Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis.

Effects of the selective adenosine A2A receptor antagonist, SCH 412348, on the parkinsonian phenotype of MitoPark mice.

Adenosine A2A receptors are predominantly localized on striatopallidal gamma-aminobutyric acid (GABA) neurons, where they are colocalized with dopamine D2 receptors and are involved in the regulation of movement. Adenosine A2A receptor antagonists have been evaluated as a novel treatment for Parkinson's disease and have demonstrated efficacy in a broad spectrum of pharmacological and toxicological rodent and primate models. Fewer studies have been performed to evaluate the efficacy of adenosine A2A receptor antagonists in genetic models of hypodopaminergic states. SCH 412348 is a potent and selective adenosine A2A receptor antagonist that shows efficacy in rodent and primate models of movement disorders. Here we evaluated the effects of SCH 412348 in the MitoPark mouse, a genetic model that displays a progressive loss of dopamine neurons. The dopamine cell loss is associated with a profound akinetic phenotype that is sensitive to levodopa (l-dopa). SCH 412348 (0.3-10mg/kg administered orally) dose dependently increased locomotor activity in the mice. Moreover, SCH 412348 retained its efficacy in the mice as motor impairment progressed (12-22 weeks of age), demonstrating that the compound was efficacious in mild to severe Parkinson's disease-like impairment in the mice. Additionally, SCH 412348 fully restored lost functionality in a measure of hind limb bradykinesia and partially restored functionality in a rotarod test. These findings provide further evidence of the anti-Parkinsonian effects of selective adenosine A2A receptor antagonists and predict that they will retain their efficacy in both mild and severe forms of motor impairment.

Dual orexin receptor antagonists show distinct effects on locomotor performance, ethanol interaction and sleep architecture relative to gamma-aminobutyric acid-A receptor modulators.

Dual orexin receptor antagonists (DORAs) are a potential treatment for insomnia that function by blocking both the orexin 1 and orexin 2 receptors. The objective of the current study was to further confirm the impact of therapeutic mechanisms targeting insomnia on locomotor coordination and ethanol interaction using DORAs and gamma-aminobutyric acid (GABA)-A receptor modulators of distinct chemical structure and pharmacological properties in the context of sleep-promoting potential. The current study compared rat motor co-ordination after administration of DORAs, DORA-12 and almorexant, and GABA-A receptor modulators, zolpidem, eszopiclone, and diazepam, alone or each in combination with ethanol. Motor performance was assessed by measuring time spent walking on a rotarod apparatus. Zolpidem, eszopiclone and diazepam [0.3-30 mg/kg administered orally (PO)] impaired rotarod performance in a dose-dependent manner. Furthermore, all three GABA-A receptor modulators potentiated ethanol- (0.25-1.5 g/kg) induced impairment on the rotarod. By contrast, neither DORA-12 (10-100 mg/kg, PO) nor almorexant (30-300 mg/kg, PO) impaired motor performance alone or in combination with ethanol. In addition, distinct differences in sleep architecture were observed between ethanol, GABA-A receptor modulators (zolpidem, eszopiclone, and diazepam) and DORA-12 in electroencephalogram studies in rats. These findings provide further evidence that orexin receptor antagonists have an improved motor side-effect profile compared with currently available sleep-promoting agents based on preclinical data and strengthen the rationale for further evaluation of these agents in clinical development.

A potent and selective metabotropic glutamate receptor 4 positive allosteric modulator improves movement in rodent models of Parkinson's disease.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGluR4) have been proposed as a novel therapeutic approach for the treatment of Parkinson's disease. However, evaluation of this proposal has been limited by the availability of appropriate pharmacological tools to interrogate the target. In this study, we describe the properties of a novel mGluR4 PAM. 5-Methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine (ADX88178) enhances glutamate-mediated activation of human and rat mGluR4 with EC(50) values of 4 and 9 nM, respectively. The compound is highly selective for mGluR4 with minimal activities at other mGluRs. Oral administration of ADX88178 in rats is associated with high bioavailability and results in cerebrospinal fluid exposure of >50-fold the in vitro EC(50) value. ADX88178 reverses haloperidol-induced catalepsy in rats at 3 and 10 mg/kg. It is noteworthy that this compound alone has no impact on forelimb akinesia resulting from a bilateral 6-hydroxydopamine lesion in rats. However, coadministration of a low dose of L-DOPA (6 mg/kg) enabled a robust, dose-dependent reversal of the forelimb akinesia deficit. ADX88178 also increased the effects of quinpirole in lesioned rats and enhanced the effects of L-DOPA in MitoPark mice. It is noteworthy that the enhancement of the actions of L-DOPA was not associated with an exacerbation of L-DOPA-induced dyskinesias in rats. ADX88178 is a novel, potent, and selective mGluR4 PAM that is a valuable tool for exploring the therapeutic potential of mGluR4 modulation. The use of this novel tool molecule supports the proposal that activation of mGluR4 may be therapeutically useful in Parkinson's disease.

