Protocol for identifying properties of ERBB receptor antagonists using the barcoded ERBBprofiler assay.

The ERBBprofiler assay measures compound effects on ERBB family receptors and key downstream signaling pathways that are implicated in cancer or other complex diseases. Here, we present a protocol for identifying properties of ERBB receptor antagonists using the barcoded ERBBprofiler assay. We describe steps for in-solution transfection, cell treatment, combined processing of samples, amplification and indexing of PCRs, sequencing, and data analysis. This approach allows for the simultaneous assessment of drug effects and cell-type-dependent effects. For complete details on the use and execution of this protocol, please refer to Popovic et al.1.

Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists.

ERBB receptor tyrosine kinases are involved in development and diseases like cancer, cardiovascular, neurodevelopmental, and mental disorders. Although existing drugs target ERBB receptors, the next generation of drugs requires enhanced selectivity and understanding of physiological pathway responses to improve efficiency and reduce side effects. To address this, we developed a multilevel barcoded reporter profiling assay, termed 'ERBBprofiler', in living cells to monitor the activity of all ERBB targets and key physiological pathways simultaneously. This assay helps differentiate on-target therapeutic effects from off-target and off-pathway side effects of ERBB antagonists. To challenge the assay, eight established ERBB antagonists were profiled. Known effects were confirmed, and previously uncharacterized properties were discovered, such as pyrotinib's preference for ERBB4 over EGFR. Additionally, two lead compounds selectively targeting ERBB4 were profiled, showing promise for clinical trials. Taken together, this multiparametric profiling approach can guide early-stage drug development and lead to improved future therapeutic interventions.

Polygenic risk for schizophrenia converges on alternative polyadenylation as molecular mechanism underlying synaptic impairment.

Schizophrenia (SCZ) is a genetically heterogenous psychiatric disorder of highly polygenic nature. Correlative evidence from genetic studies indicate that the aggregated effects of distinct genetic risk factor combinations found in each patient converge onto common molecular mechanisms. To prove this on a functional level, we employed a reductionistic cellular model system for polygenic risk by differentiating induced pluripotent stem cells (iPSCs) from 104 individuals with high polygenic risk load and controls into cortical glutamatergic neurons (iNs). Multi-omics profiling identified widespread differences in alternative polyadenylation (APA) in the 3' untranslated region of many synaptic transcripts between iNs from SCZ patients and healthy donors. On the cellular level, 3'APA was associated with a reduction in synaptic density of iNs. Importantly, differential APA was largely conserved between postmortem human prefrontal cortex from SCZ patients and healthy donors, and strongly enriched for transcripts related to synapse biology. 3'APA was highly correlated with SCZ polygenic risk and affected genes were significantly enriched for SCZ associated common genetic variation. Integrative functional genomic analysis identified the RNA binding protein and SCZ GWAS risk gene PTBP2 as a critical trans-acting factor mediating 3'APA of synaptic genes in SCZ subjects. Functional characterization of PTBP2 in iNs confirmed its key role in 3'APA of synaptic transcripts and regulation of synapse density. Jointly, our findings show that the aggregated effects of polygenic risk converge on 3'APA as one common molecular mechanism that underlies synaptic impairments in SCZ.

Comprehensive split TEV based protein-protein interaction screening reveals TAOK2 as a key modulator of Hippo signalling to limit growth.

The conserved Hippo signalling pathway plays a crucial role in tumour formation by limiting tissue growth and proliferation. At the core of this pathway are tumour suppressor kinases STK3/4 and LATS1/2, which limit the activity of the oncogene YAP1, the primary downstream effector. Here, we employed a split TEV-based protein-protein interaction screen to assess the physical interactions among 28 key Hippo pathway components and potential upstream modulators. This screen led us to the discovery of TAOK2 as pivotal modulator of Hippo signalling, as it binds to the pathway's core kinases, STK3/4 and LATS1/2, and leads to their phosphorylation. Specifically, our findings revealed that TAOK2 binds to and phosphorylates LATS1, resulting in the reduction of YAP1 phosphorylation and subsequent transcription of oncogenes. Consequently, this decrease led to a decrease in cell proliferation and migration. Interestingly, a correlation was observed between reduced TAOK2 expression and decreased patient survival time in certain types of human cancers, including lung and kidney cancer as well as glioma. Moreover, in cellular models corresponding to these cancer types the downregulation of TAOK2 by CRISPR inhibition led to reduced phosphorylation of LATS1 and increased proliferation rates, supporting TAOK2's role as tumour suppressor gene. By contrast, overexpression of TAOK2 in these cellular models lead to increased phospho-LATS1 but reduced cell proliferation. As TAOK2 is a druggable kinase, targeting TAOK2 could serve as an attractive pharmacological approach to modulate cell growth and potentially offer strategies for combating cancer.

