Dopamine D2 receptor gene variants and response to rasagiline in early Parkinson's disease:a pharmacogenetic study

The treatment of early Parkinson's disease with dopaminergic agents remains the mainstay of symptomatic therapy for this incurable neurodegenerative disorder. However, clinical responses to dopaminergic drugs vary substantially from person to person due to individual-, drug- and disease-related factors that may in part be genetically determined. Using clinical data and DNA samples ascertained through the largest placebo-controlled clinical trial of the monoamine oxidase B inhibitor, rasagiline (ClinicalTrials.gov number, NCT00256204), we examined how polymorphisms in candidate genes associate with the clinical response to rasagiline in early Parkinson's disease. Variants in genes that express proteins involved in the pharmacokinetics and pharmacodynamics of rasagiline, and genes previously associated with the risk to develop Parkinson's disease were genotyped. The LifeTechnologies OpenArray NT genotyping platform and polymerase chain reaction-based methods were used to analyse 204 single nucleotide polymorphisms and five variable number tandem repeats from 30 candidate genes in 692 available DNA samples from this clinical trial. The peak symptomatic response to rasagiline, the rate of symptom progression, and their relation to genetic variation were examined controlling for placebo effects using general linear and mixed effects models, respectively. Single nucleotide polymorphisms, rs2283265 and rs1076560, in the dopamine D2 receptor gene (DRD2) were found to be significantly associated with a favourable peak response to rasagiline at 12 weeks in early Parkinson's disease after controlling for multiple testing. From a linear regression, the betas were 2.5 and 2.38, respectively, with false discovery rate-corrected P-values of 0.032. These polymorphisms were in high linkage disequilibrium with each other (r(2) = 0.96) meaning that the same clinical response signal was identified by each of them. No polymorphisms were associated with slowing the rate of worsening in Parkinson symptoms from Weeks 12 to 36 after correction for multiple testing. This is the largest and most comprehensive pharmacogenetics study to date examining clinical response to an anti-parkinsonian drug and the first to be conducted in patients with early stage Parkinson's disease receiving monotherapy. The results indicate a clinically meaningful benefit to rasagiline in terms of the magnitude of improvement in parkinsonian symptoms for those with the favourable response genotypes. Future work is needed to elucidate the specific mechanisms through which these DRD2 variants operate in modulating the function of the nigrostriatal dopaminergic system

Rasagiline Ameliorates Olfactory Deficits in an Alpha-Synuclein Mouse Model of Parkinson's Disease

<div><p>Impaired olfaction is an early pre-motor symptom of Parkinson's disease. The neuropathology underlying olfactory dysfunction in Parkinson's disease is unknown, however α-synuclein accumulation/aggregation and altered neurogenesis might play a role. We characterized olfactory deficits in a transgenic mouse model of Parkinson's disease expressing human wild-type α-synuclein under the control of the mouse α-synuclein promoter. Preliminary clinical observations suggest that rasagiline, a monoamine oxidase-B inhibitor, improves olfaction in Parkinson's disease. We therefore examined whether rasagiline ameliorates olfactory deficits in this Parkinson's disease model and investigated the role of olfactory bulb neurogenesis. α-Synuclein mice were progressively impaired in their ability to detect odors, to discriminate between odors, and exhibited alterations in short-term olfactory memory. Rasagiline treatment rescued odor detection and odor discrimination abilities. However, rasagiline did not affect short-term olfactory memory. Finally, olfactory changes were not coupled to alterations in olfactory bulb neurogenesis. We conclude that rasagiline reverses select olfactory deficits in a transgenic mouse model of Parkinson's disease. The findings correlate with preliminary clinical observations suggesting that rasagiline ameliorates olfactory deficits in Parkinson's disease.</p> </div

Olfactory deficits in the α-syn mouse model of PD.

