Multi-omics profiling of DNA methylation and gene expression alterations in human cocaine use disorder.

Structural and functional changes of the brain are assumed to contribute to excessive cocaine intake, craving, and relapse in cocaine use disorder (CUD). Epigenetic and transcriptional changes were hypothesized as a molecular basis for CUD-associated brain alterations. Here we performed a multi-omics study of CUD by integrating epigenome-wide methylomic (N = 42) and transcriptomic (N = 25) data from the same individuals using postmortem brain tissue of Brodmann Area 9 (BA9). Of the N = 1 057 differentially expressed genes (p < 0.05), one gene, ZFAND2A, was significantly upregulated in CUD at transcriptome-wide significance (q < 0.05). Differential alternative splicing (AS) analysis revealed N = 98 alternatively spliced transcripts enriched in axon and dendrite extension pathways. Strong convergent overlap in CUD-associated expression deregulation was found between our BA9 cohort and independent replication datasets. Epigenomic, transcriptomic, and AS changes in BA9 converged at two genes, ZBTB4 and INPP5E. In pathway analyses, synaptic signaling, neuron morphogenesis, and fatty acid metabolism emerged as the most prominently deregulated biological processes. Drug repositioning analysis revealed glucocorticoid receptor targeting drugs as most potent in reversing the CUD expression profile. Our study highlights the value of multi-omics approaches for an in-depth molecular characterization and provides insights into the relationship between CUD-associated epigenomic and transcriptomic signatures in the human prefrontal cortex.

Perineuronal Net Alterations Following Early-Life Stress: Are Microglia Pulling Some Strings?

The extracellular matrix plays a key role in synapse formation and in the modulation of synaptic function in the central nervous system. Recent investigations have revealed that microglia, the resident immune cells of the brain, are involved in extracellular matrix remodeling under both physiological and pathological conditions. Moreover, the dysregulation of both innate immune responses and the extracellular matrix has been documented in stress-related psychopathologies as well as in relation to early-life stress. However, the dynamics of microglial regulation of the ECM and how it can be impacted by early-life adversity have been understudied. This brief review provides an overview of the recent literature on this topic, drawing from both animal model and human post mortem studies. Direct and indirect mechanisms through which microglia may regulate the extracellular matrix-including perineuronal nets-are presented and discussed in light of the interactions with other cell types.

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.

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan (CS-GAG) side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (N = 81) and mouse (N = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-GAGs and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress (ELS) impacts these PNN features discovered that although ELS increases WFL+ PNN density, the CS-GAG sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (n = 81) and mouse (n = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-glycosaminoglycans and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress impacts these PNN features discovered that although early-life stress increases WFL+ PNN density, the CS-glycosaminoglycan sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Adult Hippocampal Neurogenesis in the Human Brain: Updates, Challenges, and Perspectives.

The existence of neurogenesis in the adult human hippocampus has been under considerable debate within the past three decades due to the diverging conclusions originating mostly from immunohistochemistry studies. While some of these reports conclude that hippocampal neurogenesis in humans occurs throughout physiologic aging, others indicate that this phenomenon ends by early childhood. More recently, some groups have adopted next-generation sequencing technologies to characterize with more acuity the extent of this phenomenon in humans. Here, we review the current state of research on adult hippocampal neurogenesis in the human brain with an emphasis on the challenges and limitations of using immunohistochemistry and next-generation sequencing technologies for its study.

Parvalbumin interneurons in human ventromedial prefrontal cortex: a comprehensive post-mortem study of myelination and perineuronal nets in neurotypical individuals and depressed suicides with and without a history of child abuse.

Cortical parvalbumin interneurons (PV+) are major regulators of excitatory/inhibitory information processing, and their maturation is associated with the opening of developmental critical periods (CP). Recent studies reveal that cortical PV+ axons are myelinated, and that myelination along with perineuronal net (PNN) maturation around PV+ cells is associated with the closures of CP. Although PV+ interneurons are susceptible to early-life stress, their relationship between their myelination and PNN coverage remains unexplored. This study compared the fine features of PV+ interneurons in well-characterized human post-mortem ventromedial prefrontal cortex samples (n = 31) from depressed suicides with or without a history of child abuse (CA) and matched controls. In healthy controls, 81% of all sampled PV+ interneurons displayed a myelinated axon, while a subset (66%) of these cells also displayed a PNN, proposing a relationship between both attributes. Intriguingly, a 3-fold increase in the proportion of unmyelinated PV+ interneurons with a PNN was observed in CA victims, along with greater PV-immunofluorescence intensity in myelinated PV+ cells with a PNN. This study, which is the first to provide normative data on myelination and PNNs around PV+ interneurons in human neocortex, sheds further light on the cellular and molecular consequences of early-life adversity on cortical PV+ interneurons.

