Nonhuman primates: translational models for predicting antipsychotic-induced movement disorders.

Repeated haloperidol treatment administered to nonhuman primates (NHPs) over several months or even years leads to the gradual appearance of drug-induced dystonic reactions in the orofacial region (mouth opening, tongue protrusion or retraction, bar biting) and in the whole body (writhing of the limbs and trunk, bar grasping). The propensity of antipsychotics to induce dystonia in NHPs is not correlated with their propensity to induce catalepsy in rodents, suggesting that the two types of effects are dissociated and may represent distinct aspects of the extrapyramidal symptoms induced by antipsychotics. In view of the clear homology to clinically observed phenomena, antipsychotic-induced dystonias in antipsychotic-primed NHPs would appear to possess a high degree of translational validity. These NHP phenomena could therefore serve as a useful model for predicting the occurrence of similar abnormal movements with novel substances developed for the treatment of schizophrenia or other psychotic disorders. Moreover, the NHP dystonia model could possibly serve as a biomarker for substances that will eventually cause tardive dyskinesia in patients.

The age-related gliosis and accompanying deficit in spatial learning are unaffected by dimebon.

A non-selective antihistamine, dimebon, has recently emerged as a potential treatment for Alzheimer's disease and Huntington's disease. Dimebon exerts several effects in addition to its anti-histaminergic effect, and of particular interest is its ability to enhance cognitive function in several models. The mechanism underlying this is unknown though it has been suggested that it may be associated with its anti-cholinergic action. Dimebon has also been reported to be neuroprotective, perhaps as a result of its ability to stabilize mitochondria. We considered that these effects might impact on the well-described age-related impairment in spatial learning and therefore examined the effect of repeated administration of dimebon on performance of young and aged animals in the Morris water maze. Whereas a clear age-related deficit was observed, dimebon failed to exert any effect on performance. Similarly, dimebon exerted no effect on the age-related increase in hippocampal expression of several markers of microglial and astroglial activation. We conclude that, despite its cognitive enhancing effects in some models, dimebon failed to modulate the deficit in spatial learning in aged rats and the evidence suggests that the drug does not possess anti-inflammatory properties.

Dietary manipulation and caloric restriction in the development of mouse models relevant to neurological diseases.

Manipulation of diet such as increasing the level of fat or inducing insulin resistance has been shown to exacerbate the pathology in several animal models of neurological disease. Caloric restriction, however, has been demonstrated to extend the life span of many organisms. Reduced calorie consumption appears to increase the resistance of neurons to intracellular and extracellular stress and consequently improves the behavioural phenotype in animal models of neurological diseases, such as Alzheimer's disease. We review the evidence from a variety of mouse models that diet is a risk factor that can significantly contribute to the development of neurological diseases.

Discovery of GSK1997132B a novel centrally penetrant benzimidazole PPARγ partial agonist.

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor, thought to play a role in energy metabolism, glucose homeostasis and microglia-mediated neuroinflammation. A novel benzimidazole series of centrally penetrant PPARγ partial agonists has been identified. The optimization of PPARγ activity and in vivo pharmacokinetics leading to the identification of GSK1997132B a potent, metabolically stable and centrally penetrant PPARγ partial agonist, is described.

Peri-infarct depolarizations lead to loss of perfusion in ischaemic gyrencephalic cerebral cortex.

In the light of accumulating evidence for the occurrence of spontaneous cortical spreading depression and peri-infarct depolarizations in the human brain injured by trauma or aneurysmal subarachnoid haemorrhage, we used DC electrode recording and laser speckle imaging to study the relationship between depolarization events and perfusion in the ischaemic, gyrencephalic brain. In 14 adult male cats anaesthetized with chloralose, one cerebral hemisphere was exposed and the middle cerebral artery occluded. Surface cortical perfusion in core and penumbral territories was imaged semiquantitatively at intervals of 13 s for 4 h. Cortical surface DC potential was recorded. Time interval between changes in DC potential and in perfusion was examined, and this comparison was repeated using microelectrodes for DC potential in five similar experiments in a second laboratory. Mean pre-occlusion perfusion was 11707 +/- 4581 units (equivalent to CBF (cerebral blood flow) approximately 40.5 +/- SD 14.4 ml/100 g/min), and fell on occlusion to 5318 +/- 2916 (CBF approximately 17.1 +/- 8.3), 5291 +/- 3407 (CBF approximately 17.0 +/- 10.1), and 6711 +/- 3271 (CBF approximately 22.2 +/- 9.6), quickly recovering to 8704 +/- 4581 (CBF approximately 29.5 +/- 14.4), 9741 +/- 4499 (CBF approximately 33.3 +/- 14.1) and 10 314 +/- 3762 (CBF approximately 35.4 +/- 11.4) on the core, intermediate and outer penumbral gyri, respectively. Mean perfusion later fell secondarily on core and intermediate gyri but, overall, was preserved on the outer (upper level of perfusion) gyrus during the period of observation. Pattern and severity of transient changes in perfusion associated with depolarization events varied with gyral location; falls in perfusion were sometimes profound and irreversible, and followed rather than preceded depolarization. In this model of occlusive stroke, reductions in perfusion linked to peri-infarct depolarization events contribute to secondary deterioration in penumbral areas. The findings suggest that such events play a central rather than a subsidiary role in cerebral infarction in the gyrencephalic brain.

