase in pTrkA and pALK. These results demonstrate for the first time that ethanol engages TrkA signaling and that RPTPβ/ζ modulates signaling pathways activated by alcohol and behavioral responses to this drug. The data support the hypothesis that RPTPβ/ζ might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.

Keywords: ALK; Alcohol use disorder; Binge-drinking; Midkine; Pleiotrophin; TrkA.

Fig 1

Structure of the RPTPβ/ζ inhibitors MY10 and MY33-3.

Fig 2. RPTPβ/ζ inhibition attenuates binge-like ethanol consumption

(a) Ethanol consumed (g/kg) during 2 h of drinking on days 1-3 and during 4 h of drinking on day 4. On days 3 and 4, mice were administered 60 mg/kg MY10 or vehicle (n=12/group) 1 hour before each drinking session in the DID test. (b) Ethanol preference in vehicle- and MY10-treated mice, calculated as the ratio of the volume of ethanol consumed over the volume of total fluid consumed. Ethanol preference on days 1-3 was calculated for 2 h-drinking sessions and on day 4 for a 4 h-drinking session. (c) Blood ethanol concentration (BEC, mg%) in vehicle- and MY10-treated mice after the final 4 h-drinking session on day 4. (d) Volume of 2% sucrose solution (ml/kg) consumed during 2 hours of drinking on days 1–3 and 4 hours on day 4 in mice treated with vehicle (n = 5) or MY10 (n = 5). (e) Ethanol consumed (g/kg) during 2 h of drinking on days 1-3 and during 4 h of drinking on day 4. On days 3 and 4, mice were administered 60 mg/kg MY33-3 or vehicle (n=12/group) 1 hour before each drinking session in the DID test. (f) Ethanol preference score in vehicle- and MY33-3-treated mice, calculated as the ratio of the volume of ethanol consumed over the volume of total fluid consumed. Ethanol preference on days 1-3 was calculated for 2 h-drinking sessions and on day 4 for a 4 h-drinking session. (g) Blood ethanol concentration (BEC, mg%) in vehicle- and MY33-3-treated mice after the final 4 h-drinking in the dark session on day 4. (h) Total fluid consumed (water + ethanol, ml) during 2 h-drinking sessions on days 1-3 and the 4 h-drinking session on day 4. On days 3 and 4, mice were administered 60 mg/kg MY10, MY33-3 or vehicle (n=12/group) 1 hour before each drinking session in the DID test. *p < 0.05 vs. Vehicle. #p < 0.05 vs. Day 2.

Fig 3. Ethanol (2.0 g/kg)-induced place preference

(a) Scheme showing treatment conditions for the ethanol CPP test (b) Time (seconds) spent on the ethanol-paired side before conditioning (Pre-C) and after conditioning (CPP) in mice treated with vehicle or 60 mg/kg MY10. The data show that MY10 prevents ethanol CPP. (c) Scheme showing the treatment conditions for the MY10 CPP test (in the absence of ethanol). (d) Time (seconds) spent on the MY10-paired side before conditioning (Pre-C) and after conditioning (CPP) in mice conditioned with 60 mg/kg MY10 (n = 13) instead of ethanol. Time spent on the MY10-paired side did not change after conditioning. *** P < 0.001 vs. Pre-C.

Fig 4. Ethanol (2.0 g/kg)-induced ataxia

(a) Time spent on a rotarod by mice treated with the RPTPβ/ζ inhibitor MY10 (60 mg/kg) or vehicle one hour before injection of 2.0 g/kg ethanol (Eth). (b) Time spent on a rotarod by mice treated with the RPTPβ/ζ inhibitor MY10 (60 mg/kg) or vehicle (Veh) one hour before injection of saline (Sal, i.p.). (c) Blood ethanol concentration (BEC) in mice pretreated with 60 mg/kg MY10 or vehicle and treated with 2.0 g/kg ethanol. Results are presented in mg% over time.

Fig 5. Ethanol (3.6 g/kg)-induced loss of righting reflex (LORR)

Increased duration of LORR induced in mice treated with the RPTPβ/ζ inhibitor MY10 (60 mg/kg) or vehicle one hour before ethanol injection (3.6 g/kg, i.p.) (n = 8). * P < 0.05 vs. Vehicle.

