Profiling Small RNA From Brain Extracellular Vesicles in Individuals With Depression.

BACKGROUND: Major depressive disorder (MDD) is a leading cause of disability with significant mortality risk. Despite progress in our understanding of the etiology of MDD, the underlying molecular changes in the brain remain poorly understood. Extracellular vesicles (EVs) are lipid-bound particles that can reflect the molecular signatures of the tissue of origin. We aimed to optimize a streamlined EV isolation protocol from postmortem brain tissue and determine whether EV RNA cargo, particularly microRNAs (miRNAs), have an MDD-specific profile. METHODS: EVs were isolated from postmortem human brain tissue. Quality was assessed using western blots, transmission electron microscopy, and microfluidic resistive pulse sensing. EV RNA was extracted and sequenced on Illumina platforms. Functional follow-up was performed in silico. RESULTS: Quality assessment showed an enrichment of EV markers, as well as a size distribution of 30 to 200 nm in diameter, and no contamination with cellular debris. Small RNA profiling indicated the presence of several RNA biotypes, with miRNAs and transfer RNAs being the most prominent. Exploring miRNA levels between groups revealed decreased expression of miR-92a-3p and miR-129-5p, which was validated by qPCR and was specific to EVs and not seen in bulk tissue. Finally, in silico functional analyses indicate potential roles for these 2 miRNAs in neurotransmission and synaptic plasticity. CONCLUSION: We provide a streamlined isolation protocol that yields EVs of high quality that are suitable for molecular follow-up. Our findings warrant future investigations into brain EV miRNA dysregulation in MDD.

Proteomic evidence of depression-associated astrocytic dysfunction in the human male olfactory bulb.

The olfactory bulb (OB), a major structure of the limbic system, has been understudied in human investigations of psychopathologies such as depression. To explore more directly the molecular features of the OB in depression, a global comparative proteome analysis was carried out with human post-mortem OB samples from 11 males having suffered from depression and 12 healthy controls. We identified 188 differentially abundant proteins (with adjusted p < 0.05) between depressed cases and controls. Gene ontology and gene enrichment analyses suggested that these proteins are involved in biological processes including the complement and coagulation cascades. Cell type enrichment analysis displayed a significant reduction in several canonical astrocytic proteins in OBs from depressed patients. Furthermore, using RNA-fluorescence in-situ hybridization, we observed a decrease in the percentage of ALDH1L1+ cells expressing canonical astrocytic markers including ALDOC, NFIA, GJA1 (connexin 43) and SLC1A3 (EAAT1). These results are consistent with previous reports of downregulated astrocytic marker expression in other brain regions in depressed patients. We also conducted a comparative phosphoproteomic analysis of OB samples and found a dysregulation of proteins involved in neuronal and astrocytic functions. To determine whether OB astrocytic abnormalities is specific to humans, we also performed proteomics on the OB of socially defeated male mice, a commonly used model of depression. Cell-type specific analysis revealed that in socially defeated animals, the most striking OB protein alterations were associated with oligodendrocyte-lineage cells rather than with astrocytes, highlighting an important species difference. Overall, this study further highlights cerebral astrocytic abnormalities as a consistent feature of depression in humans.

Spatial transcriptomic analysis of adult hippocampal neurogenesis in the human brain.

