Fenebrutinib, a Bruton's tyrosine kinase inhibitor, blocks distinct human microglial signaling pathways.

BACKGROUND: Bruton's tyrosine kinase (BTK) is an intracellular signaling enzyme that regulates B-lymphocyte and myeloid cell functions. Due to its involvement in both innate and adaptive immune compartments, BTK inhibitors have emerged as a therapeutic option in autoimmune disorders such as multiple sclerosis (MS). Brain-penetrant, small-molecule BTK inhibitors may also address compartmentalized neuroinflammation, which is proposed to underlie MS disease progression. BTK is expressed by microglia, which are the resident innate immune cells of the brain; however, the precise roles of microglial BTK and impact of BTK inhibitors on microglial functions are still being elucidated. Research on the effects of BTK inhibitors has been limited to rodent disease models. This is the first study reporting effects in human microglia. METHODS: Here we characterize the pharmacological and functional properties of fenebrutinib, a potent, highly selective, noncovalent, reversible, brain-penetrant BTK inhibitor, in human microglia and complex human brain cell systems, including brain organoids. RESULTS: We find that fenebrutinib blocks the deleterious effects of microglial Fc gamma receptor (FcγR) activation, including cytokine and chemokine release, microglial clustering and neurite damage in diverse human brain cell systems. Gene expression analyses identified pathways linked to inflammation, matrix metalloproteinase production and cholesterol metabolism that were modulated by fenebrutinib treatment. In contrast, fenebrutinib had no significant impact on human microglial pathways linked to Toll-like receptor 4 (TLR4) and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) signaling or myelin phagocytosis. CONCLUSIONS: Our study enhances the understanding of BTK functions in human microglial signaling that are relevant to MS pathogenesis and suggests that fenebrutinib could attenuate detrimental microglial activity associated with FcγR activation in people with MS.

psupertime: supervised pseudotime analysis for time-series single-cell RNA-seq data.

MOTIVATION: Improvements in single-cell RNA-seq technologies mean that studies measuring multiple experimental conditions, such as time series, have become more common. At present, few computational methods exist to infer time series-specific transcriptome changes, and such studies have therefore typically used unsupervised pseudotime methods. While these methods identify cell subpopulations and the transitions between them, they are not appropriate for identifying the genes that vary coherently along the time series. In addition, the orderings they estimate are based only on the major sources of variation in the data, which may not correspond to the processes related to the time labels. RESULTS: We introduce psupertime, a supervised pseudotime approach based on a regression model, which explicitly uses time-series labels as input. It identifies genes that vary coherently along a time series, in addition to pseudotime values for individual cells, and a classifier that can be used to estimate labels for new data with unknown or differing labels. We show that psupertime outperforms benchmark classifiers in terms of identifying time-varying genes and provides better individual cell orderings than popular unsupervised pseudotime techniques. psupertime is applicable to any single-cell RNA-seq dataset with sequential labels (e.g. principally time series but also drug dosage and disease progression), derived from either experimental design and provides a fast, interpretable tool for targeted identification of genes varying along with specific biological processes. AVAILABILITY AND IMPLEMENTATION: R package available at github.com/wmacnair/psupertime and code for results reproduction at github.com/wmacnair/psupplementary. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dynamic distribution decomposition for single-cell snapshot time series identifies subpopulations and trajectories during iPSC reprogramming.