Safety and tolerability of high-dosage coenzyme Q10 in Huntington's disease and healthy subjects.

Coenzyme Q10 (CoQ(10)), a potential neuroprotective compound, was previously investigated at a dosage of 600 mg/day in Huntington's disease (HD) patients and demonstrated a trend toward slowing disease progression. Higher CoQ(10) dosages may prove beneficial. We investigated the tolerability and blood levels associated with 1,200, 2,400, and 3,600 mg/day of CoQ(10) in HD and healthy subjects. Twenty-eight subjects (20 HD, 8 healthy) enrolled in a 20-week open-label trial. Subjects started on 1,200 mg/day of CoQ(10), increasing every 4 weeks by 1,200 mg to a maximum dosage of 3,600 mg/day. Monthly evaluations included review of adverse events and CoQ(10) blood levels. Twenty-three subjects (82%) achieved the target dosage of 3,600 mg/day. Six subjects (2 healthy, 4 HD) withdrew prematurely (gastrointestinal (GI) symptoms in 3, worsening HD in 2, and 1 because of a fall). All three serious adverse events occurred in a single subject, and were deemed unrelated to CoQ(10). The most common adverse events seen were GI symptoms. Mean (± SD) CoQ10 blood levels achieved over the course of the trial were as follows: 1.26 ± 1.27 µg/mL (baseline, n = 28), 5.59 ± 2.24 µg/mL (1,200 mg/day, week 4, n = 26), 6.38 ± 3.25 µg/mL (2,400 mg/day, week 8, n = 25), 7.49 ± 4.09 µg/mL (3,600 mg/day, week 12, n = 23), and 6.78 ± 3.36 µg/mL (3,600 mg/day, week 20, n = 20). CoQ(10) was well tolerated with over 80% of subjects achieving the target dosage. Dosages of 2,400 mg/day may provide the best balance between tolerability and blood level achieved. Further studies examining the efficacy of 2,400 mg/day are planned.

PISA, a novel pharmacodynamic assay for assessing poly(ADP-ribose) polymerase (PARP) activity in situ.

INTRODUCTION: Poly ADP-ribose polymerase (PARP) maintains genomic integrity by repairing DNA strand breaks, however over-activation of PARP following neural tissue injury is hypothesized to cause neuronal death. Therefore, PARP inhibitors have potential for limiting neural injury under certain conditions. A reliable method for assessing PARP activity in brain is critical for development of novel inhibitors with CNS activity. We developed the PARP In Situ Activity (PISA) assay to provide a direct, quantitative assessment of CNS PARP activity in vitro or in vivo. METHODS: The assay utilized brain sections from rats with striatal kainic acid (KA) lesions and 3H- or biotinylated NAD+ as the substrate to assess PARP activity. Following optimization of the assay, it was used to assess in vitro and in vivo efficacy of known and novel PARP inhibitors. The assay also was used to assess PARP activity in male and female gonad-intact and ovariectomized rats. RESULTS: Using 3H-NAD+ as the substrate, PARP activity was greater (p<0.01) in tissue from KA-lesioned vs. non-lesioned rats. Using biotinylated NAD+ it was revealed that PARP activity was present ipsilateral to the KA injection site, and labeling was blocked by incubation with excess unlabeled NAD+ or PARP inhibitors. The PARP inhibitor, 3-aminobenzamide and several novel inhibitors reduced (p<0.01) polymerase activity in vitro. Furthermore, the inhibitor MRLSD303 reduced (p<0.001) PARP activity in vivo in both male and female rats. Finally, administration of the novel PARP inhibitor MRLIT115 dose-dependently reduced (p<0.001) polymerase activity in vivo. DISCUSSION: The PISA assay provides a direct, quantitative method for assessing PARP activity in vitro and provides critical information on factors underlying in vivo efficacy of chemical inhibitors including brain penetration and target engagement. These findings support use of the PISA assay as a screening tool for testing efficacy of PARP inhibitors in brain.

