Maree J. Webster

Many epidemiological studies have shown an association between infectious agents, particularly viruses, and psychiatric disorders including schizophrenia. However, evidence of a viral infection in the brain that associates with schizophrenia (SCZ), bipolar disorder (BPD), and major depression (MDD) has not been found. A potential reason for this discrepancy may be that viruses are more likely to infect the neuroepithelium than neural tissue. To test this hypothesis, we used viral sequence enrichment technology and performed RNA sequencing in postmortem choroid plexus (CP) isolated from 84 SCZ, 73 BPD, 23 MDD cases and 76 unaffected controls (CNT) from the Stanley Medical Research Institute brain collection. This approach enabled us to identify the presence of 13 viral species in the CP of 46 subjects. We discovered that CP samples collected from subjects with SCZ and BPD are more likely to contain viral sequences. In terms of individual viruses, the Hepatitis C Virus (HCV) was the only viral species that reached the threshold of statistical significance for an association with SCZ and BPD. Therefore, we focused on HCV to characterize the association between psychiatric disorders and viruses. Analysis of the TriNetX electronic health record database with data on 285 million patients revealed that the prevalence of chronic HCV was 3.6 and 3.9 percent in the SCZ and BPD populations respectively. The prevalence of chronic HCV in these populations was almost double that observed for MDD (1.8%) and approximately 7-fold higher than the control population (0.5%). These findings confirm previous studies that report higher HCV prevalence in SCZ and BPD and suggest that HCV infection may be associated with disease pathology rather than behaviors such as intravenous drug injection, since these behaviors are present in all three disorders. We analyzed hippocampus RNA sequencing data from the subjects identified to be HCV positive via sequence capture. We found that although the virus was absent in this tissue, HCV RNA in the CP was associated with consistent host transcriptional changes in the hippocampus that were potentially related to the innate immune response. Our results are consistent with previous studies and provide clues regarding the contribution of viruses to the pathology of psychiatric disorders.

Elevated inflammation in the midbrain of ~45% of people with schizophrenia may relate to altered trophic support for neurons. Dopamine neurons require trophic support from Brain-Derived Neurotrophic Factor (BDNF), that signals via the full-length Tropomyosin kinase B receptor (TrkB^TK+). The truncated BDNF receptor (TrkB^TK-) and the apoptosis-related p75 receptor may counteract the effects of BDNF. We hypothesised that transcriptional changes in either BDNF, and/or a transcription factor critical for the maintenance of dopamine neurons (Nuclear Receptor Related-1 protein; NURR1), and/or BDNF receptors – TrkB (TK+ or TK-) and p75, would be found in the post-mortem schizophrenia midbrain, particularly in schizophrenia cases defined as “high inflammation”. The neuroinflammatory status was delineated based on elevated expression levels of a combination of pro-inflammatory transcripts (SERPINA3, IL6, IL1β and TNFα) and defined as a subgroup (46%) by 2-step recursive clustering. Using RT-qPCR, mRNA levels of NURR1, BDNF, TrkB and p75 was quantified in schizophrenia (n = 65) and control (n = 64) ventral mesencephalon. We found significant decreases in BDNF, TrkB^TK+ and NURR1 (14–18%) and increases in TrkB^TK- and p75 (18–35%) mRNA levels in schizophrenia compared to controls (all p < 0.05), with exacerbation of changes identified in high inflammation schizophrenia. To determine whether these changes would be consistent with resulting from chronic antipsychotic treatment, we treated healthy adult rats with antipsychotics (haloperidol and risperidone) for 7 months and found all transcripts to be unaltered compared to control rats. SnRNAseq of human midbrain showed that p75 receptor mRNA is primarily localised in oligodendrocytes and pan-TrkB mRNA is in both neurons and astrocytes. We confirmed that p75 was localised to oligodendrocyte-like cells by immunohistochemistry. Altogether, we find transcriptional evidence of reduced trophic support in schizophrenia midbrain and suggest that this may directly impact dopamine neuron health, particularly when neuroinflammation is also present.