BACKGROUND: Adult hippocampal neurogenesis has been extensively characterized in rodent models, but its existence in humans remains controversial. We sought to assess the phenomenon in postmortem human hippocampal samples by combining spatial transcriptomics and multiplexed fluorescent in situ hybridization. METHODS: We computationally examined the spatial expression of various canonical neurogenesis markers in postmortem dentate gyrus (DG) sections from young and middle-aged sudden-death males. We conducted in situ assessment of markers expressed in neural stem cells, proliferative cells, and immature granule neurons in postmortem DG sections from infant, adolescent, and middle-aged males. RESULTS: We examined frozen DG tissue from infant (n = 1, age 2 yr), adolescent (n = 1, age 16 yr), young adult (n = 2, mean age 23.5 yr), and middle-aged (n = 2, mean age 42.5 yr) males, and frozen-fixed DG tissue from middle-aged males (n = 6, mean age 43.5 yr). We detected very few cells expressing neural stem cell and proliferative markers in the human DG from childhood to middle age. However, at all ages, we observed a substantial number of DG cells expressing the immature neuronal marker DCX. Most DCX + cells displayed an inhibitory phenotype, while the remainder were non-committed or excitatory in nature. LIMITATIONS: The study was limited by small sample sizes and included samples only from males. CONCLUSION: Our findings indicate very low levels of hippocampal neurogenesis throughout life and the existence of a local reserve of plasticity in the adult human hippocampus. Overall, our study provides important insight into the distribution and phenotype of cells expressing neurogenesis markers in the adult human hippocampus.

Associations of psychiatric disease and ageing with FKBP5 expression converge on superficial layer neurons of the neocortex.

Identification and characterisation of novel targets for treatment is a priority in the field of psychiatry. FKBP5 is a gene with decades of evidence suggesting its pathogenic role in a subset of psychiatric patients, with potential to be leveraged as a therapeutic target for these individuals. While it is widely reported that FKBP5/FKBP51 mRNA/protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, it is not known in which cell types and sub-anatomical areas of the human brain this occurs. This knowledge is critical to propel FKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n = 1024) of FKBP5/1, examining neocortical areas (BA9, BA11 and ventral BA24/BA24a) derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease state, ageing and genotype on cortical FKBP5/1 expression including in a cell type-specific manner. We identified consistently heightened FKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq; BA9 and BA11) and targeted histology (BA9, BA24a), we established that these disease and ageing effects on FKBP5/1 expression were most pronounced in excitatory superficial layer neurons of the neocortex, and this effect appeared to be consistent in both the granular and agranular areas examined. We then found that this increase in FKBP5 levels may impact on synaptic plasticity, as FKBP5 gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in superficial layer neurons in BA11. These findings pinpoint a novel cellular and molecular mechanism that has potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.

Child abuse associates with increased recruitment of perineuronal nets in the ventromedial prefrontal cortex: a possible implication of oligodendrocyte progenitor cells.

Child abuse (CA) is a strong predictor of psychopathologies and suicide, altering normal trajectories of brain development in areas closely linked to emotional responses such as the prefrontal cortex (PFC). Yet, the cellular underpinnings of these enduring effects are unclear. Childhood and adolescence are marked by the protracted formation of perineuronal nets (PNNs), which orchestrate the closure of developmental windows of cortical plasticity by regulating the functional integration of parvalbumin interneurons into neuronal circuits. Using well-characterized post-mortem brain samples, we show that a history of CA is specifically associated with increased densities and morphological complexity of WFL-labeled PNNs in the ventromedial PFC (BA11/12), possibly suggesting increased recruitment and maturation of PNNs. Through single-nucleus sequencing and fluorescent in situ hybridization, we found that the expression of canonical components of PNNs is enriched in oligodendrocyte progenitor cells (OPCs), and that they are upregulated in CA victims. These correlational findings suggest that early-life adversity may lead to persistent patterns of maladaptive behaviors by reducing the neuroplasticity of cortical circuits through the enhancement of developmental OPC-mediated PNN formation.

Human in vivo evidence of reduced astrocyte activation and neuroinflammation in patients with treatment-resistant depression following electroconvulsive therapy.

