Use of catechol‐O‐methyltransferase inhibition to minimize L‐3,4‐dihydroxyphenylalanine‐induced dyskinesia in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐lesioned macaque

L‐3,4‐dihydroxyphenylalanine (L‐DOPA)‐induced dyskinesia is a complication of dopaminergic treatment in Parkinson's disease. Lowering the L‐DOPA dose reduces dyskinesia but also reduces the antiparkinsonian benefit. A therapy that could enhance the antiparkinsonian action of low‐dose L‐DOPA (LDl) without exacerbating dyskinesia would thus be of considerable therapeutic benefit. This study assessed whether catechol‐O‐methyltransferase (COMT) inhibition, as an add‐on to LDl, might be a means to achieve this goal. Cynomolgus macaques were administered 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. Dyskinesia was established by chronic treatment with L‐DOPA. Two doses of L‐DOPA were identified – high‐dose L‐DOPA (LDh), which provided good antiparkinsonian benefit but was compromised by disabling dyskinesia, and LDl, which was sub‐threshold for providing significant antiparkinsonian benefit, without dyskinesia. LDh and LDl were administered in acute challenges in combination with vehicle and, for LDl, with the COMT inhibitor entacapone (5, 15 and 45 mg/kg). The duration of antiparkinsonian benefit (ON‐time), parkinsonism and dyskinesia were determined. The ON‐time after LDh was ～170 min and the ON‐time after LDl alone (～98 min) was not significantly different to vehicle (～37 min). In combination with LDl, entacapone significantly increased the ON‐time (5, 15 and 45 mg/kg being ～123, ～148 and ～180 min, respectively). The ON‐time after LDl/entacapone 45 mg/kg was not different to that after LDh. However, whereas the percentage ON‐time that was compromised by disabling dyskinesia was ～56% with LDh, it was only ～31% with LDl/entacapone 45 mg/kg. In addition to the well‐recognized action of COMT inhibition to reduce wearing‐OFF, the data presented suggest that COMT inhibition in combination with low doses of L‐DOPA has potential as a strategy to alleviate dyskinesia.     

Exercise modifies α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor expression in striatopallidal neurons in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐lesioned mouse

The α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic‐acid‐type glutamate receptor (AMPAR) plays a critical role in modulating experience‐dependent neuroplasticity, and alterations in AMPAR expression may underlie synaptic dysfunction and disease pathophysiology. Using the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of dopamine (DA) depletion, our previous work showed exercise increases total GluA2 subunit expression and the contribution of GluA2‐containing channels in MPTP mice. The purpose of this study was to determine whether exercise‐dependent changes in AMPAR expression after MPTP are specific to the striatopallidal (D₂R) or striatonigral (D₁R) medium spiny neuron (MSN) striatal projection pathways. Drd₂‐eGFP‐BAC transgenic mice were used to delineate differences in AMPAR expression between striatal D₂R‐MSNs and D₁R‐MSNs. Striatal AMPAR expression was assessed by immunohistochemical (IHC) staining, Western immunoblotting (WB) of preparations enriched for postsynaptic density (PSD), and alterations in the current–voltage relationship of MSNs. We found DA depletion results in the emergence of GluA2‐lacking AMPARs selectively in striatopallidal D₂R‐MSNs and that exercise reverses this effect in MPTP mice. Exercise‐induced changes in AMPAR channels observed after DA depletion were associated with alterations in GluA1 and GluA2 subunit expression in postsynaptic protein, D₂R‐MSN cell surface expression, and restoration of corticostriatal plasticity. Mechanisms regulating experience‐dependent changes in AMPAR expression may provide innovative therapeutic targets to increase the efficacy of treatments for basal ganglia disorders, including Parkinson's disease. © 2013 Wiley Periodicals, Inc.     