Massively parallel functional dissection of schizophrenia-associated noncoding genetic variants.

Schizophrenia (SCZ) is a highly heritable mental disorder with thousands of associated genetic variants located mostly in the noncoding space of the genome. Translating these associations into insights regarding the underlying pathomechanisms has been challenging because the causal variants, their mechanisms of action, and their target genes remain largely unknown. We implemented a massively parallel variant annotation pipeline (MVAP) to perform SCZ variant-to-function mapping at scale in disease-relevant neural cell types. This approach identified 620 functional variants (1.7%) that operate in a highly developmental context and neuronal-activity-dependent manner. Multimodal integration of epigenomic and CRISPRi screening data enabled us to link these functional variants to target genes, biological processes, and ultimately alterations of neuronal physiology. These results provide a multistage prioritization strategy to map functional single-nucleotide polymorphism (SNP)-to-gene-to-endophenotype relations and offer biological insights into the context-dependent molecular processes modulated by SCZ-associated genetic variation.

Rescue of Comorbid Behavioral and Metabolic Phenotypes of Arrhythmic Mice by Restoring Circadian Cry1/2 Expression in the Suprachiasmatic Nucleus.

Background: Psychiatric and metabolic disorders occur disproportionately often comorbidly, which poses particular hurdles for patients and therapists. However, the mechanisms that promote such comorbidities are largely unknown and therefore cannot yet be therapeutically targeted for the simultaneous treatment of both conditions. Because circadian clocks regulate most physiological processes and their disruption is a risk factor for both psychiatric and metabolic disorders, they may be considered as a potential mechanism for the development of comorbidities and a therapeutic target. In the current study, we investigated the latter assumption in Cry1/2-/- mice, which exhibit substantially disrupted endogenous circadian rhythms and marked metabolic and behavioral deficits. Methods: By targeted virus-induced restoration of circadian rhythms in their suprachiasmatic nucleus, we can restore behavioral as well as several metabolic processes of these animals to near-normal circadian rhythmicity. Results: Importantly, by rescuing suprachiasmatic nucleus rhythms, several of their anxiety-like behavioral as well as diabetes- and energy homeostasis-related deficits were significantly improved. Interestingly, however, this did not affect all deficits typical of Cry1/2-/- mice; for example, restlessness and body weight remained unaffected. Conclusions: Taken together, the results of this study demonstrate, on the one hand, that restoration of disturbed circadian rhythms can be used to simultaneously treat psychiatric and metabolic deficits. On the other hand, the results also allow us to distinguish processes that depend more on local canonical clocks from those that depend more on suprachiasmatic nucleus rhythms.

Interferon-gamma ameliorates experimental autoimmune encephalomyelitis by inducing homeostatic adaptation of microglia.

