Inhibition of Striatal Soluble Guanylyl Cyclase-cGMP Signaling Reverses Basal Ganglia Dysfunction and Akinesia in Experimental Parkinsonism

There is clearly a necessity to identify novel non-dopaminergic mechanisms as new therapeutic targets for Parkinson's disease (PD). Among these, the soluble guanylyl cyclase (sGC)-cGMP signaling cascade is emerging as a promising candidate for second messenger-based therapies for the amelioration of PD symptoms. In the present study, we examined the utility of the selective sGC inhibitor 1H-[1], [2], [4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) for reversing basal ganglia dysfunction and akinesia in animal models of PD.The utility of the selective sGC inhibitor ODQ for reversing biochemical, electrophysiological, histochemical, and behavioral correlates of experimental PD was performed in 6-OHDA-lesioned rats and mice chronically treated with MPTP.We found that one systemic administration of ODQ is sufficient to reverse the characteristic elevations in striatal cGMP levels, striatal output neuron activity, and metabolic activity in the subthalamic nucleus observed in 6-OHDA-lesioned rats. The latter outcome was reproduced after intrastriatal infusion of ODQ. Systemic administration of ODQ was also effective in improving deficits in forelimb akinesia induced by 6-OHDA and MPTP.Pharmacological inhibition of the sGC-cGMP signaling pathway is a promising non-dopaminergic treatment strategy for restoring basal ganglia dysfunction and attenuating motor symptoms associated with PD

Systemic administration of the sGC ODQ reverses the increased striatal cGMP levels and the elevations in striatal activity observed in 6-OHDA-lesioned rats.

<p>(<b>a</b>) Behavioral assessment of 6-OHDA-induced unilateral nigrostriatal DA cell lesions. Compared to sham-operated controls (n = 11), all 6-OHDA-lesioned rats included in this study (n = 19) exhibited significant impairments in contralateral forelimb adjustment steps (***<i>P<0.0005</i>, unpaired t-Test), (<b>b</b>) Cell counting of TH positive neurons in the SN at four anatomical levels indicated that the degree of DA cell loss observed in all 6-OHDA-lesioned rats included in this study was >90%. (<b>c</b>) Striatal tissue cGMP levels were assessed in sham and 6-OHDA-lesioned rats pretreated with vehicle (n = 5–6 per group) or the sGC inhibitor ODQ (20 mg/kg, i.p.). Chronic DA depletion increased striatal cGMP levels (<i>**P<0.01</i> as indicated, Tukey post-hoc test after significant one-way ANOVA) in a manner that was reversed following a single ODQ treatment (1 hour post injection). (<b>d</b>) Bar graph summarizing the increased firing activity of striatal MSNs recorded from 6-OHDA-lesioned rats (n = 25 cells/19 rats) relative to sham-operated controls (n = 13 cells/11 rats) (***<i>P<0.001</i>, unpaired t-Test). (<b>e</b>) Electrophysiological traces of simultaneously recorded striatal single-unit activity and cortical LFPs in a sham and a 6-OHDA-lesioned rat. (<b>f</b>) Time course showing the effects of one systemic administration of vehicle (n = 5 cells from 5 rats) or ODQ (20 mg/kg, i.p., n = 7 cells from 7 rats) on striatal single-unit activity recorded from 6-OHDA-lesioned rats. ODQ (but not vehicle) markedly reduced elevations in firing rate observed in cells recorded from 6-OHDA-lesioned rats (<i>***P<0.0005</i>, compared to pre-ODQ activity or to measures taken at identical time points as indicated, Tukey post-hoc test after significant ANOVA).</p

Impact of tonic cGMP signaling on corticostriatal synaptic transmission <i>in vivo</i>.

<p>(<b>a</b>) Recording arrangement employed to study the pharmacological effects of the sGC inhibitor ODQ on corticostriatal transmission <i>in vivo</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027187#s4" target="_blank">Methods section</a> for details). Cortically-evoked postsynaptic potentials (PSPs) were recorded by means of local field potential (LFP) recordings. Inset shows examples of traces of corticostriatal PSPs (calibration bars: 30 ms, 1 µV). (<b>b</b>) Time course of corticostriatal PSPs recorded before and following systemic administration of 10 mg/kg and 20 mg/kg ODQ (i.p., n = 5 rats per dose). A marked attenuation of the corticostriatal response was observed following 20 mg/kg ODQ, an effect that becomes apparent after 20 min of drug administration. (<b>c</b>) Time course of corticostriatal PSPs recorded before and following 20 mg/kg ODQ + intrastriatal administration (0.1 µl/min×10 min) of the cGMP analog 8-Br-cGMP (20 mM; n = 5 rats) or vehicle (aCSF; n = 5 rats). Note that the characteristic attenuation of corticostriatal PSPs observed after 20 min of 20 mg/kg ODQ administration was lacking following intrastriatal infusion of 8-Br-cGMP. (<b>d</b>) Bar graph depicting the averaged changes in PSP responses obtained from the last 3 data points shown in <b>c</b> (marked in gray). Intrastriatal infusion of 8-Br-cGMP completely blocked the effects of ODQ (<i>***P<0.0005</i>, unpaired t-Test).</p

