Inflammation has emerged as a prominent feature of bipolar disorder (BD) pathophysiology, drawing attention to brain barriers known to regulate immune-brain interactions. While perturbation of the blood-brain barrier has been reported in BD, the blood-cerebrospinal fluid (CSF) barrier formed largely by the choroid plexus (ChP) remains underexamined. To address this gap in knowledge, we used a multiplex array to measure cytokine protein abundance in postmortem ChP tissue from individuals with BD and unaffected controls, revealing elevated levels of CCL2 and SPP1, factors associated with monocyte and macrophage recruitment and activation. In contrast, expression of cytokines involved in tissue homeostasis, trophic support, and immune signaling, including OSM, IGF-1, CX3CL1, TGFB3, GDNF, LIF, BDNF, SCF, and FGFs, was reduced. Several cytokines, including CCL2 and PLGF, exhibited condition-specific divergent age trajectories. Bulk RNA sequencing of the same cohort revealed a modest set of differentially expressed genes, including transcripts associated with oxidative stress, mitochondrial function, and immune regulation that were upregulated in BD. Notably, the BD CSF biomarker NELL2 was downregulated in the ChP. Gene set enrichment analysis highlighted activation of inflammatory and cellular stress pathways, as well as reduced expression of junction-related gene programs. These findings suggest a shift in ChP function in BD characterized by increased pro-inflammatory signaling and reduced trophic and barrier-supportive activity. Together, these data identify the ChP as an active site of immune dysregulation in BD and support the broader notion of brain barrier dysfunction in mood disorder pathology.

Preclinical and clinical evidence has implicated inflammation in the pathophysiology of depression. Abnormal cytokine levels in blood, cerebrospinal fluid, and post-mortem brain samples have been associated with depression. To our knowledge, however, a comprehensive analysis of cytokine protein levels in brain samples from patients with depression has yet to be conducted in major components of the limbic system such as the ventromedial prefrontal cortex (vmPFC). This region plays a crucial role in depression, impacting cognitive control, emotional regulation, and executive functions. In the current exploratory study, we performed a comprehensive profiling of 72 cytokines, chemokines and growth factors in well-characterized vmPFC samples from 34 depressed suicides and 14 matched sudden-death controls. A human antibody array (RayBio®, chemiluminescent detection) was used to measure all markers. In depressed suicide samples, no significant increase in any cytokine, chemokine or growth factor was detected compared to controls. In comparison, in an independent cohort we measured the levels of 43 inflammatory markers in plasma samples from 141 depressed living subjects and 36 controls using the Mesoscale Discovery V-plex assay. Our analyses indicated no significant difference in the levels of pro- or anti-inflammatory markers in the plasma of cases vs controls. We also conducted a detailed morphological analysis of Iba1-immunostained microglia in vmPFC gray matter samples from 28 depressed suicides and 13 healthy controls. The distributions of the various morphological phenotypes assessed were similar between groups, suggesting that microglia/macrophages do not display signs of morphological changes in the vmPFC of depressed suicides. Taken together, these complementary experiments do not provide evidence of depression-associated neuroinflammatory changes in the vmPFC, at least in the samples analyzed.

Perineuronal nets (PNNs) are condensed extracellular matrix structures that regulate synaptic plasticity and neuronal stability. While their distribution and function have been well characterized in rodents, PNNs remain largely understudied in the human cerebellum. Here, we provide an anatomical characterization of PNNs in the post-mortem human cerebellum and compare their distribution and cellular phenotypes with those in mouse and macaque cerebellum. Using immunofluorescence for both Wisteria floribunda lectin (WFL) and aggrecan, we observed that PNNs in humans were exclusively localized in the deep cerebellar nuclei (DCN), with no detectable labelling in cerebellar cortex. In contrast, both macaques and mice exhibited PNNs in the DCN and cortex (granule cell layer), with interspecies differences in density and marker co-localization. Combining immunolabeling with fluorescence in situ hybridization for SLC17A7, GAD1, and PVALB, we found that in human and mouse DCN, PNNs predominantly surrounded excitatory, parvalbumin-expressing neurons, whereas in macaques, PV expression was absent from PNN-enwrapped excitatory cells. These findings highlight both conserved and divergent features of cerebellar PNNs, providing novel insights into the potential functional roles of these structures in human cerebellar circuitry, with implications for understanding cerebellar plasticity and disease vulnerability.