Compelling evidence has shown that interferon (IFN)-γ has dual effects in multiple sclerosis and in its animal model of experimental autoimmune encephalomyelitis (EAE), with results supporting both a pathogenic and beneficial function. However, the mechanisms whereby IFN-γ may promote neuroprotection in EAE and its effects on central nervous system (CNS)-resident cells have remained an enigma for more than 30 years. In this study, the impact of IFN-γ at the peak of EAE, its effects on CNS infiltrating myeloid cells (MC) and microglia (MG), and the underlying cellular and molecular mechanisms were investigated. IFN-γ administration resulted in disease amelioration and attenuation of neuroinflammation associated with significantly lower frequencies of CNS CD11b+ myeloid cells and less infiltration of inflammatory cells and demyelination. A significant reduction in activated MG and enhanced resting MG was determined by flow cytometry and immunohistrochemistry. Primary MC/MG cultures obtained from the spinal cord of IFN-γ-treated EAE mice that were ex vivo re-stimulated with a low dose (1 ng/ml) of IFN-γ and neuroantigen, promoted a significantly higher induction of CD4+ regulatory T (Treg) cells associated with increased transforming growth factor (TGF)-ß secretion. Additionally, IFN-γ-treated primary MC/MG cultures produced significantly lower nitrite in response to LPS challenge than control MC/MG. IFN-γ-treated EAE mice had a significantly higher frequency of CX3CR1high MC/MG and expressed lower levels of program death ligand 1 (PD-L1) than PBS-treated mice. Most CX3CR1highPD-L1lowCD11b+Ly6G- cells expressed MG markers (Tmem119, Sall2, and P2ry12), indicating that they represented an enriched MG subset (CX3CR1highPD-L1low MG). Amelioration of clinical symptoms and induction of CX3CR1highPD-L1low MG by IFN-γ were dependent on STAT-1. RNA-seq analyses revealed that in vivo treatment with IFN-γ promoted the induction of homeostatic CX3CR1highPD-L1low MG, upregulating the expression of genes associated with tolerogenic and anti-inflammatory roles and down-regulating pro-inflammatory genes. These analyses highlight the master role that IFN-γ plays in regulating microglial activity and provide new insights into the cellular and molecular mechanisms involved in the therapeutic activity of IFN-γ in EAE.

Disturbed Oligodendroglial Maturation Causes Cognitive Dysfunction in Schizophrenia: A New Hypothesis.

BACKGROUND AND HYPOTHESIS: Cognitive impairment is a hallmark of schizophrenia, but no effective treatment is available to date. The underlying pathophysiology includes disconnectivity between hippocampal and prefrontal brain regions. Supporting evidence comes from diffusion-weighted imaging studies that suggest abnormal organization of frontotemporal white matter pathways in schizophrenia. STUDY DESIGN: Here, we hypothesize that in schizophrenia, deficient maturation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes substantially contributes to abnormal frontotemporal macro- and micro-connectivity and subsequent cognitive deficits. STUDY RESULTS: Our postmortem studies indicate a reduced oligodendrocyte number in the cornu ammonis 4 (CA4) subregion of the hippocampus, and others have reported the same histopathological finding in the dorsolateral prefrontal cortex. Our series of studies on aerobic exercise training showed a volume increase in the hippocampus, specifically in the CA4 region, and improved cognition in individuals with schizophrenia. The cognitive effects were subsequently confirmed by meta-analyses. Cell-specific schizophrenia polygenic risk scores showed that exercise-induced CA4 volume increase significantly correlates with OPCs. From animal models, it is evident that early life stress and oligodendrocyte-related gene variants lead to schizophrenia-related behavior, cognitive deficits, impaired oligodendrocyte maturation, and reduced myelin thickness. CONCLUSIONS: Based on these findings, we propose that pro-myelinating drugs (e.g., the histamine blocker clemastine) combined with aerobic exercise training may foster the regeneration of myelin plasticity as a basis for restoring frontotemporal connectivity and cognition in schizophrenia.

Analysis of the GPR17 receptor in NG2-glia under physiological conditions unravels a new subset of oligodendrocyte progenitor cells with distinct functions.

NG2-glia comprise a heterogeneous population of cycling cells that give rise to mature, myelinating oligodendrocytes. The mechanisms that regulate the process of differentiation from NG2-glia into oligodendrocytes are still not fully understood but over the last years the G Protein-coupled Receptor 17 (GPR17) has been on the spotlight as a possible key regulator. Interestingly, GPR17-expressing NG2-glia show under physiological conditions a slower and lower level of differentiation compared to NG2-glia without GPR17. In contrast, after a CNS insult these react with proliferation and differentiation in a high rate, pointing towards a role in repair processes. However, the role of GPR17+ NG2-glia under healthy conditions in adulthood has not been addressed yet. Therefore, we aimed here to characterize the GPR17-expressing NG2-glia. Using transgenic mouse models, we showed restricted GPR17 expression in only some NG2-glia. Furthermore, we found that these cells constitute a distinct subset within the NG2-glia population, which shows a different gene expression profile and behavior when compared to the total NG2-glia population. Genetic depletion of GPR17+ cells showed that these are not contributing to the dynamic and continuous generation of new oligodendrocytes in the adult brain. Taken together, GPR17+ NG2-glia seem to play a distinct role under physiological conditions that goes beyond their classic differentiation control, that needs to be further elucidated. These results open new avenues for using the GPR17 receptor as a target to change oligodendrogenesis under physiological and pathological conditions, highlighting the importance of further characterization of this protein for future pharmacological studies.

