Chloride intracellular channel 4 (CLIC4) expression profile in the mouse medial prefrontal cortex and its regulation by ethanol.

BACKGROUND: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. METHODS: We used qRT-PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. RESULTS: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. CONCLUSIONS: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes.

Olfactory dysfunction in the 3xTg-AD model of Alzheimer's disease.

Alzheimer's disease (AD) is an incurable neurodegenerative disease in which the risk of development increases with age. People with AD are plagued with deficits in their cognition, memory, and basic social skills. Many of these deficits are believed to be caused by the formation of amyloid-ß plaques and neurofibrillary tangles in regions of the brain associated with memory, such as the hippocampus. However, one of the early, preclinical symptoms of AD is the loss of olfactory detection and discrimination. To determine if a mouse model of AD expresses the same olfactory dysfunction seen in human AD, 3xTg-AD mice were given a buried food test and, unlike previous studies, compared to their background and parental strains. Results showed that over 52 weeks, the 3xTg-AD mice took significantly longer to find the buried food than the control strains. The olfactory bulbs of the 3xTg-AD mice were removed, sliced, and stained using Congo red for histological analysis. Amyloid deposits were observed predominantly in the granule layer of the olfactory bulb beginning at 13 weeks of age in 3xTg-AD mice, but not in the control strains of mice. Further examination of the buried food test data revealed that 3xTg-AD females had a significantly longer latency to detect the buried food than males beginning at 26 weeks of age. Overall, this study provides further validation of the 3xTg-AD mouse model of AD and supports the idea that simple olfactory testing could be part of the diagnostic process for human AD.

Cross-Species Co-analysis of Prefrontal Cortex Chronic Ethanol Transcriptome Responses in Mice and Monkeys.

Despite recent extensive genomic and genetic studies on behavioral responses to ethanol, relatively few new therapeutic targets for the treatment of alcohol use disorder have been validated. Here, we describe a cross-species genomic approach focused on identifying gene networks associated with chronic ethanol consumption. To identify brain mechanisms underlying a chronic ethanol consumption phenotype highly relevant to human alcohol use disorder, and to elucidate potential future therapeutic targets, we conducted a genomic study in a non-human primate model of chronic open-access ethanol consumption. Microarray analysis of RNA expression in anterior cingulate and subgenual cortices from rhesus macaques was performed across multiple cohorts of animals. Gene networks correlating with ethanol consumption or showing enrichment for ethanol-regulated genes were identified, as were major ethanol-related hub genes within these networks. A subsequent consensus module analysis was used to co-analyze monkey data with expression data from a chronic intermittent ethanol vapor-exposure and consumption model in C57BL/6J mice. Ethanol-related gene networks conserved between primates and rodents were enriched for genes involved in discrete biological functions, including; myelination, synaptic transmission, chromatin modification, Golgi apparatus function, translation, cellular respiration, and RNA processing. The myelin-related network, in particular, showed strong correlations with ethanol consumption behavior and displayed marked network reorganization between control and ethanol-drinking animals. Further bioinformatics analysis revealed that these networks also showed highly significant overlap with other ethanol-regulated gene sets. Altogether, these studies provide robust primate and rodent cross-species validation of gene networks associated with chronic ethanol consumption. Our results also suggest potential novel focal points for future therapeutic interventions in alcohol use disorder.

Group II metabotropic glutamate receptor interactions with NHERF scaffold proteins: Implications for receptor localization in brain.