AIM: The current study aimed to investigate the neuroinflammatory hypothesis of depression and the potential anti-inflammatory effect of electroconvulsive therapy (ECT) in vivo, utilizing astrocyte-derived extracellular vesicles (ADEVs) isolated from plasma. METHODS: A total of 40 patients with treatment-resistant depression (TRD) and 35 matched healthy controls were recruited at baseline, and 34 patients with TRD completed the post-ECT visits. Blood samples were collected at baseline and post-ECT. Plasma ADEVs were isolated and confirmed, and the concentrations of two astrocyte markers (glial fibrillary acidic protein [GFAP] and S100ß), an extracellular vesicle marker cluster of differentiation 81 (CD81), and nine inflammatory markers in ADEVs were measured as main analyses. In addition, correlation analysis was conducted between clinical features and ADEV protein levels as exploratory analysis. RESULTS: At baseline, the TRD group exhibited significantly higher levels of two astrocyte markers GFAP and S100ß, as well as CD81 compared with the healthy controls. Inflammatory markers interferon γ (IFN-γ), interleukin (IL) 1ß, IL-4, IL-6, tumor necrosis factor α, IL-10, and IL-17A were also significantly higher in the TRD group. After ECT, there was a significant reduction in the levels of GFAP, S100ß, and CD81, along with a significant decrease in the levels of IFN-γ and IL-4. Furthermore, higher levels of GFAP, S100ß, CD81, and inflammatory cytokines were associated with more severe depressive symptoms and poorer cognitive function. CONCLUSION: This study provides direct insight supporting the astrocyte activation and neuroinflammatory hypothesis of depression using ADEVs. ECT may exert an anti-inflammatory effect through inhibition of such activation of astrocytes.

Potential Roles of m6A and FTO in Synaptic Connectivity and Major Depressive Disorder.

RNA modifications known as epitranscriptomics have emerged as a novel layer of transcriptomic regulation. Like the well-studied epigenetic modifications characterized in DNA and on histone-tails, they have been shown to regulate activity-dependent gene expression and play a vital role in shaping synaptic connections in response to external stimuli. Among the hundreds of known RNA modifications, N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes. Through recognition of its binding proteins, m6A can regulate various aspects of mRNA metabolism and is essential for maintaining higher brain functions. Indeed, m6A is highly enriched in synapses and is involved in neuronal plasticity, learning and memory, and adult neurogenesis. m6A can also respond to environmental stimuli, suggesting an important role in linking molecular and behavioral stress. This review summarizes key findings from fields related to major depressive disorder (MDD) including stress and learning and memory, which suggest that activity-dependent m6A changes may, directly and indirectly, contribute to synaptic connectivity changes underlying MDD. Furthermore, we will highlight the roles of m6A and FTO, a m6A eraser, in the context of depressive-like behaviors. Although we have only begun to explore m6A in the context of MDD and psychiatry, elucidating a link between m6A and MDD presents a novel molecular mechanism underlying MDD pathogenesis.

Deep feature extraction of single-cell transcriptomes by generative adversarial network.

MOTIVATION: Single-cell RNA-sequencing (scRNA-seq) offers the opportunity to dissect heterogeneous cellular compositions and interrogate the cell-type-specific gene expression patterns across diverse conditions. However, batch effects such as laboratory conditions and individual-variability hinder their usage in cross-condition designs. RESULTS: Here, we present a single-cell Generative Adversarial Network (scGAN) to simultaneously acquire patterns from raw data while minimizing the confounding effect driven by technical artifacts or other factors inherent to the data. Specifically, scGAN models the data likelihood of the raw scRNA-seq counts by projecting each cell onto a latent embedding. Meanwhile, scGAN attempts to minimize the correlation between the latent embeddings and the batch labels across all cells. We demonstrate scGAN on three public scRNA-seq datasets and show that our method confers superior performance over the state-of-the-art methods in forming clusters of known cell types and identifying known psychiatric genes that are associated with major depressive disorder. AVAILABILITYAND IMPLEMENTATION: The scGAN code and the information for the public scRNA-seq datasets are available at https://github.com/li-lab-mcgill/singlecell-deepfeature. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Sex Differences in the Behavioral, Molecular, and Structural Effects of Ketamine Treatment in Depression.

