Juan Pablo Lopez

miR-1202 is a primate-specific and brain-enriched microRNA involved in major depression and antidepressant treatment

Major depressive disorder (MDD) is a prevalent mood disorder that is associated with differential prefrontal brain expression patterns. Treatment of MDD includes a variety of biopsychosocial approaches. In medical practice, antidepressant drugs are the most common treatment for depressive episodes, and they are among the most prescribed medications in North America. Although antidepressants are clearly effective, particularly for moderate to severe depressive episodes, there is variability in how individuals respond to antidepressant treatment. Failure to respond has individual, economic and social consequences for patients and their families. Several lines of evidence demonstrate that genes are regulated through the activity of microRNAs (miRNAs), which act as fine-tuners and on-off switches of gene expression. Here we report on complementary studies using postmortem human brain samples, cellular assays and samples from clinical trials of patients with depression and show that miR-1202, a miRNA specific to primates and enriched in the human brain, is differentially expressed in individuals with depression. Additionally, miR-1202 regulates expression of the gene encoding metabotropic glutamate receptor-4 (GRM4) and predicts antidepressant response at baseline. These results suggest that miR-1202 is associated with the pathophysiology of depression and is a potential target for new antidepressant treatments.

Genome-Wide Methylation Changes in the Brains of Suicide Completers

OBJECTIVE Gene expression changes have been reported in the brains of suicide completers. More recently, differences in promoter DNA methylation between suicide completers and comparison subjects in specific genes have been associated with these changes in gene expression patterns, implicating DNA methylation alterations as a plausible component of the pathophysiology of suicide. The authors used a genome-wide approach to investigate the extent of DNA methylation alterations in the brains of suicide completers. METHOD Promoter DNA methylation was profiled using methylated DNA immunoprecipitation (MeDIP) followed by microarray hybridization in hippocampal tissue from 62 men (46 suicide completers and 16 comparison subjects). The correlation between promoter methylation and expression was investigated by comparing the MeDIP data with gene expression profiles generated through mRNA microarray. Methylation differences between groups were validated on neuronal and nonneuronal DNA fractions isolated by fluorescence-assisted cell sorting. RESULTS The authors identified 366 promoters that were differentially methylated in suicide completers relative to comparison subjects (273 hypermethylated and 93 hypomethylated). Overall, promoter methylation differences were inversely correlated with gene expression differences. Functional annotation analyses revealed an enrichment of differential methylation in the promoters of genes involved, among other functions, in cognitive processes. Validation was performed on the top genes from this category, and these differences were found to occur mainly in the neuronal cell fraction. CONCLUSIONS These results suggest broad reprogramming of promoter DNA methylation patterns in the hippocampus of suicide completers. This may help explain gene expression alterations associated with suicide and possibly behavioral changes increasing suicide risk.

Epigenetic regulation of BDNF expression according to antidepressant response

Several lines of evidence support the role of brain-derived neurotrophic factor (BDNF) in the pathophysiology and pharmacotherapy of depression.1 The neurotrophin hypothesis of depression postulates that stress and depression are associated with decreased BDNF expression, which can be reversed by antidepressant treatment.2 The goal of the present study was to investigate the effect of antidepressant treatment on the epigenetic regulation of BDNF in major depressive disorder (MDD).

Disruption of a Large Intergenic Noncoding RNA in Subjects with Neurodevelopmental Disabilities

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.

Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing

BackgroundSmall ncRNAs (sncRNAs) offer great hope as biomarkers of disease and response to treatment. This has been highlighted in the context of several medical conditions such as cancer, liver disease, cardiovascular disease, and central nervous system disorders, among many others. Here we assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing of small RNA sequencing data.MethodsA total of 45 biological samples were included in the present study. All libraries were prepared using the Illumina TruSeq Small RNA protocol and sequenced using the HiSeq2500 or MiSeq Illumina sequencers. Small RNA sequencing data was validated using qRT-PCR. At each stage, we evaluated the pros and cons of different techniques that may be suitable for different experimental designs. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.ResultsOur results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS. In addition, we describe the strengths and limitations of three commercially available library preparation methods: (1) Novex TBE PAGE gel; (2) Pippin Prep automated gel system; and (3) AMPure XP beads. We describe our bioinformatics pipeline, provide recommendations for sequencing coverage, and describe in detail the expression and distribution of all sncRNAs in four human tissues: whole-blood, brain, heart and liver.ConclusionsUltimately this study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate and effective high-throughput sequencing quantification method for various study designs, and overall generating valuable information that can contribute to our understanding of small ncRNAs as potential biomarkers and mediators of biological functions and disease.

dcc orchestrates the development of the prefrontal cortex during adolescence and is altered in psychiatric patients

Adolescence is a period of heightened susceptibility to psychiatric disorders of medial prefrontal cortex (mPFC) dysfunction and cognitive impairment. mPFC dopamine (DA) projections reach maturity only in early adulthood, when their control over cognition becomes fully functional. The mechanisms governing this protracted and unique development are unknown. Here we identify dcc as the first DA neuron gene to regulate mPFC connectivity during adolescence and dissect the mechanisms involved. Reduction or loss of dcc from DA neurons by Cre-lox recombination increased mPFC DA innervation. Underlying this was the presence of ectopic DA fibers that normally innervate non-cortical targets. Altered DA input changed the anatomy and electrophysiology of mPFC circuits, leading to enhanced cognitive flexibility. All phenotypes only emerged in adulthood. Using viral Cre, we demonstrated that dcc organizes mPFC wiring specifically during adolescence. Variations in DCC may determine differential predisposition to mPFC disorders in humans. Indeed, DCC expression is elevated in brains of antidepressant-free subjects who committed suicide.