The group II metabotropic glutamate receptors mGluR2 and mGluR3 are key modulators of glutamatergic neurotransmission. In order to identify novel Group II metabotropic glutamate receptor (mGluR)-interacting partners, we screened the C-termini of mGluR2 and mGluR3 for interactions with an array of PDZ domains. These screens identified the Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1 & -2) as candidate interacting partners. Follow-up co-immunoprecipitation studies demonstrated that both mGluR2 and mGluR3 can associate with NHERF-1 and NHERF-2 in a cellular context. Functional studies revealed that disruption of PDZ interactions with mGluR2 enhanced receptor signaling to Akt. However, further studies of mGluR2 and mGluR3 signaling in astrocytes in which NHERF expression was reduced by gene knockout (KO) and/or siRNA knockdown techniques revealed that the observed differences in signaling between WT and mutant mGluR2 were likely not due to disruption of interactions with the NHERF proteins. Electron microscopic analyses revealed that Group II mGluRs were primarily expressed in glia and unmyelinated axons in WT, NHERF-1 and NHERF-2 KO mice, but the relative proportion of labeled axons over glial processes was higher in NHERF-2 KO mice than in controls and NHERF-1 KO mice. Interestingly, our anatomical studies also revealed that loss of either NHERF protein results in ventriculomegaly, which may be related to the high incidence of hydrocephaly that has previously been observed in NHERF-1 KO mice. Together, these studies support a role for NHERF-1 and NHERF-2 in regulating the distribution of Group II mGluRs in the murine brain, while conversely the effects of the mGluR2/3 PDZ-binding motifs on receptor signaling are likely mediated by interactions with other PDZ scaffold proteins beyond the NHERF proteins.

Integrative Analysis of Genetic, Genomic, and Phenotypic Data for Ethanol Behaviors: A Network-Based Pipeline for Identifying Mechanisms and Potential Drug Targets.

Complex behavioral traits, such as alcohol abuse, are caused by an interplay of genetic and environmental factors, producing deleterious functional adaptations in the central nervous system. The long-term behavioral consequences of such changes are of substantial cost to both the individual and society. Substantial progress has been made in the last two decades in understanding elements of brain mechanisms underlying responses to ethanol in animal models and risk factors for alcohol use disorder (AUD) in humans. However, treatments for AUD remain largely ineffective and few medications for this disease state have been licensed. Genome-wide genetic polymorphism analysis (GWAS) in humans, behavioral genetic studies in animal models and brain gene expression studies produced by microarrays or RNA-seq have the potential to produce nonbiased and novel insight into the underlying neurobiology of AUD. However, the complexity of such information, both statistical and informational, has slowed progress toward identifying new targets for intervention in AUD. This chapter describes one approach for integrating behavioral, genetic, and genomic information across animal model and human studies. The goal of this approach is to identify networks of genes functioning in the brain that are most relevant to the underlying mechanisms of a complex disease such as AUD. We illustrate an example of how genomic studies in animal models can be used to produce robust gene networks that have functional implications, and to integrate such animal model genomic data with human genetic studies such as GWAS for AUD. We describe several useful analysis tools for such studies: ComBAT, WGCNA, and EW_dmGWAS. The end result of this analysis is a ranking of gene networks and identification of their cognate hub genes, which might provide eventual targets for future therapeutic development. Furthermore, this combined approach may also improve our understanding of basic mechanisms underlying gene x environmental interactions affecting brain functioning in health and disease.

Adenosine A2A receptor in the monkey basal ganglia: ultrastructural localization and colocalization with the metabotropic glutamate receptor 5 in the striatum.

The adenosine A(2A) receptor (A(2A) R) is a potential drug target for the treatment of Parkinson's disease and other neurological disorders. In rodents, the therapeutic efficacy of A(2A) R modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A(2A) R localization in the primate basal ganglia and to assess the degree of A(2A) R/mGluR5 colocalization in the striatum. A(2A) R immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A(2A) R immunoreactivity. At the electron microscopic level, striatal A(2A) R labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A(2A) R immunoreactivity. Striatal A(2A) R/mGluR5 colocalization was commonly found; 60-70% of A(2A) R-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A(2A) R in the primate basal ganglia and demonstrate that A(2A) R and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A(2A) R could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism.

Metabotropic glutamate receptor 5 antagonist protects dopaminergic and noradrenergic neurons from degeneration in MPTP-treated monkeys.