Exposure to early life adversity (ELA) poses a significant global public health concern, with profound pathophysiological implications for affected individuals. Studies suggest that ELA contributes to endothelial dysfunction, bringing into question the functional integrity of the neurovascular unit in brain regions vulnerable to chronic stress. Despite the importance of the neurovasculature in maintaining normal brain physiology, human neurovascular cells remain poorly characterized, particularly with regard to their contributory role in ELA-associated pathophysiologies. In this study, we present the first comprehensive transcriptomic analysis of microvessels isolated from postmortem ventromedial prefrontal cortex samples from adult healthy controls (CTRL) and matched depressed suicides with histories of ELA. Our findings point to substantial differences between men and women, with the latter exhibiting widespread transcriptional changes at the neurovascular unit, including key vascular nodal regulators KLF2 and KLF4, alongside a broad downregulation of immune-related pathways. These results suggest that the neurovascular unit plays a larger role in the neurobiological consequences of ELA in women.

Microglia orchestrate immunological responses in the brain and play an important role in maintaining homeostatic brain functions. Several studies have reported clock gene expression in microglia and the circadian rhythm they drive has been linked to the modulation of immune responses and neuronal functions. In the current study, complementary approaches, including immunofluorescence, multiplexed fluorescence in situ hybridization, and liquid chromatography-mass spectrometry proteomics of isolated CD11b+ microglia, were combined with publicly available transcriptomic and epigenomic datasets to investigate the expression of the core clock gene BMAL1 in human post-mortem cortical and limbic areas as well as mouse brain. The majority of BMAL1 -expressing cells were found to be neurons, with microglia representing a negligeable proportion. We also identified significantly lower chromatin accessibility or “openness” for BMAL1 gene regulatory regions (such as promoters and enhancers) in microglia compared to neurons. These regulatory regions in microglia were enriched for ETS domain transcription factor (TF) binding sites. Together, this suggests a strong role of chromatin remodeling factors in suppressing BMAL1 gene expression in microglia. Finally, while we observed a very low expression, BMAL1 TF motifs were accessible in open chromatin landscape of microglia, which may lead to downstream gene-regulatory effects upon binding, even if BMAL1 expression is constitutively low. Overall, our results reveal low or absent expression of BMAL1 in microglia and point towards potential epigenetic mechanisms regulating its expression in these cells. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research

Genetic variants associated with major depressive disorder (MDD) are enriched in the regulatory genome. Here, we investigate gene-regulatory mechanisms underlying MDD compared to neurotypical controls by combining single-cell chromatin accessibility with gene expression in over 200,000 cells from the dorsolateral prefrontal cortex of 84 individuals. MDD-associated alterations in chromatin accessibility were prominent in deep-layer excitatory neurons characterized by transcription factor (TF) motif accessibility and binding of NR4A2, an activity-dependent TF reactive to stress. The same neurons were enriched for MDD-associated genetic variants, disrupting TF binding sites linked to genes that likely affect synaptic communication. Furthermore, a gray matter microglia cluster exhibited decreased accessibility in individuals with MDD at binding sites bound by TFs known to regulate immune homeostasis. Finally, we identified gene-regulatory effects of MDD-risk variants using sequence-based accessibility predictions, donor-specific genotypes and cell-based assays. These findings shed light on the cell types and regulatory mechanisms through which genetic variation may increase the risk of MDD.

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.

ABSTRACT Using spatial transcriptomics and FISH, we detected very few cells expressing neural stem cell- and proliferation-specific genes in the human dentate gyrus (DG) from childhood to middle age. However, we observed at all ages a significant number of DG cells expressing the immature neuronal marker DCX . Across ages, the majority of these cells displayed an inhibitory phenotype, while the remainder were non-committed or excitatory in nature.