Extracellular vesicle approach to major psychiatric disorders.

Over the last few years, extracellular vesicles (EVs) have received increasing attention as potential non-invasive diagnostic and therapeutic biomarkers for various diseases. The interest in EVs is related to their structure and content, as well as to their changing cargo in response to different stimuli. One of the potential areas of use of EVs as biomarkers is the central nervous system (CNS), in particular the brain, because EVs can cross the blood-brain barrier, exist also in peripheral tissues and have a diverse cargo. Thus, they may represent "liquid biopsies" of the CNS that can reflect brain pathophysiology without the need for invasive surgical procedures. Overall, few studies to date have examined EVs in neuropsychiatric disorders, and the present evidence appears to lack reproducibility. This situation might be due to a variety of technical obstacles related to working with EVs, such as the use of different isolation strategies, which results in non-uniform vesicular and molecular outputs. Multi-omics approaches and improvements in the standardization of isolation procedures will allow highly pure EV fractions to be obtained in which the molecular cargo, particularly microRNAs and proteins, can be identified and accurately quantified. Eventually, these advances will enable researchers to decipher disease-relevant molecular signatures of the brain-derived EVs involved in synaptic plasticity, neuronal development, neuro-immune communication, and other related pathways. This narrative review summarizes the findings of studies on EVs in major psychiatric disorders, particularly in the field of biomarkers, and discusses the respective therapeutic potential of EVs.

Comparative serum proteomic analysis of a selected protein panel in individuals with schizophrenia and bipolar disorder and the impact of genetic risk burden on serum proteomic profiles.

The diagnostic criteria for schizophrenia (SCZ) and bipolar disorder (BD) are based on clinical assessments of symptoms. In this pilot study, we applied high-throughput antibody-based protein profiling to serum samples of healthy controls and individuals with SCZ and BD with the aim of identifying differentially expressed proteins in these disorders. Moreover, we explored the influence of polygenic burden for SCZ and BD on the serum levels of these proteins. Serum samples from 113 individuals with SCZ and 125 with BD from the PsyCourse Study and from 44 healthy controls were analyzed by using a set of 155 antibodies in an antibody-based assay targeting a selected panel of 95 proteins. For the cases, genotyping and imputation were conducted for DNA samples and SCZ and BD polygenic risk scores (PRS) were calculated. Univariate linear and logistic models were used for association analyses. The comparison between SCZ and BD revealed two serum proteins that were significantly elevated in BD after multiple testing adjustment: "complement C9" and "Interleukin 1 Receptor Accessory Protein". Moreover, the first principal component of variance in the proteomics dataset differed significantly between SCZ and BD. After multiple testing correction, SCZ-PRS, BD-PRS, and SCZ-vs-BD-PRS were not significantly associated with the levels of the individual proteins or the values of the proteome principal components indicating no detectable genetic effects. Overall, our findings contribute to the evidence suggesting that the analysis of circulating proteins could lead to the identification of distinctive biomarkers for SCZ and BD. Our investigation warrants replication in large-scale studies to confirm these findings.

Ketogenic diet uncovers differential metabolic plasticity of brain cells.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type-specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.

Spironolactone alleviates schizophrenia-related reversal learning in Tcf4 transgenic mice subjected to social defeat.