Major depressive disorder (MDD) is a common psychiatric illness that manifests in sex-influenced ways. Men and women may experience depression differently and also respond to various antidepressant treatments in sex-influenced ways. Ketamine, which is now being used as a rapid-acting antidepressant, is likely the same. To date, the majority of studies investigating treatment outcomes in MDD do not disaggregate the findings in males and females, and this is also true for ketamine. This review aims to highlight that gap by exploring pre-clinical data-at a behavioral, molecular, and structural level-and recent clinical trials. Sex hormones, particularly estrogen and progesterone, influence the response at all levels examined, and sex is therefore a critical factor to examine when looking at ketamine response. Taken together, the data show females are more sensitive to ketamine than males, and it might be possible to monitor the phase of the menstrual cycle to mitigate some risks associated with the use of ketamine for females with MDD. Based on the studies reviewed in this article, we suggest that ketamine should be administered adhering to sex-specific considerations.

Neuron-derived extracellular vesicles enriched from plasma show altered size and miRNA cargo as a function of antidepressant drug response.

Previous work has demonstrated that microRNAs (miRNAs) change as a function of antidepressant treatment (ADT) response. However, it is unclear how representative these peripherally detected miRNA changes are to those occurring in the brain. This study aimed to use peripherally extracted neuron-derived extracellular vesicles (NDEV) to circumvent these limitations and investigate neuronal miRNA changes associated with antidepressant response. Samples were collected at two time points (baseline and after 8 weeks of follow-up) from depressed patients who responded (N = 20) and did not respond (N = 20) to escitalopram treatment, as well as controls (N = 20). Total extracellular vesicles (EVs) were extracted from plasma, and then further enriched for NDEV by immunoprecipitation with L1CAM. EVs and NDEVs were characterized, and NDEV miRNA cargo was extracted and sequenced. Subsequently, studies in cell lines and postmortem tissue were conducted. Characterization of NDEVs revealed that they were smaller than other EVs isolated from plasma (p < 0.0001), had brain-specific neuronal markers, and contained miRNAs enriched for brain functions (p < 0.0001) Furthermore, NDEVs from depressed patients were smaller than controls (p < 0.05), and NDEV size increased with ADT response (p < 0.01). Finally, changes in NDEV cargo, specifically changes in miR-21-5p, miR-30d-5p, and miR-486-5p together (p < 0.01), were associated with ADT response. Targets of these three miRNAs were altered in brain tissue from depressed individuals (p < 0.05). Together, this study indicates that changes in peripherally isolated NDEV can act as both a clinically accessible and informative biomarker of ADT response specifically through size and cargo.

Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.

Epigenetic mechanisms, like those involving DNA methylation, are thought to mediate the relationship between chronic cocaine dependence and molecular changes in addiction-related neurocircuitry, but have been understudied in human brain. We initially used reduced representation bisulfite sequencing (RRBS) to generate a methylome-wide profile of cocaine dependence in human post-mortem caudate tissue. We focused on the Iroquois Homeobox A (IRXA) gene cluster, where hypomethylation in exon 3 of IRX2 in neuronal nuclei was associated with cocaine dependence. We replicated this finding in an independent cohort and found similar results in the dorsal striatum from cocaine self-administering mice. Using epigenome editing and 3C assays, we demonstrated a causal relationship between methylation within the IRX2 gene body, CTCF protein binding, three-dimensional (3D) chromatin interaction, and gene expression. Together, these findings suggest that cocaine-related hypomethylation of IRX2 contributes to the development and maintenance of cocaine dependence through alterations in 3D chromatin structure in the caudate nucleus.

Integrative DNA Methylation and Gene Expression Analysis in the Prefrontal Cortex of Mexicans Who Died by Suicide.