Recent high-dimensional single-cell technologies such as mass cytometry are enabling time series experiments to monitor the temporal evolution of cell state distributions and to identify dynamically important cell states, such as fate decision states in differentiation. However, these technologies are destructive, and require analysis approaches that temporally map between cell state distributions across time points. Current approaches to approximate the single-cell time series as a dynamical system suffer from too restrictive assumptions about the type of kinetics, or link together pairs of sequential measurements in a discontinuous fashion. We propose Dynamic Distribution Decomposition (DDD), an operator approximation approach to infer a continuous distribution map between time points. On the basis of single-cell snapshot time series data, DDD approximates the continuous time Perron-Frobenius operator by means of a finite set of basis functions. This procedure can be interpreted as a continuous time Markov chain over a continuum of states. By only assuming a memoryless Markov (autonomous) process, the types of dynamics represented are more general than those represented by other common models, e.g., chemical reaction networks, stochastic differential equations. Furthermore, we can a posteriori check whether the autonomy assumptions are valid by calculation of prediction error-which we show gives a measure of autonomy within the studied system. The continuity and autonomy assumptions ensure that the same dynamical system maps between all time points, not arbitrarily changing at each time point. We demonstrate the ability of DDD to reconstruct dynamically important cell states and their transitions both on synthetic data, as well as on mass cytometry time series of iPSC reprogramming of a fibroblast system. We use DDD to find previously identified subpopulations of cells and to visualise differentiation trajectories. Dynamic Distribution Decomposition allows interpretation of high-dimensional snapshot time series data as a low-dimensional Markov process, thereby enabling an interpretable dynamics analysis for a variety of biological processes by means of identifying their dynamically important cell states.

TGF-ß induces oncofetal fibronectin that, in turn, modulates TGF-ß superfamily signaling in endothelial cells.

Gene splicing profiles are frequently altered in cancer, and the splice variants of fibronectin (FN) that contain the extra-domains A (EDA) or B (EDB), referred to as EDA+FN or EDB+FN, are highly upregulated in tumor vasculature. Transforming growth factor ß (TGF-ß) signaling has been attributed a pivotal role in glioblastoma, with TGF-ß promoting angiogenesis and vessel remodeling. By using immunohistochemistry staining, we observed that the oncofetal FN isoforms EDA+FN and EDB+FN are expressed in glioblastoma vasculature. Ex vivo single-cell gene expression profiling of tumors by using CD31 and α-smooth muscle actin (αSMA) as markers for endothelial cells, and pericytes and vascular smooth muscle cells (VSMCs), respectively, confirmed the predominant expression of FN, EDA+FN and EDB+FN in the vascular compartment of glioblastoma. Specifically, within the CD31-positive cell population, we identified a positive correlation between the expression of EDA+FN and EDB+FN, and of molecules associated with TGF-ß signaling. Further, TGF-ß induced EDA+FN and EDB+FN in human cerebral microvascular endothelial cells and glioblastoma-derived endothelial cells in a SMAD3- and SMAD4-dependent manner. In turn, we found that FN modulated TGF-ß superfamily signaling in endothelial cells via the EDA and EDB, pointing towards a bidirectional influence of oncofetal FN and TGF-ß superfamily signaling.

Tree-ensemble analysis assesses presence of multifurcations in single cell data.

We introduce TreeTop, an algorithm for single cell data analysis to identify and assign a branching score to branch points in biological processes which may have multi-level branching hierarchies. We demonstrate branch point identification for processes with varying topologies, including T-cell maturation, B-cell differentiation and hematopoiesis. Our analyses are consistent with recent experimental studies suggesting a shallower hierarchy of differentiation events in hematopoiesis, rather than the classical multi-level hierarchy.

SampleQC: robust multivariate, multi-cell type, multi-sample quality control for single-cell data.

Quality control (QC) is a critical component of single-cell RNA-seq (scRNA-seq) processing pipelines. Current approaches to QC implicitly assume that datasets are comprised of one cell type, potentially resulting in biased exclusion of rare cell types. We introduce SampleQC, which robustly fits a Gaussian mixture model across multiple samples, improves sensitivity, and reduces bias compared to current approaches. We show via simulations that SampleQC is less susceptible to exclusion of rarer cell types. We also demonstrate SampleQC on a complex real dataset (867k cells over 172 samples). SampleQC is general, is implemented in R, and could be applied to other data types.

Mapping human adult hippocampal neurogenesis with single-cell transcriptomics: Reconciling controversy or fueling the debate?

