The dopamine and cAMP regulated phosphoprotein, 32 kDa (DARPP-32) signaling pathway: a novel therapeutic target in traumatic brain injury

Abstract

Traumatic brain injury (TBI) causes persistent neurologic deficits. Current therapies, predominantly focused upon cortical and hippocampal cellular survival, have limited benefit on cognitive outcomes. Striatal damage is associated with deficits in executive function, learning, and memory. Dopamine and cAMP regulated phosphoprotein 32 (DARPP-32) is expressed within striatal medium spiny neurons and regulates striatal function. We found that controlled cortical impact injury in rats produces a chronic decrease in DARPP-32 phosphorylation at threonine-34 and an increase in protein phosphatase-1 activity. There is no effect of injury on threonine-75 phosphorylation or on DARPP-32 protein. Amantadine, shown to be efficacious in treating post-TBI cognitive deficits, given daily for two weeks is able to restore the loss of DARPP-32 phosphorylation and reduce protein phosphatase-1 activity. Amantadine also decreases the phosphorylation of threonine-75 consistent with activity as a partial N-methyl-D-aspartate (NMDA) receptor antagonist and partial dopamine agonist. These data demonstrate that targeting the DARPP-32 signaling cascade represents a promising novel therapeutic approach in the treatment of persistent deficits following a TBI.

Fig. 1

Timecourse of TBI effect on DARPP-32 protein expression and phosphorylation state in rat striatum (N = 6 per group at each timepoint). A, representative western blot timecourse of DARPP-32, p-DARPP-32-Thr75, and p-DARPP-32-Thr34 protein expression. B, optical density of DARPP-32 showing no change in DARPP-32 protein expression at any examined timepoint post TBI. C, optical density of p-DARPP-32-T75 showing no significant change in p-DARPP-32-T75 following TBI. D, optical density of p-DARPP-32-Thr34 showing a significant reduction in p-DARPP-32-T34 in the striatum ipsilateral to injury at all timepoints examined and in the striatum contralateral to injury at 1 day-4 weeks post injury. **Inset image: control western with p-DARPP-32-Thr34 antibody demonstrating a single band on rat striatal tissue. *p ≤ 0.05 normalized to β-actin and compared to sham; ANOVA. Data represented as a percentage of sham following normalization to β-actin ± S.E.M. Abbreviations: SC = sham contralateral to injury; IC = injured contralateral; SI = sham ipisilateral to injury; II = injured ipsilateral; S = sham; I = injured.

Fig. 2

Timecourse of TBI effect on DARPP-32 DAB immunoreactivity in rat striatum (N = 3 per group at each timepoint; boxes indicate area of higher power view shown below). Representative sections of rat striatum at 1 day and 2 weeks post injury showing no effect of TBI on DARPP-32 expression in either the DLStr or Nacc. Abbreviations: Ipsi = ipsilateral to injury; DL = dorsolateral; NAcc = nucleus accumbens.

Fig. 3

Representative microscopic photos (N = 4 per group) of double-labeled immunofluorescent staining for p-DARPP-32-Thr34 (red, Alexa Fluor 594), and neuronal marker NeuN (green, Alexa 488) visually illustrates that TBI causes a decreased expression of p-DARPP-32-Thr34 in the ipsilateral dorsolateral striatum compared to the sham controls in rats. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Analysis of protein phosphatase-1 (PP-1) activity in homogenized rat striatum. There is a significant increase in PP-1 activity in both the contralateral (N = 6/group; p < 0.01; ANOVA) and ipsilateral (N = 6/group; p < 0.01; ANOVA) striatum compared to sham at 1 day post injury. By 2 weeks post injury there remains a significant increase in PP-1 activity in the ipsilateral (N = 6/group; p < 0.01; ANOVA) striatum compared to sham. Increases in PP-1 activity are consistent with decreases in p-DARPP-32-T34. Data represents means ± S.E.M.

Fig. 5

Analysis of AMH effects on DARPP-32 signaling post TBI. A, representative western blot of ipsilateral striatum following 2 weeks of 10 mg/kg i.p. AMH on p-DARPP-32-Thr34, p-DARPP-32-Thr75, and β-actin control. B, C, optical density of p-DARPP-32-Thr34 and p-DARPP-32-Thr75. (N = 6/group; p < 0.01 following normalization to β-actin compared to TBI with vehicle; ANOVA). Data represented as a percentage of sham following normalization to β-actin ± S.E.M. D, Effect of chronic AMH (10 mg/kg i.p. daily) on striatal PP-1 activity. Consistent with higher p-DARPP-32-Thr34 levels, following chronic AMH administration there is a relative reduction in PP-1 activity compared to TBI with vehicle. (N = 6/group; p < 0.01; ANOVA). Data represents means ± standard deviation. Abbreviations: AMH = amantadine hydrochloride, DARPP-32 = dopamine and cAMP regulated phosphoprotein 32; TBI = traumatic brain injury.