MAPT H2 haplotype and risk of Pick's disease in the Pick's disease International Consortium: a genetic association study.

BACKGROUND: Pick's disease is a rare and predominantly sporadic form of frontotemporal dementia that is classified as a primary tauopathy. Pick's disease is pathologically defined by the presence in the frontal and temporal lobes of Pick bodies, composed of hyperphosphorylated, three-repeat tau protein, encoded by the MAPT gene. MAPT has two distinct haplotypes, H1 and H2; the MAPT H1 haplotype is the major genetic risk factor for four-repeat tauopathies (eg, progressive supranuclear palsy and corticobasal degeneration), and the MAPT H2 haplotype is protective for these disorders. The primary aim of this study was to evaluate the association of MAPT H2 with Pick's disease risk, age at onset, and disease duration. METHODS: In this genetic association study, we used data from the Pick's disease International Consortium, which we established to enable collection of data from individuals with pathologically confirmed Pick's disease worldwide. For this analysis, we collected brain samples from individuals with pathologically confirmed Pick's disease from 35 sites (brainbanks and hospitals) in North America, Europe, and Australia between Jan 1, 2020, and Jan 31, 2023. Neurologically healthy controls were recruited from the Mayo Clinic (FL, USA, or MN, USA between March 1, 1998, and Sept 1, 2019). For the primary analysis, individuals were directly genotyped for the MAPT H1-H2 haplotype-defining variant rs8070723. In a secondary analysis, we genotyped and constructed the six-variant-defined (rs1467967-rs242557-rs3785883-rs2471738-rs8070723-rs7521) MAPT H1 subhaplotypes. Associations of MAPT variants and MAPT haplotypes with Pick's disease risk, age at onset, and disease duration were examined using logistic and linear regression models; odds ratios (ORs) and ß coefficients were estimated and correspond to each additional minor allele or each additional copy of the given haplotype. FINDINGS: We obtained brain samples from 338 people with pathologically confirmed Pick's disease (205 [61%] male and 133 [39%] female; 338 [100%] White) and 1312 neurologically healthy controls (611 [47%] male and 701 [53%] female; 1312 [100%] White). The MAPT H2 haplotype was associated with increased risk of Pick's disease compared with the H1 haplotype (OR 1·35 [95% CI 1·12 to 1·64], p=0·0021). MAPT H2 was not associated with age at onset (ß -0·54 [95% CI -1·94 to 0·87], p=0·45) or disease duration (ß 0·05 [-0·06 to 0·16], p=0·35). Although not significant after correcting for multiple testing, associations were observed at p less than 0·05: with risk of Pick's disease for the H1f subhaplotype (OR 0·11 [0·01 to 0·99], p=0·049); with age at onset for H1b (ß 2·66 [0·63 to 4·70], p=0·011), H1i (ß -3·66 [-6·83 to -0·48], p=0·025), and H1u (ß -5·25 [-10·42 to -0·07], p=0·048); and with disease duration for H1x (ß -0·57 [-1·07 to -0·07], p=0·026). INTERPRETATION: The Pick's disease International Consortium provides an opportunity to do large studies to enhance our understanding of the pathobiology of Pick's disease. This study shows that, in contrast to the decreased risk of four-repeat tauopathies, the MAPT H2 haplotype is associated with an increased risk of Pick's disease in people of European ancestry. This finding could inform development of isoform-related therapeutics for tauopathies. FUNDING: Wellcome Trust, Rotha Abraham Trust, Brain Research UK, the Dolby Fund, Dementia Research Institute (Medical Research Council), US National Institutes of Health, and the Mayo Clinic Foundation.

SNORA69 is up-regulated in the lateral habenula of individuals with major depressive disorder.

