SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

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.

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A.

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.

Retromer dysfunction in amyotrophic lateral sclerosis.

Retromer is a heteropentameric complex that plays a specialized role in endosomal protein sorting and trafficking. Here, we report a reduction in the retromer proteins-vacuolar protein sorting 35 (VPS35), VPS26A, and VPS29-in patients with amyotrophic lateral sclerosis (ALS) and in the ALS model provided by transgenic (Tg) mice expressing the mutant superoxide dismutase-1 G93A. These changes are accompanied by a reduction of levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA1, a proxy of retromer function, in spinal cords from Tg SOD1G93A mice. Correction of the retromer deficit by a viral vector expressing VPS35 exacerbates the paralytic phenotype in Tg SOD1G93A mice. Conversely, lowering Vps35 levels in Tg SOD1G93A mice ameliorates the disease phenotype. In light of these findings, we propose that mild alterations in retromer inversely modulate neurodegeneration propensity in ALS.

Rare variant analyses validate known ALS genes in a multi-ethnic population and identifies ANTXR2 as a candidate in PLS.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting over 300,000 people worldwide. It is characterized by the progressive decline of the nervous system that leads to the weakening of muscles which impacts physical function. Approximately, 15% of individuals diagnosed with ALS have a known genetic variant that contributes to their disease. As therapies that slow or prevent symptoms continue to develop, such as antisense oligonucleotides, it is important to discover novel genes that could be targets for treatment. Additionally, as cohorts continue to grow, performing analyses in ALS subtypes, such as primary lateral sclerosis (PLS), becomes possible due to an increase in power. These analyses could highlight novel pathways in disease manifestation. METHODS: Building on our previous discoveries using rare variant association analyses, we conducted rare variant burden testing on a substantially larger multi-ethnic cohort of 6,970 ALS patients, 166 PLS patients, and 22,524 controls. We used intolerant domain percentiles based on sub-region Residual Variation Intolerance Score (subRVIS) that have been described previously in conjunction with gene based collapsing approaches to conduct burden testing to identify genes that associate with ALS and PLS. RESULTS: A gene based collapsing model showed significant associations with SOD1, TARDBP, and TBK1 (OR = 19.18, p = 3.67 × 10-39; OR = 4.73, p = 2 × 10-10; OR = 2.3, p = 7.49 × 10-9, respectively). These genes have been previously associated with ALS. Additionally, a significant novel control enriched gene, ALKBH3 (p = 4.88 × 10-7), was protective for ALS in this model. An intolerant domain-based collapsing model showed a significant improvement in identifying regions in TARDBP that associated with ALS (OR = 10.08, p = 3.62 × 10-16). Our PLS protein truncating variant collapsing analysis demonstrated significant case enrichment in ANTXR2 (p = 8.38 × 10-6). CONCLUSIONS: In a large multi-ethnic cohort of 6,970 ALS patients, collapsing analyses validated known ALS genes and identified a novel potentially protective gene, ALKBH3. A first-ever analysis in 166 patients with PLS found a candidate association with loss-of-function mutations in ANTXR2.

Unexpected frequency of the pathogenic AR CAG repeat expansion in the general population.

CAG repeat expansions in exon 1 of the AR gene on the X chromosome cause spinal and bulbar muscular atrophy, a male-specific progressive neuromuscular disorder associated with a variety of extra-neurological symptoms. The disease has a reported male prevalence of approximately 1:30 000 or less, but the AR repeat expansion frequency is unknown. We established a pipeline, which combines the use of the ExpansionHunter tool and visual validation, to detect AR CAG expansion on whole-genome sequencing data, benchmarked it to fragment PCR sizing, and applied it to 74 277 unrelated individuals from four large cohorts. Our pipeline showed sensitivity of 100% [95% confidence interval (CI) 90.8-100%], specificity of 99% (95% CI 94.2-99.7%), and a positive predictive value of 97.4% (95% CI 84.4-99.6%). We found the mutation frequency to be 1:3182 (95% CI 1:2309-1:4386, n = 117 734) X chromosomes-10 times more frequent than the reported disease prevalence. Modelling using the novel mutation frequency led to estimate disease prevalence of 1:6887 males, more than four times more frequent than the reported disease prevalence. This discrepancy is possibly due to underdiagnosis of this neuromuscular condition, reduced penetrance, and/or pleomorphic clinical manifestations.

A new approach for rare variation collapsing on functional protein domains implicates specific genic regions in ALS.

Large-scale sequencing efforts in amyotrophic lateral sclerosis (ALS) have implicated novel genes using gene-based collapsing methods. However, pathogenic mutations may be concentrated in specific genic regions. To address this, we developed two collapsing strategies: One focuses rare variation collapsing on homology-based protein domains as the unit for collapsing, and the other is a gene-level approach that, unlike standard methods, leverages existing evidence of purifying selection against missense variation on said domains. The application of these two collapsing methods to 3093 ALS cases and 8186 controls of European ancestry, and also 3239 cases and 11,808 controls of diversified populations, pinpoints risk regions of ALS genes, including SOD1, NEK1, TARDBP, and FUS While not clearly implicating novel ALS genes, the new analyses not only pinpoint risk regions in known genes but also highlight candidate genes as well.

CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1.

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.

Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy.

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 transactive 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.

Intricate interplay between astrocytes and motor neurons in ALS.