Repeated administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) modulates neuroinflammation and amyloid plaque load in mice bearing amyloid precursor protein and presenilin-1 mutant transgenes.

BACKGROUND: Data indicates anti-oxidant, anti-inflammatory and pro-cognitive properties of noradrenaline and analyses of post-mortem brain of Alzheimer's disease (AD) patients reveal major neuronal loss in the noradrenergic locus coeruleus (LC), the main source of CNS noradrenaline (NA). The LC has projections to brain regions vulnerable to amyloid deposition and lack of LC derived NA could play a role in the progression of neuroinflammation in AD. Previous studies reveal that intraperitoneal (IP) injection of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) can modulate neuroinflammation in amyloid over-expressing mice and in one study, DSP-4 exacerbated existing neurodegeneration. METHODS: TASTPM mice over-express human APP and beta amyloid protein and show age related cognitive decline and neuroinflammation. In the present studies, 5 month old C57/BL6 and TASTPM mice were injected once monthly for 6 months with a low dose of DSP-4 (5 mg kg-1) or vehicle. At 8 and 11 months of age, mice were tested for cognitive ability and brains were examined for amyloid load and neuroinflammation. RESULTS: At 8 months of age there was no difference in LC tyrosine hydroxylase (TH) across all groups and cortical NA levels of TASTPM/DSP-4, WT/Vehicle and WT/DSP-4 were similar. NA levels were lowest in TASTPM/Vehicle. Messenger ribonucleic acid (mRNA) for various inflammatory markers were significantly increased in TASTPM/Vehicle compared with WT/Vehicle and by 8 months of age DSP-4 treatment modified this by reducing the levels of some of these markers in TASTPM. TASTPM/Vehicle showed increased astrocytosis and a significantly larger area of cortical amyloid plaque compared with TASTPM/DSP-4. However, by 11 months, NA levels were lowest in TASTPM/DSP-4 and there was a significant reduction in LC TH of TASTPM/DSP-4 only. Both TASTPM groups had comparable levels of amyloid, microglial activation and astrocytosis and mRNA for inflammatory markers was similar except for interleukin-1 beta which was increased by DSP-4. TASTPM mice were cognitively impaired at 8 and 11 months but DSP-4 did not modify this. CONCLUSION: These data reveal that a low dose of DSP-4 can have varied effects on the modulation of amyloid plaque deposition and neuroinflammation in TASTPM mice dependent on the duration of dosing.

Rosiglitazone induces mitochondrial biogenesis in mouse brain.

Rosiglitazone was found to simulate mitochondrial biogenesis in mouse brain in an apolipoprotein (Apo) E isozyme-independent manner. Rosiglitazone induced both mitochondrial DNA (mtDNA) and estrogen-stimulated related receptor alpha (ESRRA) mRNA, a key regulator of mitochondrial biogenesis. Transcriptomics and proteomics analysis suggested the mitochondria produced in the presence of human ApoE3 and E4 were not as metabolically efficient as those in the wild type or ApoE knockout mice. Thus, we propose that PPARgamma agonism induces neuronal mitochondrial biogenesis and improves glucose utilization leading to improved cellular function and provides mechanistic support for the improvement in cognition observed in treatment of Alzheimer's patients with rosiglitazone.

A beam-walking apparatus to assess behavioural impairments in MPTP-treated mice: pharmacological validation with R-(-)-deprenyl.

