Golexanolone reduces glial activation in the striatum and improves non-motor and some motor alterations in a rat model of Parkinson's disease

Abstract

Background: Parkinson's disease (PD) affects more than 6 million people worldwide. Along with motor impairments, patients and animal models exhibiting PD symptoms also experience cognitive impairment, fatigue, anxiety, and depression. Currently, there are no drugs available for PD that alter the progression of the disease. A body of evidence suggests that increased GABA levels contribute to the reduced expression of tyrosine hydroxylase (TH) and accompanying behavioral deficits. TH expression may be restored by blocking GABA_A receptors. We hypothesized that golexanolone (GR3027), a well-tolerated GABA_A receptor-modulating steroid antagonist (GAMSA), may improve Parkinson's symptoms in a rat model of PD.

Objectives: The aims of this study were to assess whether golexanolone can ameliorate motor and non-motor symptoms in a rat model of PD and to identify some underlying mechanisms.

Methods: We used the unilateral 6-OHDA rat model of PD. The golexanolone treatment started 4 weeks after surgery. Motor symptoms were assessed using Motorater and CatWalk tests. We also analyzed fatigue (using a treadmill test), anhedonia (via the sucrose preference test), anxiety (with an open field test), and short-term memory (using a Y maze). Glial activation and key proteins involved in PD pathogenesis were analyzed using immunohistochemistry and Western blot.

Results: Rats with PD showed motor incoordination and impaired locomotor gait, increased fatigue, anxiety, depression, and impaired short-term memory. Golexanolone treatment led to improvements in motor incoordination, certain aspects of locomotor gait, fatigue, anxiety, depression, and short-term memory. Notably, golexanolone reduced the activation of microglia and astrocytes, mitigated TH loss at 5 weeks after surgery, and prevented the increase of α-synuclein levels at 10 weeks.

Conclusions: Golexanolone may be useful in improving both motor and non-motor symptoms that adversely affect the quality of life in PD patients, such as anxiety, depression, fatigue, motor coordination, locomotor gait, and certain cognitive alterations.

Keywords: 6-OHDA model; GABAergic neurotransmission; Parkinson's disease; cognitive impairment; glial activation; motor impairment.

Figure 1

Experimental design. The unilateral injection of 6-OHDA was used a model of Parkinson's diseases. The apomorphine-induced rotation test was performed 3 weeks after surgery to select successfully operated rats. (A) In one set of rats (n = 8–10 per group), golexanolone treatment started at week 4 after surgery and different motor and non-motor parameters were evaluated: fatigue (treadmill), anxiety and ambulatory activity (open field test), object location memory, anhedonia (sucrose preference test), motor coordination (Motorater), locomotor gait (CatWalk) and short-term memory. Rats were sacrificed 10 weeks after surgery to extract the brains for immunohistochemistry and western blot analysis. (B) A different set of rats (n = 6 per group) didn't perform the behavioral tests and was sacrificed at 5 weeks after surgery to evaluate glial activation and TH parameters by immunohistochemistry at a shorter time. (C) A third set of rats (n = 12 per group) started golexanolone and DOPA treatment at weeks 6 after surgery and the induction of dyskinesia was evaluated during 5 weeks.

Figure 2

Golexanolone treatment reverses fatigue, anxiety, anhedonia and short-term memory deficits in 6-OHDA rats. Fatigue was evaluated in the treadmill: (A) time in treadmill [6-OHDA effect was significant (p = 0.024) whereas treatment effect (p = 0.094) and interaction were not statistically significant; Tukey's test was used for multiple comparisons] (B) time in shock zone, expressed as percentage of total time of the test (effect of 6-OHDA (p = 0.0002), of golexanolone treatment (p = 0.024), and interaction (p = 0.032) were all statistically significant; Tukey's test was used for multiple comparisons). Anxiety was evaluated in the open field during 5 min: time spent in the central zone of the box [effect of 6-OHDA injection was significant (p = 0.045), as well as interaction (p = 0.027), whereas treatment effect was not statistically significant; Fisher's LSD test was used for multiple comparisons] (C), ambulatory activity, total distance traveled, was also evaluated in the open field (only effect of 6-OHDA injection was significant (p < 0.0001); Tukey's test was used for multiple comparisons) (D) and distance in the central zone [effect of 6-OHDA injection was significant (p = 0.001), but treatment effect does not reach significance (p = 0.051) and interaction was not significant; Tukey's test was used for multiple comparisons] (E). Anhedonia, evaluated by the sucrose preference test, was expressed as percentage of sucrose intake [effect of 6-OHDA was statistically significant (p = 0.0009) as well as interaction effect (p = 0.023), whereas treatment effect not reached significance (p = 0.058); Tukey's test was used for multiple comparisons] (F). Short-term memory evaluated in a Y-maze was measured with a discrimination ratio [injection of 6-OHDA do not induce significant effect but there is a significant interaction (p = 0.011) and treatment effect not reach significance (p = 0.085); Tukey's test was used for multiple comparisons] (G). In (H) is shown the time spent in each arm. In all groups except in the 6-OHDA group without golexanolone treatment, time spent in the new arm is significantly higher than in the old arm (one-way ANOVA followed by Tukey's test was used). In (I) we also show number of entries in each arm, which was higher to the new arm in shams groups but not in 6-OHDA groups, in which total number of entries is significant lower due to lower activity (one-way ANOVA followed by Tukey's test was used). Data are the mean ± SEM of 7–12 rats per group. Values significantly different from SHAM-VH rats are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001), values significantly different from 6-OHDA-VH are indicated by a (a = p < 0.05) and values significant different between new and old arm in the Y-maze are indicated with # (^## p < 0.01; ^### p < 0.001) (VH, vehicle and Golex, golexanolone).

