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BackgroundNeuronal ceroid lipofuscinoses (NCLs) comprise at least eight genetically characterized neurodegenerative disorders of childhood. Despite of genetic heterogeneity, the high similarity of clinical symptoms and pathology of different NCL disorders suggest cooperation between different NCL proteins and common mechanisms of pathogenesis. Here, we have studied molecular interactions between NCL proteins, concentrating specifically on the interactions of CLN5, the protein underlying the Finnish variant late infantile form of NCL (vLINCLFin).ResultsWe found that CLN5 interacts with several other NCL proteins namely, CLN1/PPT1, CLN2/TPP1, CLN3, CLN6 and CLN8. Furthermore, analysis of the intracellular targeting of CLN5 together with the interacting NCL proteins revealed that over-expression of PPT1 can facilitate the lysosomal transport of mutated CLN5FinMajor, normally residing in the ER and in the Golgi complex. The significance of the novel interaction between CLN5 and PPT1 was further supported by the finding that CLN5 was also able to bind the F1-ATPase, earlier shown to interact with PPT1.ConclusionWe have described novel interactions between CLN5 and several NCL proteins, suggesting a modifying role for these proteins in the pathogenesis of individual NCL disorders. Among these novel interactions, binding of CLN5 to CLN1/PPT1 is suggested to be the most significant one, since over-expression of PPT1 was shown to influence on the intracellular trafficking of mutated CLN5, and they were shown to share a binding partner outside the NCL protein spectrum.

MicroRNAs (miRNAs) are small regulatory molecules that cause post-transcriptional gene silencing. Although some miRNAs are known to have region-specific expression patterns in the adult brain, the functional consequences of the region-specificity to the gene regulatory networks of the brain nuclei are not clear. Therefore, we studied miRNA expression patterns by miRNA-Seq and microarrays in two brain regions, frontal cortex (FCx) and hippocampus (HP), which have separate biological functions. We identified 354 miRNAs from FCx and 408 from HP using miRNA-Seq, and 245 from FCx and 238 from HP with microarrays. Several miRNA families and clusters were differentially expressed between FCx and HP, including the miR-8 family, miR-182|miR-96|miR-183 cluster, and miR-212|miR-312 cluster overexpressed in FCx and miR-34 family overexpressed in HP. To visualize the clusters, we developed support for viewing genomic alignments of miRNA-Seq reads in the Chipster genome browser. We carried out pathway analysis of the predicted target genes of differentially expressed miRNA families and clusters to assess their putative biological functions. Interestingly, several miRNAs from the same family/cluster were predicted to regulate specific biological pathways. We have developed a miRNA-Seq approach with a bioinformatic analysis workflow that is suitable for studying miRNA expression patterns from specific brain nuclei. FCx and HP were shown to have distinct miRNA expression patterns which were reflected in the predicted gene regulatory pathways. This methodology can be applied for the identification of brain region-specific and phenotype-specific miRNA-mRNA-regulatory networks from the adult and developing rodent brain.

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Genetic mapping efforts have identified putative susceptibility genes for human anxiety disorders. The most intensively studied genes are involved in neurotransmitter metabolism and signaling or stress response. In addition, neuropeptides and targets of anxiolytics have been examined. It has become apparent that gene × environment interactions may explain individual variation in stress resilience and predisposition to mental disorders. We aimed to replicate previous genetic findings in 16 putative anxiety susceptibility genes and further test whether they modulate the risk for developing an anxiety disorder in adulthood after childhood stress exposure. We tested 93 single-nucleotide polymorphisms (SNPs) for genetic association to anxiety disorders in the Finnish population-based Health 2000 sample (282 cases and 575 matched controls). In addition, we examined by logistic regression modeling whether the SNP genotypes modified the effect of the number of self-reported childhood adversities on anxiety disorder risk. The most significant evidence for association was observed in glutamate decarboxylase 1 (GAD1) with phobias (P = 0.0005). A subsequent meta-analysis (N = 1985) incorporating previously published findings supported involvement of a single GAD1 risk haplotype in determining susceptibility to a broad range of internalizing disorders (P = 0.0009). We additionally found that SNPs and haplotypes in neuropeptide Y (NPY) modified the effect of childhood adversities on anxiety susceptibility (P = 0.003). In conclusion, we provide further support for involvement of mainly GAD1, but also NPY in determining predisposition to anxiety disorders.

Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p < 0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1a and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.

Diversity in the structure and expression of microRNAs, important regulators of gene expression, arises from SNPs, duplications followed by divergence, production of isomiRs, and RNA editing. Inbred mouse strains and crosses using them are important reference populations for genetic mapping, and as models of human disease. We determined the nature and extent of interstrain miRNA variation by (i) identifying miRNA SNPs in whole-genome sequence data from 36 strains, and (ii) examining miRNA editing and expression in hippocampus (Hpc) and frontal cortex (FCx) of six strains, to facilitate the study of miRNAs in neurobehavioral phenotypes. miRNA loci were strongly conserved among the 36 strains, but even the highly conserved seed region contained 16 SNPs. In contrast, we identified RNA editing in 58.9% of miRNAs, including 11 consistent editing events in the seed region. We confirmed the functional significance of three conserved edits in the miR-379/410 cluster, demonstrating that edited miRNAs gained novel target mRNAs not recognized by the unedited miRNAs. We found significant interstrain differences in miRNA and isomiR expression: Of 779 miRNAs expressed in Hpc and 719 in FCx, 262 were differentially expressed (190 in Hpc, 126 in FCx, 54 in both). We also identified 32 novel miRNA candidates using miRNA prediction tools. Our studies provide the first comprehensive analysis of SNP, isomiR, and RNA editing variation in miRNA loci across inbred mouse strains, and a detailed catalog of expressed miRNAs in Hpc and FCx in six commonly used strains. These findings will facilitate the molecular analysis of neurological and behavioral phenotypes in this model organism.

Anxiety is a normal response to a threatening situation. When anxiety is excessive and disturbs daily life and functioning, it can be diagnosed as an anxiety disorder. Anxiety disorders are typical complex disorders that are triggered by environmental factors in genetically susceptible individuals. Genetic variants in many genes have been shown to predispose to anxiety disorders, but the results have been difficult to replicate, most likely due to a small effect size of individual variants. Anxiety is an evolutionarily conserved response, and can therefore be fairly reliably modelled in rodents. Quantitative trait locus mapping and functional genomics and proteomics approaches in mouse models of anxiety have led to the identification of novel genes and pathways that are involved in the regulation of anxiety‐like behaviour. Some of these genes are expected to regulate anxiety in humans as well, and variants in them might predispose to anxiety disorders. Key Concepts: Anxiety disorders are complex diseases triggered by environmental factors in genetically susceptible individuals. Genetic variants that predispose to anxiety disorders have been identified by candidate gene and genome‐wide association studies. The effect size of the identified susceptibility variants for anxiety disorders seems to be small. Features of human anxiety disorders can be fairly well modelled in animals, and different approaches have revealed novel genes and pathways that regulate anxiety‐like behaviour. Combined information from several approaches, including human and mouse genetics, functional genomics, proteomics, epigenetics and neuroimaging are expected to reveal molecular mechanisms that regulate anxiety.