A beam-walking apparatus has been evaluated for its ability to detect motor impairments in mice acutely treated with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg, s.c., single or double administration). Mice subjected to MPTP lesioning showed deficits in motor performance on the beam-walking task, for up to 6 days post-MPTP administration, as compared to saline-treated controls. In addition, MPTP-treated mice were detected to have a marked depletion in striatal dopamine levels and a concomitant reduction in substantia nigra (SN) tyrosine hydroxylase (TH) immunoreactivity, at 7 days post-MPTP administration, indicative of dopaminergic neuronal loss. Pre-administration of the potent MAO-B inhibitor R-(-)-deprenyl at 3 or 10 mg/kg, 30 min, s.c, significantly inhibited the MPTP-induced reduction in SN TH-immunoreactivity, striatal dopamine depletions and impairments in mouse motor function. The data described in the present study provides further evidence that functional deficits following an acute MPTP dosing schedule in mice can be quantified and are related to nigro-striatal dopamine function.

Further validation of LABORAS using various dopaminergic manipulations in mice including MPTP-induced nigro-striatal degeneration.

The automated behavioural apparatus, LABORAS (Laboratory Animal Behaviour Observation, Registration and Analysis System), has been further validated with respect to the ability of the system to detect behavioural impairments in mice, following various dopaminergic manipulations. Initially data were obtained from mice administered with amphetamine, haloperidol, SCH23390, apomorphine and L-DOPA, with the focus on locomotor and grooming activities. The data recorded by LABORAS on administration of these pharmacological tool compounds, is comparable with published findings using standard LMA systems and conventional observer methods. In addition the home cage behaviour of mice administered with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) using an acute dosing regimen was also investigated. In LABORAS, mice subjected to MPTP lesioning showed deficits in spontaneous motor activity at day 6-7 post-MPTP administration, over a 24 h test period, as compared to saline treated controls. The data captured and analysed using LABORAS, suggests that the automated system is able to detect both pharmacologically and lesion-induced changes in behaviour of mice, reliably and efficiently.

Identification of an epidermal keratinocyte AMPA glutamate receptor involved in dermatopathies associated with sensory abnormalities.

INTRODUCTION: Epidermal keratinocytes are increasingly recognized as active participants in the sensory transduction of itch and pain, processes known to involve primary afferent glutamatergic neurons. However the role of keratinocyte glutamate signaling in sensory functioning is not fully understood. Here, we present the observation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid type glutamate receptors (AMPAR) in epidermal keratinocytes. METHODS: Immunohistochemical and in situ hybridization analyses were conducted to assess the expression of AMPAR subunits in epidermal keratinocytes in mouse and human skin samples, and in organotypic cultures of human keratinocytes. In addition, RTPCR further confirmed the expression of GluA4-containing AMPAR in epidermal keratinocytes. RESULTS: We found prominent immunolabeling (IL) for the GluA4 subunit of AMPAR in keratinocytes of glabrous and hairy skin of mouse epidermis, as well as in human epidermal keratinocytes. RTPCR confirmed Gria4 transcript expression in epidermal mouse keratinocytes. In addition, expression of GRIA4 mRNA was confirmed in epidermal human keratinocytes by in situ hybridization. Immunohistochemical studies conducted in human skin biopsies from patients with atopic dermatitis (AD) and postherpetic neuralgia (PHN) demonstrate that keratinocyte expression of GluA4 can be altered under pathological conditions. Moreover, a decrease of GluA4 expression was observed in organotypic cultures of human keratinocytes after direct application of algogenic agents. CONCLUSIONS: We provide evidence that GluA4-containing AMPAR are expressed in epidermal keratinocytes, that human pruritic and painful dermatopathologies have alterations in the keratinocyte expression levels of GluA4-containing AMPAR, and that itch and pain producing substances can directly regulate their production in keratinocytes.

Identification of neuroprotective properties of anti-MAG antibody: a novel approach for the treatment of stroke?

The inhibitory activity of myelin-associated glycoprotein (MAG) on neurons is thought to contribute to the lack of regenerative capacity of the CNS after injury. The interaction of MAG and its neuronal receptors mediates bidirectional signaling between neurons and oligodendrocytes. The novel finding that an anti-MAG monoclonal antibody not only possesses the ability to neutralise the inhibitory effect of MAG on neurons but also directly protects oligodendrocytes from glutamate-mediated oxidative stress-induced cell death is reported here. Furthermore, administration of anti-MAG antibody (centrally and systemically) starting 1 hour after middle cerebral artery occlusion in the rat significantly reduced lesion volume at 7 days. This neuroprotection was associated with a robust improvement in motor function compared with animals receiving control IgG1. Together, these data highlight the potential for the use of anti-MAG antibodies as therapeutic agents for the treatment of stroke.

Choice, methodology, and characterization of focal ischemic stroke models: the search for clinical relevance.