Major depressive disorder (MDD) is a complex and potentially debilitating illness whose etiology and pathology remains unclear. Non-coding RNAs have been implicated in MDD, where they display differential expression in the brain and the periphery. In this study, we quantified small nucleolar RNA (snoRNA) expression by small RNA sequencing in the lateral habenula (LHb) of individuals with MDD (n = 15) and psychiatrically-healthy controls (n = 15). We uncovered five snoRNAs that exhibited differential expression between MDD and controls (FDR < 0.01). Specifically, SNORA69 showed increased expression in MDD and was technically validated via RT-qPCR. We further investigated the expression of Snora69 in the LHb and peripheral blood of an unpredicted chronic mild stress (UCMS) mouse model of depression. Snora69 was specifically up-regulated in mice that underwent the UCMS paradigm. SNORA69 is known to guide pseudouridylation onto 5.8S and 18S rRNAs. We quantified the relative abundance of pseudouridines on 5.8S and 18S rRNA in human post-mortem LHb samples and found increased abundance of pseudouridines in the MDD group. Overall, our findings indicate the importance of brain snoRNAs in the pathology of MDD. Future studies characterizing SNORA69's role in MDD pathology is warranted.

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.

Substance P, NPY, CCK and their receptors in five brain regions in major depressive disorder with transcriptomic analysis of locus coeruleus neurons.

Major depressive disorder (MDD) is a serious disease and a burden to patients, families and society. Rodent experiments and human studies suggest that several neuropeptide systems are involved in mood regulation. The aim of this study is two-fold: (i) to monitor, with qPCR, transcript levels of the substance P/tachykinin (TAC), NPY and CCK systems in bulk samples from control and suicide subjects, targeting five postmortem brain regions including locus coeruleus (LC); and (ii) to analyse expression of neuropeptide family transcripts in LC neurons of 'normal' postmortem brains by using laser capture microdissection with Smart-Seq2 RNA sequencing. qPCR revealed distinct regional expression patterns in male and female controls with higher levels for the TAC system in the dorsal raphe nucleus and LC, versus higher transcripts levels of the NPY and CCK systems in prefrontal cortex. In suicide patients, TAC, TAC receptors and a few NPY family transcript levels were increased mainly in prefrontal cortex and LC. The second study on 'normal' noradrenergic LC neurons revealed expression of transcripts for GAL, NPY, TAC1, CCK, and TACR1 and many other peptides (e.g. Cerebellin4 and CARTPT) and receptors (e.g. Adcyap1R1 and GPR173). These data and our previous results on suicide brains indicates that the tachykinin and galanin systems may be valid targets for developing antidepressant medicines. Moreover, the perturbation of neuropeptide systems in MDD patients, and the detection of further neuropeptide and receptor transcripts in LC, shed new light on signalling in noradrenergic LC neurons and on mechanisms possibly associated with mood disorders.

Enhancing HIF-1α-P2X2 signaling in dorsal raphe serotonergic neurons promotes psychological resilience.

Major depressive disorder (MDD) is a devastating condition. Although progress has been made in the past seven decades, patients with MDD continue to receive an inadequate treatment, primarily due to the late onset of first-line antidepressant drugs and to their acute withdrawal symptoms. Resilience is the ability to rebound from adversity in a healthy manner and many people have psychological resilience. Revealing the mechanisms and identifying methods promoting resilience will hopefully lead to more effective prevention strategies and treatments for depression. In this study, we found that intermittent hypobaric hypoxia training (IHHT), a method for training pilots and mountaineers, enhanced psychological resilience in adult mice. IHHT produced a sustained antidepressant-like effect in mouse models of depression by inducing long-term (up to 3 months after this treatment) overexpression of hypoxia-inducible factor (HIF)-1α in the dorsal raphe nucleus (DRN) of adult mice. Moreover, DRN-infusion of cobalt chloride, which mimics hypoxia increasing HIF-1α expression, triggered a rapid and long-lasting antidepressant-like effect. Down-regulation of HIF-1α in the DRN serotonergic (DRN5-HT) neurons attenuated the effects of IHHT. HIF-1α translationally regulated the expression of P2X2, and conditionally knocking out P2rx2 (encodes P2X2 receptors) in DRN5-HT neurons, in turn, attenuated the sustained antidepressant-like effect of IHHT, but not its acute effect. In line with these results, a single sub-anesthetic dose of ketamine enhanced HIF-1α-P2X2 signaling, which is essential for its rapid and long-lasting antidepressant-like effect. Notably, we found that P2X2 protein levels were significantly lower in the DRN of patients with MDD than that of control subjects. Together, these findings elucidate the molecular mechanism underlying IHHT promoting psychological resilience and highlight enhancing HIF-1α-P2X2 signaling in DRN5-HT neurons as a potential avenue for screening novel therapeutic treatments for MDD.

Universal method for the isolation of microvessels from frozen brain tissue: A proof-of-concept multiomic investigation of the neurovasculature.