Fig. 6. Ethanol increases phosphorylation of anaplastic lymphoma kinase (ALK) and TrkA in SH-SY5Y cells

(a) Representative western blots showing phosphorylated ALK (pALK, Tyr 1278) 140 and phosphorylated TrkA (p-TrkA, Tyr 490) within 15 min of 0-100 mM ethanol treatment. Total ALK and TrkA western blots are shown below each phosphorylated protein blot for comparison. Both proteins were detected at ~ 140 kDa. (b) Graph shows the ratio p-TrkA/TrkA of optical density measurements corresponding to the p-TrkA and total TrkA protein levels respectively. (c) Graph shows the ratio TrkA/Actin of optical density measurements corresponding to the total TrkA and actin protein levels respectively. (d) Graph shows the ratio p-ALK140/ALK140 of optical density measurements corresponding to the p-ALK140 and total ALK140 protein levels respectively. (e) Graph shows the ratio ALK140/Actin of optical density measurements corresponding to the total ALK140 and actin protein levels respectively. (f) Representative western blots showing phosphorylated ALK (p-ALK) 140 and phosphorylated TrkA (p-TrkA) within 0 to 30 min of 50 mM ethanol treatment. Total ALK and TrkA western blots are shown below each phosphorylated protein blot for comparison. Both proteins were detected at ~ 140 kDa. (g) Graph shows the ratio p-TrkA/TrkA of optical density measurements corresponding to the p-TrkA and total TrkA protein levels respectively. (h) Graph shows the ratio TrkA/Actin of optical density measurements corresponding to the total TrkA and actin protein levels respectively. (i) Graph shows the ratio p-ALK140/ALK140 of optical density measurements corresponding to the p-ALK140 and total ALK140 protein levels respectively. (j) Graph shows the ratio ALK140/Actin of optical density measurements corresponding to the total ALK140 and actin protein levels respectively. Data are presented as the mean ± SEM. * p < 0.05 vs. 0 mM ethanol. # p < 0.05 vs. 0 min.

Fig. 7. RPTPβ/ζ inhibitors (MY10 and MY33-3) block ethanol-induced activation of TrkA and ALK in SH-SY5Y cells

(a) Representative western blots showing phosphorylated ALK (p-ALK, Tyr 1278) 140 and phosphorylated TrkA (p-TrkA, Tyr 490) in cells incubated for 5 min with 1 μM MY10 or DMSO (control) and then for 15 min with 50 mM ethanol. Total ALK and TrkA western blots are shown below each phosphorylated protein blot for comparison. Both proteins were detected at ~ 140 kDa. (b) Graph shows the ratio p-TrkA/TrkA of optical density measurements corresponding to the p-TrkA and total TrkA protein levels respectively. (c) Graph shows the ratio TrkA/Actin of optical density measurements corresponding to the total TrkA and actin protein levels respectively. (d) Graph shows the ratio p-ALK140/ALK140 of optical density measurements corresponding to the p-ALK140 and total ALK140 protein levels respectively. (e) Graph shows the ratio ALK140/Actin of optical density measurements corresponding to the total ALK140 and actin protein levels respectively. (f) Representative western blots showing phosphorylated ALK (p-ALK) 140 and phosphorylated TrkA (p-TrkA) in cells incubated for 5 min with 1 μM MY33-3 or DMSO (control) and then for 15 min with 50 mM ethanol. Total ALK and TrkA western blots are shown below each phosphorylated protein blot for comparison. Both proteins were detected at ~ 140 kDa. (g) Graph shows the ratio p-TrkA/TrkA of optical density measurements corresponding to the p-TrkA and total TrkA protein levels respectively. (h) Graph shows the ratio TrkA/Actin of optical density measurements corresponding to the total TrkA and actin protein levels respectively. (i) Graph shows the ratio p-ALK140/ALK140 of optical density measurements corresponding to the p-ALK140 and total ALK140 protein levels respectively. (j) Graph shows the ratio ALK140/Actin of optical density measurements corresponding to the total ALK140 and actin protein levels respectively. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 vs. unstimulated cells (controls, lane 1).

Fig. 8. Schematic showing RPTPβ/ζ and ALK interaction and possible involvement in ethanol consumption and reward

(a) Under normal conditions, RPTPβ/ζ dephosphorylates ALK and maintains ALK in an inactive state. (b) Inhibition of RPTPβ/ζ by inhibitors such as MY10 leads to constitutive ALK phosphorylation and activation. (c) Activated ALK would undergo endocytosis to desensitize the receptor and render it unavailable for ethanol-induced activation. (d) ALK inhibitors also block ethanol-induced activation of ALK. Inactivation of ALK reduces ethanol consumption and reward.