Impaired myelin ultrastructure is reversed by citalopram treatment in a mouse model for major depressive disorder.

Major depressive disorder (MDD) is the most common and widespread mental disorder. Selective serotonin reuptake inhibitors (SSRIs) are the first-line treatment for MDD. The relation between the inhibition of serotonin reuptake in the central nervous system and remission from MDD remains controversial, as reuptake inhibition occurs rapidly, but remission from MDD takes weeks to months. Myelination-related deficits and white matter abnormalities were shown to be involved in psychiatric disorders such as MDD. This may explain the delay in remission following SSRI administration. The raphe nuclei (RN), located in the brain stem, consist of clusters of serotonergic (5-HT) neurons that project to almost all regions of the brain. Thus, the RN are an intriguing area for research of the potential effect of SSRI on myelination, and their involvement in MDD. MicroRNAs (miRNAs) regulate many biological features that might be altered by antidepressants. Two cohorts of chronic unpredictable stress (CUS) mouse model for depression underwent behavioral tests for evaluating stress, anxiety, and depression levels. Following application of the CUS protocol and treatment with the SSRI, citalopram, 48 mice of the second cohort were tested via magnetic resonance imaging and diffusion tensor imaging for differences in brain white matter tracts. RN and superior colliculus were excised from both cohorts and measured for changes in miRNAs, mRNA, and protein levels of candidate genes. Using MRI-DTI scans we found lower fractional anisotropy and axial diffusivity in brains of stressed mice. Moreover, both miR-30b-5p and miR-101a-3p were found to be downregulated in the RN following CUS, and upregulated following CUS and citalopram treatment. The direct binding of these miRNAs to Qki, and the subsequent effects on mRNA and protein levels of myelin basic protein (Mbp), indicated involvement of these miRNAs in myelination ultrastructure processes in the RN, in response to CUS followed by SSRI treatment. We suggest that SSRIs are implicated in repairing myelin deficits resulting from chronic stress that leads to depression.

The antipsychotropic drug Duloxetine rescues PAX6 haploinsufficiency of mutant limbal stem cells through inhibition of the MEK/ERK signaling pathway.

Aniridia is a panocular disease causing progressive severe visual impairment and blindness due to PAX-6 haploinsufficiency. One of the most disabling ocular symptoms is aniridia-related keratopathy (ARK), a progressive corneal opacification due to epithelial impairment, vascular and conjunctival pathologies. There is currently no available treatment to prevent progressive visual loss. For this aim, we have used mutant limbal cells for phenotypic screening using FDA-approved and bio-actives drug library and found Duloxetine, a serotonin and norepinephrine reuptake inhibitor used against severe depression as able to enhance endogenous PAX6 expression and target genes, which returned fairly to amounts found in normal limbal cells. In addition, Duloxetine could restore cell migration of the mutant cells. Furthermore, we show that Duloxetine activates PAX6 through inhibition of the ERK pathway on limbal mutant cells. This observation fits the recent report that MEK inhibitors enhance PAX6 in vivo, partially rescuing aniridia developmental phenotype of Pax6+/- mice. The discovery of an unique compound able to enhance PAX6 activity and that could be locally administered using eye drops associated with drug repurposing is expected to lead to rapid development of applicable drugs for the topical (eye drops) treatment of aniridia.

Ritanserin, a potent serotonin 2A receptor antagonist, represses MEK/ERK signalling pathway to restore PAX6 production and function in aniridia-like cellular model.

Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency.

Synergy of oxytocin and citalopram in modulating Itgb3/Chl1 interplay: Relevance to sensitivity to SSRI therapy.