Mitochondria and Huntington's disease pathogenesis: insight from genetic and chemical models.

A mechanistic link between cellular energetic defects and the pathogenesis of Huntington's disease (HD) has long been hypothesized based on the cardinal observations of progressive weight loss in patients and metabolic defects in brain and muscle. Identification of respiratory chain deficits in HD postmortem brain led to the use of mitochondrial complex II inhibitors to generate acute toxicity models that replicate aspects of HD striatal pathology in vivo. Subsequently, the generation of progressive genetic animal models has enabled characterization of numerous cellular and systematic changes over disease etiology, including mitochondrial modifications that impact cerebral metabolism, calcium handling, oxidative damage, and apoptotic cascades. This review focuses on how HD animal models have influenced our understanding of mechanisms underlying HD pathogenesis, concentrating on insight gained into the roles of mitochondria in disease etiology. One outstanding question concerns the hierarchy of mitochondrial alterations in the cascade of events following mutant huntingtin (mhtt)-induced toxicity. One hypothesis is that a direct interaction of mhtt with mitochondria may trigger the neuronal damage and degeneration that occurs in HD. While there is evidence that mhtt associates with mitochondria, deleterious consequences of this interaction have not yet been established. Contrary evidence suggests that a primary nuclear action of mhtt may detrimentally influence mitochondrial function via effects on gene transcription. Irrespective of whether the principal toxic action of mhtt directly or secondarily impacts mitochondria, the repercussions of sufficient mitochondrial dysfunction are catastrophic to cells and may arguably underlie many of the other disruptions in cellular processes that evolve during HD pathogenesis.

A pivotal role of matrix metalloproteinase-3 activity in dopaminergic neuronal degeneration via microglial activation.

Recent studies have demonstrated that activated microglia play an important role in dopamine (DA) neuronal degeneration in Parkinson disease (PD) by generating NADPH-oxidase (NADPHO)-derived superoxide. However, the molecular mechanisms that underlie microglial activation in DA cell death are still disputed. We report here that matrix metalloproteinase-3 (MMP-3) was newly induced and activated in stressed DA cells, and the active form of MMP-3 (actMMP-3) was released into the medium. The released actMMP-3, as well as catalytically active recombinant MMP-3 (cMMP-3) led to microglial activation and superoxide generation in microglia and enhanced DA cell death. cMMP-3 caused DA cell death in mesencephalic neuron-glia mixed culture of wild-type (WT) mice, but this was attenuated in the culture of NADPHO subunit null mice (gp91(phox-/-)), suggesting that NADPHO mediated the cMMP-3-induced microglial production of superoxide and DA cell death. Furthermore, in the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected animal model of PD, nigrostriatal DA neuronal degeneration, microglial activation, and superoxide generation were largely attenuated in MMP-3-/- mice. These results indicate that actMMP-3 released from stressed DA neurons is responsible for microglial activation and generation of NADPHO-derived superoxide and eventually enhances nigrostriatal DA neuronal degeneration. Our results could lead to a novel therapeutic approach to PD.

Oral uridine pro-drug PN401 is neuroprotective in the R6/2 and N171-82Q mouse models of Huntington's disease.

Previously, uridine pro-drug 2',3',5'-tri-O-acetyluridine (PN401) was shown to be protective in the mitochondrial complex II inhibitor 3-nitropropionic acid model of Huntington's disease (HD). In this study, PN401 increased survival and improved motor function on the rotarod in both R6/2 and N171-82Q polyglutamine repeat mouse models of HD. PN401 significantly decreased neurodegeneration in both the piriform cortex and striatum although PN401 decreased huntingtin protein aggregates only in the striatum. Cortical and striatal brain-derived neurotrophic factor (BDNF) protein levels were reduced in the +/- compared to the -/- N171-82Q mice and PN401 treatment significantly increased cortical BDNF in both +/- and -/- mice, but PN401 did not affect striatal BDNF. These results suggest that PN401 may have beneficial effects in the treatment of neurodegenerative diseases such as HD.