<p><b>A-B: </b><b>Odor detection test. A.</b> Description of the protocol composed of 3 sessions (S). In each 5-min session, mice were exposed to 2 cartridges, one filled with water, the other with increasing odor concentrations from 1∶10⁸ to 1∶10⁴. <b>B.</b> Percentage of time sniffing the odor for the different concentrations. WT mice start detecting the odor at the concentration 1∶10⁶ when percentage of time sniffing the odor is significantly different from the chance level (50%, where mice spent same time sniffing water and odor cartridges) (°°°p<0.001, °°p<0.01, one-sample t-test). α-Syn mice can detect the odor only at 1∶10⁴ (°°°p<0.001, one-sample t-test). At the concentration 1∶10⁶, α-syn mice are significantly impaired compared to WT. N = 10 for each group aged 10–11 months. Statistics: One-sample t-test to compare each value to chance level (50%), (°°°p<0.001, °°p<0.01). Two-way RM ANOVA: odor concentration, p = 0.0001, F(2,36) = 11.96; genotype, p = 0.015, F(1,36) = 7.2; odor concentration×genotype, p = 0.0073, F(2,36) = 5.65; Bonferroni post-hoc (***p<0.001). <b>C-D: Short-term olfactory memory test. C.</b> Description of the protocol composed of 3 sessions (S). Each session consisted of two 5 min-trials (T) where mice are exposed to a novel odor separated by an increasing inter-trial time (ITI) from 60 s to 120 s. <b>D.</b> Percentage of time sniffing the odor during T2 compared to the total time spent sniffing during both trials. WT mice remember the odor during the 2^nd exposure for the 3 ITI tested and their percentage of time sniffing the odor during T2 was significantly different from the chance level (50%, where mice spent same time sniffing the odor during T1 and T2) (°°p<0.01, °°°p<0.001, one-sample t-test). By contrast, short-term olfactory memory of α-syn mice was impaired from an ITI of 120 s (p>0.05, one-sample t-test). However, it was significantly different from chance level at 60 s and 90 s (°°°p<0.001 and °p<0.05 respectively, one-sample t-tests). N = 10 for each group aged 10–11 months. Statistics: One-sample t-test to compare each value to chance level (50%), (°°°p<0.001, °°p<0.01, °p<0.05). <b>E-H: Odor discrimination test. E and G:</b> Social odor discrimination test. <b>E.</b> Description of the protocol composed of 6 habituation trials where mice are exposed to a familiar odor (F, odor of the tested mouse); and one odor discrimination trial, where one familiar odor is replaced by a novel odor (N, another mouse's odor). This test was performed with low or high odor intensities (wood blocks impregnated with mouse's odor for 2 or 7 days respectively). Each trial lasted 2 min and was separated by 1 min. <b>G.</b> Percentage of time sniffing novel odor. For both low and high odor intensities, α-syn mice have impaired odor discrimination with the percentage of time sniffing the odor significantly lower than WT. N = 19–21 for each group aged 10–11 months. Statistics: Two-way RM ANOVA: odor intensity, p = 0.55, F(1,38) = 0.37; genotype, p<0.0001, F(1,38) = 27.1; odor intensity×genotype, p = 0.63, F(1,38) = 0.23; Bonferroni post-hoc (***p<0.001). <b>F and H:</b> Non-social odor discrimination test. <b>F.</b> Description of the protocol based on the same principle of the social odor discrimination test but using non-social odors (lemon and lime). In the 8^th 2 min-trial, an item discrimination trial was added where the usual cartridge, with the novel odor (lime), was replaced by a novel item (a novel type of cartridge associated with the same novel odor, lime). <b>H.</b> Percentage of time sniffing the novel odor during the odor discrimination trial and percentage of time exploring the novel item in the item discrimination trial. α-syn mice had significantly impaired odor discrimination of the social odor. By contrast, the ability to discriminate the novel item was similar between WT and α-syn mice suggesting that the discrimination deficit is specific to olfaction. Statistics: unpaired t-test, non-social odor discrimination p<0.0001, N = 19–21 for each group aged 10–11 months; item discrimination p = 0.16, N = 10 for each group aged 10-11 months (***p<0.001).</p

Overexpression of α-synuclein in the olfactory bulb of the α-syn transgenic mice.

<p>Immunostaining of human wild-type α-synuclein in OB of <b>A.</b> WT mice and <b>B-D.</b> α-syn mice aged 12 months. <b>A-B.</b> Scale bars: 500 µm. <b>C-D.</b> High magnification of <b>C.</b> the glomerular layer (Gl) and <b>D.</b> the granule cell layer (GCL). Scale bars: 50 µm. α-Syn mice exhibit high expression of human α-synuclein in the different layers of the OB. α-Synuclein immunoreactivity indicates large profiles (arrows) as well as numerous small α-synuclein immunoreactive puncta (arrow heads).</p

Behavioral experiments: design and parameters analyzed.