Degeneration of the dopaminergic nigrostriatal system and of noradrenergic neurons in the locus coeruleus are important pathological features of Parkinson's disease. There is an urgent need to develop therapies that slow down the progression of neurodegeneration in Parkinson's disease. In the present study, we tested whether the highly specific metabotropic glutamate receptor 5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, reduces dopaminergic and noradrenergic neuronal loss in monkeys rendered parkinsonian by chronic treatment with low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Weekly intramuscular 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections (0.2-0.5 mg/kg body weight), in combination with daily administration of 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine or vehicle, were performed until the development of parkinsonian motor symptoms in either of the two experimental groups (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine versus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle). After 21 weeks of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment, all 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated animals displayed parkinsonian symptoms, whereas none of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated monkeys were significantly affected. These behavioural observations were consistent with in vivo positron emission tomography dopamine transporter imaging data, and with post-mortem stereological counts of midbrain dopaminergic neurons, as well as striatal intensity measurements of dopamine transporter and tyrosine hydroxylase immunoreactivity, which were all significantly higher in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated animals than in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated monkeys. The 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine treatment also had a significant effect on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of norepinephrine neurons in the locus coeruleus and adjoining A5 and A7 noradrenaline cell groups. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/vehicle-treated animals, almost 40% loss of tyrosine hydroxylase-positive norepinephrine neurons was found in locus coeruleus/A5/A7 noradrenaline cell groups, whereas the extent of neuronal loss was lower than 15% of control values in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine-treated monkeys. Our data demonstrate that chronic treatment with the metabotropic glutamate receptor 5 antagonist, 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, significantly reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity towards dopaminergic and noradrenergic cell groups in non-human primates. This suggests that the use of metabotropic glutamate receptor 5 antagonists may be a useful strategy to reduce degeneration of catecholaminergic neurons in Parkinson's disease.

Behavioral responses of dopamine beta-hydroxylase knockout mice to modafinil suggest a dual noradrenergic-dopaminergic mechanism of action.

Modafinil is approved for use in the treatment of excessive daytime sleepiness. The precise mechanism of modafinil action has not been elucidated, although both dopamine (DA) and norepinephrine (NE) systems have been implicated. To explore the roles of DA and NE in the mechanism of modafinil-induced arousal, dopamine beta-hydroxylase knockout (Dbh -/-) mice were examined in behavioral paradigms of arousal (photobeam breaks and behavioral scoring of sleep latency). Dbh -/- mice completely lack NE but have hypersensitive DA signaling. It was hypothesized that Dbh -/- mice would be unresponsive to modafinil if the compound acts primarily via NE, but would be hypersensitive to modafinil if it acts primarily via DA. Dbh -/- mice had increased sensitivity to the locomotor-activating and wake-promoting effects of modafinil. Paradoxically, the alpha1-adrenergic receptor antagonist, prazosin, attenuated the effects of modafinil in control mice, but not in Dbh -/- mice. Blockade of DA receptors with flupenthixol decreased modafinil-induced locomotion and wake in both control and Dbh -/- mice. These results suggest that both NE and DA are involved in the behavioral effects of modafinil in control mice, but the requirement for NE can be bypassed by hypersensitive DA signaling.

The imprinted gene Magel2 regulates normal circadian output.

Mammalian circadian rhythms of activity are generated within the suprachiasmatic nucleus (SCN). Transcripts from the imprinted, paternally expressed Magel2 gene, which maps to the chromosomal region associated with Prader-Willi Syndrome (PWS), are highly enriched in the SCN. The Magel2 message is circadianly expressed and peaks during the subjective day. Mice deficient in Magel2 expression entrain to light cycles and express normal running-wheel rhythms, but with markedly reduced amplitude of activity and increased daytime activity. These changes are associated with reductions in food intake and male fertility. Orexin levels and orexin-positive neurons in the lateral hypothalamus are substantially reduced, suggesting that some of the consequences of Magel2 loss are mediated through changes in orexin signaling. The robust rhythmicity of Magel2 expression in the SCN and the altered behavioral rhythmicity of null mice reveal Magel2 to be a clock-controlled circadian output gene whose disruption results in some of the phenotypes characteristic of PWS.