Systemic response to rupture of intracranial aneurysms involves expression of specific gene isoforms.

BACKGROUND: Rupture of an intracranial aneurysm (IA) causes a systemic response that involves an immune/inflammatory reaction. Our previous study revealed a downregulation of genes related to T lymphocytes and an upregulation of genes related to monocytes and neutrophils after IA rupture. It remains unknown whether that resulted from alterations in transcription or cell count. We sought to characterize the systemic response to IA rupture through analysis of transcript expression profiles in peripheral blood cells. We also investigated effects of IA rupture on the composition of mononuclear cells in peripheral blood. METHODS: We included 19 patients in the acute phase of IA rupture (RAA, first 72 h), 20 patients in the chronic phase (RAC, 3-15 months), and 20 controls. Using deep transcriptome sequencing, we analyzed the expression of protein-coding and noncoding RNAs. Expression levels, transcript biotypes, alternative splicing and other features of the regulated transcripts were studied. A functional analysis was performed to determine overrepresented ontological groups among gene expression profiles. Flow cytometry was used to analyze alterations in the level of mononuclear leukocyte subpopulations. RESULTS: Comparing RAA and controls, we identified 491 differentially expressed transcripts (303 were downregulated, and 188 were upregulated in RAA). The results indicate that the molecular changes in response to IA rupture occur at the level of individual transcripts. Functional analysis revealed that the most impacted biological processes are related to regulation of lymphocyte activation and toll-like receptor signaling pathway. Differences between RAC and controls were less prominent. Analysis of leukocyte subsets revealed a significantly decreased number of CD4+ lymphocytes and increase of classical and intermediate monocytes in RAA patients compared to controls. CONCLUSIONS: IA rupture in the acute phase strongly influences the transcription profiles of peripheral blood cells as well as the composition of mononuclear cells. A specific pattern of gene expression alteration was found, suggesting a depression of lymphocyte response and enhancement of monocyte activity.

Expression of alternatively spliced variants of the Dclk1 gene is regulated by psychotropic drugs.

BACKGROUND: The long-term effects of psychotropic drugs are associated with the reversal of disease-related alterations through the reorganization and normalization of neuronal connections. Molecular factors that trigger drug-induced brain plasticity remain only partly understood. Doublecortin-like kinase 1 (Dclk1) possesses microtubule-polymerizing activity during synaptic plasticity and neurogenesis. However, the Dclk1 gene shows a complex profile of transcriptional regulation, with two alternative promoters and exon splicing patterns that suggest the expression of multiple isoforms with different kinase activities. RESULTS: Here, we applied next-generation sequencing to analyze changes in the expression of Dclk1 gene isoforms in the brain in response to several psychoactive drugs with diverse pharmacological mechanisms of action. We used bioinformatics tools to define the range and levels of Dclk1 transcriptional regulation in the mouse nucleus accumbens and prefrontal cortex. We also sought to investigate the presence of DCLK1-derived peptides using mass spectrometry. We detected 15 transcripts expressed from the Dclk1 locus (FPKM > 1), including 2 drug-regulated variants (fold change > 2). Drugs that act on serotonin receptors (5-HT2A/C) regulate a subset of Dclk1 isoforms in a brain-region-specific manner. The strongest influence was observed for the mianserin-induced expression of an isoform with intron retention. The drug-activated expression of novel alternative Dclk1 isoforms was validated using qPCR. The drug-regulated isoform contains genetic variants of DCLK1 that have been previously associated with schizophrenia and hyperactivity disorder in humans. We identified a short peptide that might originate from the novel DCLK1 protein product. Moreover, protein domains encoded by the regulated variant indicate their potential involvement in the negative regulation of the canonical DCLK1 protein. CONCLUSIONS: In summary, we identified novel isoforms of the neuroplasticity-related gene Dclk1 that are expressed in the brain in response to psychotropic drug treatments.

Molecular profile of dissociative drug ketamine in relation to its rapid antidepressant action.