Oxidative damage in Huntington's disease pathogenesis.

Huntington's disease (HD) is a devastating neurodegenerative disorder characterized by the progressive development of involuntary choreiform movements, cognitive impairment, neuropsychiatric symptoms, and premature death. These phenotypes reflect neuronal dysfunction and ultimately death in selected brain regions, the striatum and cerebral cortex being principal targets. The genetic mutation responsible for the HD phenotype is known, and its protein product, mutant huntingtin (mhtt), identified. HD is one of several "triplet repeat" diseases, in which abnormal expansions in trinucleotide repeat domains lead to elongated polyglutamine stretches in the affected gene's protein product. Mutant htt-mediated toxicity in the brain disrupts a number of vital cellular processes in the course of disease progression, including energy metabolism, gene transcription, clathrin-dependent endocytosis, intraneuronal trafficking, and postsynaptic signaling, but the crucial initiation mechanism induced by mhtt is still unclear. A large body of evidence, however, supports an early and critical involvement of defects in mitochondrial function and CNS energy metabolism in the disease trigger. Thus, downstream death-effector mechanisms, including excitotoxicity, apoptosis, and oxidative damage, have been implicated in the mechanism of selective neuronal damage in HD. Here we review the current evidence supporting a role for oxidative damage in the etiology of neuronal damage and degeneration in HD.

Bioenergetic abnormalities in discrete cerebral motor pathways presage spinal cord pathology in the G93A SOD1 mouse model of ALS.

Multiple cell death pathways are implicated in the etiology of amyotrophic lateral sclerosis (ALS), but the cause of the characteristic motor neuron degeneration remains unknown. To determine whether CNS metabolic defects are critical for ALS pathogenesis, we examined the temporal evolution of energetic defects in the G93A SOD1 mouse model of familial ALS. [14C]-2-deoxyglucose in vivo autoradiography in G93A mice showed that glucose utilization is impaired in components of the corticospinal and bulbospinal motor tracts prior to either pathologic or bioenergetic changes in the spinal cord. This was accompanied by significant depletions in cortical ATP content in presymptomatic mice, which was partially ameliorated by creatine administration. Findings suggest that bioenergetic defects are involved in the initial stages of mSOD1-induced toxicity in G93A mice and imply that the selective dysfunction and degeneration of spinal cord motor neurons in this model may be secondary to dysfunction within cerebral motor pathways.

Mitochondrial aconitase is a transglutaminase 2 substrate: transglutamination is a probable mechanism contributing to high-molecular-weight aggregates of aconitase and loss of aconitase activity in Huntington disease brain.

Transglutaminase activity was found to be present in highly purified non-synaptosomal rat brain mitochondria. A 78-kDa protein in these organelles was shown to be a transglutaminase 2 substrate, and incubation of a non-synaptosomal mitochondrial lysate with transglutaminase 2 yielded high-Mr proteins. The 78-kDa protein was identified as mitochondrial aconitase by MALDI-TOF analysis. Aconitase activity was decreased in a dose-dependent manner when non-synaptosomal rat brain mitochondria were incubated with transglutaminase 2. Transglutaminase activity is increased about 2-fold in the mitochondrial fraction of HD caudate. Moreover, Western blotting of the mitochondrial fraction revealed that most of the mitochondrial aconitase in HD caudate is present as high-Mr aggregates. Aconitase activity was previously shown to be decreased in Huntington disease (HD) caudate (a region severely damaged by the disease). The present findings suggest that an increase of transglutaminase activity in HD caudate may contribute to mitochondrial dysfunction by incorporating aconitase into inactive polymers.