Cognitive deficits are a hallmark of schizophrenia, for which no convincing pharmacological treatment option is currently available. Here, we tested spironolactone as a repurposed compound in Tcf4 transgenic mice subjected to psychosocial stress. In this '2-hit' gene by environment mouse (GxE) model, the animals showed schizophrenia-related cognitive deficits. We had previously shown that spironolactone ameliorates working memory deficits and hyperactivity in a mouse model of cortical excitatory/inhibitory (E/I) dysbalance caused by an overactive NRG1-ERBB4 signaling pathway. In an add-on clinical study design, we used spironolactone as adjuvant medication to the standard antipsychotic drug aripiprazole. We characterized the compound effects using our previously established Platform for Systematic Semi-Automated Behavioral and Cognitive Profiling (PsyCoP). PsyCoP is a widely applicable analysis pipeline based on the Research Domain Criteria (RDoC) framework aiming at facilitating translation into the clinic. In addition, we use dimensional reduction to analyze and visualize overall treatment effect profiles. We found that spironolactone and aripiprazole improve deficits of several cognitive domains in Tcf4tg x SD mice but partially interfere with each other's effect in the combination therapy. A similar interaction was detected for the modulation of novelty-induced activity. In addition to its strong activity-dampening effects, we found an increase in negative valence measures as a side effect of aripiprazole treatment in mice. We suggest that repurposed drug candidates should first be tested in an adequate preclinical setting before initiating clinical trials. In addition, a more specific and effective NRG1-ERBB4 pathway inhibitor or more potent E/I balancing drug might enhance the ameliorating effect on cognition even further.

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation.

Oligodendrocytes (OLs) are critical for myelination and are implicated in several brain disorders. Directed differentiation of human-induced OLs (iOLs) from pluripotent stem cells can be achieved by forced expression of different combinations of the transcription factors SOX10 (S), OLIG2 (O), and NKX6.2 (N). Here, we applied quantitative image analysis and single-cell transcriptomics to compare different transcription factor (TF) combinations for their efficacy towards robust OL lineage conversion. Compared with S alone, the combination of SON increases the number of iOLs and generates iOLs with a more complex morphology and higher expression levels of myelin-marker genes. RNA velocity analysis of individual cells reveals that S generates a population of oligodendrocyte-precursor cells (OPCs) that appear to be more immature than those generated by SON and to display distinct molecular properties. Our work highlights that TFs for generating iOPCs or iOLs should be chosen depending on the intended application or research question, and that SON might be beneficial to study more mature iOLs while S might be better suited to investigate iOPC biology.

Dissecting intercellular and intracellular signaling networks with barcoded genetic tools.

The power of next-generation sequencing has stimulated the development of many analysis techniques for transcriptomics and genomics. More recently, the concept of 'molecular barcoding' has broadened the spectrum of sequencing-based applications to dissect different aspects of intracellular and intercellular signaling. In these assay formats, barcode reporters replace standard reporter genes. The virtually infinitive number of expressed barcode sequences allows high levels of multiplexing, hence accelerating experimental progress. Furthermore, reporter barcodes are used to quantitatively monitor a variety of biological events in living cells which has already provided much insight into complex cellular signaling and will further increase our knowledge in the future.

Circadian gene × environment perturbations influence alcohol drinking in Cryptochrome-deficient mice.

Alcohol use disorder (AUD) is a widespread addiction disorder with severe consequences for health. AUD patients often suffer from sleep disturbances and irregular daily patterns. Conversely, disruptions of circadian rhythms are considered a risk factor for AUD and alcohol relapses. In this study, we investigated the extent to which circadian genetic and environmental disruptions and their interaction alter alcohol drinking behaviour in mice. As a model of genetic circadian disruption, we used Cryptochrome1/2-deficient (Cry1/2-/- ) mice with strongly suppressed circadian rhythms and found that they exhibit significantly reduced preference for alcohol but increased incentive motivation to obtain it. Similarly, we found that low circadian SCN amplitude correlates with reduced alcohol preference in WT mice. Moreover, we show that the low alcohol preference of Cry1/2-/- mice concurs with high corticosterone and low levels of the orexin precursor prepro-orexin and that WT and Cry1/2-/- mice respond differently to alcohol withdrawal. As a model of environmentally induced disruption of circadian rhythms, we exposed mice to a "shift work" light/dark regimen, which also leads to a reduction in their alcohol preference. Interestingly, this effect is even more pronounced when genetic and environmental circadian perturbations interact in Cry1/2-/- mice under "shift work" conditions. In conclusion, our study demonstrates that in mice, disturbances in circadian rhythms have pronounced effects on alcohol consumption as well as on physiological factors and other behaviours associated with AUD and that the interaction between circadian genetic and environmental disturbances further alters alcohol consumption behaviour.

Improved Split TEV GPCR ß-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and ß-arrestin Binding Motif Variants.

