Cognitive and inflammatory heterogeneity in severe mental illness: Translating findings from blood to brain.

Recent findings link cognitive impairment and inflammatory-immune dysregulation in schizophrenia (SZ) and bipolar (BD) spectrum disorders. However, heterogeneity and translation between the periphery and central (blood-to-brain) mechanisms remains a challenge. Starting with a large SZ, BD and healthy control cohort (n = 1235), we aimed to i) identify candidate peripheral markers (n = 25) associated with cognitive domains (n = 9) and elucidate heterogenous immune-cognitive patterns, ii) evaluate the regulation of candidate markers using human induced pluripotent stem cell (iPSC)-derived astrocytes and neural progenitor cells (n = 10), and iii) evaluate candidate marker messenger RNA expression in leukocytes using microarray in available data from a subsample of the main cohort (n = 776), and in available RNA-sequencing deconvolution analysis of postmortem brain samples (n = 474) from the CommonMind Consortium (CMC). We identified transdiagnostic subgroups based on covariance between cognitive domains (measures of speed and verbal learning) and peripheral markers reflecting inflammatory response (CRP, sTNFR1, YKL-40), innate immune activation (MIF) and extracellular matrix remodelling (YKL-40, CatS). Of the candidate markers there was considerable variance in secretion of YKL-40 in iPSC-derived astrocytes and neural progenitor cells in SZ compared to HC. Further, we provide evidence of dysregulated RNA expression of genes encoding YKL-40 and related signalling pathways in a high neuroinflammatory subgroup in the postmortem brain samples. Our findings suggest a relationship between peripheral inflammatory-immune activity and cognitive impairment, and highlight YKL-40 as a potential marker of cognitive functioning in a subgroup of individuals with severe mental illness.

Increased ferroptosis in leukocytes from preeclamptic women involving the long non-coding taurine upregulated gene 1 (TUG1).

BACKGROUND: Ferroptosis plays a key role in placental development and physiology, and abnormal ferroptosis has been implicated in trophoblast injury leading to preeclampsia (PE). We hypothesize that leukocytes isolated from PE exhibit increased ferroptosis and that extracellular vesicles contain long non-coding (lnc) RNA/mRNAs that modulate oxidative stress and iron toxicity in vascular endothelial cells. METHODS: We measured the expression of key regulators of ferroptosis in leukocytes and extracellular vesicles as well as circulating biomarkers of iron homeostasis and oxidative stress in plasma from women with/without PE at different timepoints during pregnancy. For markers that were dysregulated, we assessed their temporal correlation with established markers of disease activity and marker of endothelial activation. For markers dysregulated in early pregnancy, we assessed their ability to predict the development of PE. RESULTS: We found decreased lncRNA/mRNAs in leukocytes, but not extracellular vesicles, in PE that may modulate oxidative stress and iron toxicity. This decrease in anti-ferroptotic markers does not appear to be related to maternal disease activity or plasma oxidative stress status but rather to attenuated anti-inflammatory expression in these cells. Circulating ferritin was elevated in PE, supporting the hypothesis that PE represents a disbalance in iron homeostasis. Low lncRNA taurine upregulated gene 1 RNA levels in leukocytes at 22-24 weeks were strongly associated with the development of PE. CONCLUSIONS: Our findings suggest that maternal leukocytes in PE show decreased anti-ferroptotic activity that correlates with anti-inflammatory expression. Moreover, some of these changes in ferroptotic activity appear to precede the development of PE.

Longitudinal Transcriptomic Analysis of Human Cortical Spheroids Identifies Axonal Dysregulation in the Prenatal Brain as a Mediator of Genetic Risk for Schizophrenia.