MicroRNA regulation of central glial cell line-derived neurotrophic factor (GDNF) signalling in depression

Although multiple studies have reported that peripheral glial cell line-derived neurotrophic factor (GDNF) is reduced in depression, cerebral GDNF signalling has yet to be examined in this condition. Here, we report an isoform-specific decrease in GDNF family receptor alpha 1 (GFRA1) mRNA expression, resulting in lowered GFRα1a protein levels in basolateral amygdala (BLA) samples from depressed subjects. Downregulation of GFRα1a was associated with increased expression of microRNAs, including miR-511, predicted to bind to long 3’ untranslated region (3’-UTR)-containing transcripts (GFRA1-L) coding for GFRα1a. Transfection of human neural progenitor cells (NPCs) with a miR-511 mimic was sufficient to repress GFRA1-L/GFRα1a without altering GFRα1b, and resulted in pathway-specific changes in immediate early gene activity. Unexpectedly, GFRα1a knockdown did not reduce NPC responses to GDNF. Rather, it greatly enhanced mitogen-activated protein kinase signalling. This effect appeared to be mediated by GDNF/soluble GFRα1/neural cell adhesion molecule binding, and substituting the soluble GFRα1a/GFRα1b content of miR-511-transfected NPCs with that of controls rescued signalling. In light of previous reports suggesting that GFRα1b can inhibit GFRα1a-induced neuroplasticity, we also assessed the association between GFRα1 and doublecortin (DCX; a hyperplastic marker) in human BLA. Although controls displayed coordinated expression of GFRα1a and b isoforms and these correlated positively with DCX, the only significant association observed among depressed subjects was a strongly negative correlation between GFRα1b and DCX. Taken together, these results suggest that microRNA-mediated reductions of GFRα1a in depression change the quality, rather than the quantity, of GDNF signalling. They also suggest that central GDNF signalling may represent a novel target for antidepressant treatment.

DCC Confers Susceptibility to Depression-like Behaviors in Humans and Mice and Is Regulated by miR-218

BACKGROUD Variations in the expression of the Netrin-1 guidance cue receptor DCC (deleted in colorectal cancer) appear to confer resilience or susceptibility to psychopathologies involving prefrontal cortex (PFC) dysfunction. METHODS With the use of postmortem brain tissue, mouse models of defeat stress, and in vitro analysis, we assessed microRNA (miRNA) regulation of DCC and whether changes in DCC levels in the PFC lead to vulnerability to depression-like behaviors. RESULTS We identified miR-218 as a posttranscriptional repressor of DCC and detected coexpression of DCC and miR-218 in pyramidal neurons of human and mouse PFC. We found that exaggerated expression of DCC and reduced levels of miR-218 in the PFC are consistent traits of mice susceptible to chronic stress and of major depressive disorder in humans. Remarkably, upregulation of Dcc in mouse PFC pyramidal neurons causes vulnerability to stress-induced social avoidance and anhedonia. CONCLUSIONS These data are the first demonstration of microRNA regulation of DCC and suggest that, by regulating DCC, miR-218 may be a switch of susceptibility versus resilience to stress-related disorders.

Effects of Postmortem Interval on Biomolecule Integrity in the Brain

Abstract Postmortem brain research is invaluable to the study of neurologic and neuropsychiatric disorders, including Alzheimer disease, schizophrenia, and major depression. A major confounder in molecular studies using human brain tissue is postmortem interval (i.e. the amount of time between a subject’s death and processing of tissue). We examined the integrity of biomolecules that were of interest to molecular studies of neurologic disorders, including RNA, microRNA, histone modifications, and proteins, at various postmortem intervals in an animal model to assess their robustness and suitability for experimentation. Sprague-Dawley rats were selected as model and subjected to 2 conditions: a variable postmortem interval at room temperature and a fixed time of 24hours at 4°C, which simulates the period commonly spent in the morgue before brain collection. Eight time points were investigated. MicroRNA was impressively resistant to postmortem intervals; methylated histone modifications showed a threshold between 72 and 96 hours, mirroring results from histone proteins at 72 hours. RNA degradation was transcript-specific, with housekeeping genes being more robust than genes with lower expression. Our results suggest that molecules commonly investigated in genetic and epigenetic studies were highly stable through the postmortem intervals investigated. These results support the continued use of postmortem tissue for neuropsychiatric research.

MicroRNAs 146a/b-5 and 425-3p and 24-3p are markers of antidepressant response and regulate MAPK/Wnt-system genes

Antidepressants (ADs) are the most common treatment for major depressive disorder (MDD). However, only ∼30% of patients experience adequate response after a single AD trial, and this variability remains poorly understood. Here, we investigated microRNAs (miRNAs) as biomarkers of AD response using small RNA-sequencing in paired samples from MDD patients enrolled in a large, randomized placebo-controlled trial of duloxetine collected before and 8 weeks after treatment. Our results revealed differential expression of miR-146a-5p, miR-146b-5p, miR-425-3p and miR-24-3p according to treatment response. These results were replicated in two independent clinical trials of MDD, a well-characterized animal model of depression, and post-mortem human brains. Furthermore, using a combination of bioinformatics, mRNA studies and functional in vitro experiments, we showed significant dysregulation of genes involved in MAPK/Wnt signalling pathways. Together, our results indicate that these miRNAs are consistent markers of treatment response and regulators of the MAPK/Wnt systems.