Systemic ODQ administration improves stepping performance in chronic MPTP-treated mice.

<p>(<b>a</b>) Chronic MPTP treatment significantly reduced the number of TH positive neurons in the SN. The data were collected at four anatomical levels from vehicle or MPTP-treated mice 3 weeks following the last injection (**<i>P<0.005</i> compared to vehicle). (<b>b</b>) Images of TH immunostaining showing the degree of reduction of TH positive cells in the SN at four anatomical levels (mm from bregma): −3.1 (<i>i, v</i>), −3.3 (<i>ii, vi</i>), −3.5 (<i>iii, vii</i>) and −3.7 (<i>iv, viii</i>). (<b>c</b>) Summary graph depicting the effect of chronic saline (n = 9) or MPTP (20 mg/kg, s.c., n = 13) injections on forelimb stepping performance. A significant decrease in the number of adjusting steps was observed after 6 weeks of MPTP injection (**<i>P<0.005</i> vs. vehicle, Tukey post-hoc test after significant ANOVA). (<b>d</b>) Time course showing the effect of a single s.c. injection of vehicle and ODQ (5 and 10 mg/kg) on MPTP-induced stepping deficits (n = 5–7 mice per group). A transient improvement in stepping performance was detected 30–90 min after ODQ administration (**<i>P<0.005</i>, compared to measures taken at time 0 or identical time points in vehicle, Tukey post-hoc test after significant two-way ANOVA).</p

Role of the indirect striatopallidal pathway in mediating the ODQ-dependent reversal of STN hyperactivity.

<p>(<b>a</b>) Bar graph showing levels of CO-I staining in the STN of sham-operated and 6-OHDA lesioned rats following systemic administration of vehicle or ODQ (20 mg/kg, i.p.). ODQ significantly reduced the increase in CO-I levels observed in the STN of 6-OHDA-lesioned rats (n = 10–11 rats/group; ***<i>P<0.0005</i> as indicated, Tukey post-hoc test after significant one-way ANOVA). <i>Inset:</i> Examples of coronal sections of STN derived from CO-I staining showing the reversal effect of ODQ. (<b>b</b>) Summary of the effect of single intrastriatal infusion (0.1 µl/min for 10 min) of vehicle (0.5% DMSO in aCSF) or ODQ (50 µM) on STN CO-I staining. Intrastriatal ODQ also normalized the increased STN CO-I observed in 6-OHDA-lesioned rats (n = 5–7 rats/group; *<i>P<0.05</i>, **<i>P<0.005</i> as indicated, Tukey post-hoc test after significant one-way ANOVA). <i>Inset:</i> Sections of CO-I staining showing the reversal effect of intrastriatal ODQ on STN activity.</p

Dose dependent effects of ODQ on reducing forelimb stepping deficits observed in 6-OHDA-lesioned rats.

<p>(<b>a–c</b>) Bar graphs showing the effect of systemic ODQ administration (10, 20, 40 mg/kg, i.p.) on deficits in forelimb use in 6-OHDA lesioned rats assessed with the stepping test. ODQ treatment transiently attenuated stepping deficits observed in 6-OHDA lesioned rats in a dose-dependent manner (n = 8 rats/group; *<i>P<0.05</i>, **<i>P<0.005</i>, ***<i>P<0.0005</i>, compared to baseline or time 0, Tukey post-hoc test after significant two-way ANOVA). (<b>d</b>) Summary graph (% change to baseline or time 0) depicting the impact of ODQ administration (10, 20, 40 mg/kg, i.p.) on deficits in forelimb use observed in 6-OHDA lesioned rats (*,⁺<i>P<0.05</i>, **,⁺⁺<i>P<0.005</i>, ***<i>P<0.0005</i>, compared to measures taken at identical time points as indicated, Tukey post-hoc test after significant two-way ANOVA).</p