BACKGROUND: Suicide represents a major health concern, especially in developing countries. While many demographic risk factors have been proposed, the underlying molecular pathology of suicide remains poorly understood. A body of evidence suggests that aberrant DNA methylation and expression is involved. In this study, we examined DNA methylation profiles and concordant gene expression changes in the prefrontal cortex of Mexicans who died by suicide. METHODS: In collaboration with the coroner's office in Mexico City, brain samples of males who died by suicide (n = 35) and age-matched sudden death controls (n = 13) were collected. DNA and RNA were extracted from prefrontal cortex tissue and analyzed with the Infinium Methylation480k and the HumanHT-12 v4 Expression Beadchips, respectively. RESULTS: We report evidence of altered DNA methylation profiles at 4430 genomic regions together with 622 genes characterized by differential expression in cases vs controls. Seventy genes were found to have concordant methylation and expression changes. Metacore-enriched analysis identified 10 genes with biological relevance to psychiatric phenotypes and suicide (ADCY9, CRH, NFATC4, ABCC8, HMGA1, KAT2A, EPHA2, TRRAP, CD22, and CBLN1) and highlighted the association that ADCY9 has with various pathways, including signal transduction regulated by the cAMP-responsive element modulator, neurophysiological process regulated by the corticotrophin-releasing hormone, and synaptic plasticity. We therefore went on to validate the observed hypomethylation of ADCY9 in cases vs control through targeted bisulfite sequencing. CONCLUSION: Our study represents the first, to our knowledge, analysis of DNA methylation and gene expression associated with suicide in a Mexican population using postmortem brain, providing novel insights for convergent molecular alterations associated with suicide.

Chromatin Profiling Techniques: Exploring the Chromatin Environment and Its Contributions to Complex Traits.

The genetic architecture of complex traits is multifactorial. Genome-wide association studies (GWASs) have identified risk loci for complex traits and diseases that are disproportionately located at the non-coding regions of the genome. On the other hand, we have just begun to understand the regulatory roles of the non-coding genome, making it challenging to precisely interpret the functions of non-coding variants associated with complex diseases. Additionally, the epigenome plays an active role in mediating cellular responses to fluctuations of sensory or environmental stimuli. However, it remains unclear how exactly non-coding elements associate with epigenetic modifications to regulate gene expression changes and mediate phenotypic outcomes. Therefore, finer interrogations of the human epigenomic landscape in associating with non-coding variants are warranted. Recently, chromatin-profiling techniques have vastly improved our understanding of the numerous functions mediated by the epigenome and DNA structure. Here, we review various chromatin-profiling techniques, such as assays of chromatin accessibility, nucleosome distribution, histone modifications, and chromatin topology, and discuss their applications in unraveling the brain epigenome and etiology of complex traits at tissue homogenate and single-cell resolution. These techniques have elucidated compositional and structural organizing principles of the chromatin environment. Taken together, we believe that high-resolution epigenomic and DNA structure profiling will be one of the best ways to elucidate how non-coding genetic variations impact complex diseases, ultimately allowing us to pinpoint cell-type targets with therapeutic potential.

The interaction between oxytocin receptor gene methylation and maternal behavior on children's early theory of mind abilities.

Theory of mind, the ability to represent the mental states of others, is an important social cognitive process, which contributes to the development of social competence. Recent research suggests that interactions between gene and environmental factors, such as oxytocin receptor gene (OXTR) polymorphisms and maternal parenting behavior, may underlie individual differences in children's theory of mind. However, the potential influence of DNA methylation of OXTR remains unclear. The current study investigated the roles of OXTR methylation, maternal behavior, and their statistical interaction on toddlers' early emerging theory of mind abilities. Participants included a community sample of 189 dyads of mothers and their 2- to 3-year-old children, whose salivary DNA was analyzed. Results indicated that more maternal structuring behavior was associated with better performance, on a battery of three theory of mind tasks, while higher OXTR methylation within exon 3 was associated with poorer performance. A significant interaction also emerged, such that OXTR methylation was related to theory of mind among children whose mothers displayed less structuring, when controlling for children's age, sex, ethnicity, number of child-aged siblings, verbal ability, and maternal education. Maternal structuring behavior may buffer the potential negative impact of hypermethylation on OXTR gene expression and function.