BACKGROUND: Schizophrenia (SCZ) has a known neurodevelopmental etiology, but limited access to human prenatal brain tissue hampers the investigation of basic disease mechanisms in early brain development. Here, we elucidate the molecular mechanisms contributing to SCZ risk in a disease-relevant model of the prenatal human brain. METHODS: We generated induced pluripotent stem cell-derived organoids, termed human cortical spheroids (hCSs), from a large, genetically stratified sample of 14 SCZ cases and 14 age- and sex-matched controls. The hCSs were differentiated for 150 days, and comprehensive molecular characterization across 4 time points was carried out. RESULTS: The transcriptional and cellular architecture of hCSs closely resembled that of fetal brain tissue at 10 to 24 postconception weeks, showing strongest spatial overlap with frontal regions of the cerebral cortex. A total of 3520 genes were differentially modulated between SCZ and control hCSs across organoid maturation, displaying a significant contribution of genetic loading, an overrepresentation of risk genes for autism spectrum disorder and SCZ, and the strongest enrichment for axonal processes in all hCS stages. The two axon guidance genes SEMA7A and SEMA5A, the first a promoter of synaptic functions and the second a repressor, were downregulated and upregulated, respectively, in SCZ hCSs. This expression pattern was confirmed at the protein level and replicated in a large postmortem sample. CONCLUSIONS: Applying a disease-relevant model of the developing fetal brain, we identified consistent dysregulation of axonal genes as an early risk factor for SCZ, providing novel insights into the effects of genetic predisposition on the neurodevelopmental origins of the disorder.

Polygenic overlap with body-mass index improves prediction of treatment-resistant schizophrenia.

Treatment resistant schizophrenia (TRS) is characterized by repeated treatment failure with antipsychotics. A recent genome-wide association study (GWAS) of TRS showed a polygenic architecture, but no significant loci were identified. Clozapine is shown to be the superior drug in terms of clinical effect in TRS; at the same time it has a serious side effect profile, including weight gain. Here, we sought to increase power for genetic discovery and improve polygenic prediction of TRS, by leveraging genetic overlap with Body Mass Index (BMI). We analysed GWAS summary statistics for TRS and BMI applying the conditional false discovery rate (cFDR) framework. We observed cross-trait polygenic enrichment for TRS conditioned on associations with BMI. Leveraging this cross-trait enrichment, we identified 2 novel loci for TRS at cFDR <0.01, suggesting a role of MAP2K1 and ZDBF2. Further, polygenic prediction based on the cFDR analysis explained more variance in TRS when compared to the standard TRS GWAS. These findings highlight putative molecular pathways which may distinguish TRS patients from treatment responsive patients. Moreover, these findings confirm that shared genetic mechanisms influence both TRS and BMI and provide new insights into the biological underpinnings of metabolic dysfunction and antipsychotic treatment.

Transcriptional and functional effects of lithium in bipolar disorder iPSC-derived cortical spheroids.

Lithium (Li) is recommended for long-term treatment of bipolar disorder (BD). However, its mechanism of action is still poorly understood. Induced pluripotent stem cell (iPSC)-derived brain organoids have emerged as a powerful tool for modeling BD-related disease mechanisms. We studied the effects of 1 mM Li treatment for 1 month in iPSC-derived human cortical spheroids (hCS) from 10 healthy controls (CTRL) and 11 BD patients (6 Li-responders, Li-R, and 5 Li non-treated, Li-N). At day 180 of differentiation, BD hCS showed smaller size, reduced proportion of neurons, decreased neuronal excitability and reduced neural network activity compared to CTRL hCS. Li rescued excitability of BD hCS neurons by exerting an opposite effect in the two diagnostic groups, increasing excitability in BD hCS and decreasing it in CTRL hCS. We identified 132 Li-associated differentially expressed genes (DEGs), which were overrepresented in sodium ion homeostasis and kidney-related pathways. Moreover, Li regulated secretion of pro-inflammatory cytokines and increased mitochondrial reserve capacity in BD hCS. Through long-term Li treatment of a human 3D brain model, this study partly elucidates the functional and transcriptional mechanisms underlying the clinical effects of Li, such as rescue of neuronal excitability and neuroprotection. Our results also underscore the substantial influence of treatment duration in Li studies. Lastly, this study illustrates the potential of patient iPSC-derived 3D brain models for precision medicine in psychiatry.

Systemic Cell Adhesion Molecules in Severe Mental Illness: Potential Role of Intercellular CAM-1 in Linking Peripheral and Neuroinflammation.

