Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia

Abstract

Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future.

Keywords: Brain-derived neurotrophic factor; Depression; ERK1/2; Levodopa; Parkinson's disease; TrkB; mTOR.

Fig. 1.

Schemes for Experiments and post hoc analyses. (A) Scheme of the ketamine injection paradigm for Experiment 1 during development of LID (daily L-DOPA injections). FAS = Forelimb adjusting steps test. (B) Scheme of the injection paradigm in PD rats for Experiment 2. AR = amphetamine-rotation test; RR = RotaRod test. (C) Scheme of the injection paradigm for Experiment 3 during development of LID (daily L-DOPA injections). (D) Verification of unilateral 6-OHDA lesion and evaluation of striatal dopamine (DA) levels after ketamine in the rats from the study shown in (A). Electrochemical detection of striatal DA content (mean ± SEM) is reduced by>95% in the lesioned side. Striatal DA content was unchanged by a 10-h-treatment of either ketamine (K; n=9), R-ketamine (R-K; n=9) vs. vehicle (V; n = 9) 1-h before rats were euthanized, showing no effect on overall striatal DA levels by ketamine or R-ketamine-treatment compared to vehicle in either the lesioned (Lx) or the intact hemisphere. (E) Verification of unilateral 6-OHDA lesion from the study shown in (B) using semi-quantitative TH western analysis in striatal tissue plotting % loss (mean ± SEM) in Lx vs. intact (In) hemisphere (n=9). Two-tailed t-test, ***p < .001. (F) Verification of unilateral 6-OHDA lesions in the rats from the study depicted in (C). The graph shows the quantification of the TH-ir plotting the % loss (mean ± SEM) in the Lx vs. intact SN hemispheres (n =10/group; V =vehicle, K =ketamine, K +A = ketamine+ ANA-12). (G) Example photomicrograph of a SN in Experiment 3 shows the unilateral reduction in TH-ir post-lesion. Two-way ANOVAs, Bonferroni post hoc tests, ***p < .001. (H) Verification of unilateral 6-OHDA lesion from the ANA-12-only control study, the negative control for Experiment 3, using semi-quantitative TH western analysis in striatal tissue, plotting % loss (mean ± SEM) in Lx vs. intact hemisphere (n = 10). Two-tailed t-test, ***p < .001.

Fig. 2.

Low-dose racemic ketamine treatment once a week attenuates the development of LID in the preclinical model. In Experiment 1 6-OHDA-lesioned PD rats were injected daily with L-DOPA (days 0–13: 6 mg/kg; days 14–28: 12 mg/kg; i.p.) to induce dyskinesia and tested for LAO-AIMs twice a week for 3 h by blinded investigators. (A) The mean LAO AIMs scores±SEM are plotted showing a 50% reduction after racemic low-dose ketamine treatments (K) when compared to the vehicle group (V) and a group treated with R-ketamine (R-K), to test for contribution of the stereospecific ketamine isomer. The blue arrows point to the days of the 10-h racemic ketamine (20 mg/kg; i.p.), R-ketamine (10 mg/kg; i.p.) or vehicle treatment paradigm; n = 9 per group, *p < .05, **p < .01, Kruskal-Wallis test with Dunn’s multiple comparisons post hoc tests. (B) Example time course of the LAO-AIMs data showed in (A) for day 11. (C) Example time course of the LAO-AIMs data showed in (A) for day 25. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

