Mari-Anne Philips

Cell type- and brain region-resolved mouse brain proteome

Brain transcriptome and connectome maps are being generated, but an equivalent effort on the proteome is currently lacking. We performed high-resolution mass spectrometry–based proteomics for in-depth analysis of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type–specific proteins defined as tenfold more abundant than average expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.

Sociability trait and serotonin metabolism in the rat social interaction test

Social behaviour is the basis of one of the most generally accepted independent dimensions of personality. The purpose of the present study was to find out whether the social activity of individual rats, expressed in the social interaction test of anxiety, is consistent, and associated with monoamine levels. Four social interaction tests with 10 days intervals were carried out in 20 rats, and the animals were decapitated 4 days after the last test. There was no consistent correlation between performances in single tests, but the social interaction time in each test correlated strongly with the mean values of social activity in all or the other three tests. Social interaction time of rats correlated moderately but significantly with their partners social activity in the test. The average social interaction time correlated strongly with 5-HIAA levels in the frontal cortex (r = -0.67, P < 0.01). Neither exposure of rats singly to the social interaction test box nor the test procedure had any effect on monoamine levels. When animals were decapitated immediately after a single social interaction test, there was a negative correlation between the social interaction time and 5-HIAA and 5-HT levels in the septum, but not in the frontal cortex or hippocampus. Thus, social behaviour is a stable trait, expression of which depends in part upon the partners social behaviour. This trait is negatively associated with 5-HT metabolism in the frontal cortex. Social activity of rats in a particular test situation may rather be related to 5-HT metabolism in the septum.

Lower anxiety and a decrease in agonistic behaviour in Lsamp-deficient mice.

In rodents, the Lsamp gene has been implicated in trait anxiety, fear reaction and fear conditioning. Human data link the LSAMP gene to several psychiatric disorders. In this study, we presented a general phenotypic characterization of Lsamp gene-deficient mouse line, created by deleting exon 1b. These mice displayed no gross sensory-motor deficiencies, no overt abnormalities and performed normally in memory and learning tests. However, they responded with increased activity to new environments. Moreover, they displayed reduced anxiety and notable deviations in social behaviour, such as lack of whisker trimming, reduced aggressiveness and reduced dominance. One possible explanation for the anxiolytic-like effect of the deletion of the Lsamp gene is a shift in balance in the Gabra1 and Gabra2 genes in the temporal lobe in favor of the Gabra2 transcript, encoding α2 subunit of GABA(A) receptors that mediate the stimulating effect of GABA agonists. The overall phenotype of Lsamp-deficient mice, characterized by decreased anxiety and several alterations in social behaviour, makes them a good model for studying the molecular mechanisms behind inadequate social behaviours observed in several psychiatric disorders.

Alterations in opioid system of the rat brain after cat odor exposure

The effect of cat odor exposure was studied on morphine-induced increase of exploratory behavior and on the expression of opioid genes in forebrain structures of male Wistar rats. Treatment with morphine (1 mg/kg) induced a significant increase in exploratory behavior in an unfamiliar environment in rats. Previous exposure of animals to cat odor completely abolished this stimulating action of mu-opioid receptor agonist on exploratory activity. Cat odor exposure induced a significant increase in the expression of pro-opio-melanocortin (POMC) and mu-opioid receptor (MOR) genes in the brain structures related to anxiety and motivation. This study clearly demonstrates that cat odor exposure increases the activity of opioid system in rat forebrain structures.

Deletion of the Lsamp gene lowers sensitivity to stressful environmental manipulations in mice.

The Lsamp gene gives rise to limbic system-associated membrane protein (LAMP), which is expressed on the surface of somata and proximal dendrites of neurons. Lsamp-deficient mice have been shown to be slightly hyperactive in novel environments and less anxious, and they display alterations in swimming speed, fear reaction, fear conditioning and social behaviour. In human studies, links between the LSAMP gene and several psychiatric disorders have been found and LSAMP has been established as a tumour suppressor gene. To study the impact of environmental manipulations on the phenotype, we exposed male Lsamp-deficient mice to environmental enrichment (EE), a technique that has often been shown to abolish phenotypic deviations in knockout mice, and to social isolation, a stressful manipulation, after which all the mice were tested in a behavioural battery. EE abolished differences between the genotypes in body weight and anogenital sniffing, a behaviour related to aggressiveness, and amplified the anxiolytic-like phenotype of Lsamp-deficient mice both in the plus maze and motility box. Isolation abolished differences between the genotypes in body weight and anxiety and amplified the differences in swimming speed and anogenital sniffing. EE and isolation failed to modify the results as compared to standard housing in whisker trimming, locomotor activity, marble burying and corticosterone levels. In conclusion, Lsamp-deficient mice were less sensitive to isolation stress than their wild-type littermates. Lack of LAMP protein seemingly leads to a deterioration in the ability to adapt to novel stressful environments and stimuli.