BACKGROUND: Cell adhesion molecules (CAMs) orchestrate leukocyte trafficking and could link peripheral and neuroinflammation in patients with severe mental illness (SMI), by promoting inflammatory and immune-mediated responses and mediating signals across blood-brain barrier. We hypothesized that CAMs would be dysregulated in SMI and evaluated plasma levels of different vascular and neural CAMs. Dysregulated CAMs in plasma were further evaluated in vivo in leukocytes and brain tissue and in vitro in induced pluripotent stem cells. METHODS: We compared plasma soluble levels of different vascular (VCAM-1, ICAM-1, P-SEL) and neural (JAM-A, NCAD) CAMs in circulating leukocytes in a large SMI sample of schizophrenia (SCZ) spectrum disorder (n = 895) and affective disorder (n = 737) and healthy control participants (n = 1070) controlling for age, sex, body mass index, C-reactive protein, and freezer storage time. We also evaluated messenger RNA expression of ICAM1 and related genes encoding ICAM-1 receptors in leukocytes using microarray (n = 842) and in available RNA sequencing data from the CommonMind Consortium (CMC) in postmortem samples from the dorsolateral prefrontal cortex (n = 474). The regulation of soluble ICAM-1 in induced pluripotent stem cell-derived neurons and astrocytes was assessed in patients with SCZ and healthy control participants (n = 8 of each). RESULTS: Our major findings were 1) increased soluble ICAM-1 in patients with SMI compared with healthy control participants; 2) increased ITGB2 messenger RNA, encoding the beta chain of the ICAM-1 receptor, in circulating leukocytes from patients with SMI and increased prefrontal cortex messenger RNA expression of ICAM1 in SCZ; and 3) enhanced soluble ICAM-1 release in induced pluripotent stem cell-derived neurons from patients with SCZ. CONCLUSIONS: Our results support a systemic and cerebral dysregulation of soluble ICAM-1 expression in SMI and especially in patients with SCZ.

Elevated Systemic Levels of Markers Reflecting Intestinal Barrier Dysfunction and Inflammasome Activation Are Correlated in Severe Mental Illness.

BACKGROUND AND HYPOTHESIS: Gut microbiota alterations have been reported in severe mental illness (SMI) but fewer studies have probed for signs of gut barrier disruption and inflammation. We hypothesized that gut leakage of microbial products due to intestinal inflammation could contribute to systemic inflammasome activation in SMI. STUDY DESIGN: We measured plasma levels of the chemokine CCL25 and soluble mucosal vascular addressin cell adhesion molecule-1 (sMAdCAM-1) as markers of T cell homing, adhesion and inflammation in the gut, lipopolysaccharide binding protein (LBP) and intestinal fatty acid binding protein (I-FABP) as markers of bacterial translocation and gut barrier dysfunction, in a large SMI cohort (n = 567) including schizophrenia (SCZ, n = 389) and affective disorder (AFF, n = 178), relative to healthy controls (HC, n = 418). We assessed associations with plasma IL-18 and IL-18BPa and leukocyte mRNA expression of NLRP3 and NLRC4 as markers of inflammasome activation. STUDY RESULTS: Our main findings were: (1) higher levels of sMAdCAM-1 (P = .002), I-FABP (P = 7.6E-11), CCL25 (P = 9.6E-05) and LBP (P = 2.6E-04) in SMI compared to HC in age, sex, BMI, CRP and freezer storage time adjusted analysis; (2) the highest levels of sMAdCAM-1 and CCL25 (both P = 2.6E-04) were observed in SCZ and I-FABP (P = 2.5E-10) and LBP (3) in AFF; and (3), I-FABP correlated with IL-18BPa levels and LBP correlated with NLRC4. CONCLUSIONS: Our findings support that intestinal barrier inflammation and dysfunction in SMI could contribute to systemic inflammation through inflammasome activation.

Derivation and Molecular Characterization of a Morphological Subpopulation of Human iPSC Astrocytes Reveal a Potential Role in Schizophrenia and Clozapine Response.