LAO-AIMs scores from Experiment 1 separated by sub-type of dyskinesia. Low-dose racemic ketamine treatment once a week attenuates the development of (A) limb (B) axial (C) orolingual AIMs in the preclinical model. 6-OHDA-lesioned PD rats were injected daily with L-DOPA (days 0–13: 6 mg/kg; days 14–28: 12 mg/kg; i.p.) to induce dyskinesia and tested for LAO AIMs twice a week for 3 h by blinded investigators. The mean LAO AIMs scores ± SEM are plotted showing a 50% reduction after racemic low-dose ketamine treatments (K) when compared to the vehicle group (V) and a group treated with R-ketamine (R-K), to test for contribution of the stereospecific ketamine isomer. The blue arrows point to the days of the 10-h racemic ketamine (20 mg/kg; i.p.), R-ketamine (10 mg/kg; i.p.) or vehicle treatment paradigm; n = 9 per group, *p < .05, **p < .01, Kruskal-Wallis tests with Dunn’s post hoc tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Ketamine does not interfere with the anti-PD effect of L-DOPA and reduces PD-motor behavior post-6-OHDA-lesion (Post-Lx) by itself. (A) Mean % contralateral/ipsilateral ratios of steps ± SEM using the FAS test paradigm in the LID cohort of Experiment 1, are plotted after normalization to pre-lesion (Pre-Lx) indicate a significant anti-PD effect of ketamine *p < .05; repeated measures ANOVA. Ketamine does also not interfere with the anti-PD effect of L-DOPA, and a significant increase of stepping contralateral to the lesioned side after either L-DOPA alone or L-DOPA + Ketamine vs. all Post-Lx time points is seen: ***p < .001; one-way ANOVA on data prior to normalization, Tukey-Kramer corrected post hoc tests; n = 9 per group. (B) In Experiment 2 we tested ketamine treatment in a separate cohort of hemi-parkinsonian 6-OHDA-lesioned rats and used the RotaRod test to evaluate the deficit. The graph shows the mean latency to fall ± SEM, normalized to pre-lesion baseline (Pre-Lx). Post-lesion (Post-Lx) the latency to fall was reduced by 50% in these PD animals. This motor deficit was reversed by ketamine treatment (blue bars), already at the 1st injection, and the animals performed as good as at baseline. One-way ANOVA, with Tukey-Kramer corrected post hoc tests, on raw data before normalization. n = 9, *p < .05, **p < .01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

Ketamine did activate striatal mTOR and ERK1/2 pathways. (A, B) Involvement of the mTOR pathway in the effects of ketamine. We show an increased phosphorylation level of striatal mTOR in striatal tissue using a multiplex immunoassay. Mean % p-mTOR / mTOR level ± SEM normalized to vehicle control is plotted for the intact (A) and lesioned (B) striatum for vehicle control (V) and ketamine (K) conditions. n = 9 per group, *p < .05, ***p < .001; two-tailed t-tests on data prior to normalization. (C, D) Western analysis of levels of striatal ERK1/2 phosphorylation. Mean % p-ERK1/2 / ERK1/2 level ± SEM normalized to vehicle control is plotted for the intact (C) and lesioned (D) hemispheres. *p < .05; two-tailed t-tests on data prior to normalization. (E) Example western blots testing for pERK1/2 and total ERK1/2 are shown. I = intact hemisphere; Lx = lesioned hemisphere; Neg = negative control; Pos = positive control.

Fig. 6.

Ketamine’s long-term anti-dyskinetic activity was driven by BDNF signaling. (A) The mean LAO-AIMs scores ± SEM of Experiment 3 are plotted. The sustained anti-dyskinetic effect of low-dose ketamine is reduced by blocking the BDNF receptor TrkB, with co-injection of the TrkB antagonist ANA-12 (0.5 mg/kg; i.p.) with ketamine (K + A). The blue arrows point to the days of the 10-h racemic (K) ketamine (20 mg/kg; i.p.), or vehicle treatment paradigm (V). ANA-12 co-injection (green bars) did reduce the sustained anti-dyskinetic effect seen in ketamine-only injected LID (blue bars) leading to LAO AIMs comparable to those of the vehicle group (grey bars), indicating an involvement of BDNF in the sustained anti-dyskinetic effects of ketamine. n = 10 per group, *p < .05, **p < .01, ANOVAs, Tukey-Kramer corrected post hoc tests. (B) Example time course of the LAO-AIMs for day 14 showed in (A). (C) A control study using 10-h ANA-12-only treatments on days 0 and 7 of daily L-DOPA-treatment (6 mg/kg; i.p.) verified that, while systemic TrkB antagonism does block the ketamine effect, it does not change development of LID in this model, and serves as an important negative control for the data shown in (A). The graph depicts the mean LAO-AIMs scores ± SEM from the vehicle control groups in Experiment 1 (V-E1; n = 9) and Experiment 3 (V-E3; n = 10), as well as the ANA-12-only control study (ANA-12; n = 10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