Co‐expression of C‐terminal truncated alpha‐synuclein enhances full‐length alpha‐synuclein‐induced pathology

Lewy bodies, which are a pathological hallmark of Parkinson’s disease, contain insoluble polymers of alpha‐synuclein (αsyn). Among the different modifications that can promote the formation of toxic αsyn species, C‐terminal truncation is among the most abundant alterations in patients with Parkinson’s disease. In vitro, C‐terminal truncated αsyn aggregates faster and sub‐stoichiometric amounts of C‐terminal truncated αsyn promote aggregation of the full‐length αsyn (αsynFL) and induce neuronal toxicity. To address in vivo the putative stimulation of αsyn‐induced pathology by the presence of truncated αsyn, we used recombinant adeno‐associated virus to express either αsynFL or a C‐terminal truncated αsyn (1‐110) in rats. We adjusted the recombinant adeno‐associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of αsyn were co‐expressed at these pre‐determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C‐terminal truncated αsyn (1‐110) but only αsynFL, we demonstrated that the co‐expressed truncated protein promoted the progressive accumulation of αsynFL and formation of larger pathological accumulations. Moreover, in the co‐expression group, three of the eight animals showed apomorphine‐induced turning, suggesting prominent post‐synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C‐terminal truncated αsyn can interact with and exacerbate the formation of pathological accumulations containing αsynFL in vivo.     

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid receptor activation protects against phencyclidine‐induced caspase‐3 activity by activating voltage‐gated calcium channels

Phencyclidine (PCP) is a noncompetitive, open channel blocker of the N‐methyl‐D‐aspartate (NMDA) receptor–ion channel complex. When administered to immature animals, it is known to cause apoptotic neurodegeneration in several regions, and this is followed by olanzapine‐sensitive, schizophrenia‐like behaviors in late adolescence and adulthood. Clarification of its mechanism of action could yield data that would help to inform the treatment of schizophrenia. In our initial experiments, we found that α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid (AMPA) inhibited PCP‐induced apoptosis in organotypic neonatal rat brain slices in a concentration‐dependent and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione‐sensitive manner. Calcium signaling pathways are widely implicated in apoptosis, and PCP prevents calcium influx through NMDA receptor channels. We therefore hypothesized that AMPA could protect against this effect by activation of voltage‐dependent calcium channels (VDCCs). In support of this hypothesis, pretreatment with the calcium channel blocker cadmium chloride eliminated AMPA‐mediated protection against PCP. Furthermore, the L‐type VDCC inhibitor nifedipine (10 µM) fully abrogated the effects of AMPA, suggesting that L‐type VDCCs are required for AMPA‐mediated protection against PCP‐induced neurotoxicity. Whereas the P/Q‐type inhibitor ω‐agatoxin TK (200 nM) reduced AMPA protection by 51.7%, the N‐type VDCC inhibitor ω‐conotoxin (2 µM) had no effect. Decreased AMPA‐mediated protection following cotreatment with K252a, a TrkB inhibitor, suggests that brain‐derived neurotrophic factor signaling plays an important role. By analogy, these results suggest that activation of L‐type, and to a lesser extent P/Q‐type, VDCCs might be advantageous in treating conditions associated with diminished NMDAergic activity during early development. © 2014 Wiley Periodicals, Inc.     

Evaluation of N‐benzyl‐N‐[11C]methyl‐2‐ (7‐methyl‐8‐oxo‐2‐phenyl‐7,8‐dihydro‐9H‐purin‐9‐yl)acetamide ([11C]DAC) as a novel translocator protein (18 kDa) radioligand in kainic acid‐lesioned rat