Astrocytes are the most abundant cell type in the human brain and are important regulators of several critical cellular functions, including synaptic transmission. Although astrocytes are known to play a central role in the etiology and pathophysiology of schizophrenia, little is known about their potential involvement in clinical response to the antipsychotic clozapine. Moreover, astrocytes display a remarkable degree of morphological diversity, but the potential contribution of astrocytic subtypes to disease biology and drug response has received little attention. Here, we used state-of-the-art human induced pluripotent stem cell (hiPSC) technology to derive a morphological subtype of astrocytes from healthy individuals and individuals with schizophrenia, including responders and nonresponders to clozapine. Using functional assays and transcriptional profiling, we identified a distinct gene expression signature highly specific to schizophrenia as shown by disease association analysis of more than 10 000 diseases. We further found reduced levels of both glutamate and the NMDA receptor coagonist d-serine in subtype astrocytes derived from schizophrenia patients, and that exposure to clozapine only rescued this deficiency in cells from clozapine responders, providing further evidence that d-serine in particular, and NMDA receptor-mediated glutamatergic neurotransmission in general, could play an important role in disease pathophysiology and clozapine action. Our study represents a first attempt to explore the potential contribution of astrocyte diversity to schizophrenia pathophysiology using a human cellular model. Our findings suggest that specialized subtypes of astrocytes could be important modulators of disease pathophysiology and clinical drug response, and warrant further investigations.

A human iPSC-astroglia neurodevelopmental model reveals divergent transcriptomic patterns in schizophrenia.

While neurodevelopmental abnormalities have been associated with schizophrenia (SCZ), the role of astroglia in disease pathophysiology remains poorly understood. In the present study, we used a human induced pluripotent stem cell (iPSC)-derived astrocyte model to investigate the temporal patterns of astroglia differentiation during developmental stages critical for SCZ using RNA sequencing. The model generated astrocyte-specific gene expression patterns during differentiation that corresponded well to astroglia-specific expression signatures of in vivo cortical fetal development. Using this model we identified SCZ-specific expression dynamics, and found that SCZ-associated differentially expressed genes were significantly enriched in the medial prefrontal cortex, striatum, and temporal lobe, targeting VWA5A and ADAMTS19. In addition, SCZ astrocytes displayed alterations in calcium signaling, and significantly decreased glutamate uptake and metalloproteinase activity relative to controls. These results implicate novel transcriptional dynamics in astrocyte differentiation in SCZ together with functional changes that are potentially important biological components of SCZ pathology.

All-Optical Electrophysiology in hiPSC-Derived Neurons With Synthetic Voltage Sensors.

Voltage imaging and "all-optical electrophysiology" in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.

Lithium increases mitochondrial respiration in iPSC-derived neural precursor cells from lithium responders.

Lithium (Li), valproate (VPA) and lamotrigine (LTG) are commonly used to treat bipolar disorder (BD). While their clinical efficacy is well established, the mechanisms of action at the molecular level are still incompletely understood. Here we investigated the molecular effects of Li, LTG and VPA treatment in induced pluripotent stem cell (iPSC)-derived neural precursor cells (NPCs) generated from 3 healthy controls (CTRL), 3 affective disorder Li responsive patients (Li-R) and 3 Li non-treated patients (Li-N) after 6 h and 1 week of exposure. Differential expression (DE) analysis after 6 h of treatment revealed a transcriptional signature that was associated with all three drugs and most significantly enriched for ribosome and oxidative phosphorylation (OXPHOS) pathways. In addition to the shared DE genes, we found that Li exposure was associated with 554 genes uniquely regulated in Li-R NPCs and enriched for spliceosome, OXPHOS and thermogenesis pathways. In-depth analysis of the treatment-associated transcripts uncovered a significant decrease in intron retention rate, suggesting that the beneficial influence of these drugs might partly be related to splicing. We examined the mitochondrial respiratory function of the NPCs by exploring the drugs' effects on oxygen consumption rate (OCR) and glycolytic rate (ECAR). Li improved OCR levels only in Li-R NPCs by enhancing maximal respiration and reserve capacity, while VPA enhanced maximal respiration and reserve capacity in Li-N NPCs. Overall, our findings further support the involvement of mitochondrial functions in the molecular mechanisms of mood stabilizers and suggest novel mechanisms related to the spliceosome, which warrant further investigation.

Transcriptome analysis reveals disparate expression of inflammation-related miRNAs and their gene targets in iPSC-astrocytes from people with schizophrenia.