LAO-AIMs scores from Experiment 3 separated by sub-type of dyskinesia. Low-dose ketamine treatment (blue arrows) once a week attenuates the development of individual AIMs scores. 6-OHDA-lesioned PD rats were injected daily with L-DOPA (days 0–14: 6 mg/kg; i.p.) to induce dyskinesia and tested for LAO-AIMs twice a week for 3 h by blinded investigators. The anti-dyskinetic effect of low-dose ketamine (K, blue bars) reduced the individual (A) limb (B) axial (C) and orolingual AIMs (mean ± SEM) scores, compared to the vehicle (V, grey bars) group and a group treated with the TrkB antagonist, ANA-12 (K + A, green bars). The blue arrows point to the days of the 10-h racemic ketamine (20 mg/kg; i.p.), R-ketamine (10 mg/kg; i.p.) n = 10 per group, *p < .05, **p < .01, Kruskal-Wallis tests with Dunn’s post hoc tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8.

Ketamine reduced dendritic mushroom spines in the dyskinetic striatum. (A) The mean dendritic length of the MSN neurons ( ± SEM) is plotted for intact and lesioned hemisphere for all the treatment groups. No significant difference was found between groups for dendritic length. (B) Ketamine-treated rats (K) showed a significant reduction of mushroom spine density in medium spiny neurons (MSNs) of the dorsal striatum in both the intact and lesioned hemispheres, as compared to vehicle controls (V). Mushroom spines were at control levels in rats co-treated with ketamine and the TrkB receptor antagonist, ANA-12 (K + A). Example photomicrographs of lesioned hemispheres with * indicating mushroom spines are shown for vehicle treatment (C), ketamine treatment (D), and co-treatment with ketamine and ANA-12 (E). Scale bar = 10 μm. n = 10 rats/group, and n = 3 dendrites/hemisphere, **p < .01, ***p < .001. One-way ANOVA with Sidak’s multiple comparisons test.

Fig. 9.

LAO-AIMs are correlated with mushroom spines in the lesioned dyskinetic striatum. Correlation analyses of mushroom spine density (mean ± SEM) as compared to total LAO-AIMs (mean ± SEM) are shown for (A) vehicle, intact, (B) ketamine, intact hemisphere, (C) ketamine + ANA-12, intact hemisphere, (D) vehicle, lesioned hemisphere, (E) ketamine, lesioned hemisphere, (F) and ketamine + ANA-12, lesioned hemisphere. There was a high correlation between mushroom spines and LAO-AIMs in the lesioned hemispheres of all treatment groups, indicating the importance of striatal mushroom spine density for LID. n = 10 rats/group, and n = 3 dendrites/hemisphere, Pearson’s correlation test.

Fig. 10.

Schematic for the proposed molecular mechanisms for the prolonged anti-dyskinetic effects of ketamine. Cortical disinhibition hypothesis: Ketamine (K) causes a burst of glutamate thought to occur via cortical parvalbumin (PV)-positive gamma-aminobutyric acid (GABA)-interneurons in the motor cortex; the tonic firing of the interneuron is driven by NMDARs, and the active, open-channel state allows ketamine to enter and block activity. The release of inhibition activates glutamatergic principal cells, stimulates α-amino-113-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and depolarizes downstream striatal MSN neurons. In addition, release of cortical BDNF leads to stimulation of TrkB on striatal MSNs, activating mTOR and ERK1/2, followed by an increase in transcriptional regulation and as a result maladaptive multisynaptic mushroom spines in the dyskinetic striatum are replaced by monosynaptic thin spines.