The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for the proper function of the central nervous system. The neurovascular unit gatekeeps blood-brain barrier properties, which experiences impairment in several central nervous system diseases associated with neuroinflammation and contributes to pathogenesis. To better understand function and dysfunction at the neurovascular unit and how it may confer inflammatory processes within the brain, isolation and characterization of the neurovascular unit is needed. Here, we describe a singular, standardized protocol to enrich and isolate microvessels from archived snap-frozen human and frozen mouse cerebral cortex using mechanical homogenization and centrifugation-separation that preserves the structural integrity and multicellular composition of microvessel fragments. For the first time, microvessels are isolated from postmortem ventromedial prefrontal cortex tissue and are comprehensively investigated as a structural unit using both RNA sequencing and Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Both the transcriptome and proteome are obtained and compared, demonstrating that the isolated brain microvessel is a robust model for the NVU and can be used to generate highly informative datasets in both physiological and disease contexts.

SNORD90 induces glutamatergic signaling following treatment with monoaminergic antidepressants.

Pharmacotherapies for the treatment of major depressive disorder were serendipitously discovered almost seven decades ago. From this discovery, scientists pinpointed the monoaminergic system as the primary target associated with symptom alleviation. As a result, most antidepressants have been engineered to act on the monoaminergic system more selectively, primarily on serotonin, in an effort to increase treatment response and reduce unfavorable side effects. However, slow and inconsistent clinical responses continue to be observed with these available treatments. Recent findings point to the glutamatergic system as a target for rapid acting antidepressants. Investigating different cohorts of depressed individuals treated with serotonergic and other monoaminergic antidepressants, we found that the expression of a small nucleolar RNA, SNORD90, was elevated following treatment response. When we increased Snord90 levels in the mouse anterior cingulate cortex (ACC), a brain region regulating mood responses, we observed antidepressive-like behaviors. We identified neuregulin 3 (NRG3) as one of the targets of SNORD90, which we show is regulated through the accumulation of N6-methyladenosine modifications leading to YTHDF2-mediated RNA decay. We further demonstrate that a decrease in NRG3 expression resulted in increased glutamatergic release in the mouse ACC. These findings support a molecular link between monoaminergic antidepressant treatment and glutamatergic neurotransmission.

Cell type specific transcriptomic differences in depression show similar patterns between males and females but implicate distinct cell types and genes.

Major depressive disorder (MDD) is a common, heterogenous, and potentially serious psychiatric illness. Diverse brain cell types have been implicated in MDD etiology. Significant sexual differences exist in MDD clinical presentation and outcome, and recent evidence suggests different molecular bases for male and female MDD. We evaluated over 160,000 nuclei from 71 female and male donors, leveraging new and pre-existing single-nucleus RNA-sequencing data from the dorsolateral prefrontal cortex. Cell type specific transcriptome-wide threshold-free MDD-associated gene expression patterns were similar between the sexes, but significant differentially expressed genes (DEGs) diverged. Among 7 broad cell types and 41 clusters evaluated, microglia and parvalbumin interneurons contributed the most DEGs in females, while deep layer excitatory neurons, astrocytes, and oligodendrocyte precursors were the major contributors in males. Further, the Mic1 cluster with 38% of female DEGs and the ExN10_L46 cluster with 53% of male DEGs, stood out in the meta-analysis of both sexes.

DNA methylation in cocaine use disorder-An epigenome-wide approach in the human prefrontal cortex.