Repression of Astrocytic Connexins in Cortical and Subcortical Brain Regions and Prefrontal Enrichment of H3K9me3 in Depression and Suicide.

Background: Major depressive disorder has been associated with dysfunctional astrocytic networks. The underlying causes, extent, and consequences of such dysfunctions remain to be characterized. Astrocyte-astrocyte communication occurs principally through gap junction channels primarily formed by connexin 30 and 43 (CX30 and CX43). We previously reported decreased connexin expression in the prefrontal cortex of depressed suicides. In the present study, we investigated whether these changes are mediated by epigenetic regulation, and expanded gene expression quantifications to other cortical and subcortical regions to assess the regional distribution of connexion disruptions in depressed suicides. Methods: The expression of CX30 and CX43 was measured by real-time PCR in samples of neocortex (Brodmann areas 4 and 17), cerebellar cortex, mediodorsal thalamus, and caudate nucleus of 22 depressed suicides and 22 matched sudden-death controls. Chromatin immunoprecipitation was used to measure enrichment levels of the repressive chromatin mark H3K9me3 in the prefrontal cortex. Results: We found a consistent downregulation of connexin genes in all regions examined, except in the cerebellum where an increase in the expression of CX30 was measured and using chromatin immunoprecipitation we observed an enrichment of H3K9me3 for both Cx30 and Cx43 in the prefrontal cortex. Conclusions: Our study shows widespread astrocytic CX gene repression in depressed suicides that is mediated, at least in part, through epigenetic mechanisms. Taken together, these findings support the notion of widespread cerebral astrocytic dysfunction in major depressive disorder.

Perinatal depression and DNA methylation of oxytocin-related genes: a study of mothers and their children.

The present study investigated the association of perinatal depression (PD) with differential methylation of 3 genomic regions among mother and child dyads: exon 3 within the oxytocin receptor (OXTR) gene and 2 intergenic regions (IGR) between the oxytocin (OXT) and vasopressin (AVP) genes. Maternal PD was assessed at 5 time-points during pregnancy and postpartum. Four groups were established based on Edinburgh Postnatal Depression Scale (EPDS) cut-off scores: no PD, prenatal or postpartum depressive symptoms only and persistent PD (depressive symptoms both prenatally and postpartum). Salivary DNA was collected from mothers and children at the final time-point, 2.9years postpartum. Mothers with persistent PD had significantly higher overall OXTR methylation than the other groups and this pattern extended to 16/22 individual CpG sites. For the IGR, only the region closer to the AVP gene (AVP IGR) showed significant differential methylation, with the persistent PD group displaying the lowest levels of methylation overall, but not for individual CpG sites. These results suggest that transient episodes of depression may not be associated with OXTR hypermethylation. Validation studies need to confirm the downstream biological effects of AVP IGR hypomethylation as it relates to persistent PD. Differential methylation of the OXTR and IGR regions was not observed among children exposed to maternal PD. The consequences of OXTR hypermethylation and AVP IGR hypomethylation found in mothers with persistent PDS may not only impact the OXT system, but may also compromise maternal behavior, potentially resulting in negative outcomes for the developing child.

WITHDRAWN: Global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine after extended post-mortem interval.

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DNA hypomethylation of Synapsin II CpG islands associates with increased gene expression in bipolar disorder and major depression.