The aim of this study was to evaluate N‐benzyl‐N‐[¹¹C]methyl‐2‐(7‐methyl‐8‐oxo‐2‐phenyl‐7,8‐dihydro‐9H‐purin‐9‐yl)acetamide ([¹¹C]DAC) as a new translocator protein (18 kDa) [TSPO, formerly known as the peripheral‐type benzodiazepine receptor (PBR)] positron emission tomography (PET) ligand in normal mice and unilateral kainic acid (KA)‐lesioned rats. DAC is a derivative of AC‐5216, which is a potent and selective PET ligand for the clinical investigation of TSPO. The binding affinity and selectivity of DAC for TSPO were similar to those of AC‐5216, and DAC was less lipophilic than AC‐5216. The distribution pattern of [¹¹C]DAC was in agreement with TSPO distribution in rodents. No radioactive metabolite of [¹¹C]DAC was found in the mouse brain, although it was metabolized rapidly in mouse plasma. Using small‐animal PET, we examined the in vivo binding of [¹¹C]DAC for TSPO in KA‐lesioned rats. [¹¹C]DAC and [¹¹C]AC‐5216 exhibited similar brain uptake in the lesioned and nonlesioned striatum, respectively. The binding of [¹¹C]DAC to TSPO was increased significantly in the lesioned striatum, and [¹¹C]DAC showed good contrast between the lesioned and nonlesioned striatum (the maximum ratio was about threefold). In displacement experiments, the uptake of [¹¹C]DAC in the lesioned striatum was eventually blocked using an excess of either unlabeled DAC or PK11195 injected. [¹¹C]DAC had high in vivo specific binding to TSPO in the injured rat brain. Therefore, [¹¹C]DAC is a useful PET ligand for TSPO imaging, and its specific binding to TSPO is suitable as a new biomarker for brain injury. Synapse 63:961–971, 2009. © 2009 Wiley‐Liss, Inc.     

Non‐classical mechanism of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor channel block by fluoxetine

Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca²⁺‐permeable and Ca²⁺‐impermeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca²⁺‐impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC₅₀ value for the inhibition of Ca²⁺‐permeable AMPA receptors in giant striatal interneurons was 43 ± 7 μm . The inhibition of Ca²⁺‐permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open‐channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane‐impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open‐channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.     

Mechanisms of status epilepticus: α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor hypothesis

Prolonged seizures of status epilepticus (SE) result from failure of mechanisms of seizure termination or activation of mechanisms that sustain seizures. Reduced γ‐aminobutyric acid type A receptor–mediated synaptic transmission contributes to impairment of seizure termination. However, mechanisms that sustain prolonged seizures are not known. We propose that insertion of GluA1 subunits at the glutamatergic synapses causes potentiation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic receptor (AMPAR)‐mediated neurotransmission, which helps to spread and sustain seizures. The AMPAR‐mediated neurotransmission of CA1 pyramidal neurons was increased in animals in SE induced by pilocarpine. The surface membrane expression of GluA1 subunit–containing AMPARs on CA1 pyramidal neurons was also increased. Blockade of N‐methyl‐d ‐aspartate receptors 10 minutes after the onset of continuous electrographic seizure activity prevented the increase in the surface expression of GluA1 subunits. N‐methyl‐d ‐aspartate receptor antagonist MK‐801 in conjunction with diazepam also terminated seizures that were refractory to MK‐801 or diazepam alone. Future studies using mice lacking the GluA1 subunit expression will provide further insights into the role of GluA1 subunit–containing AMPAR plasticity in sustaining seizures of SE.     

Evidence of oligodendrogliosis in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinsonism

V. Annese, C. Barcia, F. Ros‐Bernal, A. Gómez, C. M. Ros, V. De Pablos, E. Fernández‐Villalba, M.‐E. De Stefano and M. T. Herrero (2013) Neuropathology and Applied Neurobiology 39, 132–143 Evidence of oligodendrogliosis in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinsonism Aims: Mice and nonhuman primates administered with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) represent elective experimental models of Parkinsonism, in which degeneration of the nigrostriatal dopaminergic pathway is associated with prominent neuroinflammation, characterized by activated microglia and astrocytes in both substantia nigra (SN) and striatum. To date, it is unknown whether oligodendrocytes play a role in these events. Methods: We performed a detailed qualitative and quantitative analysis of oligodendrocyte‐associated changes induced by acute and chronic MPTP treatment, in the SN and striatum of mice and macaques respectively. Oligodendrocytes were immunolabelled by cell‐specific markers and analysed by confocal microscopy. Results: In both experimental models, MPTP treatment induces an increase in oligodendrocyte cell number and average size, as well as in the total area occupied by this cell type per tissue section, accompanied by evident morphological changes. This multifaceted array of changes, herein referred to as oligodendrogliosis, significantly correlates with the reduction in the level of dopaminergic innervation to the striatum. Conclusions: This event, associated with early damage of the dopaminergic neurone axons and of the complex striatal circuits of which they are part, may result in an important, although neglected, aspect in the onset and progression of Parkinsonism.     

