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.

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.

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.

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.

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.

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.

Oligodendrocytes Provide Antioxidant Defense Function for Neurons by Secreting Ferritin Heavy Chain.

An evolutionarily conserved function of glia is to provide metabolic and structural support for neurons. To identify molecules generated by glia and with vital functions for neurons, we used Drosophila melanogaster as a screening tool, and subsequently translated the findings to mice. We found that a cargo receptor operating in the secretory pathway of glia was essential to maintain axonal integrity by regulating iron buffering. Ferritin heavy chain was identified as the critical secretory cargo, required for the protection against iron-mediated ferroptotic axonal damage. In mice, ferritin heavy chain is highly expressed by oligodendrocytes and secreted by employing an unconventional secretion pathway involving extracellular vesicles. Disrupting the release of extracellular vesicles or the expression of ferritin heavy chain in oligodendrocytes causes neuronal loss and oxidative damage in mice. Our data point to a role of oligodendrocytes in providing an antioxidant defense system to support neurons against iron-mediated cytotoxicity.

Proteome profile of peripheral myelin in healthy mice and in a neuropathy model.

Proteome and transcriptome analyses aim at comprehending the molecular profiles of the brain, its cell-types and subcellular compartments including myelin. Despite the relevance of the peripheral nervous system for normal sensory and motor capabilities, analogous approaches to peripheral nerves and peripheral myelin have fallen behind evolving technical standards. Here we assess the peripheral myelin proteome by gel-free, label-free mass-spectrometry for deep quantitative coverage. Integration with RNA-Sequencing-based developmental mRNA-abundance profiles and neuropathy disease genes illustrates the utility of this resource. Notably, the periaxin-deficient mouse model of the neuropathy Charcot-Marie-Tooth 4F displays a highly pathological myelin proteome profile, exemplified by the discovery of reduced levels of the monocarboxylate transporter MCT1/SLC16A1 as a novel facet of the neuropathology. This work provides the most comprehensive proteome resource thus far to approach development, function and pathology of peripheral myelin, and a straightforward, accurate and sensitive workflow to address myelin diversity in health and disease.

Pathway sensor-based functional genomics screening identifies modulators of neuronal activity.

Neuronal signal transduction shapes brain function and malfunction may cause mental disorders. Despite the existence of functional genomics screens for proliferation and toxicity, neuronal signalling has been difficult to address so far. To overcome this limitation, we developed a pooled screening assay which combines barcoded activity reporters with pooled genetic perturbation in a dual-expression adeno-associated virus (AAV) library. With this approach, termed pathScreener, we comprehensively dissect signalling pathways in postmitotic neurons. This overcomes several limitations of lentiviral-based screens. By applying first a barcoded and multiplexed reporter assay, termed cisProfiler, we identified the synaptic-activity responsive element (SARE) as top performance sensor of neuronal activity. Next, we targeted more than 4,400 genes and screened for modulatory effects on SARE activity in primary cortical neurons. We identified with high replicability many known genes involved in glutamatergic synapse-to-nucleus signalling of which a subset was validated in orthogonal assays. Several others have not yet been associated with the regulation of neuronal activity such as the hedgehog signalling members Ptch2 and Ift57. This assay thus enhances the toolbox for analysing regulatory processes during neuronal signalling and may help identifying novel targets for brain disorders.

Age-related myelin degradation burdens the clearance function of microglia during aging.

Myelin is synthesized as a multilamellar membrane, but the mechanisms of membrane turnover are unknown. We found that myelin pieces were gradually released from aging myelin sheaths and were subsequently cleared by microglia. Myelin fragmentation increased with age and led to the formation of insoluble, lipofuscin-like lysosomal inclusions in microglia. Thus, age-related myelin fragmentation is substantial, leading to lysosomal storage and contributing to microglial senescence and immune dysfunction in aging.

Cell type- and brain region-resolved mouse brain proteome.

Brain transcriptome and connectome maps are being generated, but an equivalent effort on the proteome is currently lacking. We performed high-resolution mass spectrometry-based proteomics for in-depth analysis of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type-specific proteins defined as tenfold more abundant than average expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.