Cat odour exposure decreases exploratory activity and alters neuropeptide gene expression in CCK2 receptor deficient mice, but not in their wild-type littermates

An attempt was made to establish whether the anxiogenic effect of cat odour differs in female wild-type and CCK(2) receptor deficient mice, having different exploratory activity in the elevated plus-maze. The exposure of wild-type and homozygous CCK(2) receptor deficient mice to cat odour did not reveal substantial differences between the two genotypes. The number of contacts with the cat odour impregnated cloth was reduced and the frequency of stretch-attend postures was increased similarly in wild-type and homozygous mice. However, the following exposure of mice to the elevated plus-maze established differences as homozygous mice displayed increased exploratory activity in the plus-maze. The cat odour exposure significantly reduced exploratory activity only in homozygous mice. Together with the increased exploratory activity we established in homozygous mice significantly increased expression of the Oprm1 gene in the frontal cortex and mesencephalon. The exposure of mice to cat odour caused only minor changes in the gene expression of wild-type mice, whereas in homozygous animals a significantly increased expression of the Mc3r gene in the frontal cortex and temporal lobe, and the Pomc1 gene in the temporal lobe, mesencephalon and mesolimbic area was established. In conclusion, CCK(2) receptor deficient mice displayed reduced anxiety compared to their wild-type littermates in the plus-maze test. This behavioural effect seems to be related, at least partly, to an increased tone of opioid system in the brain. Moreover, homozygous mice respond to the exposure of cat odour with an increased anxiety. This effect seems to be related to the increased function of the melanocortin system in the brain structures of genetically modified mice.

Limbic system-associated membrane protein as a potential target for neuropsychiatric disorders

The studies performed in laboratory animals and psychiatric patients suggest a possible role of limbic system-associated membrane protein (LAMP) in the mechanisms of psychiatric disorders. Stressful manipulations and genetic invalidation have revealed a role of the Lsamp gene in the regulation of anxiety in rodents. Besides that, Lsamp-deficient mice display reduced aggressiveness and impaired adaptation in novel and stressful environments. The behavioral effects of amphetamine were blunted in genetically modified mice. Recent pharmacological and biochemical studies point toward altered function of GABA-, 5-hydroxytryptamine-, and dopaminergic systems in Lsamp-deficient mice. Moreover, we found an association between the gene polymorphisms of LSAMP and major depressive disorder (MDD). Patients suffering from MDD had significantly increased ratio between risk and protective haplotypes of the LSAMP gene compared to healthy volunteers. However, the impact of these haplotypes for the function of LAMP is not clear and remains to be elucidated in future studies.

Promoter polymorphism -119C/G in MYG1 (C12orf10) gene is related to vitiligo susceptibility and Arg4Gln affects mitochondrial entrance of Myg1

