GSK249320, A Monoclonal Antibody Against the Axon Outgrowth Inhibition Molecule Myelin-Associated Glycoprotein, Improves Outcome of Rodents with Experimental Stroke.

Myelin-associated glycoprotein (MAG) is an inhibitor of axon growth. MAG levels increase after stroke. GSK249320 is a monoclonal antibody that neutralizes MAG-mediated inhibition and so may promote axon outgrowth and improve post-stroke outcomes. The current study tested the hypothesis that GSK249320 initiated 24 hours or 7 days after experimental stroke improves behavioural outcomes. Rats with right middle cerebral artery occlusion for 90 minutes were randomized to receive 6 weeks of intravenous (a) GSK249320 starting 24 hours post-stroke, (b) GSK249320 starting 7 days post-stroke, or (c) vehicle. Behavioral testing was performed over 7 weeks. Serial MRI demonstrated no differences in infarct volume across groups. Animals treated with GSK249320 24 hours post-stroke showed larger increases in Neuroscore (time X group, p = 0.0008) and staircase test (main effect of group, p = 0.0214) as compared to controls, but animals treated 7 days post-stroke showed no significant behavioral benefit. No significant results were found for the sticky tape or cylinder tests. A separate set of animals with experimental stroke received a single intravenous dose of GSK249320 or vehicle at 1 hour, 24 hours, 48 hours or 1 week post-stroke, and immunohistochemistry methods were used to measure GSK249320 distribution; GSK249320 was found in the ipsilesional hemisphere only, the extent of which increased with later times of injection. These data suggest that intravenous GSK249320 penetrates the lesion site and is associated with a small effect on functional outcomes when initiated 24 hours post-stroke and so support the translational potential of this monoclonal antibody as a restorative therapy for patients with stroke.

A translational systems biology approach in both animals and humans identifies a functionally related module of accumbal genes involved in the regulation of reward processing and binge drinking in males.

BACKGROUND: The mesolimbic dopamine system, composed primarily of dopaminergic neurons in the ventral tegmental area that project to striatal structures, is considered to be the key mediator of reinforcement-related mechanisms in the brain. Prompted by a genome-wide association meta-analysis implicating the Ras-specific guanine nucleotide-releasing factor 2 (RASGRF2) gene in the regulation of alcohol intake in men, we have recently shown that male Rasgrf2(-/-) mice exhibit reduced ethanol intake and preference accompanied by a perturbed mesolimbic dopamine system. We therefore propose that these mice represent a valid model to further elucidate the precise genes and mechanisms regulating mesolimbic dopamine functioning. METHODS: Transcriptomic data from the nucleus accumbens (NAcc) of male Rasgrf2(-/-) mice and wild-type controls were analyzed by weighted gene coexpression network analysis (WGCNA). We performed follow-up genetic association tests in humans using a sample of male adolescents from the IMAGEN study characterized for binge drinking (n = 905) and ventral striatal activation during an fMRI reward task (n = 608). RESULTS: The WGCNA analyses using accumbal transcriptomic data revealed 37 distinct "modules," or functionally related groups of genes. Two of these modules were significantly associated with Rasgrf2 knockout status: M5 (p < 0.001) and M6 (p < 0.001). In follow-up translational analyses we found that human orthologues for the M5 module were significantly (p < 0.01) enriched with genetic association signals for binge drinking in male adolescents. Furthermore, the most significant locus, originating from the EH-domain containing 4 (EHD4) gene (p < 0.001), was also significantly associated with altered ventral striatal activity in male adolescents performing an fMRI reward task (pempirical < 0.001). LIMITATIONS: It was not possible to determine the extent to which the M5 module was dysregulated in Rasgrf2(-/-) mice by perturbed mesolimbic dopamine signalling or by the loss of Rasgrf2 function in the NAcc. CONCLUSION: Taken together, our findings indicate that the accumbal M5 module, initially identified as being dysregulated in male Rasgrf2(-/-) mice, is also relevant for human alcohol-related phenotypes potentially through the modulation of reinforcement mechanisms in the NAcc. We therefore propose that the genes comprising this module represent important candidates for further elucidation within the context of alcohol-related phenotypes.