Intranasal treatment with oxytocin showed beneficial effects in post-traumatic stress disorder and autism spectrum disorders; however, it was not investigated as much in depression. Keeping in mind the favorable effects of oxytocin on animal models of anxiety and depression, we postulated that synergy between prescribed first choice drugs, selective serotonin reuptake inhibitors (SSRIs) and oxytocin could improve the treatment outcome compared with SSRI monotherapy. Our previous in vitro genome-wide transcriptomic study on human lymphoblastoid cell lines exposed to paroxetine resulted in increase of integrin ß3 (ITGB3) gene expression, and further, ITGB3/CHL1 expression ratio was hypothesized to influence the sensitivity to SSRIs. The aim of this report was to explore molecular mechanisms behind the antidepressant-like oxytocin effect, alone and in synergy with citalopram, on behavioral and molecular level in corticosterone treated rats, a paradigm used to model anxiety and depression in animals. Oxytocin treatment (1) ameliorated corticosterone-induced reduction of neurogenesis and number of parvalbumin-positive interneurons in the hippocampal CA1 region, (2) enhanced anxiolytic- and antidepressant-like effects of citalopram in the open field test, and (3) the SSRI/oxytocin synergy persisted in reversing the reduction of the Itgb3 gene expression and increased Itgb3/Chl1 ratio in the prefrontal cortices. These results support the existence of synergy between citalopram and oxytocin in reversing the molecular and behavioral changes induced by corticosterone treatment and point to possible molecular mechanisms behind antidepressant-like effect of oxytocin.

Antifungal activity, mode of action variability, and subcellular distribution of coumarin-based antifungal azoles.

Azole antifungals inhibit the biosynthesis of ergosterol, the fungal equivalent of cholesterol in mammalian cells. Here we report an investigation of the activity of coumarin-substituted azole antifungals. Screening against a panel of Candida pathogens, including a mutant lacking CYP51, the target of antifungal azoles, revealed that this enzyme is inhibited by triazole-based antifungals, whereas imidazole-based derivatives have more than one mode of action. The imidazole-bearing antifungals more effectively reduced trailing growth associated with persistence and/or recurrence of fungal infections than triazole-based derivatives. The imidazole derivatives were more toxic to mammalian cells and more potently inhibited the activity of CYP3A4, which is one of the main causes of azole toxicity. Using live cell imaging, we showed that regardless of the type of azole ring fluorescent 7-diethylaminocoumarin-based azoles localized to the endoplasmic reticulum, the organelle that harbors CYP51. This study suggests that the coumarin is a promising scaffold for development of novel azole-based antifungals that effectively localize to the fungal cell endoplasmic reticulum.

MicroRNA-Mediated Regulation of ITGB3 and CHL1 Is Implicated in SSRI Action.

Background: Selective serotonin reuptake inhibitor (SSRI) antidepressant drugs are the first-line of treatment for major depressive disorder (MDD) but are effective in <70% of patients. Our earlier genome-wide studies indicated that two genes encoding for cell adhesion proteins, close homolog of L1 (CHL1) and integrin beta-3 (ITGB3), and microRNAs, miR-151a-3p and miR-221/222, are implicated in the variable sensitivity and response of human lymphoblastoid cell lines (LCL) from unrelated individuals to SSRI drugs. Methods: The microRNAs miR-221, miR-222, and miR-151-a-3p, along with their target gene binding sites, were explored in silico using miRBase, TargetScan, microRNAviewer, and the UCSC Genome Browser. Luciferase reporter assays were conducted for demonstrating the direct functional regulation of ITGB3 and CHL1 expression by miR-221/222 and miR-151a-3p, respectively. A human LCL exhibiting low sensitivity to paroxetine was utilized for studying the phenotypic effect of CHL1 regulation by miR-151a-3p on SSRI response. Results: By showing direct regulation of CHL1 and ITGB3 by miR-151a-3p and miR-221/222, respectively, we link these microRNAs and genes with cellular SSRI sensitivity phenotypes. We report that miR-151a-3p increases cell sensitivity to paroxetine via down-regulating CHL1 expression. Conclusions: miR-151a-3p, miR-221/222 and their (here confirmed) respective target-genes, CHL1 and ITGB3, are implicated in SSRI responsiveness, and possibly in the clinical response to antidepressant drugs.

Sex differences in human lymphoblastoid cells sensitivities to antipsychotic drugs.