BackgroundMYG1 (Melanocyte proliferating gene 1, also C12orf10 in human) is a ubiquitous nucleo-mitochondrial protein, involved in early developmental processes and in adult stress/illness conditions. We recently showed that MYG1 mRNA expression is elevated in the skin of vitiligo patients. Our aim was to examine nine known polymorphisms in the MYG1 gene, to investigate their functionality, and to study their association with vitiligo susceptibility.MethodsNine single nucleotide polymorphisms (SNPs) in the MYG1 locus were investigated by SNPlex assay and/or sequencing in vitiligo patients (n = 124) and controls (n = 325). MYG1 expression in skin biopsies was detected by quantitative-real time PCR (Q-RT-PCR) and polymorphisms were further analysed using luciferase and YFP reporters in the cell culture.ResultsControl subjects with -119G promoter allele (rs1465073) exhibited significantly higher MYG1 mRNA levels than controls with -119C allele (P = 0.01). Higher activity of -119G promoter was confirmed by luciferase assay. Single marker association analysis showed that the -119G allele was more frequent in vitiligo patients (47.1%) compared to controls (39.3%, P < 0.05, OR 1.37, 95%CI 1.02-1.85). Analysis based on the stage of progression of the vitiligo revealed that the increased frequency of -119G allele occurred prevalently in the group of patients with active vitiligo (n = 86) compared to the control group (48.2% versus 39.3%, P < 0.05; OR 1.44, 95%CI 1.02-2.03). Additionally, we showed that glutamine in the fourth position (in Arg4Gln polymorphism) completely eliminated mitochondrial entrance of YFP-tagged Myg1 protein in cell culture. The analysis of available EST, cDNA and genomic DNA sequences revealed that Myg1 4Gln allele is remarkably present in human populations but is never detected in homozygous state according to the HapMap database.ConclusionsOur study demonstrated that both MYG1 promoter polymorphism -119C/G and Arg4Gln polymorphism in the mitochondrial signal of Myg1 have a functional impact on the regulation of the MYG1 gene and promoter polymorphism (-119C/G) is related with suspectibility for actively progressing vitiligo.

Lsamp is implicated in the regulation of emotional and social behavior by use of alternative promoters in the brain.

Limbic system-associated membrane protein (LSAMP) is a neural cell adhesion molecule involved in neurite formation and outgrowth. The purpose of the present study was to characterize the distribution of alternatively transcribed Lsamp isoforms in the mouse brain and its implications on the regulation of behavior. Limbic system-associated membrane protein 1b transcript was visualized by using a mouse strain expressing beta-galactosidase under the control of Lsamp 1b promoter. The distribution of Lsamp 1a transcript and summarized expression of the Lsamp transcripts was investigated by non-radioactive in situ RNA hybridization analysis. Cross-validation was performed by using radioactive in situ hybridization with oligonucleotide probes. Quantitative RT-PCR was used to study correlations between the expression of Lsamp isoforms and behavioral parameters. The expression pattern of two promoters differs remarkably from the developmental initiation at embryonic day 12.5. Limbic system-associated membrane protein 1a promoter is active in “classic” limbic structures where the hippocampus and amygdaloid area display the highest expression. Promoter 1b is mostly active in the thalamic sensory nuclei and cortical sensory areas, but also in areas that regulate stress and arousal. Higher levels of Lsamp 1a transcript had significant correlations with all of the measures indicating higher trait anxiety in the elevated plus-maze test. Limbic system-associated membrane protein transcript levels in the hippocampus and ventral striatum correlated with behavioral parameters in the social interaction test. The data are in line with decreased anxiety and alterations in social behavior in Lsamp-deficient mice. We propose that Lsamp is involved in emotional and social operating systems by complex regulation of two alternative promoters.

Enrichment and individual housing reinforce the differences in aggressiveness and amphetamine response in 129S6/SvEv and C57BL/6 strains

As different kinds of enrichment equipment are applied to standard rodent laboratory housing conditions in Europe (Directive 2010/63/EU) and worldwide, it is essential to understand how it may influence the brain and behaviour of animals. We observed common inbred mouse strains 129S6/SvEv/Tac (129) and C57BL/6 Bkl (B6) reared in 3 different environments: standard housing (SH), individual housing (IH) and enriched environment (EE). We measured common behavioural parameters, social behaviour and BDNF mRNA expression in hippocampus and frontal cortex. Our results demonstrate that the robust behavioural differences between B6 and 129 mouse strains which are well studied in the literature persist in varied housing conditions, but the response to these conditions has different directionality in studied strains. EE appears to reinforce the existing coping strategies in both strains: B6 became more agile, sensitive and venturous in behavioural models, whereas 129 became even more inhibited than they are in standard conditions. The stimulating effect of amphetamine is decreased in 129 animals that have been reared in EE. This may indicate desensitisation or inhibition of their dopamine system by EE. A frequently used biomarker in enrichment studies, BDNF, is a likely mediator for the long-term phenotype effects of EE. In this paper we demonstrate the differences of BDNF expression pattern in 129 and B6 mice.