αCaMKII autophosphorylation mediates neuronal activation in the hippocampal dentate gyrus after alcohol and cocaine in mice.

Psychoactive drug-induced cellular activation is a key mechanism to promote neuronal plasticity and addiction. Alpha Ca(2+)/calmodulin-dependent protein kinase II (αCaMKII) and its autophosphorylation play a key role in the development of drug use associated behaviours. It has been suggested that αCaMKII autophosphorylation is necessary for drug-induced neuronal activation in the mesolimbic system. Here, we show an alcohol- and cocaine-induced increase in c-fos expression in the hippocampal dentate gyrus, which is absent in αCaMKII(T286A) autophosphorylation deficient mice. These findings may suggest a role in hippocampal αCaMKII autophosphorylation in the acute neuroplastic effects of alcohol and cocaine.

Rasgrf2 controls dopaminergic adaptations to alcohol in mice.

Alcohol abuse leads to serious health problems with no effective treatment available. Recent evidence suggests a role for ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) in alcoholism. Rasgrf2 is a calcium sensor and MAPK/ERK activating protein, which has been linked to neurotransmitter release and monoaminergic receptor adaptations. Rasgrf2 knock out (KO) mice do not develop a dopamine response in the nucleus accumbens after an alcohol challenge and show a reduced consumption of alcohol. The present study aims to further characterise the role of Rasgrf2 in dopaminergic activation beyond the nucleus accumbens following alcohol treatment. Using in vivo microdialysis we found that alcohol induces alterations in dopamine levels in the dorsal striatum between wildtype (WT) and Rasgrf2 KO mice. There was no difference in the expression of dopamine transporter (DAT), dopamine receptor regulating factor (DRRF), or dopamine D2 receptor (DRD2) mRNA in the brain between Rasgrf2 KO and WT mice. After sub-chronic alcohol treatment, DAT and DRRF, but not DRD2 mRNA expression differed between WT and Rasgrf2 KO mice. Brain adaptations were positively correlated with splenic expression levels. These data suggest that Rasgrf2 controls dopaminergic signalling and adaptations to alcohol also in other brain regions, beyond the nucleus accumbens.

Rasgrf2 controls noradrenergic involvement in the acute and subchronic effects of alcohol in the brain.

RATIONALE: Alcohol addiction is a major psychiatric disease, and yet, the underlying molecular adaptations in the brain remain unclear. Recent evidence suggests a functional role for the ras-specific guanine-nucleotide releasing factor 2 (Rasgrf2) in alcoholism. Rasgrf2(-/-) mice consume less alcohol and show entirely absent dopamine responses to an alcohol challenge compared to wild types (WT). OBJECTIVE: In order to further investigate how Rasgrf2 modifies the acute and subchronic effects of alcohol in the brain, we investigated its effects on the noradrenergic and serotonergic systems. METHODS: We measured noradrenaline and serotonin activity in the brain by in vivo microdialysis and RNA expression by chip analysis and RT-PCR after acute and sub-chronic alcohol exposure in Rasgrf2(-/-) and WT mice. RESULTS: In vivo microdialysis showed a significantly reduced noradrenergic response and an absent serotonergic response in the nucleus accumbens (NAcc) and caudate putamen (CPu) after an alcohol challenge in Rasgrf2(-/-) mice. A co-expression analysis showed that there is a high correlation between Rasgrf2 and α2 adrenoceptor RNA expression in the ventral striatum in naïve animals. Accordingly, we further assessed the role of Rasgrf2 in the response of the noradrenergic system to subchronic alcohol exposure. A decrease in ß1 adrenoceptor gene expression was seen in Rasgrf2(+/+), but not Rasgrf2(-/-) mice following alcohol exposure. Conversely, alcohol resulted in a decrease in both ß2 and α2 adrenoceptor gene expression in knockout but not WT Rasgrf2 mice. CONCLUSIONS: These findings suggest that adaptations in the noradrenergic system contribute to the Rasgrf2 enhanced risk of alcoholism.