Elevated inflammation in the midbrain of ~ 45% of people with schizophrenia may relate to altered trophic support for neurons in schizophrenia. Dopamine neurons require trophic support from Brain-Derived Neurotrophic Factor (BDNF), that signals via the full-length Tropomyosin kinase B receptor (TrkB^TK+). The truncated BDNF receptor (TrkB^TK−) may perpetuate neuroinflammation and the apoptosis-related p75 receptor may counteract the effects of BDNF. We hypothesised that transcriptional changes in either BDNF, and/or a transcription factor critical for the maintenance of dopamine neurons (Nuclear Receptor Related-1 protein; NURR1), and/or BDNF receptors – TrkB (TK + or TK-) and p75, would be found in the post-mortem schizophrenia midbrain, particularly in high inflammation cases. Using RT-qPCR, mRNA levels of NURR1, BDNF, TrkB and p75 were quantified from schizophrenia (n = 65) and control (n = 64) ventral mesencephalon. We found significant decreases in BDNF IV, TrkB^TK+ and NURR1 (14–18%) and increases in TrkB^TK− and p75 (18–35%) mRNA levels in schizophrenia compared to controls (all p < 0.05), with exacerbation in high inflammation schizophrenia. To determine whether these changes result from chronic antipsychotic treatment, we treated healthy adult rats with antipsychotics and found all corresponding trophic mRNAs to be unaltered. SnRNAseq of human midbrain showed that p75 receptor mRNA is primarily localised in oligodendrocytes and pan-TrkB mRNA is distributed to both neurons and astrocytes. We confirmed that p75 was localised to oligodendrocyte-like cells by immunohistochemistry. Altogether, we find transcriptional evidence of reduced levels of trophic support in the midbrain in schizophrenia and suggest that this may directly impact dopamine neuron health, particularly in neuroinflammatory conditions.

A subgroup of schizophrenia cases with elevated inflammation have reduced neurogenesis markers and increased macrophage density in the human subependymal zone (SEZ; also termed subventricular zone or SVZ) neurogenic niche. Inflammation can impair neurogenesis; however, it is unclear which other pathways are associated with reduced neurogenesis. This research aimed to discover transcriptomic differences between inflammatory subgroups of schizophrenia in the SEZ. Total RNA sequencing was performed on SEZ tissue from schizophrenia cases, designated into low inflammation (n = 13) and high inflammation (n = 14) subgroups, based on cluster analysis of inflammation marker gene expression. 718 genes were differentially expressed in high compared to low inflammation schizophrenia (FDR p < 0.05) and were most significantly over-represented in the pathway ‘Hepatic Fibrosis/Hepatic Stellate-Cell Activation’. Genes in this pathway relate to extracellular matrix stability (including ten collagens) and vascular remodelling suggesting increased angiogenesis. Collagen-IV, a key element of the basement membrane and fractones, had elevated gene expression. Immunohistochemistry revealed novel collagen-IV+ fractone bulbs within the human SEZ hypocellular gap. Considering the extracellular matrix’s regulatory role in SEZ neurogenesis, fibrosis-related alterations in high inflammation schizophrenia may disrupt neurogenesis. Increased angiogenesis could facilitate immune cell transmigration, potentially explaining elevated macrophages in high inflammation schizophrenia. This discovery-driven analysis sheds light on how inflammation may contribute to schizophrenia neuropathology in the neurogenic niche.

Schizophrenia genome-wide association studies (GWAS) have reported many genomic risk loci, but it is unclear how they affect schizophrenia susceptibility through interactions of multiple SNPs. We propose a stepwise deep learning technique with multi-precision data (SLEM) to explore the SNP combination effects on schizophrenia through intermediate molecular and cellular functions. The SLEM technique utilizes two levels of precision data for learning. It constructs initial backbone networks with more precise but small amount of multilevel assay data. Then, it learns strengths of intermediate interactions with the less precise but massive amount of GWAS data. The learned networks facilitate identifying effective SNP interactions from the intractably large space of all possible SNP combinations. We have shown that the extracted SNP combinations show higher accuracy than any single SNPs and preserve the accuracy in an independent dataset. The learned networks also provide interpretations of molecular and cellular interactions of SNP combinations toward schizophrenia etiology.

Cytomegalovirus (CMV) is a common, neurotrophic herpesvirus that can be reactivated by inflammation and cause central nervous system disease. We hypothesize that CMV may contribute to the neuroinflammation that underlies some psychiatric disorders by (1) exacerbating inflammation through the induction of anti-viral immune responses, and (2) translating peripheral inflammation into neuroinflammation. We investigated whether the presence of anti-CMV antibodies in blood were associated with mental illness, suicide, neuroinflammation, and microglial density in the dorsolateral prefrontal cortex (DLPFC) in postmortem samples. Data (n = 114 with schizophrenia; n = 78 with bipolar disorder; n = 87 with depression; n = 85 controls) were obtained from the Stanley Medical Research Institute. DLPFC gene expression data from a subset of 82 samples were categorized into “high” (n = 30), and “low” (n = 52) inflammation groups based on a recursive two-step cluster analysis using expression data for four inflammation-related genes. Measurements of the ratio of non-ramified to ramified microglia, a proxy of microglial activation, were available for a subset of 49 samples. All analyses controlled for age, sex, and ethnicity, as well as postmortem interval, and pH for gene expression and microglial outcomes. CMV seropositivity significantly increased the odds of a mood disorder diagnosis (bipolar disorder: OR = 2.45; major depression: OR = 3.70) and among the psychiatric samples, of suicide (OR = 2.09). Samples in the upper tercile of anti-CMV antibody titers were more likely to be members of the “high” inflammation group (OR = 4.41, an effect driven by schizophrenia and bipolar disorder samples). CMV positive samples also showed an increased ratio of non-ramified to ramified microglia in layer I of the DLPFC (Cohen’s d = 0.81) as well as a non-significant increase in this ratio for the DLPFC as a whole (d = 0.56). The results raise the possibility that the reactivation of CMV contributes to the neuroinflammation that underlies some cases of psychiatric disorders.

