Sensitization to amphetamine psychostimulant effect: A key role for ventral tegmental area neurotensin type 2 receptors and MAP kinase pathway.

Neurotensin is an endogenous neuropeptide that acts as a potent modulator of ventral tegmental area (VTA) neurotransmission. The present study was aimed at determining VTA cell population and neurotensin receptor subtype responsible for the initiation of amphetamine-induced psychomotor activity and extracellular signal-regulated kinases (ERK1/2) sensitization. During an induction phase, rats were injected intra-VTA on two occasions, every second day, with [D-Tyr11 ]-neurotensin (D-Tyr-NT), SR142948 (a mix Ntsr1/Ntsr2 receptor subtype antagonist), SR48692 (a Ntsr1 antagonist), D-Tyr-NT + SR142498, D-Tyr-NT + SR48692, or the vehicle. Effects of intra-VTA drugs were evaluated at locomotor activity and ERK1/2 phosphorylation. Five days after the last VTA microinjection, the effect of a systemic injection of amphetamine was tested (sensitization test). Results show that D-Tyr-NT stimulated locomotor activity during the induction phase, an effect that was blocked by SR142948, but not SR48692. Amphetamine also induced significantly higher ambulatory activity in rats preinjected with D-Tyr-NT than in rats preinjected with the vehicle. This sensitization effect was again attenuated by SR142948, but not SR48692, hence suggesting that this effect is mediated by Ntsr2 receptors. To confirm this, we tested a highly selective Ntsr2 peptide-peptoid hybrid ligand, NT150. At the concentration tested, NT150 stimulated locomotor activity and lead to sensitized locomotor activity and a selective neurochemical (pERK1/2) response in tyrosine hydroxylase-positive neurons of the VTA. Both effects were prevented by SR142948. Taken together, these results show that neurotensin, acting on Ntsr2 receptor subtypes, stimulates locomotor activity and initiates neural changes (ERK1/2 phosphorylation) that lead to amphetamine-induced sensitization.

Nonlinear pharmacodynamics of levodopa through Parkinson's disease progression.

The effect of levodopa in alleviating the symptoms of Parkinson's disease is altered in a highly nonlinear manner as the disease progresses. This can be attributed to different compensation mechanisms taking place in the basal ganglia where the dopaminergic neurons are progressively lost. This alteration in the effect of levodopa complicates the optimization of a drug regimen. The present work aims at investigating the nonlinear dynamics of Parkinson's disease and its therapy through mechanistic mathematical modeling. Using a holistic approach, a pharmacokinetic model of levodopa was combined to a dopamine dynamics and a neurocomputational model of basal ganglia. The influence of neuronal death on these different mechanisms was also integrated. Using this model, we were able to investigate the nonlinear relationships between the levodopa plasma concentration, the dopamine brain concentration, and a response to a motor task. Variations in dopamine concentrations in the brain for different levodopa doses were also studied. Finally, we investigated the narrowing of a levodopa therapeutic index with the progression of the disease as a result of these nonlinearities. In conclusion, various consequences of nonlinear dynamics in Parkinson's disease treatment were studied by developing an integrative model. This model paves the way toward individualization of a dosing regimen. Using sensor based information, the parameters of the model could be fitted to individual data to propose optimal individual regimens.

The Stress-Induced Transcription Factor NR4A1 Adjusts Mitochondrial Function and Synapse Number in Prefrontal Cortex.

The energetic costs of behavioral chronic stress are unlikely to be sustainable without neuronal plasticity. Mitochondria have the capacity to handle synaptic activity up to a limit before energetic depletion occurs. Protective mechanisms driven by the induction of neuronal genes likely evolved to buffer the consequences of chronic stress on excitatory neurons in prefrontal cortex (PFC), as this circuitry is vulnerable to excitotoxic insults. Little is known about the genes involved in mitochondrial adaptation to the buildup of chronic stress. Using combinations of genetic manipulations and stress for analyzing structural, transcriptional, mitochondrial, and behavioral outcomes, we characterized NR4A1 as a stress-inducible modifier of mitochondrial energetic competence and dendritic spine number in PFC. NR4A1 acted as a transcription factor for changing the expression of target genes previously involved in mitochondrial uncoupling, AMP-activated protein kinase activation, and synaptic growth. Maintenance of NR4A1 activity by chronic stress played a critical role in the regressive synaptic organization in PFC of mouse models of stress (male only). Knockdown, dominant-negative approach, and knockout of Nr4a1 in mice and rats (male only) protected pyramidal neurons against the adverse effects of chronic stress. In human PFC tissues of men and women, high levels of the transcriptionally active NR4A1 correlated with measures of synaptic loss and cognitive impairment. In the context of chronic stress, prolonged expression and activity of NR4A1 may lead to responses of mitochondria and synaptic connectivity that do not match environmental demand, resulting in circuit malfunction between PFC and other brain regions, constituting a pathological feature across disorders.SIGNIFICANCE STATEMENT The bioenergetic cost of chronic stress is too high to be sustainable by pyramidal prefrontal neurons. Cellular checkpoints have evolved to adjust the responses of mitochondria and synapses to the buildup of chronic stress. NR4A1 plays such a role by controlling the energetic competence of mitochondria with respect to synapse number. As an immediate-early gene, Nr4a1 promotes neuronal plasticity, but sustained expression or activity can be detrimental. NR4A1 expression and activity is sustained by chronic stress in animal models and in human studies of neuropathologies sensitive to the buildup of chronic stress. Therefore, antagonism of NR4A1 is a promising avenue for preventing the regressive synaptic reorganization in cortical systems in the context of chronic stress.