<p><b>A.</b> Experimental design of the behavioral study. <b>B</b>. Olfactory- and control tests and the parameters analyzed from these experiments.</p

Olfactory deficits are age-dependent. A-C: Odor detection test.

<p>Description of the protocol consisting of 2 sessions (S). <b>B.</b> Percentage of time spent sniffing the odor at the concentration of 1∶10⁶ (session 1). WT mice aged 3, 11 and 18 months could detect the odor and the percentage of time sniffing the odor was significantly different from the chance level (50%) (°°°p<0.001). On the contrary, α-syn mice are progressively impaired in detecting the odor. Whereas at 3 months transgenic mice spent more time sniffing the odor compared to the chance level (p<0.05), from 11 months of age their scores no longer differed from the chance level (p>0.05) and the percentage of time spent by α-syn mice to sniff the odor is significantly different from WT mice (two way ANOVA). Statistics: One-sample t-tests to compare each value to chance level (50%) (°p<0.05, °°°p<0.001). Two-way ANOVA: age, p = 0.49, F(2,70) = 0.71; genotype, p<0.0001, F(1,70) = 40.21; age×genotype, p = 0.016, F(2,70) = 4.42; Bonferroni post-hoc (***p<0.001). <b>C.</b> Percentage of time spent sniffing the odor at the concentration of 1∶10⁴ (session 2). Both WT and α-syn mice aged 3, 11 and 18 months can detect the odor at the concentration of 1∶10⁴ and their percentage of time sniffing the odor is significantly different from the chance level (°°°p<0.001). Moreover, there is no significant difference between the genotypes (two-way ANOVA p>0.05). Statistics: One-sample t-tests to compare each value to chance level (50%) (°°°p<0.001). Two-way ANOVA: age, p = 0.12, F(2,70) = 2.15; genotype, p = 0.83, F(1,70) = 0.045; age×genotype, p = 0.64, F(2,70) = 0.45. <b>D-F: Short-term olfactory memory test. D.</b> Description of the protocol consisting of 2 sessions (S). <b>E.</b> Session 1 with an inter-trial interval of 60 s. Percentage of time spent sniffing the odor during T2 (trial 2) compared to the total time spent sniffing during both trials. All groups of WT mice aged 3, 11 and 18 months as well as α-syn mice aged 3 and 11 months remember the odor during the 2^nd exposure and their percentage of time sniffing the odor during T2 is significantly different from the chance level (50%) (°°°p<0.001). However, from 18 months of age, α-syn mice are impaired in remembering the odor during the 2^nd exposure (one-sample t-test, p>0.05) and the percentage of time spent sniffing the odor during T2 is significantly higher compared to 18 month-old WT mice (two-way ANOVA, p<0.001). Statistics: One-sample t-test to compare each value to chance level (50%) (°°°p<0.001). Two-way ANOVA: age, p = 0.0010, F(2,70) = 7.63; genotype, p = 0.0032, F(1,70) = 9.32, age×genotype, p = 0.011, F(2,70) = 4.78; Bonferroni post-hoc (***p<0.001). <b>F.</b> Session 2 with an inter-trial interval of 120 s. Percentage of time spent sniffing the odor during T2 compared to the total time spent sniffing during both trials. All groups of WT mice aged 3, 11 and 18 months remember the odor during the 2^nd exposure and their percentage of time spent sniffing the odor during T2 is significantly different from the chance level (one-sample t-tests, °°°p<0.001). On the contrary, α-syn mice aged 3, 11 and 18 months, are all impaired in remembering the odor during the 2^nd exposure (one-sample t-tests, p>0.05) and the percentage of time spent sniffing the odor during T2 is significantly higher compared to WT mice of the same age (two way ANOVA, *p<0.05, **p<0.01). Statistics: One-sample t-tests to compare each value to chance level (50%) (°°p<0.01, °°°p<0.001). Two-way ANOVA: age, p = 0.13, F(2,70) = 2.12; genotype, p<0.0001, F(1,70) = 26.86; age×genotype, p = 0.53, F(2,70) = 0.64; Bonferroni post-hoc (*p<0.05, **p<0.01). <b>G-H: Odor discrimination test. G.</b> Description of the protocol consisting of 6 habituation trials and one odor discrimination trial. <b>H.</b> Percentage of time spent sniffing the novel odor. At 3, 11 and 18 months, α-syn mice spend significantly less time compared to age-matched control mice to sniff the novel odor suggesting that they are impaired in their ability to discriminate the novel odor (two-way ANOVA, ***p<0.001). Statistics: Two-way ANOVA: age, p = 0.0028, F(2,70) = 6.42; genotype, p<0.0001, F(1,70) = 77.78; age×genotype, p = 0.077, F(2,70) = 2.66; Bonferroni post-hoc (***p<0.001). For all tests, N = 14 for group aged 3 and 18 months; N = 10 for group aged 11 months.</p

Rasagiline improved some aspects of olfaction in α-syn mice. A. Effect of rasagiline on odor detection deficit in α-syn mice.