To develop novel neuroprotective or neurorestorative agents for clinical application, the appropriate selection and characterization of preclinical focal stroke models is required to provide confidence in predicting therapeutic efficacy. Compelling evidence for novel therapies derived from the pathological and functional consequences of models of cerebral ischemia in the rat (and higher species) is an essential prerequisite before large expensive clinical trials are begun. This chapter provides an overview of focal ischemic models, with an emphasis on objective functional assessment of pathological mechanisms and efficacy of novel therapeutic strategies. The ability to predict functional consequences from structural abnormalities is a critical theme that can be extrapolated from the preclinical to the clinical setting, in that certain brain regions are inextricably linked to specific behavioral functions. This underlying approach is highly relevant, as monitoring the dynamic pathological and functional changes attributed to focal stroke will reveal new insights into novel mechanisms and targets that play a role in the evolution of cell death and impaired function. The utility of novel genomic technologies that are aligned with methods to determine structure-function relationships in preclinical models will facilitate a greater understanding of the pathophysiological process and potentially generate new targets that may ultimately be used to predict or offer clinical benefit.

A new primate model of focal stroke: endothelin-1-induced middle cerebral artery occlusion and reperfusion in the common marmoset.

The purpose of the present set of studies was to develop a new primate model of focal ischemia with reperfusion for long-term functional assessment in the common marmoset. Initially, the cerebral vascular anatomy of the marmoset was interrogated by Araldite-cast and ink-perfusion methods to determine the feasibility of an intravascular surgical approach. The methods showed that the internal carotid artery was highly tortuous in its passage, precluding the development of an extracranial method of inducing temporary middle cerebral artery occlusion in the marmoset. A pilot dose-response study investigated an intracranial approach of topically applying endothelin-1 (ET-1) to the M2 portion of the middle cerebral artery in a small sample of marmosets for up to 6 hours (n = 2 or 3 per group). Dose-dependent reductions in middle cerebral artery vessel caliber followed by gradual reperfusion were inversely related to increases in corrected lesion volume after ET-1 treatment, relative to vehicle control application. Finally, the functional consequences of ET-1-induced lesions to the M2 vascular territory were assessed up to 24 hours after surgery using the optimal dose established in the pilot study (2.5 nmol/25 microL). ET-1-treated marmosets (n = 4) showed marked contralateral motor deficits in grip strength and retrieval of food rewards and contralateral sensory/motor neglect towards tactile stimulation, relative to their ipsilateral side and vehicle-treated marmosets (n = 4). Strong correlations were shown between contralateral impairments and histopathologic parameters, which revealed unilateral putamen and cortical damage to the middle cerebral artery territory. No deficits were shown on general mobility, and self-care was promptly resumed in ET-1 marmosets after surgery. These results show that this novel model of ischemia with reperfusion in the marmoset has the potential to assess long-term function and to gauge the efficacy of novel therapeutic strategies targeted for clinical stroke.

Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

An optimistic view for quantifying mRNA in post-mortem human brain.

Quantitative human mRNA data are derived from post-mortem or biopsied tissue. RNA degradation, poor replication, a large mRNA variance and confounding factors such as brain pH and age of death are often cited, however, as objections to the data's reliability. A central question is whether post-mortem human mRNA can be treated as a statistically ordered system. TaqMan real-time RT-PCR was used to measure seven mRNAs in 513 cortical samples taken from 90 Alzheimer's disease and 81 control brains. Despite a high mRNA variance strong correlations were found between the mRNA transcripts in a single brain. Where a brain has a high/low level of one mRNA, the same brain invariably has a high/low level of other mRNAs; correlated order is present and allows removal of that source of variation common to all genes. Although levels of mRNA are highly variable between subjects (>1000-fold), quantitative order is present in post-mortem human mRNA, allowing effects due to pathology or gender to be isolated and tested for significance.

Beta-secretase (BACE) and GSK-3 mRNA levels in Alzheimer's disease.

Beta-secretase (BACE) and glycogen synthase kinase (GSK 3) are two enzymes thought to play a role in Alzheimer's disease. We extracted mRNA from 90 Alzheimer and 81 control brains. Levels of mRNA were quantified for BACE and GSK 3 with TaqMan real-time RT-PCR. We found no change in the Alzheimer's disease brains relative to controls for either the BACE or the GSK 3alpha mRNA levels.

The quantification of gene expression in an animal model of brain ischaemia using TaqMan real-time RT-PCR.