Neuroprotective effects of phenylbutyrate in the N171-82Q transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is caused by an expansion of exonic CAG triplet repeats in the gene encoding the huntingtin protein (Htt), however, the means by which neurodegeneration occurs remains obscure. There is evidence that mutant Htt interacts with transcription factors leading to reduced histone acetylation. We report that administration of the histone deacetylase inhibitor phenylbutyrate after onset of symptoms in a transgenic mouse model of HD significantly extends survival and attenuates both gross brain and neuronal atrophy. Administration of phenylbutyrate increased brain histone acetylation and decreased histone methylation levels as assessed by both immunocytochemistry and Western blots. Phenylbutyrate increased mRNA for components of the ubiquitin-proteosomal pathway and down-regulated caspases implicated in apoptotic cell death, and active caspase 3 immunoreactivity in the striatum. These results show that administration of phenylbutyrate, at doses that are well tolerated in man, exerts significant neuroprotective effects in a transgenic mouse model of HD, and therefore represents a very promising therapeutic approach for HD.

Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species.

Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

The energetics of Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder that gradually robs sufferers of the ability to control movements and induces psychological and cognitive impairments. This devastating, lethal disease is one of several neurological disorders caused by trinucleotide expansions in affected genes, including spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy, and spinal bulbar muscular atrophy. HD symptoms are associated with region-specific neuronal loss within the central nervous system, but to date the mechanism of this selective cell death remains unknown. Strong evidence from studies in humans and animal models suggests the involvement of energy metabolism defects, which may contribute to excitotoxic processes, oxidative dmage, and altered gene regulation. The development of transgenic mouse models expressing the human HD mutation has provided novel opportunities to explore events underlying selective neuronal death in HD, which has hitherto been impossible in humans. Here we discuss how animal models are redefining the role of energy metabolism in HD etiology.

Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.

Ataxia-telangiectasia is caused by mutations in the ATM gene, the protein product of which is essential for effective response to double-stranded DNA breaks. Loss of ATM function explains most aspects of the disease, but not the cerebellar neurodegeneration characteristic of the disease. Mice lacking ATM provide an excellent model of the human disorder. In addition to deficient response to DNA damage, these mice exhibit oxidative stress, which we hypothesized is the cause of cerebellar dysfunction. We show that treatment with a catalytic antioxidant corrects the neurobehavioral deficit in these mice.

Mice deficient in dihydrolipoamide dehydrogenase show increased vulnerability to MPTP, malonate and 3-nitropropionic acid neurotoxicity.

Altered energy metabolism, including reductions in activities of the key mitochondrial enzymes alpha-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC), are characteristic of many neurodegenerative disorders including Alzheimer's Disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Dihydrolipoamide dehydrogenase is a critical subunit of KGDHC and PDHC. We tested whether mice that are deficient in dihydrolipoamide dehydrogenase (Dld+/-) show increased vulnerability to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), malonate and 3-nitropropionic acid (3-NP), which have been proposed for use in models of PD and HD. Administration of MPTP resulted in significantly greater depletion of tyrosine hydroxylase-positive neurons in the substantia nigra of Dld+/- mice than that seen in wild-type littermate controls. Striatal lesion volumes produced by malonate and 3-NP were significantly increased in Dld+/- mice. Studies of isolated brain mitochondria treated with 3-NP showed that both succinate-supported respiration and membrane potential were suppressed to a greater extent in Dld+/- mice. KGDHC activity was also found to be reduced in putamen from patients with HD. These findings provide further evidence that mitochondrial defects may contribute to the pathogenesis of neurodegenerative diseases.

CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation.

Molecular chaperones, ubiquitin ligases and proteasome impairment have been implicated in several neurodegenerative diseases, including Alzheimer's and Parkinson's disease, which are characterized by accumulation of abnormal protein aggregates (e.g. tau and alpha-synuclein respectively). Here we report that CHIP, an ubiquitin ligase that interacts directly with Hsp70/90, induces ubiquitination of the microtubule associated protein, tau. CHIP also increases tau aggregation. Consistent with this observation, diverse of tau lesions in human postmortem tissue were found to be immunopositive for CHIP. Conversely, induction of Hsp70 through treatment with either geldanamycin or heat shock factor 1 leads to a decrease in tau steady-state levels and a selective reduction in detergent insoluble tau. Furthermore, 30-month-old mice overexpressing inducible Hsp70 show a significant reduction in tau levels. Together these data demonstrate that the Hsp70/CHIP chaperone system plays an important role in the regulation of tau turnover and the selective elimination of abnormal tau species. Hsp70/CHIP may therefore play an important role in the pathogenesis of tauopathies and also represents a potential therapeutic target.