Figure 3

Golexanolone treatment reverses motor incoordination and some locomotor gait deficits in 6-OHDA rats. (A) Motor incoordination: average of errors/run for each rat in the Motorater test. Two-way ANOVA indicated significant effects of 6-OHDA (p = 0.0012), of golexanolone treatment (p = 0.0001) and also a significant interaction (p = 0.027); Tukey's test was used for multiple comparisons. Different parameters evaluating locomotor gait were measured in the CatWalk: (B) regularity index [the interaction was significant (0.0005), although 6-OHDA and golexanolone treatment did not induce significant effects; Fisher's LSD test was used for multiple comparisons], (C) print positions were increased in 6-OHDA groups [group effect was significant (p = 0.0001) but not treatment effect or interaction; Tukey's test was used for multiple comparisons], (D) stand index [only 6-OHDA effect was significant (p = 0.0001); Tukey's test was used for multiple comparisons], (E) stand is shown separated for front and hind paws, as the effects were different; for front paws there is a significant effect of 6-OHDA injection but not of treatment, but interaction was close of significance (p = 0.077); Fisher's LSD test was used for multiple comparisons, (F) for hind paws only group effect was significant (p < 0.0001); Fisher's LSD test was used for multiple comparisons, (G) Max Intensity At as percentage of stand is also increased in 6-OHDA injected rats [only significant effect of 6-OHDA injection was found (p = 0.0001); Tukey's test was used for multiple comparisons], (H) swing (as percentage of the step cycle), statistic data were: p = 0.05 for 6-OHDA effect, p = 0.079 for treatment effect and no significant interaction; Fisher's LSD test was used for multiple comparisons, (I) swing speed is clearly decreased in 6-OHDA injected rats and not affected by golexanolone [only 6-OHDA effect was significant (p < 0.0001); Tukey's test was used for multiple comparisons], (J) stride length is decreased in 6-OHDA injected groups (p < 0.0001 for 6-OHDA effect and treatment effect or interaction were no significant; Tukey's test was used for multiple comparisons) (K) duty cycle, as percentage of the step cycle [effect of 6-OHDA injection is significant (p = 0.0043) and also effect of golexanolone treatment (p = 0.018) but not significant interaction was found; Fisher's LSD test was used for multiple comparisons], (L) initial dual stance [significant effect of 6-OHDA (p < 0.0001) and of golexanolone treatment (p = 0.028) were found, as well as a significant interaction (P = 0.034); Tukey's test was used for multiple comparisons]. Data are the mean ± SEM of 8–10 rats per group. Values significantly different from SHAM-VH rats are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and values significantly different from 6-OHDA-VH are indicated by a (a = p < 0.05, aaa = p < 0.001) (VH, vehicle and Golex, golexanolone).

Figure 4

Golexanolone treatment only induces mild orolingual dyskinesias in 6-OHDA rats, unlike the general and strong dyskinesias induced by L-DOPA. Different types of dyskinesia were followed after golexanolone or L-DOPA treatment for 5 weeks, 3 sessions/week, for a total of 15 sessions. The score/session is showed for each of the four subtypes of AIMs evaluated: (A) axial, (B) limb, (C) orolingual, and (D) locomotive. Data are the mean ± SEM of 10–12 rats per group.

Figure 5

Tyrosine hydroxylase content is reduced in the striatum of 6-OHDA rats and golexanolone treatment partially restores it at 5 weeks after surgery. Representative images of immunohistochemistry staining against tyrosine hydroxylase (TH) in the ipsilateral (right) and contralateral (left) striatum at 5 (A) and 10 (C) weeks after surgery. Scale bar = 100 μm, as indicated in the figure. TH staining optical density in striatum, expressed as percentage of shams at 5 (B) and 10 (D) weeks after surgery. Data are the mean ± SEM of 6 rats per group in B and 3-4 rats per group in (D). Data were analyzed by Two-way ANOVA, independently in the right and the left striatum: (B) in the injected striatum 6-OHDA effect was significant (p < 0.0001) and there is also significant interaction between 6-OHDA injection and treatment (p = 0.02) but treatment effect was not significant; in the contralateral striatum no significant differences were found; multiple comparisons were performed with Fisher's LSD test, (D) in the lesioned part 6-OHDA induced significant effect (p < 0.0001), but not golexanolone treatment, although interaction was almost significant (p = 0.053); Tukey's test was used for multiple comparisons; in the contralateral striatum both 6-OHDA effect (p < 0.0001) and treatment effect (p = 0.0003) were significant and interaction p-value was 0.055; multiple comparisons were performed with Fisher's LSD test. TH content was also analyzed by western blot (E), in the injected hemisphere 6-OHDA effect (p < 0.0001) was significant as well as treatment effect (p = 0.014) and interaction p-value was p = 0.06; Tukey's test was used for multiple comparisons; in the contralateral hemisphere also 6-OHDA effect (p < 0.0001) and treatment effect (p = 0.0057) were significant but not the interaction; Tukey's test was used for multiple comparisons. Values significantly different from SHAM-VH rats are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and values significantly different from 6-OHDA-VH are indicated by a (a p < 0.05).