Despite the high heritability of schizophrenia (SCZ), details of its pathophysiology and etiology are still unknown. Recent findings suggest that aberrant inflammatory regulation and microRNAs (miRNAs) are involved. Here we performed a comparative analysis of the global miRNome of human induced pluripotent stem cell (iPSC)-astrocytes, derived from SCZ patients and healthy controls (CTRLs), at baseline and following inflammatory modulation using IL-1ß. We identified four differentially expressed miRNAs (miR-337-3p, miR-127-5p, miR-206, miR-1185-1-3p) in SCZ astrocytes that exhibited significantly lower baseline expression relative to CTRLs. Group-specific differential expression (DE) analyses exploring possible distinctions in the modulatory capacity of IL-1ß on miRNA expression in SCZ versus CTRL astroglia revealed trends toward altered miRNA expressions. In addition, we analyzed peripheral blood samples from a large cohort of SCZ patients (n = 484) and CTRLs (n = 496) screening for the expression of specific gene targets of the four DE miRNAs that were identified in our baseline astrocyte setup. Three of these genes, LAMTOR4, IL23R, and ERBB3, had a significantly lower expression in the blood of SCZ patients compared to CTRLs after multiple testing correction. We also found nominally significant differences for ERBB2 and IRAK1, which similarly displayed lower expressions in SCZ versus CTRL. Furthermore, we found matching patterns between the expressions of identified miRNAs and their target genes when comparing our in vitro and in vivo results. The current results further our understanding of the pathobiological basis of SCZ.

Cannabis Use Is Associated With Increased Levels of Soluble gp130 in Schizophrenia but Not in Bipolar Disorder.

The complex effects of plant cannabinoids on human physiology is not yet fully understood, but include a wide spectrum of effects on immune modulation. The immune system and its inflammatory effector pathways are recently emerging as possible causative factors in psychotic disorders. The present study aimed to investigate whether self-administered Cannabis use was associated with changes in circulating immune and neuroendocrine markers in schizophrenia (SCZ) and bipolar disorder (BD) patients. A screening of 13 plasma markers reflecting different inflammatory pathways was performed in SCZ (n = 401) and BD patients (n = 242) after subdividing each group into Cannabis user and non-user subgroups. We found that i) soluble gp130 (sgp130) concentrations were significantly elevated among Cannabis users in the SCZ group (p = 0.002) after multiple testing correction, but not in BD. ii) Nominally significant differences were observed in the levels of IL-1RA (p = 0.0059), YKL40 (p = 0.0069), CatS (p = 0.013), sTNFR1 (p = 0.031), and BDNF (p = 0.020), where these factors exhibited higher plasma levels in Cannabis user SCZ patients than in non-users. iii) These differences in systemic levels were not reflected by altered mRNA expression of genes encoding sgp130, IL-1RA, YKL40, CatS, sTNFR1, and BDNF in whole blood. Our results show that Cannabis self-administration is associated with markedly higher sgp130 levels in SCZ, but not in BD, and that this phenomenon is independent of the modulation of peripheral immune cells. These findings warrant further investigation into the potential IL-6 trans-signaling modulatory, anti-inflammatory, neuroimmune, and biobehavioral-cognitive effects of Cannabis use in SCZ.

Decreased IL-1ß-induced CCL20 response in human iPSC-astrocytes in schizophrenia: Potential attenuating effects on recruitment of regulatory T cells.

Schizophrenia (SCZ) is a severe mental disorder with a high heritability. Although its pathophysiology is mainly unknown, dysregulated immune activation and inflammation have recently emerged as possible candidates in the underlying mechanisms of SCZ. Previous studies suggest that aberrant inflammasome activation, glia dysregulation, and brain inflammation may be involved in the pathophysiology of the disorder. Here, we studied the effects of inflammatory modulation on human induced pluripotent stem cell (iPSC)-derived astrocytes generated from SCZ patients and healthy controls (CTRL). Inflammasome activation was mimicked by short-term IL-1ß exposure, and gene expression were measured with high-coverage RNA-Seq to ensure a global characterization of the transcriptional effects of the treatment. IL-1ß exposure modulated several pathways involved in innate immune responses, cell cycle regulation, and metabolism in both SCZ and CTRL astrocytes. Significant differences were found in the expression of HILPDA and CCL20 genes, both of which had reduced up-regulation upon IL-1ß treatment in SCZ astrocytes compared to CTRL astrocytes. CCL20 data were further validated and confirmed using qPCR, ELISA, and regulatory T lymphocyte (Treg) migration assays. Additionally, we found significantly decreased mRNA expression of the Treg-specific marker FOXP3 in the blood of a large cohort of SCZ patients (n = 484) compared to CTRL (n = 472). Since CCL20 is a specific chemoattractant for CD4+CD25+CCR6+ Tregs, which are crucially involved in anti-inflammatory responses during brain (auto)inflammation, our results imply a plausible role for an altered astroglia-CCL20-CCR6-Treg axis in SCZ pathophysiology.