BACKGROUND: The NMDA receptor antagonist ketamine was found to act as a fast-acting antidepressant. The effects of single treatment were reported to persist for days to weeks, even in otherwise treatment-refractory cases. Identification of the mechanisms underlying ketamine's antidepressant action may permit development of novel drugs, with similar clinical properties but lacking psychotomimetic, sedative and other side effects. METHODS: We applied whole-genome microarray profiling to analyze detailed time-course (1, 2, 4 and 8 h) of transcriptome alterations in the striatum and hippocampus following acute administration of ketamine, memantine and phencyclidine in C57BL/6 J mice. The transcriptional effects of ketamine were further analyzed using next-generation sequencing and quantitative PCR. Gene expression alterations induced by the NMDA antagonists were compared to the molecular profiles of psychotropic drugs: antidepressants, antipsychotics, anxiolytics, psychostimulants and opioids. RESULTS: We identified 52 transcripts (e.g. Dusp1, Per1 and Fkbp5) with altered expression (FDR < 1 %) in response to treatment with NMDA receptor antagonists. Functional links that connect expression of the regulated genes to the MAPK, IL-6 and insulin signaling pathways were indicated. Moreover, ketamine-regulated expression of specific gene isoforms was detected (e.g. Tsc22d3, Sgk1 and Hif3a). The comparison with other psychotropic drugs revealed that the molecular effects of ketamine are most similar to memantine and phencyclidine. Clustering based on expression profiles placed the NMDA antagonists among fluoxetine, tianeptine, as well as opioids and ethanol. CONCLUSIONS: The identified patterns of gene expression alteration in the brain provided novel molecular classification of ketamine. The transcriptional profile of ketamine reflects its multi-target pharmacological nature. The results reveal similarities between the effects of ketamine and monoaminergic antidepressants that may explain the mechanisms of its rapid antidepressant action.

Decoding the transcriptional programs activated by psychotropic drugs in the brain.

Analysis of drug-induced gene expression in the brain has long held the promise of revealing the molecular mechanisms of drug actions as well as predicting their long-term clinical efficacy. However, despite some successes, this promise has yet to be fulfilled. Here, we present an overview of the current state of understanding of drug-induced gene expression in the brain and consider the obstacles to achieving a robust prediction of the properties of psychoactive compounds based on gene expression profiles. We begin with a comprehensive overview of the mechanisms controlling drug-inducible transcription and the complexity resulting from expression of noncoding RNAs and alternative gene isoforms. Particular interest is placed on studies that examine the associations within drug classes with regard to the effects on gene transcription, alterations in cell signaling and neuropharmacological drug properties. While the ability of gene expression signatures to distinguish specific clinical classes of psychotropic and addictive drugs remains unclear, some reports show that under specific constraints, drug properties can be predicted based on gene expression. Such signatures offer a simple and effective way to classify psychotropic drugs and screen novel psychoactive compounds. Finally, we note that the amount of data regarding molecular programs activated in the brain by drug treatment has grown exponentially in recent years and that future advances may therefore come in large part from integrating the currently available high-throughput data sets.

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages.

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on Mϕ. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived Mϕs (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (α and ß) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria Staphylococcus aureus (SA) and Porphyromonas gingivalis (PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a "signaling complex chaperone," with activity that is essential for cytokine response but not for target engulfment by Mϕ.

NMDA Receptors on Dopaminoceptive Neurons Are Essential for Drug-Induced Conditioned Place Preference.

Plasticity of the brain's dopamine system plays a crucial role in adaptive behavior by regulating appetitive motivation and the control of reinforcement learning. In this study, we investigated drug- and natural-reward conditioned behaviors in a mouse model in which the NMDA receptor-dependent plasticity of dopaminoceptive neurons was disrupted. We generated a transgenic mouse line with inducible selective inactivation of the NR1 subunit in neurons expressing dopamine D1 receptors (the NR1(D1CreERT2) mice). Whole-cell recordings of spontaneous EPSCs on neurons in the nucleus accumbens confirmed that a population of neurons lacked the NMDA receptor-dependent component of the current. This effect was accompanied by impaired long-term potentiation in the nucleus accumbens and in the CA1 area of the ventral, but not the dorsal, hippocampus. Mutant mice did not differ from control animals when tested for pavlovian or instrumental conditioning. However, NR1(D1CreERT2) mice acquired no preference for a context associated with administration of drugs of abuse. In the conditioned place preference paradigm, mutant mice did not spend more time in the context paired with cocaine, morphine, or ethanol, although these mice acquired a preference for sucrose jelly and an aversion to naloxone injections, as normal. Thus, we observed that the selective inducible ablation of the NMDA receptors specifically blocks drug-associated context memory with no effect on positive reinforcement in general.