Prolyl Oligopeptidase Regulates Dopamine Transporter Phosphorylation in the Nigrostriatal Pathway of Mouse

Alpha-synuclein is the main component of Lewy bodies, a histopathological finding of Parkinson's disease. Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein and accelerates its aggregation in vitro. PREP enzyme inhibitors have been shown to block the alpha-synuclein aggregation process in vitro and in cellular models, and also to enhance the clearance of alpha-synuclein aggregates in transgenic mouse models. Moreover, PREP inhibitors have induced alterations in dopamine and metabolite levels, and dopamine transporter immunoreactivity in the nigrostriatal tissue. In this study, we characterized the role of PREP in the nigrostriatal dopaminergic and GABAergic systems of wild-type C57Bl/6 and PREP knockout mice, and the effects of PREP overexpression on these systems. Extracellular concentrations of dopamine and protein levels of phosphorylated dopamine transporter were increased and dopamine reuptake was decreased in the striatum of PREP knockout mice, suggesting increased internalization of dopamine transporter from the presynaptic membrane. Furthermore, PREP overexpression increased the level of dopamine transporters in the nigrostriatal tissue but decreased phosphorylated dopamine transporters in the striatum in wild-type mice. Our results suggest that PREP regulates the function of dopamine transporter, possibly by controlling the phosphorylation and transport of dopamine transporter into the striatum or synaptic membrane.Peer reviewe

Importance of GluA1 Subunit-Containing AMPA Glutamate Receptors for Morphine State-Dependency

Peer reviewe

Morphine-induced locomotor sensitization in GluA1−/− and GluA1+/+ mice.

<p>(<b>A–B</b>), Distance moved in the CS+ (morphine, or saline for saline control animals) trials is shown. The scores indicate 1) significant morphine effects and dose-response (^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001, compared to the previous dose, Bonferroni test), 2) significant locomotor sensitization to morphine (^&<i>p</i><0.05, ^&&<i>p</i><0.01, ^&&&<i>p</i><0.001, compared to the previous day, Bonferroni test), 3) significant habituation in GluA1—mice (saline control group) (^HH<i>p</i><0.01, compared to the first trial, Bonferroni test). Data represent means ± SEM, <i>n</i> = 24–49. ^*<i>p</i><0.05, ^**<i>p</i><0.01, ^***<i>p</i><0.001, between the genotypes, <i>t</i>-test. The scores were derived from the raw data depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038325#pone-0038325-g001" target="_blank">Fig. 1</a>. (<b>C–D</b>), The difference in distance moved between trials 1 and 2 (CS+ minus CS−) on each conditioning day. The data indicate 1) that the activity of GluA1−/− is strongly reduced, in comparison to GluA1+/+ mice, during the second trial of the first conditioning day after both saline control and morphine injections (^*<i>p</i><0.05, ^**<i>p</i><0.01, ^***<i>p</i><0.001, between the genotypes, <i>t</i>-test), 2) that during the following days, GluA1−/− and GluA1+/+ mice were equally activated by 10 mg/kg morphine and 3) that morphine 20 mg/kg produced incremental locomotor activity in both mouse lines (^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001, compared to the previous dose, Bonferroni test). Data represent means ± SEM, <i>n</i> = 24–49. The scores were derived from the raw data depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038325#pone-0038325-g001" target="_blank">Fig. 1</a>.</p

Unaltered detection of opioid stimulus in GluA1−/− mice.

<p>Mice were trained to discriminate 5 mg/kg morphine from saline and then tested for various doses of morphine in a 20-min drug discrimination test session. (<b>A</b>) Dose-response curves for discriminative stimulus effects of morphine; data show morphine-appropriate lever responses expressed as percentage of total responses on both levers. (<b>B</b>) Dose-response curves for response rate-lowering effects of morphine during the dose-response tests. Data are shown as means ± SEM, <i>n</i> = 5–6.</p

Lack of morphine-induced increase in the ventral tegmental area dopamine neuron AMPA/NMDA ratios in GluA1−/− mice.

<p>A single morphine (10 mg/kg) or saline injection was administered one day before <i>ex vivo</i> electrophysiological recordings from slices. (<b>A</b>) Representative traces displaying AMPA and NMDA receptor-mediated current components. (<b>B</b>) Quantification of the electrophysiological data, which is represented as means ± SEM, <i>n</i> = 6–11. ^*<i>p</i><0.05, between the genotypes; ^##<i>p</i><0.01, compared to the corresponding saline control, Bonferroni test.</p

EC₅₀ and maximal stimulation values for DAMGO stimulation of GTPγ[³⁵S] binding in various brain regions of GluA1+/+ and GluA1−/− mice.

<p>Values are mean ± SEM, n = 5–7, in µM for EC₅₀ values and as percentage of basal for maximal stimulation.</p

State-dependency is impaired in GluA1−/− mice in morphine-conditioned place preference.

<p>(<b>A</b>) Time spent in the morphine-associated zone during 15 min (900 s). Animals were tested either in Sal-state (priming injection of saline) or in Mor10-state (priming injection of 10 mg/kg morphine). (<b>B</b>) Distance moved during the 15 min preference test. Data represent means ± SEM, <i>n</i> = 8–33. ^*<i>p</i><0.05, ^***<i>p</i><0.001, between the genotypes, <i>t</i>-test; ^$<i>p</i><0.05, ^$$<i>p</i><0.01, ^$$$<i>p</i><0.001, between testing states, <i>t</i>-test; ^#<i>p</i><0.05, ^##<i>p</i><0.01, between conditioning doses, compared to saline control, Bonferroni test; ^&<i>p</i><0.05, between conditioning doses, compared to morphine 10 mg/kg conditioning dose, Bonferroni test. Sal = Saline, Mor = Morphine.</p

Distance moved during each of the eight conditioning trials for the GluA1−/− (−/−) mice and their littermate wildtype GluA1+/+ mice (+/+).

<p>In CS+ trials, animals were injected with saline (saline control animals) (<b>A</b>), morphine 10 mg/kg (<b>B</b>) or morphine 20 mg/kg (<b>C</b>) immediately before transferring them into cages, in which their locomotor activity was recorded for 30 min. Saline was injected in CS− trials. Data represent means ± SEM, <i>n</i> = 24–49. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, between the genotypes, <i>t</i>-test. S = Saline; 10 and 20 denote doses of morphine in mg/kg.</p