αCaMKII autophosphorylation controls the establishment of alcohol-induced conditioned place preference in mice.

The autophosphorylation of alpha Ca2+ /calmodulin dependent protein kinase II (αCaMKII) is important for memory formation and is becoming increasingly implicated in the development of drug addiction. Previous work suggests that αCaMKII acts via the monoaminergic systems to facilitate the establishment of alcohol drinking behaviour. The present study aims to investigate whether αCaMKII autophosphorylation deficient αCaMKII(T286A) mice show a difference in the rewarding properties of alcohol (2 g/kg, i.p.), as measured by conditioned place preference (CPP). We found that alcohol-induced CPP could be established at an accelerated rate in αCaMKII(T286A) compared to wild type (WT) mice. Hyperactivity/hyper-arousal induced by the test environment was normalised by alcohol in the αCaMKII(T286A), but not WT mice. This effect could be conditioned to the test environment and may suggest enhanced negative reinforcing action of alcohol in αCaMKII autophosphorylation deficient mice.

αCaMKII autophosphorylation controls the establishment of alcohol drinking behavior.

The α-Ca(2+)/calmodulin-dependent protein kinase II (αCaMKII) is a crucial enzyme controlling plasticity in the brain. The autophosphorylation of αCaMKII works as a 'molecular memory' for a transient calcium activation, thereby accelerating learning. We investigated the role of αCaMKII autophosphorylation in the establishment of alcohol drinking as an addiction-related behavior in mice. We found that alcohol drinking was initially diminished in αCaMKII autophosphorylation-deficient αCaMKII(T286A) mice, but could be established at wild-type level after repeated withdrawals. The locomotor activating effects of a low-dose alcohol (2 g/kg) were absent in αCaMKII(T286A) mice, whereas the sedating effects of high-dose (3.5 g/kg) were preserved after acute and subchronic administration. The in vivo microdialysis revealed that αCaMKII(T286A) mice showed no dopamine (DA) response in the nucleus accumbens to acute or subchronic alcohol administration, but enhanced serotonin (5-HT) responses in the prefrontal cortex. The attenuated DA response in αCaMKII(T286A) mice was in line with altered c-Fos activation in the ventral tegmental area after acute and subchronic alcohol administration. In order to compare findings in mice with the human condition, we tested 23 single-nucleotide polymorphisms (SNPs) in the CAMK2A gene for their association with alcohol dependence in a population of 1333 male patients with severe alcohol dependence and 939 controls. We found seven significant associations between CAMK2A SNPs and alcohol dependence, one of which in an autophosphorylation-related area of the gene. Together, our data suggest αCaMKII autophosphorylation as a facilitating mechanism in the establishment of alcohol drinking behavior with changing the DA-5-HT balance as a putative mechanism.

RASGRF2 regulates alcohol-induced reinforcement by influencing mesolimbic dopamine neuron activity and dopamine release.