The notion of exploiting the regenerative potential of the human brain in physiological aging or neurological diseases represents a particularly attractive alternative to conventional strategies for enhancing or restoring brain function. However, a major first question to address is whether the human brain does possess the ability to regenerate. The existence of human adult hippocampal neurogenesis (AHN) has been at the center of a fierce scientific debate for many years. The advent of single-cell transcriptomic technologies was initially viewed as a panacea to resolving this controversy. However, recent single-cell RNA sequencing studies in the human hippocampus yielded conflicting results. Here, we critically discuss and re-analyze previously published AHN-related single-cell transcriptomic datasets. We argue that, although promising, the single-cell transcriptomic profiling of AHN in the human brain can be confounded by methodological, conceptual, and biological factors that need to be consistently addressed across studies and openly discussed within the scientific community.

Cell-type-specific cis-eQTLs in eight human brain cell types identify novel risk genes for psychiatric and neurological disorders.

To date, most expression quantitative trait loci (eQTL) studies, which investigate how genetic variants contribute to gene expression, have been performed in heterogeneous brain tissues rather than specific cell types. In this study, we performed an eQTL analysis using single-nuclei RNA sequencing from 192 individuals in eight brain cell types derived from the prefrontal cortex, temporal cortex and white matter. We identified 7,607 eGenes, a substantial fraction (46%, 3,537/7,607) of which show cell-type-specific effects, with strongest effects in microglia. Cell-type-level eQTLs affected more constrained genes and had larger effect sizes than tissue-level eQTLs. Integration of brain cell type eQTLs with genome-wide association studies (GWAS) revealed novel relationships between expression and disease risk for neuropsychiatric and neurodegenerative diseases. For most GWAS loci, a single gene co-localized in a single cell type, providing new clues into disease etiology. Our findings demonstrate substantial contrast in genetic regulation of gene expression among brain cell types and reveal potential mechanisms by which disease risk genes influence brain disorders.

Doublet identification in single-cell sequencing data using scDblFinder.

Doublets are prevalent in single-cell sequencing data and can lead to artifactual findings. A number of strategies have therefore been proposed to detect them. Building on the strengths of existing approaches, we developed scDblFinder, a fast, flexible and accurate Bioconductor-based doublet detection method. Here we present the method, justify its design choices, demonstrate its performance on both single-cell RNA and accessibility sequencing data, and provide some observations on doublet formation, detection, and enrichment analysis. Even in complex datasets, scDblFinder can accurately identify most heterotypic doublets, and was already found by an independent benchmark to outcompete alternatives.

Negative allosteric modulators of metabotropic glutamate receptor 3 target the stem-like phenotype of glioblastoma.

Glioblastoma is an invariably deadly disease. A subpopulation of glioma stem-like cells (GSCs) drives tumor progression and treatment resistance. Two recent studies demonstrated that neurons form oncogenic glutamatergic electrochemical synapses with post-synaptic GSCs. This led us to explore whether glutamate signaling through G protein-coupled metabotropic receptors would also contribute to the malignancy of glioblastoma. We found that glutamate metabotropic receptor (Grm)3 is the predominantly expressed Grm in glioblastoma. Associations of GRM3 gene expression levels with survival are confined to the proneural gene expression subtype, which is associated with enrichment of GSCs. Using multiplexed single-cell qRT-PCR, GSC marker-based cell sorting, database interrogations, and functional assays in GSCs derived from patients' tumors, we establish Grm3 as a novel marker and potential therapeutic target in GSCs. We confirm that Grm3 inhibits adenylyl cyclase and regulates extracellular signal-regulated kinase. Targeting Grm3 disrupts self-renewal and promotes differentiation of GSCs. Thus, we hypothesize that Grm3 signaling may complement oncogenic functions of glutamatergic ionotropic receptor activity in neuroglial synapses, supporting a link between neuronal activity and the GSC phenotype. The novel class of highly specific Grm3 inhibitors that we characterize herein have been clinically tested as cognitive enhancers in humans with a favorable safety profile.