Dopamine depletion alters phosphorylation of striatal proteins in a model of Parkinsonism

Abstract

Nigrostriatal dopamine depletion disrupts striatal medium spiny neuron morphology in Parkinson's disease and modulates striatal synaptic plasticity in animal models of parkinsonism. We demonstrate that long-term nigrostriatal dopamine depletion in the rat induces evolving changes in the phosphorylation of striatal proteins critical for synaptic plasticity. Dopamine depletion increased the phosphorylation of the alpha isoform of calcium-calmodulin-dependent protein kinase II (CaMKIIalpha) at Thr286, a site associated with enhanced autonomous kinase activity, but did not alter total levels of CaMKIIalpha or other synaptic proteins. Dopamine depletion decreased CaMKIIalpha levels in postsynaptic density-enriched fractions without significant changes in other proteins. The activity of protein phosphatase 1 (PP1), a postsynaptic phosphatase that dephosphorylates CaMKII, is regulated by DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa). Dopamine depletion had no effect on DARPP-32 phosphorylation at Thr34, but increased DARPP-32 phosphorylation at Thr75. Levodopa administration reversed the increased phosphorylation of both CaMKIIalpha and DARPP-32. Normal ageing increased the levels of PP1(gamma1 isoform) but decreased levels of the PP1gamma1-targeting proteins spinophilin and neurabin. Elevated phosphorylations of CaMKIIalpha and DARPP-32 were maintained for up to 20 months after dopamine depletion. However, phosphorylation of the CaMKII-PP1 substrate, Ser831 in the glutamate receptor GluR1 subunit, was increased only after sustained (9-20 months) dopamine depletion. Interaction of ageing-related changes in PP1 with the dopamine depletion-induced changes in CaMKIIalpha may account for enhanced GluR1 phosphorylation only after long-term dopamine depletion. These evolving changes may impact striatal synaptic plasticity, Parkinson's disease progression and the changing efficacy and side-effects associated with dopamine replacement therapy.

Fig. 1

Short-term 6-OHDA lesion increased CaMKIIα phosphorylation at Thr²⁸⁶. (A and B) Representative blots and summary graphs quantitating total striatal protein levels or phosphorylation (mean ± SEM) in samples harvested 3–12 weeks postoperatively. The number of rats analysed is indicated within or above each bar. ‘L’ (lesion) and ‘I’ (intact) indicate samples ipsilateral and contralateral to the lesion, respectively. ‘C’ indicates tissue from sham-lesioned rats. Only the decrease in TH (F_2,49 = 37.66, P < 0.0001) and the increase of P-Thr²⁸⁶-CaMKIIα (F_2,21 = 11.98, P = 0.0003) were significantly altered in dopamine-depleted striatum. (C) The increase in Thr²⁸⁶ phosphorylation of CaMKIIα was reversed by L-DOPA administration (F_1,27 = 5.61, P = 0.026).

Fig. 2

6-OHDA lesions changed PSD-associated CaMKIIα. PSD-enriched P2 fractions taken from rats 3 weeks after 6-OHDA lesion surgery were analysed by immunoblotting. The top panel shows representative blots from contralateral (intact) and lesioned hemispheres of two representative animals. The levels in samples from 6-OHDA-lesioned striatum are expressed as a percentage of levels in samples from the contralateral (intact) striatum (mean ± SEM). Total CaMKIIα was significantly decreased (t₁₈ = 2.92, P = 0.009) and Thr²⁸⁶-phosphorylated CaMKIIα was elevated (t₁₉ = 0.33, P = 0.006) following dopamine depletion, as determined by anova followed by post hoc t-test.

Fig. 3

Effects of 6-OHDA lesion on striatal protein phosphatases. (A and B) Representative immunoblots and summary graphs quantifying total striatal levels of protein phosphatase catalytic subunits and PP1 targeting and regulatory proteins. Phosphorylation of DARPP-32 at Thr⁷⁵ was significantly elevated in dopamine-depleted striatal samples (F_2,45 = 3.5, P = 0.038). (C) The increase in Thr⁷⁵ phosphorylation of DARPP-32 was reversed by L-DOPA administration (F_1,27 = 22.13, P = 0.0005).

Fig. 4

Changes in PP1γ1 and PP1-regulatory proteins during normal ageing. Quantification of protein levels in dorsolateral striatal homogenates from normal rats at 4–6, 12–14 or 21–23 months of age. PP1γ1 was elevated only at 21–23 months (F_2,47 = 12.02, P < 0.0001), DARPP-32 was decreased only at 12–14 months (F_2,45 = 7.98, P = 0.001), while both spinophilin (F_2,45 = 7.77, P = 0.001) and neurabin (F_2,43 = 10.46, P = 0.0002) were decreased at 21–23 months.

Fig. 5

Effects of chronic dopamine depletion on striatal proteins. (A–C) Long-term dopamine depletion caused an enduring decrease in TH at 9–11 months (t₁₂ = 6.82, P = 0.0001) and at 18–20 months (t₅ = 7.99, P = 0.0005). This was paralleled by an enduring increase in phosphorylation of both CaMKIIα at Thr²⁸⁶ at 9–11 months (t₁₁ = 2.28, P = 0.043) and 18–20 months (t₅ = 3.50, P = 0.017) and of DARPP-32 at Thr⁷⁵ at 9–11 months (t₆ = 5.03, P = 0.0024) and at 18–20 months (t₅ = 3.46, P = 0.018).

Fig. 6

Chronic dopamine depletion selectively increased GluR1 phosphorylation at Ser⁸³¹. Total GluR1 levels and levels of GluR1 phosphorylated at Ser⁸³¹ or Ser⁸⁴⁵ at 3–12 weeks, 9–11 months or 18–20 months following 6-OHDA lesion surgery. There was a significant effect of age (F_2,48 = 4.10, P = 0.0322 for Ser⁸³¹); post hoc tests revealed that Ser⁸³¹ phosphorylation was increased at both 9–11 months (t₆ = 2.495, P = 0.047) and at 18–20 months (t₄ = 4.738, P = 0.009) following dopamine depletion. In contrast, there was a trend for increased phosphorylation at Ser⁸⁴⁵ only 18–20 months following surgery (t₅ = 2.490, P = 0.068).