Cytomegalovirus (CMV) is a common, neurotrophic herpesvirus that can be reactivated by inflammation and cause neurological disease. CMV replication can in turn worsen inflammation, raising the possibility that poorly controlled CMV infections may contribute to the neuroinflammation underlying some psychiatric disorders. We investigated whether the presence of anti-CMV antibodies in blood were associated with mental illness, suicide, neuroinflammation, and microglial density in the dorsolateral prefrontal cortex in postmortem samples. Data (n = 114 with schizophrenia; n = 78 with bipolar disorder; n = 87 with depression; n = 85 controls) were obtained from the Stanley Medical Research Institute. Gene expression data from a subset of 82 cases were categorized into “high” (n = 30), and “low” (n = 52) inflammation groups based on a recursive two-step cluster analysis using expression data for four inflammation-related genes. Measurements of the density of non-ramified and ramified microglia were available for an independent subset of 51 samples. All analyses controlled for age, sex, ethnicity, postmortem interval, and pH. CMV seropositivity significantly increased the odds of a mood disorder diagnosis (bipolar disorder: OR = 2.49; major depression: OR = 2.62) and among the psychiatric cases, of suicide (OR = 2.21). Samples in the upper tercile of anti-CMV antibody titers were more likely to be members of the “high” inflammation group (OR = 4.41). CMV positive cases also showed an increased ratio of non-ramified to ramified microglia in layer I of the dorsolateral prefrontal cortex (Cohen’s d = 0.81). The results raise the possibility that the reactivation of CMV contributes to the neuroinflammation that underlies some cases of psychiatric disorders.

Upregulation of genes and coexpression networks related to immune function and inflammation have been repeatedly reported in the brain of individuals with schizophrenia. However, a causal relationship between the abnormal immune/inflammation-related gene expression and schizophrenia has not been determined. We conducted co-expression networks using publicly available RNA-seq data from prefrontal cortex (PFC) and hippocampus (HP) of 64 individuals with schizophrenia and 64 unaffected controls from the SMRI tissue collections. We identified proinflammatory cytokine, transmembrane tumor necrosis factor-α (tmTNFα), as a potential regulator in the module of co-expressed genes that we find related to the immune/inflammation response in endothelial cells (ECs) and/or microglia of the brain of individuals with schizophrenia. The immune/inflammation-related modules associated with schizophrenia and the TNF signaling pathway that regulate the network were replicated in an independent cohort of brain samples from 68 individuals with schizophrenia and 135 unaffected controls. To investigate the association between the overexpression of tmTNFα in brain ECs and schizophrenia-like behaviors, we induced short-term overexpression of the uncleavable form of (uc)-tmTNFα in ECs of mouse brain for 7 weeks. We found schizophrenia-relevant behavioral deficits in these mice, including cognitive impairment, abnormal sensorimotor gating, and sensitization to methamphetamine (METH) induced locomotor activity and METH-induced neurotransmitter levels. These uc-tmTNFα effects were mediated by TNF receptor2 (TNFR2) and induced activation of TNFR2 signaling in astrocytes and neurons. A neuronal module including neurotransmitter signaling pathways was down-regulated in the brain of mice by the short-term overexpression of the gene, while an immune/inflammation-related module was up-regulated in the brain of mice after long-term expression of 22 weeks. Our results indicate that tmTNFα may play a direct role in regulating neurotransmitter signaling pathways that contribute to the clinical features of schizophrenia.

The midbrain is an extensively studied brain region in schizophrenia, in view of its reported dopamine pathophysiology and neuroimmune changes associated with this disease. Besides the dopaminergic system, the midbrain contains other cell types that may be involved in schizophrenia pathophysiology. The neurovascular hypothesis of schizophrenia postulates that both the neurovasculature structure and the functioning of the blood-brain barrier (BBB) are compromised in schizophrenia. In the present study, potential alteration in the BBB of patients with schizophrenia was investigated by single-nucleus RNA sequencing of post-mortem midbrain tissue (15 schizophrenia cases and 14 matched controls). We did not identify changes in the relative abundance of the major BBB cell types, nor in the sub-populations, associated with schizophrenia. However, we identified 14 differentially expressed genes in the cells of the BBB in schizophrenia as compared to controls, including genes that have previously been related to schizophrenia, such as FOXP2 and PDE4D. These transcriptional changes were limited to the ependymal cells and pericytes, suggesting that the cells of the BBB are not broadly affected in schizophrenia.