Iterative focused screening with biological fingerprints identifies selective Asc-1 inhibitors distinct from traditional high throughput screening

N-methyl-d-aspartate receptors (NMDARs) mediate glutamatergic signaling that is critical to cognitive processes in the central nervous system, and NMDAR hypofunction is thought to contribute to cognitive impairment observed in both schizophrenia and Alzheimer’s disease. One approach to enhance the function of NMDAR is to increase the concentration of an NMDAR coagonist, such as glycine or d-serine, in the synaptic cleft. Inhibition of alanine–serine–cysteine transporter-1 (Asc-1), the primary transporter of d-serine, is attractive because the transporter is localized to neurons in brain regions critical to cognitive function, including the hippocampus and cortical layers III and IV, and is colocalized with d-serine and NMDARs. To identify novel Asc-1 inhibitors, two different screening approaches were performed with whole-cell amino acid uptake in heterologous cells stably expressing human Asc-1: (1) a high-throughput screen (HTS) of 3 M compounds measuring 35S l-cysteine uptake into cells attached to scintillation proximity assay beads in a 1536 well format and (2) an iterative focused screen (IFS) of a 45 000 compound diversity set using a 3H d-serine uptake assay with a liquid scintillation plate reader in a 384 well format. Critically important for both screening approaches was the implementation of counter screens to remove nonspecific inhibitors of radioactive amino acid uptake. Furthermore, a 15 000 compound expansion step incorporating both on- and off-target data into chemical and biological fingerprint-based models for selection of additional hits enabled the identification of novel Asc-1-selective chemical matter from the IFS that was not identified in the full-collection HTS

Defective adult neurogenesis in CB1 cannabinoid receptor knockout mice.

ABSTRACT Pharmacological studies suggest a role for CB1 cannabinoid receptors (CB1R) in regulating neurogenesis in the adult brain. To investigate this possibility, we measured neurogenesis by intraperitoneal injection of bromodeoxyuridine (BrdU), which labels newborn neurons, in wild-type and CB1R-knockout (CB1R-KO) mice. CB1R-KO mice showed reductions in the number of BrdU-labeled cells to ϳ50% of wild-type (WT) levels in dentate gyrus and subventricular zone (SVZ), suggesting that CB1R activation promotes neurogenesis. To test this further, WT mice were given the CB1R antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A) before measuring neurogenesis with BrdU

Partial change in EphA4 knockout mouse phenotype: Loss of diminished GFAP upregulation following spinal cord injury

In a previous study we found that the EphA4 receptor inhibits regeneration following spinal cord injury by blocking regrowth of axons and regulation of astrocyte reactivity. In our original studies using EphA4 null mice [Goldshmit et al., J. Neurosci., 2004] we found attenuated astrocyte reactivity following spinal cord injury. Several other studies have now supported the role of EphA4 in regulating neural regeneration but a recent study [Herrmann et al., Exp. Neurol., 2010] did not find an effect of EphA4 on astrocyte reactivity. Re-examination of astrocytic gliosis following injury in our current cohort of EphA4 null mice revealed that they no longer showed attenuation of astrocyte reactivity, however other EphA4 null mouse phenotypes, such as decreased size of the dorsal funiculus were unaltered. We hypothesised that long-term breeding on the C57Bl/6 background may influence the EphA4-mediated astrocyte phenotype and compared astrocytic gliosis at 4 days following spinal cord injury in wildtype and EphA4 null mice on the C57Bl/6 background and backcrossed C57Bl/6×129Sv(F2) mice, as well as wildtype 129Sv mice. 129Sv mice had increased GFAP expression and increased numbers of reactive GFAP astrocytes compared to C57Bl/6 mice. There was no significant effect of EphA4 deletion on GFAP expression in C57Bl/6 mice or the F2 crosses other than a moderately decreased number of EphA4 null astrocytes in C57Bl/6 mice using one of two antibodies. Therefore, there has been an apparent change in EphA4-mediated astroglial phenotype associated with long term breeding of the EphA4 colony but it does not appear to be influenced by background mouse strain

Differential Gene Expression in the EphA4 Knockout Spinal Cord and Analysis of the Inflammatory Response Following Spinal Cord Injury

Mice lacking the axon guidance molecule EphA4 have been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips™. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wildtype spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage/microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages/activated microglia in injured EphA4 knockout compared to wild-type spinal cords at 2, 4 or 14 days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages/activated microglia was observed in EphA4 knockout spinal cords at 4 days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury

Involvement of the endocannabinoid system in the physiological response to transient common carotid artery occlusion and reperfusion