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.

Inflammatory bowel disease (IBD), a common term for Crohn's disease and ulcerative colitis, is a chronic, relapse-remitting condition of the gastrointestinal tract that is increasing worldwide. Psychiatric comorbidities, including depression and anxiety, are more prevalent in IBD patients than in healthy individuals. Evidence suggests that varying levels of neuroinflammation might underlie these states in IBD patients. Within this context, microglia are the crucial non-neural cells in the brain responsible for innate immune responses following inflammatory insults. Alterations in microglia's functions, such as secretory profile, phagocytic activity, and synaptic pruning, might play significant roles in mediating psychiatric manifestations of IBD. In this review, we discuss the role played by microglia in IBD-associated comorbidities.

Microglia, known for their diverse roles in the central nervous system, have recently been recognized for their involvement in degrading the extracellular matrix. Perineuronal nets (PNNs), a specialized form of this matrix, are crucial for stabilizing neuronal connections and constraining plasticity. Our group recently reported increased PNN densities in the ventromedial prefrontal cortex (vmPFC) of depressed individuals that died by suicide in adulthood after experiencing childhood abuse (DS-CA) compared to matched controls. To explore potential underlying mechanisms, we employed a comprehensive approach in similar postmortem vmPFC samples, combining a human matrix metalloproteinase and chemokine array, isolation of CD11b-positive microglia and enzyme-linked immunosorbent assays (ELISA). Our findings indicate a significant downregulation of matrix metalloproteinase (MMP)-9 and tissue inhibitors of metalloproteinases (TIMP)-2 in both whole vmPFC grey matter and isolated microglial cells from DS-CA samples. Furthermore, our experiments reveal that a history of child abuse is associated with diminished levels of microglial CX3CR1 and IL33R in both vmPFC whole lysate and CD11b isolated cells. However, levels of the CX3CR1 ligand, CX3CL1 (Fractalkine), did not differ between groups. While these data suggest potential long-lasting alterations in microglial markers in the vmPFC of individuals exposed to severe childhood adversity, direct functional assessments were not conducted. Nonetheless, these findings offer insight into how childhood abuse may contribute to PNN alterations via microglial-related mechanisms.

A novel radiotracer, [ 11 C]SL25.1188 targets monoamine oxidase-B (MAO-B) enzyme, which metabolizes monoamines (including dopamine) primarily found in astrocytes. Altered astrocyte function in schizophrenia is supported by convergent evidence from post-mortem, genetic, transcriptomic, peripheral and preclinical findings. However, this has never been tested in living brains of early psychosis. Thirty-eight participants including antipsychotic-free/minimally exposed patients with first-episode psychosis (FEP), clinical high-risk (CHR) individuals and healthy volunteers (HVs) underwent a 90-minute positron emission tomography (PET) scan with [ 11 C]SL25.1188, to measure MAO-B V T , an index of MAO-B concentration. Participants were excluded if tested positive on urine drug screen (except for cannabis). This study of 14 FEP (mean[SD] age, 25.7[5.7] years; 6 F), 7 CHR (mean[SD] age, 20.9[3.7] years; 4 F) and 17 HV (mean[SD] age, 31.2[13.9] years; 9 F) demonstrated significant group differences in regional MAO-B V T (F (2,37.46) = 4.56, p = 0.02, Cohen’s f = 0.49), controlling for tobacco (F (1,37.46) = 5.50 p = 0.02) and cannabis use (F (1,37.46) = 5.05, p = 0.03) with significant reductions in CHR compared to HV (Cohen’s d = 0.99). We report a significant cannabis effect on MAO-B V T (F (1,39.47) = 12.45, p = 0.001, Cohen’s f = 0.56), with a significant group-by-cannabis interaction (F (2,37.35) = 3.81, p = 0.03, Cohen’s f = 0.45), indicating lower MAO-B V T in cannabis-using patients. Decreased MAO-B V T levels was more robust in striatal than cortical regions, in both clinical groups (F (12,46.07) = 2.00, p = 0.046, Cohen’s f = 0.72) and in cannabis users (F (6,46.07) = 6.01, p < 0.001, Cohen’s f = 0.89). Reduced MAO-B concentration supports astrocyte dysfunction in cannabis-using CHR and FEP patients. Reduced MAO-B is consistent with replicated striatal dopamine elevation in psychosis, as well as astrocyte dysfunction in schizophrenia.