Background: Cocaine use disorder (CUD) is characterized by a loss of control over cocaine intake and is associated with structural, functional, and molecular alterations in the human brain. At the molecular level, epigenetic alterations are hypothesized to contribute to the higher-level functional and structural brain changes observed in CUD. Most evidence of cocaine-associated epigenetic changes comes from animal studies while only a few studies have been performed using human tissue. Methods: We investigated epigenome-wide DNA methylation (DNAm) signatures of CUD in human post-mortem brain tissue of Brodmann area 9 (BA9). A total of N = 42 BA9 brain samples were obtained from N = 21 individuals with CUD and N = 21 individuals without a CUD diagnosis. We performed an epigenome-wide association study (EWAS) and analyzed CUD-associated differentially methylated regions (DMRs). To assess the functional role of CUD-associated differential methylation, we performed Gene Ontology (GO) enrichment analyses and characterized co-methylation networks using a weighted correlation network analysis. We further investigated epigenetic age in CUD using epigenetic clocks for the assessment of biological age. Results: While no cytosine-phosphate-guanine (CpG) site was associated with CUD at epigenome-wide significance in BA9, we detected a total of 20 CUD-associated DMRs. After annotation of DMRs to genes, we identified Neuropeptide FF Receptor 2 (NPFFR2) and Kalirin RhoGEF Kinase (KALRN) for which a previous role in the behavioral response to cocaine in rodents is known. Three of the four identified CUD-associated co-methylation modules were functionally related to neurotransmission and neuroplasticity. Protein-protein interaction (PPI) networks derived from module hub genes revealed several addiction-related genes as highly connected nodes such as Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1), and Jun Proto-Oncogene, AP-1 Transcription Factor Subunit (JUN). In BA9, we observed a trend toward epigenetic age acceleration (EAA) in individuals with CUD remaining stable even after adjustment for covariates. Conclusion: Results from our study highlight that CUD is associated with epigenome-wide differences in DNAm levels in BA9 particularly related to synaptic signaling and neuroplasticity. This supports findings from previous studies that report on the strong impact of cocaine on neurocircuits in the human prefrontal cortex (PFC). Further studies are needed to follow up on the role of epigenetic alterations in CUD focusing on the integration of epigenetic signatures with transcriptomic and proteomic data.

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.

Quantitative and carbon isotope ratio analysis of fatty acids isolated from human brain hemispheres.

Our knowledge surrounding the overall fatty acid profile of the adult human brain has been largely limited to extrapolations from brain regions in which the distribution of fatty acids varies. This is especially problematic when modeling brain fatty acid metabolism, therefore, an updated estimate of whole-brain fatty acid concentration is necessitated. Here, we sought to conduct a comprehensive quantitative analysis of fatty acids from entire well-characterized human brain hemispheres (n = 6) provided by the Douglas-Bell Canada Brain Bank. Additionally, exploratory natural abundance carbon isotope ratio (CIR; δ13 C, 13 C/12 C) analysis was performed to assess the origin of brain fatty acids. Brain fatty acid methyl esters (FAMEs) were quantified by gas chromatography (GC)-flame ionization detection and minor n-6 and n-3 polyunsaturated fatty acid pentafluorobenzyl esters by GC-mass spectrometry. Carbon isotope ratio values of identifiable FAMEs were measured by GC-combustion-isotope ratio mass spectrometry. Overall, the most abundant fatty acid in the human brain was oleic acid, followed by stearic acid (STA), palmitic acid (PAM), docosahexaenoic acid (DHA), and arachidonic acid (ARA). Interestingly, cholesterol as well as saturates including PAM and STA were most enriched in 13 C, while PUFAs including DHA and ARA were most depleted in 13 C. These findings suggest a contribution of endogenous synthesis utilizing dietary sugar substrates rich in 13 C, and a combination of marine, animal, and terrestrial PUFA sources more depleted in 13 C, respectively. These results provide novel insights on cerebral fatty acid origin and concentration, the latter serving as a valuable resource for future modeling of fatty acid metabolism in the human brain.

Early-life stress lastingly impacts microglial transcriptome and function under basal and immune-challenged conditions.

Early-life stress (ELS) leads to increased vulnerability to psychiatric disorders including depression later in life. Neuroinflammatory processes have been implicated in ELS-induced negative health outcomes, but how ELS impacts microglia, the main tissue-resident macrophages of the central nervous system, is unknown. Here, we determined the effects of ELS-induced by limited bedding and nesting material during the first week of life (postnatal days [P]2-9) on microglial (i) morphology; (ii) hippocampal gene expression; and (iii) synaptosome phagocytic capacity in male pups (P9) and adult (P200) mice. The hippocampus of ELS-exposed adult mice displayed altered proportions of morphological subtypes of microglia, as well as microglial transcriptomic changes related to the tumor necrosis factor response and protein ubiquitination. ELS exposure leads to distinct gene expression profiles during microglial development from P9 to P200 and in response to an LPS challenge at P200. Functionally, synaptosomes from ELS-exposed mice were phagocytosed less by age-matched microglia. At P200, but not P9, ELS microglia showed reduced synaptosome phagocytic capacity when compared to control microglia. Lastly, we confirmed the ELS-induced increased expression of the phagocytosis-related gene GAS6 that we observed in mice, in the dentate gyrus of individuals with a history of child abuse using in situ hybridization. These findings reveal persistent effects of ELS on microglial function and suggest that altered microglial phagocytic capacity is a key contributor to ELS-induced phenotypes.