Avoidance‐based human Pavlovian‐to‐instrumental transfer

The Pavlovian‐to‐instrumental transfer (PIT) paradigm probes the influence of Pavlovian cues over instrumentally learned behavior. The paradigm has been used extensively to probe basic cognitive and motivational processes in studies of animal learning. More recently, PIT and its underlying neural basis have been extended to investigations in humans. These initial neuroimaging studies of PIT have focused on the influence of appetitively conditioned stimuli on instrumental responses maintained by positive reinforcement, and highlight the involvement of the striatum. In the current study, we sought to understand the neural correlates of PIT in an aversive Pavlovian learning situation when instrumental responding was maintained through negative reinforcement. Participants exhibited specific PIT, wherein selective increases in instrumental responding to conditioned stimuli occurred when the stimulus signaled a specific aversive outcome whose omission negatively reinforced the instrumental response. Additionally, a general PIT effect was observed such that when a stimulus was associated with a different aversive outcome than was used to negatively reinforce instrumental behavior, the presence of that stimulus caused a non‐selective increase in overall instrumental responding. Both specific and general PIT behavioral effects correlated with increased activation in corticostriatal circuitry, particularly in the striatum, a region involved in cognitive and motivational processes. These results suggest that avoidance‐based PIT utilizes a similar neural mechanism to that seen with PIT in an appetitive context, which has implications for understanding mechanisms of drug‐seeking behavior during addiction and relapse.     

Bacillus Calmette‐Guerin vaccine‐mediated neuroprotection is associated with regulatory T‐cell induction in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine mouse model of Parkinson's disease

We previously showed that, in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), vaccination with bacillus Calmette‐Guerin (BCG) prior to MPTP exposure limited the loss of striatal dopamine (DA) and dopamine transporter (DAT) and prevented the activation of nigral microglia. Here, we conducted BCG dose studies and investigated the mechanisms underlying BCG vaccination's neuroprotective effects in this model. We found that a dose of 1 × 10⁶ cfu BCG led to higher levels of striatal DA and DAT ligand binding (28% and 42%, respectively) in BCG‐vaccinated vs. unvaccinated MPTP‐treated mice, but without a significant increase in substantia nigra tyrosine hydroxylase‐staining neurons. Previous studies showed that BCG can induce regulatory T cells (Tregs) and that Tregs are neuroprotective in models of neurodegenerative diseases. However, MPTP is lymphotoxic, so it was unclear whether Tregs were maintained after MPTP treatment and whether a relationship existed between Tregs and the preservation of striatal DA system integrity. We found that, 21 days post‐MPTP treatment, Treg levels in mice that had received BCG prior to MPTP were threefold greater than those in MPTP‐only‐treated mice and elevated above those in saline‐only‐treated mice, suggesting that the persistent BCG infection continually promoted Treg responses. Notably, the magnitude of the Treg response correlated positively with both striatal DA levels and DAT ligand binding. Therefore, BCG vaccine‐mediated neuroprotection is associated with Treg levels in this mouse model. Our results suggest that BCG‐induced Tregs could provide a new adjunctive therapeutic approach to ameliorating pathology associated with PD and other neurodegenerative diseases. © 2013 Wiley Periodicals, Inc.