Background: The transient global cerebral hypoperfusion/reperfusion achieved by induction of Bilateral Common Carotid Artery Occlusion followed by Reperfusion (BCCAO/R) may trigger a physiological response in an attempt to preserve tissue and function integrity. There are several candidate molecules among which the endocannabinoid system (ECS) and/or peroxisome-proliferator activated receptor-alpha (PPAR-alpha) may play a role in modulating oxidative stress and inflammation. The aims of the present study are to evaluate whether the ECS, the enzyme cyclooxygenase-2 (COX-2) and PPAR-alpha are involved during BCCAO/R in rat brain, and to identify possible markers of the ongoing BCCAO/R-induced challenge in plasma. Methods: Adult Wistar rats underwent BCCAO/R with 30 min hypoperfusion followed by 60 min reperfusion. The frontal and temporal-occipital cortices and plasma were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to determine concentrations of endocannabinoids (eCBs) and related molecules behaving as ligands of PPAR-alpha, and of oxidative-stress markers such as lipoperoxides, while Western Blot and immunohistochemistry were used to study protein expression of cannabinoid receptors, COX-2 and PPAR-alpha. Unpaired Student's t-test was used to evaluate statistical differences between groups. Results: The acute BCCAO/R procedure is followed by increased brain tissue levels of the eCBs 2-arachidonoylglycerol and anandamide, palmitoylethanolamide, an avid ligand of PPAR-alpha, lipoperoxides, type 1 (CB1) and type 2 (CB2) cannabinoid receptors, and COX-2, and decreased brain tissue concentrations of docosahexaenoic acid (DHA), one of the major targets of lipid peroxidation. In plasma, increased levels of anandamide and lipoperoxides were observed. Conclusions: The BCCAO/R stimulated early molecular changes that can be easily traced in brain tissue and plasma, and that are indicative of the tissue physiological response to the reperfusion-induced oxidative stress and inflammation. The observed variations suggest that the positive modulation of the ECS and the increase of proinflammatory substances are directly correlated events. Increase of plasmatic levels of anandamide and lipoperoxides further suggests that dysregulation of these molecules may be taken as an indicator of an ongoing hypoperfusion/reperfusion challenge

Cannabinoids and Dementia: A Review of Clinical and Preclinical Data

The endocannabinoid system has been shown to be associated with neurodegenerative diseases and dementia. We review the preclinical and clinical data on cannabinoids and four neurodegenerative diseases: Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD) and vascular dementia (VD). Numerous studies have demonstrated an involvement of the cannabinoid system in neurotransmission, neuropathology and neurobiology of dementias. In addition, several candidate compounds have demonstrated efficacy in vitro. However, some of the substances produced inconclusive results in vivo. Therefore, only few trials have aimed to replicate the effects seen in animal studies in patients. Indeed, the literature on cannabinoid administration in patients is scarce. While preclinical findings suggest causal treatment strategies involving cannabinoids, clinical trials have only assessed the suitability of cannabinoid receptor agonists, antagonists and cannabidiol for the symptomatic treatment of dementia. Further research is needed, including in vivo models of dementia and human studies

Pharmacological activation of mGlu5 receptors with the positive allosteric modulator, VU0360172 modulates thalamic GABAergic transmission

Previous studies have shown that injection of the mGlu5 receptor positive allosteric modulator (PAM) VU0360172 into either the thalamus or somatosensory cortex markedly reduces the frequency of spike-and-wave discharges (SWDs) in the WAG/Rij model of absence epilepsy. Here we have investigated the effects of VU0360172 on GABA transport in the thalamus and somatosensory cortex, as possible modes of action underlying the suppression of SWDs. Systemic VU0360172 injections increase GABA uptake in thalamic synaptosomes from epileptic WAG/Rij rats. Consistent with this observation, VU0360172 could also enhance thalamic GAT-1 protein expression, depending on the dosing regimen. This increase in GAT-1 expression was also observed in the thalamus from non-epileptic rats (presymptomatic WAG/Rij and Wistar) and appeared to occur selectively in neurons. The tonic GABAA receptor current present in ventrobasal thalamocortical neurons was significantly reduced by VU0360172 consistent with changes in GAT-1 and GABA uptake. The in vivo effects of VU0360172 (reduction in tonic GABA current and increase in GAT-1 expression) could be reproduced in vitro by treating thalamic slices with VU0360172 for at least 1 hour and appeared to be dependent on the activation of PLC. Thus, the effects of VU0360172 do not require an intact thalamocortical circuit. In the somatosensory cortex, VU0360172 reduced GABA uptake but did not cause significant changes in GAT-1 protein levels. These findings reveal a novel mechanism of regulation mediated by mGlu5 receptors, which could underlie the powerful anti-absence effect of mGlu5 receptor enhancers in animal models

The anti-absence effect of mGlu5 receptor amplification with VU0360172 is maintained during and after antiepileptogenesis

Ethosuximide (ETX) has become the drug of choice in the treatment of patients with absence seizures taking into account both its efficacy, tolerability and antiepileptogenic properties. However, 47% of subjects treated with ETX failed in therapy, and most antiepileptic drugs have cognitive side effects. VU0360172, a positive allosteric modulator (PAM) of mGluR5, acutely and chronically administered decreased seizures dose dependently in rats of the WAG/Rij strain, a genetic absence model. Here it is investigated whether anti-epileptogenesis induced by ETX alters the sensitivity of VU0360172 as an anti-absence drug, and cognition is affected during and after chronic ETX treatment. Method: Male WAG/Rij rats were chronically treated with ETX for 4 months. EEG’s were recorded during and after treatment as well as challenged with VU0360172. Rats were also periodically exposed to a cue discrimination learning task in a Y-maze. mGlu5 receptors were quantified with Western Blot. Results: Antiepileptogenesis was successfully induced by ETX and VU0360172 showed a time and dose dependent anti-absence action. However, chronic ETX treated rats showed a decrease in absences both during and after the end treatment, without clear time and dose related effects. The decrease of sensitivity for VU0360172 was not accompanied by a change in mGluR5 expression in cortex and thalamus. Chronic ETX enhanced motivation to collect sucrose pallets and this was followed by an increase in cued discrimination learning. It is concluded that VU0360172 keeps its antiabsence effects after chronic treatment. Moreover, its differential effects in the two groups cannot be explained by a simple receptor down regulation suggesting a more downstream interaction between ETX and mGluR5. The cognitive enhancing effects of ETX, as found at the end of the experiment might be mediated to the antidepressant action of ETX as expressed by an increase in the rewarding properties of sucrose pallets