Comparative analysis of Parkinson's disease-associated genes in mice reveals altered survival and bioenergetics of Parkin-deficient dopamine neurons.

Many mutations in genes encoding proteins such as Parkin, PTEN-induced putative kinase 1 (PINK1), protein deglycase DJ-1 (DJ-1 or PARK7), leucine-rich repeat kinase 2 (LRRK2), and α-synuclein have been linked to familial forms of Parkinson's disease (PD). The consequences of these mutations, such as altered mitochondrial function and pathological protein aggregation, are starting to be better understood. However, little is known about the mechanisms explaining why alterations in such diverse cellular processes lead to the selective loss of dopamine (DA) neurons in the substantia nigra (SNc) in the brain of individuals with PD. Recent work has shown that one of the reasons for the high vulnerability of SNc DA neurons is their high basal rate of mitochondrial oxidative phosphorylation (OXPHOS), resulting from their highly complex axonal arborization. Here, we examined whether axonal growth and basal mitochondrial function are altered in SNc DA neurons from Parkin-, Pink1-, or DJ-1-KO mice. We provide evidence for increased basal OXPHOS in Parkin-KO DA neurons and for reduced survival of DA neurons that have a complex axonal arbor. The surviving smaller neurons exhibited reduced vulnerability to the DA neurotoxin and mitochondrial complex I inhibitor MPP+, and this reduction was associated with reduced expression of the DA transporter. Finally, we found that glial cells play a role in the reduced resilience of DA neurons in these mice and that WT Parkin overexpression rescues this phenotype. Our results provide critical insights into the complex relationship between mitochondrial function, axonal growth, and genetic risk factors for PD.

Tardive dyskinesia is associated with altered putamen Akt/GSK-3ß signaling in nonhuman primates.

BACKGROUND: Tardive dyskinesia is a delayed and potentially irreversible motor complication arising from chronic exposure to antipsychotic drugs. Interaction of antipsychotic drugs with G protein-coupled receptors triggers multiple intracellular events. Nevertheless, signaling pathways that might be associated with chronic unwanted effects of antipsychotic drugs remain elusive. In this study, we aimed to better understand kinase signaling associated with the expression of tardive dyskinesia in nonhuman primates. METHODS: We exposed capuchin monkeys to prolonged haloperidol (n = 10) or clozapine (n = 6) treatments. Untreated animals were used as controls (n = 6). Half of haloperidol-treated animals (5) developed mild tardive dyskinesia similar to that found in humans. Using Western blots and immunochemistry, we measured putamen total and phosphorylated protein kinase levels associated with canonical and noncanonical signaling cascades of G protein-coupled receptors. RESULTS: Antipsychotic drugs enhanced pDARPP-32 and pERK1/2, but no difference ws observed in phosphoprotein kinase levels between dyskinetic and nondyskinetic monkeys. On the other hand, comparison of kinase levels between haloperidol-treated dyskinetic and nondyskinetic monkeys indicated that dyskinetic animals had lower GRK6 and ß-arrestin2 levels. Levels of pAkt and pGSK-3ß were also reduced, but only haloperidol-treated monkeys that developed tardive dyskinesia had reduced pGSK-3ß levels, whereas pAkt levels in dyskinetic animals positively correlated with dyskinetic scores. Interestingly, double immunofluorescence labeling showed that putamen dopamine D3 receptor levels were upregulated and that D3/pAkt colocalization was enriched in haloperidol-treated animals displaying tardive dyskinesia. CONCLUSIONS: Our results suggest that upregulation of putamen dopamine D3 receptor and alterations along the noncanonical GRK6/ß-arrestin2/Akt/GSK-3ß molecular cascade are associated with the development of tardive dyskinesia in nonhuman primates. © 2019 International Parkinson and Movement Disorder Society.

Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion.

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.

Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors.

Retinoid X receptors (RXRs) play a role as master regulators because of their capacity to form heterodimers with other nuclear receptors (NRs). Accordingly, retinoid signaling is involved in multiple biologic processes, including development, cell differentiation, metabolism, and cell death. However, the role and function of RXRs in different heterodimer complexes remain unidentified, mainly because most RXR drugs (called rexinoids) are not selective of specific heterodimer complexes. The lack of selectivity strongly limits the use of rexinoids for specific therapeutic approaches. To better characterize rexinoids at specific NR complexes, we have developed and optimized luciferase (Luc) protein complementation(PCA)-based bioluminescence resonance energy transfer (BRET) assays that can directly measure recruitment of a coactivator (CoA) motif fused to yellow fluorescent protein (YFP) by specific NR dimers. To validate the assays, we compared rexinoid modulation of CoA recruitment by the RXR homodimer and by the heterodimers Nur77/RXR and Nurr1/RXR. Results revealed that some rexinoids display selective CoA recruitment activities with homo- or heterodimer complexes. In particular, SR11237 (BMS649) has stronger potency for recruitment of CoA motif and transcriptional activity with the heterodimer Nur77/RXR than other complexes. This technology should be useful in identifying new compounds with specificity for individual dimeric species formed by NRs.

Upregulation of dopamine D3, not D2, receptors correlates with tardive dyskinesia in a primate model.

Tardive dyskinesia (TD) is a delayed and potentially irreversible motor complication arising in patients chronically exposed to centrally active dopamine D2 receptor antagonists, including antipsychotic drugs and metoclopramide. The classical dopamine D2 receptor supersensitivity hypothesis in TD, which stemmed from rodent studies, lacks strong support in humans. To investigate the neurochemical basis of TD, we chronically exposed adult capuchin monkeys to haloperidol (median, 18.5 months; n = 11) or clozapine (median, 6 months; n = 6). Six unmedicated animals were used as controls. Five haloperidol-treated animals developed mild TD movements, and no TD was observed in the clozapine group. Using receptor autoradiography, we measured striatal dopamine D1, D2, and D3 receptor levels. We also examined the D3 receptor/preprotachykinin messenger RNA (mRNA) co-expression, and quantified preproenkephalin mRNA levels, in striatal sections. Unlike clozapine, haloperidol strongly induced dopamine D3 receptor binding sites in the anterior caudate-putamen, particularly in TD animals, and binding levels positively correlated with TD intensity. Interestingly, the D3 receptor upregulation was observed in striatonigral neurons. In contrast, D2 receptor binding was comparable to controls, and dopamine D1 receptor binding was reduced in the anterior putamen. Enkephalin mRNA widely increased in all animals, but to a greater extent in TD-free animals. These results suggest for the first time that upregulated striatal D3 receptors correlate with TD in nonhuman primates, adding new insights to the dopamine receptor supersensitivity hypothesis. The D3 receptor could provide a novel target for drug intervention in human TD.

Haloperidol-induced striatal Nur77 expression in a non-human primate model of tardive dyskinesia.

Tardive dyskinesia (TD) is a delayed and potentially irreversible motor complication arising in patients chronically exposed to antipsychotic drugs. As several modern (so-called atypical) antipsychotic drugs are common offenders, combined with the widening clinical indications for prescription as well as exposure of vulnerable individuals, TD will remain a significant drug-induced unwanted side effect. In addition, the pathophysiology of TD remains elusive and therapeutics are difficult. Based on rodent experiments, we have previously shown that the transcriptional factor Nur77 (also known as nerve growth factor inducible gene B or Nr4a1) is induced in the striatum following antipsychotic drug exposure as part of a long-term neuroadaptive process. To confirm this, we exposed adult capuchin (Cebus apella) monkeys to prolonged treatments with haloperidol (median 18.5 months, N = 11) or clozapine (median 6 months, N = 6). Six untreated animals were used as controls. Five haloperidol-treated animals developed mild TD movements similar to those found in humans. No TD was observed in the clozapine group. Postmortem analysis of Nur77 expression measured by in situ hybridization revealed a stark contrast between the two drugs, as Nur77 mRNA levels in the caudate-putamen were strongly upregulated in animals exposed to haloperidol but were spared following clozapine treatment. Interestingly, within the haloperidol-treated group, TD-free animals showed higher Nur77 expression in putamen subterritories compared with dyskinetic animals. This suggests that Nur77 expression might be associated with a reduced risk of TD in this experimental model and could provide a novel target for drug intervention.