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.

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.

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.

We recently described a novel ribosome-based regulatory mechanism/checkpoint that controls innate immune gene translation and microglial activation in non-sterile inflammation orchestrated by RNA binding protein SRSF3. Here we describe a role of SRSF3 in the regulation of microglia/macrophage activation phenotypes after experimental stroke. Using a model-system for analysis of the dynamic translational state of microglial ribosomes we show that 24 h after stroke highly upregulated immune mRNAs are not translated resulting in a marked dissociation of mRNA and protein networks in activated microglia/macrophages. Next, microglial activation after stroke was characterized by a robust increase in pSRSF3/SRSF3 expression levels. Targeted knockdown of SRSF3 using intranasal delivery of siRNA 24 h after stroke caused a marked knockdown of endogenous protein. Further analyses revealed that treatment with SRSF3-siRNA alleviated translational arrest of selected genes and induced a transient but significant increase in innate immune signaling and IBA1+ immunoreactivity peaking 5 days after initial injury. Importantly, delayed SRSF3-mediated increase in immune signaling markedly reduced the size of ischemic lesion measured 7 days after stroke. Together, our findings suggest that targeting SRSF3 and immune mRNA translation may open new avenues for molecular/therapeutic reprogramming of innate immune response after ischemic injury.

Brain regions involved in olfaction, including the olfactory bulb, have been implicated in the etiology of major depression mainly on the basis of rodent models of the illness. To explore more directly the molecular features of OB in major depression, a global comparative proteome analysis was carried out with human OB samples from 12 middle-aged male depressed suicides and 12 matched controls. Cases displayed a significant reduction in astrocytic proteins. 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 suicides. 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 can be observed in an animal model of depression, we also performed proteomics on the OB of socially defeated mice. 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 and suicide in humans, and suggests that animal models of depression may not display all cellular and molecular features of depression, at least in the OB.

Major depressive disorder (MDD) associated genetic variants reside primarily in the non-coding, regulatory genome. Here we investigate genome-wide regulatory differences and putative gene-regulatory effects of disease risk-variants by examining chromatin accessibility combined with single-cell gene-expression profiles in over 200,000 cells from the dorsolateral prefrontal cortex (DLPFC) of 84 individuals with MDD and neurotypical controls. MDD-associated accessibility alterations were prominent in deep-layer excitatory neurons characterized by transcription factor (TF) motif accessibility and binding of nuclear receptor (NR)4A2, an activity-dependent TF responsive to pathological stress. The same neurons were significantly enriched for MDD-associated genetic variation disrupting cis-regulatory sites and TF binding associated with genes involved in synaptic communication. Furthermore, a grey matter microglial cluster exhibited differentially closed chromatin in MDD affecting binding sites bound by TFs known to regulate immune homeostasis. In summary, our study points to specific cell types and regulatory mechanisms whereby genetic variation may increase predisposition to MDD.

The neurovascular unit (NVU), comprised of endothelial cells, pericytes, smooth muscle cells, astrocytic endfeet and microglia together with neurons, is paramount for the proper function of the central nervous system. The NVU gatekeeps blood-brain barrier (BBB) properties which, as a system, experiences impairment in several neurological and psychiatric diseases, and contributes to pathogenesis. To better understand function and dysfunction at the NVU, isolation and characterization of the NVU 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 vmPFC tissue and are comprehensively investigated using both RNA sequencing and Liquid Chromatography with tandem mass spectrometry (LC-MS-MS). Both the transcriptome and proteome are elucidated 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.

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.