BACKGROUND: The Synapsins (SYN1, SYN2, and SYN3) are important players in the adult brain, given their involvement in synaptic transmission and plasticity, as well as in the developing brain through roles in axon outgrowth and synaptogenesis. We and others previously reported gene expression dysregulation, both as increases and decreases, of Synapsins in mood disorders, but little is known about the regulatory mechanisms leading to these differences. Thus, we proposed to study DNA methylation at theses genes' promoter regions, under the assumption that altered epigenetic marks at key regulatory sites would be the cause of gene expression changes and thus part of the mood disorder etiology. METHODS: We performed CpG methylation mapping focusing on the three genes' predicted CpG islands using the Sequenom EpiTYPER platform. DNA extracted from post-mortem brain tissue (BA10) from individuals who had lived with bipolar disorder (BD), major depressive disorder (MDD), as well as psychiatrically healthy individuals was used. Differences in methylation across all CpGs within a CpG island and between the three diagnostic groups were assessed by 2-way mixed model analyses of variance. RESULTS: We found no significant results for SYN1 or SYN3, but there was a significant group difference in SYN2 methylation, as well as an overall pattern of hypomethylation across the CpG island. Furthermore, we found a significant inverse correlation of DNA methylation with SYN2a mRNA expression. CONCLUSIONS: These findings contribute to previous work showing dysregulation of Synapsins, particularly SYN2, in mood disorders and improve our understanding of the regulatory mechanisms that precipitate these changes likely leading to the BD or MDD phenotype.

BisQC: an operational pipeline for multiplexed bisulfite sequencing.

BACKGROUND: Bisulfite sequencing is the most efficient single nucleotide resolution method for analysis of methylation status at whole genome scale, but improved quality control metrics are needed to better standardize experiments. RESULTS: We describe BisQC, a step-by-step method for multiplexed bisulfite-converted DNA library construction, pooling, spike-in content, and bioinformatics. We demonstrate technical improvements for library preparation and bioinformatic analyses that can be done in standard laboratories. We find that decoupling amplification of bisulfite converted (bis) DNA from the indexing reaction is an advantage, specifically in reducing total PCR cycle number and pre-selecting high quality bis-libraries. We also introduce a progressive PCR method for optimal library amplification and size-selection. At the sequencing stage, we thoroughly test the benefits of pooling non-bis DNA library with bis-libraries and find that BisSeq libraries can be pooled with a high proportion of non-bis DNA libraries with minimal impact on BisSeq output. For informatics analysis, we propose a series of optimization steps including the utilization of the mitochondrial genome as a QC standard, and we assess the validity of using duplicate reads for coverage statistics. CONCLUSION: We demonstrate several quality control checkpoints at the library preparation, pre-sequencing, post-sequencing, and post-alignment stages, which should prove useful in determining sample and processing quality. We also determine that including a significant portion of non-bisulfite converted DNA with bisulfite converted DNA has a minimal impact on usable bisulfite read output.

Altered in vivo early neurogenesis traits in patients with depression: Evidence from neuron-derived extracellular vesicles and electroconvulsive therapy.

BACKGROUND: The neurogenesis hypothesis is a promising candidate etiologic hypothesis for depression, and it is associated with electroconvulsive therapy (ECT). However, human in vivo molecular-level evidence is lacking. OBJECTIVE: We used neuron-derived extracellular vesicles (NDEVs) as a "window to the neurons" to explore the in vivo neurogenesis status associated with ECT in patients with treatment-resistant depression (TRD). METHODS: In this study, we enrolled 40 patients with TRD and 35 healthy controls (HCs). We isolated NDEVs from the plasma of each participant to test the levels of doublecortin (DCX), a marker of neurogenesis, and cluster of differentiation (CD) 81, a marker of EVs. We also assessed the plasma levels of brain-derived neurotrophic factor (BDNF), a protein that is known to be associated with ECT and neuroplastic processes. RESULTS: Our findings indicated that both the levels of DCX in NDEVs and BDNF in plasma were significantly lower in TRD patients compared to HCs at baseline, but increased following ECTs. Conversely, levels of CD81 in NDEVs were found higher in TRD patients at baseline, but did not change after the ECT treatments. Exploratory analyses revealed that lower levels of BDNF in plasma and DCX in NDEVs, along with higher CD81 levels in NDEVs, were associated with more severe depressive symptoms and reduced cognitive function at baseline. Furthermore, higher baseline CD81 concentrations in NDEVs were correlated with greater decreases in depression symptoms. CONCLUSIONS: We first present human in vivo evidence of early neurogenesis using DCX through NDEVs: decreased in TRD patients, increased after ECTs.