Expression levels of mRNA are commonly measured as a ratio of test to reference gene. The assumption is that reference genes such as beta-actin or cyclophilin are unaffected by treatment and act as steady-state controls. TaqMan real-time RT-PCR was used to test these assumptions in a rat model of cerebral ischaemia (tMCAO). Following measurement of 24 genes, we show that reference genes in this animal model fail the criteria for steady-state controls. Neuronal loss, glial proliferation and an influx of leukocytes into the lesioned brain result in major disturbance to cell populations. The mRNA for reference genes, as for test genes, reflects these changes. Specific mRNA levels vary according to the choice of reference gene to which they are normalised. In the process of resolving reference gene issues, mRNA increases were discovered for leukaemia inhibitory factor, nestin and galanin in rat brain hemispheres affected by ischaemia. Results are reported for a further 21 genes and mathematical and statistical methods are described that allow in this study fraction-fold changes in mRNA to be detected.

High-performance liquid chromatography/tandem mass spectrometry assay for the determination of 1-methyl-4-phenyl pyridinium (MPP+) in brain tissue homogenates.

A high-throughput liquid chromatography/tandem mass spectrometry method has been developed for the quantitative assessment of 1-methyl-4-phenylpyridinium (MPP+) in brain tissue samples. This separation is based on reversed phase chromatography using formic acid and acetonitrile as the mobile phase. Using gradient separation conditions, MPP+ was resolved within 5 min and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for MPP+ was found to be 1 fmol on column with a signal to noise ratio of 3:1. The assay has been used routinely in our laboratory for the measurement of MPP+ levels in brain tissue from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and can be used to distinguish neuroprotective efficacy and monoamine oxidase inhibition.

Differential behavioral profiling of stimulant substances in the rat using the LABORAS™ system.

Preclinical testing requires rapid and reliable evaluation of the main in vivo effects of novel test substances usually in rodents. Nevertheless, the techniques primarily used up to now involve either automated measurement of motor activity or direct observation of behavioral effects by extensively trained investigators. The advantages of these approaches are respectively high-throughput and comprehensive behavioral assessment. Nevertheless, motor activity is only one aspect of animal behavior and it cannot predict the full neurobehavioral profile of a substance, whereas direct observation is time-consuming. There is thus a need for novel approaches that combine the advantages of both automatic detection and comprehensive behavioral analysis. In the present study, we used the LABORAS™ system to analyze motor and non-motor behavior in rats administered various stimulant substances with or without known psychotomimetic properties or abuse liability (amphetamine, cocaine dizocilpine (MK-801), ketamine, modafinil and nicotine). The data show that LABORAS™ clearly detects the stimulating effects on motor behaviors of amphetamine, cocaine, dizocilpine and ketamine in a dose- and time-dependent manner. Differential effects of these test substances on non-motor behaviors, such as grooming, eating and drinking could also be detected. Nicotine displayed only slight stimulating effects on locomotion, whereas modafinil was virtually without effect on the behaviors evaluated by the system. These data with different stimulant substances suggest that LABORAS™ presents an advantage over classical methods performing automated measurements restricted to locomotion. Furthermore, the procedure is considerably more rapid than behavioral observation procedures. Characterization of the behavioral profile of test substances using LABORAS™ should therefore accelerate preclinical studies. In addition, the multi-faceted parameters measured by LABORAS™ permit a more detailed comparison of the behavioral profiles of novel substances with standard reference substances, thereby providing important indicators for orienting further substance evaluation and supporting drug development.

The neuroprotective effect of a specific P2X7 receptor antagonist derives from its ability to inhibit assembly of the NLRP3 inflammasome in glial cells.

Release of interleukin (IL)-1ß from immunocompetent cells requires formation of the NACHT, LLR and PYD domains-containing protein 3 (NLRP3) inflammasome and caspase 1 activation. Adenosine 5'-triphosphate (ATP), acting on the P2X(7) receptor, is one factor that stimulates inflammasome assembly. We show that a novel specific P2X(7) receptor antagonist, GSK1370319A, inhibits ATP-induced increase in IL-1ß release and caspase 1 activation in lipopolysaccharide (LPS)-primed mixed glia by blocking assembly of the inflammasome in a pannexin 1-dependent manner. GSK1370319A also inhibits ATP-induced subregion-specific neuronal loss in hippocampal organotypic slice cultures, which is dependent on its ability to prevent inflammasome assembly in glia. Significantly, GSK1370319A attenuates age-related deficits in long-term potentiation (LTP) and inhibits the accompanying age-related caspase 1 activity. We conclude that inhibiting P2X(7) receptor-activated NLRP3 inflammasome formation and the consequent IL-1ß release from glia preserve neuronal viability and synaptic activity.