<p>Rasagiline rescued the odor detection deficit in α-syn mice. At a concentration of 1∶10⁶, non-treated α-syn mice do not detect the odor and the percentage of time spent sniffing the odor was close to chance level, whereas rasagiline treated mice were significantly higher than the chance level. Moreover, rasagiline treated mice spent a similar time sniffing the odor compared to control mice. N = 9–10 for each group aged 10–11 months. Statistics: One-sample t-test to compare each value to chance level (50%), (°p<0.05, °°p<0.01, °°°p<0.001). Two-way RM ANOVA: odor concentration, p<0.0001, F(2,66) = 29; group, p = 0.19, F(3,66) = 1.67; odor concentration×group, p = 0.06, F(6,66) = 2.12; Bonferroni post-hoc (*p<0.05, **p<0.001). <b>B. Effect of rasagiline on short-term olfactory memory impairment in α-syn mice.</b> For the 120 s-ITI, percentage of time spent sniffing the odor in T2 was not different from chance level for both α-syn mice groups, treated or not treated with rasagiline. Rasagiline did not improve the short-term olfactory memory in α-syn mice. N =  9–10 for each group aged 10–11 months. Statistics: One-sample t-test compare to chance level (50%), (°p<0.05 and °°°p<0.001). Two-way RM ANOVA: ITI, p<0.0001, F(2,68) = 15.65; group, p = 0.13, F(3,68) = 2.04; ITI×group, p = 0.23, F(6,68) = 1.39; Bonferroni post-hoc. <b>C. Effect of rasagiline on odor discrimination deficit in α-syn mice.</b> Percentage of time spent sniffing the novel odor of α-syn mice was increased by rasagiline treatment for both intensities of the social odor as well as for the non-social odor. α-Syn mice treated with rasagiline were similar to control mice (p>0.05). Rasagiline rescued the odor discrimination deficit of α-syn mice. N =  18–21 for each group aged 10–11 months. Statistics for social odor discrimination: Two-way RM ANOVA, odor intensity, p = 0.032, F(1,74) = 4.78; group, p<0.0001, F(3,74) = 13.3; odor intensity×group, p = 0.034, F(3,74) = 3.04; Bonferroni post-hoc (*p<0.05, **p<0.01, ***p<0.001). Statistics for non-social odor discrimination: one-way ANOVA, p<0.001, F(3,73) = 18.16; Bonferroni post-hoc (***p<0.001).</p

Neurogenesis changes are not involved in the olfactory deficit of α-syn mice and rasagiline-induced improvement.

<p><b>A.</b> Quantification of newborn cells in the granule cell layer of the OB. Total number of BrdU positive cells was assessed every sixth section by stereology (counting frame 100 µm×100 µm; counting grid: 300 µm×300 µm). No difference between control and α-syn mice as well as no effect of rasagiline treatment was observed. N = 4-6 for each group aged 12 months. Statistic: one-way ANOVA, p = 0.66, F(3,14) = 0.54. <b>B.</b> BrdU staining in the olfactory bulb of WT and α-syn mice. Scale bars: 100 µm. <b>C. </b>Quantification of newborn neurons in the granule cell layer of the OB. The proportion of BrdU positive cells, which are also NeuN positive, was assessed by confocal microscopy. No difference between control and α-syn mice as well as no effect of rasagiline treatment was observed. On average, we analyzed 100 BrdU-positive cells in each animal, N = 3 mice in each group aged 12 months. Statistic: one-way ANOVA, p = 0.61, F(3,8) = 0.65. <b>D.</b> NeuN (green) and BrdU (red) double staining in the OB. Examples of NeuN-positive/BrdU positive-cells observed in WT and α-syn mice. Scale bars: 55.5 µm.</p