Figure 6

α-synuclein content is increased in the striatum of 6-OHDA rats and golexanolone treatment normalizes it at 10 weeks after surgery. (A) Representative images of immunohistochemistry staining against α-synuclein in the ipsilateral (right) and contralateral (left) striatum at 10 weeks after surgery. Scale bar = 100 μm, as indicated in the figure. Quantification of α-synuclein content (optical density in the immunohistochemistry) in 3–4 rats per group at ten (B) and five (D) weeks after surgery. Data are the mean ± SEM and were analyzed by Two-way ANOVA; at ten weeks, in the injected hemisphere not significant effect of 6-OHDA or treatment was found but interaction was significant (p = 0.0006); in the contralateral striatum no significant effects were found; multiple comparisons were performed with Fisher's LSD test; at 5 weeks, no significant difference was found between the groups. The content of α-synuclein at 10 weeks was also analyzed by Western blot in 8–9 rats per group (C) expressed as percentage of shams (mean ± SEM); in the injected striatum 6-OHDA effect (p = 0.035), treatment effect (p = 0.029) and interaction (p = 0.0057) were all significant; in the unlesioned striatum, also significant effects of 6-OHDA (p < 0.0001) and of treatment with golexanolone (p < 0.0001) were found but not significant interaction; Tukey's test was used for multiple comparisons. Values significantly different from SHAM-VH rats are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and values significantly different from 6-OHDA-VH are indicated by a (a p < 0.05, aa p < 0.01).

Figure 7

Microglia is activated in the striatum of 6-OHDA rats and golexanolone treatment normalizes it at 5 weeks after surgery. Representative images of immunohistochemistry staining against Iba1 in the ipsilateral (right) and contralateral (left) striatum at 5 (A) and 10 (B) weeks after surgery. Scale bar = 25 μm, as indicated in the figure. Arrows indicate examples of activated (ameboid) microglia cell in 6-OHDA group at 5 weeks and of a more branched (non-activated) microglial cell in the 6-OHDA group treated with golexanolone, indicating reversal of microglia activation by golexanolone. Perimeter of the Iba1 stained cells at 5 (C) and 10 (D) weeks after surgery. Data are the mean ± SEM of 3–6 rats per group and were analyzed by Two-way ANOVA. In (C), injected hemisphere, no significant effects of 6-OHDA or treatment, or interaction, were found, but significant effects results from Fisher's LSD multiple comparisons test; in the contralateral hemisphere interaction was significant (p = 0.033) and multiple comparisons were also performed with Fisher's LSD test. In (D), 6-OHDA injection has significant effect in the ipsilateral striatum (p < 0.0001), as well as treatment (p = 0.032) and interaction between group and treatment was also significant (p = 0.036). In the contralateral striatum the effect of 6-OHDA was also significant (p = 0.0012) and the treatment (p = 0.021), but not significant interaction exists; Tukey's test was used for multiple comparisons. Values significantly different from SHAM-VH rats are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001) and values significantly different from 6-OHDA-VH are indicated by a (a p < 0.05).

Figure 8

Astrocytes are activated in the striatum of 6-OHDA rats at 5 weeks after surgery and golexanolone treatment reduces this activation. Representative images of immunohistochemistry staining against GFAP in the ipsilateral (right) and contralateral (left) striatum at 5 (A) and 10 (B) weeks after surgery. Scale bar = 50 μm, as indicated in the figure. GFAP stained area in striatum, expressed as percentage of shams at (C) 5 and (D) 10 weeks after surgery. Data are the mean ± SEM of 3–5 rats per group and were analyzed by Two-way ANOVA; in C, in the injected striatum, significant effect of golexanolone treatment (p = 0.035) was found, but not of 6-OHDA injection, and interaction p-value was p = 0.054; in the contralateral striatum no significant differences were found; in (D) no significant differences were found in any hemisphere; multiple comparisons were performed with Fisher's LSD test. Values significantly different from SHAM-VH rats are indicated by asterisks (*p < 0.05) and values significantly different from 6-OHDA-VH are indicated by a (a p < 0.05).