The firing of mesolimbic dopamine neurons is important for drug-induced reinforcement, although underlying genetic factors remain poorly understood. In a recent genome-wide association metaanalysis of alcohol intake, we identified a suggestive association of SNP rs26907 in the ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) gene, encoding a protein that mediates Ca(2+)-dependent activation of the ERK pathway. We performed functional characterization of this gene in relation to alcohol-related phenotypes and mesolimbic dopamine function in both mice and adolescent humans. Ethanol intake and preference were decreased in Rasgrf2(-/-) mice relative to WT controls. Accordingly, ethanol-induced dopamine release in the ventral striatum was blunted in Rasgrf2(-/-) mice. Recording of dopamine neurons in the ventral tegmental area revealed reduced excitability in the absence of Ras-GRF2, likely because of lack of inhibition of the I(A) potassium current by ERK. This deficit provided an explanation for the altered dopamine release, presumably linked to impaired activation of dopamine neurons firing. Functional neuroimaging analysis of a monetary incentive-delay task in 663 adolescent boys revealed significant association of ventral striatal activity during reward anticipation with a RASGRF2 haplotype containing rs26907, the SNP associated with alcohol intake in our previous metaanalysis. This finding suggests a link between the RASGRF2 haplotype and reward sensitivity, a known risk factor for alcohol and drug addiction. Indeed, follow-up of these same boys at age 16 y revealed an association between this haplotype and number of drinking episodes. Together, these combined animal and human data indicate a role for RASGRF2 in the regulation of mesolimbic dopamine neuron activity, reward response, and alcohol use and abuse.

Lentiviral-mediated gene delivery reveals distinct roles of nucleus accumbens dopamine D2 and D3 receptors in novelty- and light-induced locomotor activity.

The importance of the dopaminergic system for proper brain activity is demonstrated by findings that alterations in this system lead to severe disabilities, including motor impairment observed in various neurological and psychiatric disorders. Although the roles of specific dopamine receptors in behaviour have been extensively investigated using pharmacological agents and knockout mice, non-specificity of ligands and compensatory molecular adaptations in mutated animals restrict the interpretation of the results. To overcome these limitations and further explore the role of the dopamine D2 and D3 receptors (D2R and D3R) in rats, we used lentivirus-mediated gene knockdown and overexpression to specifically manipulate expression levels of these genes in the rat nucleus accumbens (NAcc), a brain area important for spontaneous and induced locomotor responses. Lentiviruses, inducing expression of rat D2R or D3R, or efficient knockdown of either receptor by small hairpin (sh)RNAs, were stereotaxically injected into the NAcc. While knockdown of either receptor significantly reduced spontaneous locomotor activity in a novel but not in a habituated environment, D2R and D3R appeared to contribute in opposite ways to light-induced locomotor activity. D2R knockdown increased while D3R knockdown decreased locomotor activity in this test. Altogether, our findings suggest that D2R and D3R, expressed in the NAcc, have both shared and non-overlapping roles in transduction of alerting signals elicited by potentially important sensory and environmental cues.

αCaMKII autophosphorylation controls exploratory activity to threatening novel stimuli.

Autophosphorylation of αCaMKII is regarded as a 'molecular memory' for Ca(2+) transients and a crucial mechanism in aversely, but less so in appetitively, motivated learning and memory. While there is a growing body of research implicating αCaMKII in general in behavioral responses to threat or fearful stimuli, little is known about the contribution of the autophosphorylation. The present study asked how αCaMKII autophosphorylation controls anxiety-like behavioral responses toward novel, potentially threatening stimuli. We tested homozygous and heterozygous T286A αCaMKII autophosphorylation deficient mice and wild types in a systematic series of behavioral tests. Homozygous mutants were more active in the open field test and showed reduced anxiety-related behavior in the light/dark test, but these findings were confounded by a hyperlocomotor phenotype. The analysis of elevated plus maze showed significantly reduced anxiety-related behavior in the αCaMKII autophosphorylation-deficient mice which appeared to mediate a hyperlocomotor response. An analysis of home cage behavior, where neither novel nor threatening stimuli were present, showed no differences in locomotor activity between genotypes. Increased locomotion was not observed in the novel object exploration test in the αCaMKII autophosphorylation-deficient mice, implying that hyperactivity does not occur in response to discrete novel stimuli. The present data suggest that the behavior of αCaMKII autophosphorylation-deficient mice cannot simply be described as a low anxiety phenotype. Instead it is suggested that αCaMKII autophosphorylation influences locomotor reactivity to novel environments that are potentially, but not necessarily threatening.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.