G protein-coupled receptors (GPCRs) are major disease-relevant drug targets; robust monitoring of their activities upon drug treatment is key to drug discovery. The split TEV cell-based assay technique monitors the interaction of an activated GPCR with ß-arrestin-2 through TEV protein fragment complementation using a luminescent signal as the readout. In this work, split TEV GPCR ß-arrestin-2 recruitment assays were optimized to monitor the endogenous ligand-induced activities of six GPCRs (DRD1, DRD2, HTR2A, GCGR, AVPR2, and GLP1R). Each GPCR was tested in four forms; i.e., its wildtype form, a variant with a signal peptide (SP) to facilitate receptor expression, a variant containing the C-terminal tail from the V2 vasopressin receptor (V2R tail) to promote ß-arrestin-2 recruitment, and a variant containing both the SP and V2R tail. These 24 GPCR variants were systematically tested for assay performance in four cell lines (HEK-293, PC12 Tet-Off, U-2 OS, and HeLa). We found that the assay performance differed significantly for each GPCR variant and was dependent on the cell line. We found that V2R improved the DRD2 split TEV assays and that HEK-293 cells were the preferred cell line across the GPCRs tested. When taking these considerations into account, the defined selection of assay modifications and conditions may improve the performance of drug development campaigns that apply the split TEV technique as a screening tool.

Differential gene regulation in the anterior cingulate cortex and superior temporal cortex in schizophrenia: A molecular network approach.

The closely connected anterior cingulate cortex (ACC) and superior temporal cortex (STC) are important for higher cognitive functions. Both brain regions are disturbed in schizophrenia, i.e., functional and structural alterations have been reported. This postmortem investigation in brains from patients with schizophrenia and controls compared gene expression in the left ACC and left STC. Most differentially expressed genes were unique to each brain region, but some clusters of genes were equally dysregulated in both, giving rise to a more general disease-specific pattern of gene regulation. The data was used to construct a molecular network of the genes identically expressed in both regions as primary nodes and the metabolically connected genes as secondary nodes. The network analysis identified downregulated clusters of immune-associated gene products and upregulated clusters belonging to the ubiquitin-proteasome system. These findings could help to identify new potential therapeutic targets for future approaches.

Hyperactivity is a Core Endophenotype of Elevated Neuregulin-1 Signaling in Embryonic Glutamatergic Networks.

The neuregulin 1 (NRG1) ErbB4 module is at the core of an "at risk" signaling pathway in schizophrenia. Several human studies suggest hyperstimulation of NRG1-ErbB4 signaling as a plausible pathomechanism; however, little is known about the significance of stage-, brain area-, or neural cell type-specific NRG1-ErbB4 hyperactivity for disease-relevant brain endophenotypes. To address these spatiotemporal aspects, we generated transgenic mice for Cre recombinase-mediated overexpression of cystein-rich domain (CRD) NRG1, the most prominent NRG1 isoform in the brain. A comparison of "brain-wide" vs cell type-specific CRD-NRG1 overexpressing mice revealed that pathogenic CRD-NRG1 signals for ventricular enlargement and neuroinflammation originate outside glutamatergic neurons and suggests a subcortical function of CRD-NRG1 in the control of body weight. Embryonic onset of CRD-NRG1 in glutamatergic cortical networks resulted in reduced inhibitory neurotransmission and locomotor hyperactivity. Our findings identify ventricular enlargement and locomotor hyperactivity, 2 main endophenotypes of schizophrenia, as specific consequences of spatiotemporally distinct expression profiles of hyperactivated CRD-NRG1 signaling.

White matter aging drives microglial diversity.

Aging results in gray and white matter degeneration, but the specific microglial responses are unknown. Using single-cell RNA sequencing from white and gray matter separately, we identified white matter-associated microglia (WAMs), which share parts of the disease-associated microglia (DAM) gene signature and are characterized by activation of genes implicated in phagocytic activity and lipid metabolism. WAMs depend on triggering receptor expressed on myeloid cells 2 (TREM2) signaling and are aging dependent. In the aged brain, WAMs form independent of apolipoprotein E (APOE), in contrast to mouse models of Alzheimer's disease, in which microglia with the WAM gene signature are generated prematurely and in an APOE-dependent pathway similar to DAMs. Within the white matter, microglia frequently cluster in nodules, where they are engaged in clearing degenerated myelin. Thus, WAMs may represent a potentially protective response required to clear degenerated myelin accumulating during white matter aging and disease.