Exploring lithium's transcriptional mechanisms of action in bipolar disorder: a multi-step study.

Lithium has been the first-line treatment for bipolar disorder (BD) for more than six decades. Although the molecular effects of lithium have been studied extensively and gene expression changes are generally believed to be involved, the specific mechanisms of action that mediate mood regulation are still not known. In this study, a multi-step approach was used to explore the transcriptional changes that may underlie lithium's therapeutic efficacy. First, we identified genes that are associated both with lithium exposure and with BD, and second, we performed differential expression analysis of these genes in brain tissue samples from BD patients (n = 42) and healthy controls (n = 42). To identify genes that are regulated by lithium exposure, we used high-sensitivity RNA-sequencing of corpus callosum (CC) tissue samples from lithium-treated (n = 8) and non-treated (n = 9) rats. We found that lithium exposure significantly affected 1108 genes (FDR < 0.05), 702 up-regulated and 406 down-regulated. These genes were mostly enriched for molecular functions related to signal transduction, including well-established lithium-related pathways such as mTOR and Wnt signaling. To identify genes with differential expression in BD, we performed expression quantitative trait loci (eQTL) analysis on BD-associated genetic variants from the most recent genome-wide association study (GWAS) using three different gene expression databases. We found 307 unique eQTL genes regulated by BD-associated variants, of which 12 were also significantly modulated by lithium treatment in rats. Two of these showed differential expression in the CC of BD cases: RPS23 was significantly down-regulated (p = 0.0036, fc = 0.80), while GRIN2A showed suggestive evidence of down-regulation in BD (p = 0.056, fc = 0.65). Crucially, GRIN2A was also significantly up-regulated by lithium in the rat brains (p = 2.2e-5, fc = 1.6), which suggests that modulation of GRIN2A expression may be a part of the therapeutic effect of the drug. These results indicate that the recent upsurge in research on this central component of the glutamatergic system, as a target of novel therapeutic agents for affective disorders, is warranted and should be intensified.

Expression of TCN1 in Blood is Negatively Associated with Verbal Declarative Memory Performance.

Memory is indispensable for normal cognitive functioning, and the ability to store and retrieve information is central to mental health and disease. The molecular mechanisms underlying complex memory functions are largely unknown, but multiple genome-wide association studies suggest that gene regulation may play a role in memory dysfunction. We performed a global gene expression analysis using a large and balanced case-control sample (n = 754) consisting of healthy controls and schizophrenia and bipolar disorder patients. Our aim was to discover genes that are differentially expressed in relation to memory performance. Gene expression in blood was measured using Illumina HumanHT-12 v4 Expression BeadChip and memory performance was assessed with the updated California Verbal Learning Test (CVLT-II). We found that elevated expression of the vitamin B12-related gene TCN1 (haptocorrin) was significantly associated with poorer memory performance after correcting for multiple testing (ß = -1.50, p = 3.75e-08). This finding was validated by quantitative real-time PCR and followed up with additional analyses adjusting for confounding variables. We also attempted to replicate the finding in an independent case-control sample (n = 578). The relationship between TCN1 expression and memory impairment was comparable to that of important determinants of memory function such as age and sex, suggesting that TCN1 could be a clinically relevant marker of memory performance. Thus, we identify TCN1 as a novel genetic finding associated with poor memory function. This finding may have important implications for the diagnosis and treatment of vitamin B12-related conditions.