Adverse drug reactions (ADRs) are a major concern in pharmacotherapy and are more common among women. Immortalized human lymphoblastoid cell lines (LCLs) are emerging as a novel tool for studying interindividual variability in drug response, including ADRs. In the present study, we compared sensitivities of LCLs from unrelated healthy male and female donors to growth inhibition by a panel of common drugs. We observed large interindividual drug sensitivity variations with similar mean sensitivities recorded for LCLs from male and female donors for most tested drugs. A notable exception was observed for the typical antipsychotic haloperidol and the atypical antipsychotic risperidone, which exhibited, on average, more robust in vitro growth inhibition in male as compared with female LCLs. An opposite finding was observed for the antidepressant paroxetine, which was more potent for inhibiting the growth of female as compared with male LCLs. These observations are discussed in the context of the higher incidence of dystonia reported for male schizophrenia patients treated with haloperidol and the higher efficacy of paroxetine in female major depression patients.

Genome-wide transcriptomic variations of human lymphoblastoid cell lines: insights from pairwise gene-expression correlations.

AIMS: Human lymphoblastoid cell lines (LCLs) are a rich resource of information on human interindividual genomic, transcriptomic, proteomic and phenomic variations, and are therefore gaining popularity for pharmacogenomic studies. In the present study we demonstrate that genome-wide transcriptomic data from a small LCL panel from unrelated individuals is sufficient for detecting pairs of genes that exhibit highly correlated expression levels and may thus convey insights about coregulated genes. MATERIALS & METHODS: RNA samples were prepared from LCLs representing 12 unrelated healthy adult female Caucasian donors. Transcript levels were determined with the Affymetrix Human Gene arrays. Expression-level correlations were searched using Partek(®) Genomics Suite™ and the R environment. Sequences of detected correlated gene pairs were compared for shared conserved 3´-UTR miRNA binding. RESULTS: Most of the approximately 33,000 transcripts covered by the Affymetrix arrays showed closely similar expression levels in LCLs from unrelated donors. However, the expression levels of some transcripts showed large inter-individual variations. When comparing the expression levels of each of the top 1000 genes showing the largest interindividual expression variations against the others, two sets containing 156 and 4438 correlated gene pairs with false-discovery rates of 0.01 and 0.05 were detected, respectively. Similar analysis of another gene-expression data set from LCLs (GSE11582) indicated that 61 and 39% of identified pairs matched the pairs detected from our transcriptomic data, respectively. Shared conserved 3´-UTR miRNA binding sites were noted for 14-17% of the top 100 gene pairs, suggesting that regulation by miRNA may contribute to their coordinated expression. CONCLUSION: Probing genome-wide transcriptomic data sets of LCLs from unrelated individuals may detect coregulated genes, adding insights on cellular regulation by miRNAs.

Genome-wide miRNA expression profiling of human lymphoblastoid cell lines identifies tentative SSRI antidepressant response biomarkers.

AIM: Over 30% of patients with major depression do not respond well to first-line treatment with selective serotonin reuptake inhibitors (SSRIs). Using genome-wide expression profiling of human lymphoblastoid cell lines (LCLs) CHL1 was identified as a tentative SSRI sensitivity biomarker. This study reports on miRNAs implicated in SSRI sensitivity of LCLs. METHODS: Eighty LCLs were screened from healthy adult female individuals for growth inhibition by paroxetine. Eight LCLs exhibiting high or low sensitivities to paroxetine were chosen for genome-wide expression profiling with miRNA microarrays. RESULTS: The miRNA miR-151-3p had 6.7-fold higher basal expression in paroxetine-sensitive LCLs. This corresponds with lower expression of CHL1, a target of miR-151-3p. The additional miRNAs miR-212, miR-132, miR-30b*, let-7b and let-7c also differed by >1.5-fold (p < 0.05) between the two LCL groups. CONCLUSION: The potential value of these miRNAs as tentative SSRI response biomarkers awaits validation with lymphocyte samples of major depression patients.