Spatially resolved transcriptomics (SRT) offers the promise of understanding cells and their modes of dysfunction in the context of intact tissues. Technologies for SRT have advanced rapidly with a ...large number being published in recent years. Diverse methods for SRT produce data at widely varying depth, throughput, accessibility and cost. Many published SRT methods have been demonstrated only in their labs of origin, while others have matured to the point of commercialization and widespread availability. Here we review technologies for SRT, and their application in studies of tumor heterogeneity.
Paralysis occurring in amyotrophic lateral sclerosis (ALS) results from denervation of skeletal muscle as a consequence of motor neuron degeneration. Interactions between motor neurons and glia ...contribute to motor neuron loss, but the spatiotemporal ordering of molecular events that drive these processes in intact spinal tissue remains poorly understood. Here, we use spatial transcriptomics to obtain gene expression measurements of mouse spinal cords over the course of disease, as well as of postmortem tissue from ALS patients, to characterize the underlying molecular mechanisms in ALS. We identify pathway dynamics, distinguish regional differences between microglia and astrocyte populations at early time points, and discern perturbations in several transcriptional pathways shared between murine models of ALS and human postmortem spinal cords.
Microglia are resident immune cells of the CNS that are activated by infection, neuronal injury, and inflammation. Here, we utilize flow cytometry and deep RNA sequencing of acutely isolated spinal ...cord microglia to define their activation in vivo. Analysis of resting microglia identified 29 genes that distinguish microglia from other CNS cells and peripheral macrophages/monocytes. We then analyzed molecular changes in microglia during neurodegenerative disease activation using the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS). We found that SOD1G93A microglia are not derived from infiltrating monocytes, and that both potentially neuroprotective and toxic factors, including Alzheimer’s disease genes, are concurrently upregulated. Mutant microglia differed from SOD1WT, lipopolysaccharide-activated microglia, and M1/M2 macrophages, defining an ALS-specific phenotype. Concurrent messenger RNA/fluorescence-activated cell sorting analysis revealed posttranscriptional regulation of microglia surface receptors and T cell-associated changes in the transcriptome. These results provide insights into microglia biology and establish a resource for future studies of neuroinflammation.
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•Identification of specific marker genes for acutely isolated microglia•Progressive resident microglia transcriptome changes reveal in vivo activation phenotype•Microglial ALS disease activation signature distinct from M1/M2 macrophages•Parallel transcriptome and FACS analyses reveal T cell/microglia crosstalk
Microglia are resident immune cells of the brain that are activated by infection or tissue damage. In this study, Maniatis and colleagues report the acute isolation, transcriptional profiling, and immunological analysis of microglia during disease activation in an ALS mouse model. A neurodegeneration-specific gene-expression signature is identified that includes induction of both neuroprotective and toxic factors and is distinct from that associated with M1/M2 macrophages. The data also provide a resource for future studies of microglia activation in neurodegenerative diseases.
The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise ...these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.
The C-terminal repeat domain (CTD), an unusual extension appended to the C terminus of the largest subunit of RNA polymerase II, serves as a flexible binding scaffold for numerous nuclear factors; ...which factors bind is determined by the phosphorylation patterns on the CTD repeats. Changes in phosphorylation patterns, as polymerase transcribes a gene, are thought to orchestrate the association of different sets of factors with the transcriptase and strongly influence functional organization of the nucleus. In this review we appraise what is known, and what is not known, about patterns of phosphorylation on the CTD of RNA polymerases II at the beginning, the middle, and the end of genes; the proposal that doubly phosphorylated repeats are present on elongating polymerase is explored. We discuss briefly proteins known to associate with the phosphorylated CTD at the beginning and ends of genes; we explore in more detail proteins that are recruited to the body of genes, the diversity of their functions, and the potential consequences of tethering these functions to elongating RNA polymerase II. We also discuss accumulating structural information on phosphoCTD-binding proteins and how it illustrates the variety of binding domains and interaction modes, emphasizing the structural flexibility of the CTD. We end with a number of open questions that highlight the extent of what remains to be learned about the phosphorylation and functions of the CTD.
Amyotrophic lateral sclerosis (ALS) is the most common adult-onset paralytic disorder, characterized mainly by a loss of motor neurons (MNs) in the CNS. Over the past decades, thanks to intense ...investigations performed in both in vivo and in vitro models of ALS, major progress has been made toward gaining insights into the pathobiology of this incurable, fatal disorder. Among these advances is the growing recognition that non-neuronal cells participate in the degeneration of MNs in ALS, which could transform our understanding of the neurobiology of disease and the ability to devise effective disease-modifying therapies. In this review, we examine the contribution of non-cell-autonomous processes to the pathogenesis of ALS, with a focus on glial cells and in particular on astrocytes.
Whereas the pathophysiology of amyotrophic lateral sclerosis (ALS) involves the selective degeneration of motor neurons (MNs), non-neuronal cells are increasingly acknowledged as critical determinants of MN death and survival.Extensive co-culture experiments suggest that the deleterious effects of astrocytes on MNs involve loss of neuroprotective function as well as gain of toxic function.Astrocytic heterogeneity may contribute to the mix of neuroprotective and neurotoxic effects imposed on neighboring MNs and can itself be influenced by other cell types.The intricate communication between neurons and astrocytes bridges cell-intrinsic and -extrinsic factors, and this crosstalk is perturbed by mutant superoxide dismutase 1 (SOD1).
To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required ...for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.
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•Genome-wide CRISPR knockout screen identifies host factors for SARS-CoV-2 infection•Top-ranked genes include vacuolar ATPases, Retromer, Commander, and Arp2/3 complex•Validation using CRISPR knockout, RNA interference, and small molecule inhibitors•Reduced infection via increased cholesterol biosynthesis and sequestration of ACE2
To identify potential therapeutic targets for SARS-CoV-2, Daniloski et al. conduct a genome-wide CRISPR screen in human lung epithelial cells. They identify genes and pathways required for SARS-CoV-2 infection, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. Using single-cell transcriptomics, they identify upregulation of cholesterol biosynthesis as a common mechanism underlying viral resistance, in addition to ACE2 sequestration.
Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase ...C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 3' end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 ↔ CDK9 and Ctk1 ↔ CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.
Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to ...address the question of cell autonomy in tran s active response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.
Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each ...of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument, comprising an epifluorescence microscope with integrated fluidics, into an open platform for programmable applications without need for specialized engineering or software development expertise. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. Given the widespread availability of HiSeq 2500 platforms and the simplicity of the modifications required to repurpose these systems, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.