ALS results from the selective and progressive degeneration of motor neurons. Although the underlying disease mechanisms remain unknown, glial cells have been implicated in ALS disease progression. Here, we examine the effects of glial cell/motor neuron interactions on gene expression using the hSOD1(G93A) (the G93A allele of the human superoxide dismutase gene) mouse model of ALS. We detect striking cell autonomous and nonautonomous changes in gene expression in cocultured motor neurons and glia, revealing that the two cell types profoundly affect each other. In addition, we found a remarkable concordance between the cell culture data and expression profiles of whole spinal cords and acutely isolated spinal cord cells during disease progression in the G93A mouse model, providing validation of the cell culture approach. Bioinformatics analyses identified changes in the expression of specific genes and signaling pathways that may contribute to motor neuron degeneration in ALS, among which are TGF-ß signaling pathways.

An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.

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.

Stochastic expression of the interferon-ß gene.

Virus infection of mammalian cells induces the production of high levels of type I interferons (IFNα and ß), cytokines that orchestrate antiviral innate and adaptive immunity. Previous studies have shown that only a fraction of the infected cells produce IFN. However, the mechanisms responsible for this stochastic expression are poorly understood. Here we report an in depth analysis of IFN-expressing and non-expressing mouse cells infected with Sendai virus. Mouse embryonic fibroblasts in which an internal ribosome entry site/yellow fluorescent protein gene was inserted downstream from the endogenous IFNß gene were used to distinguish between the two cell types, and they were isolated from each other using fluorescence-activated cell sorting methods. Analysis of the separated cells revealed that stochastic IFNß expression is a consequence of cell-to-cell variability in the levels and/or activities of limiting components at every level of the virus induction process, ranging from viral replication and expression, to the sensing of viral RNA by host factors, to activation of the signaling pathway, to the levels of activated transcription factors. We propose that this highly complex stochastic IFNß gene expression evolved to optimize both the level and distribution of type I IFNs in response to virus infection.

Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability.

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening.

Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease.

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.

The broken Alzheimer's disease genome.

The complex pathobiology of late-onset Alzheimer's disease (AD) poses significant challenges to therapeutic and preventative interventions. Despite these difficulties, genomics and related disciplines are allowing fundamental mechanistic insights to emerge with clarity, particularly with the introduction of high-resolution sequencing technologies. After all, the disrupted processes at the interface between DNA and gene expression, which we call the broken AD genome, offer detailed quantitative evidence unrestrained by preconceived notions about the disease. In addition to highlighting biological pathways beyond the classical pathology hallmarks, these advances have revitalized drug discovery efforts and are driving improvements in clinical tools. We review genetic, epigenomic, and gene expression findings related to AD pathogenesis and explore how their integration enables a better understanding of the multicellular imbalances contributing to this heterogeneous condition. The frontiers opening on the back of these research milestones promise a future of AD care that is both more personalized and predictive.

Identifying FUS amyotrophic lateral sclerosis disease signatures in patient dermal fibroblasts.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, highly heterogeneous neurodegenerative disease, underscoring the importance of obtaining information to personalize clinical decisions quickly after diagnosis. Here, we investigated whether ALS-relevant signatures can be detected directly from biopsied patient fibroblasts. We profiled familial ALS (fALS) fibroblasts, representing a range of mutations in the fused in sarcoma (FUS) gene and ages of onset. To differentiate FUS fALS and healthy control fibroblasts, machine-learning classifiers were trained separately on high-content imaging and transcriptional profiles. "Molecular ALS phenotype" scores, derived from these classifiers, captured a spectrum from disease to health. Interestingly, these scores negatively correlated with age of onset, identified several pre-symptomatic individuals and sporadic ALS (sALS) patients with FUS-like fibroblasts, and quantified "movement" of FUS fALS and "FUS-like" sALS toward health upon FUS ASO treatment. Taken together, these findings provide evidence that non-neuronal patient fibroblasts can be used for rapid, personalized assessment in ALS.

Tissue and cellular spatiotemporal dynamics in colon aging.

Tissue structure and molecular circuitry in the colon can be profoundly impacted by systemic age-related effects, but many of the underlying molecular cues remain unclear. Here, we built a cellular and spatial atlas of the colon across three anatomical regions and 11 age groups, encompassing ~1,500 mouse gut tissues profiled by spatial transcriptomics and ~400,000 single nucleus RNA-seq profiles. We developed a new computational framework, cSplotch, which learns a hierarchical Bayesian model of spatially resolved cellular expression associated with age, tissue region, and sex, by leveraging histological features to share information across tissue samples and data modalities. Using this model, we identified cellular and molecular gradients along the adult colonic tract and across the main crypt axis, and multicellular programs associated with aging in the large intestine. Our multi-modal framework for the investigation of cell and tissue organization can aid in the understanding of cellular roles in tissue-level pathology.

Integrative transcriptomic analysis of the amyotrophic lateral sclerosis spinal cord implicates glial activation and suggests new risk genes.

Amyotrophic lateral sclerosis (ALS) is a progressively fatal neurodegenerative disease affecting motor neurons in the brain and spinal cord. In this study, we investigated gene expression changes in ALS via RNA sequencing in 380 postmortem samples from cervical, thoracic and lumbar spinal cord segments from 154 individuals with ALS and 49 control individuals. We observed an increase in microglia and astrocyte gene expression, accompanied by a decrease in oligodendrocyte gene expression. By creating a gene co-expression network in the ALS samples, we identified several activated microglia modules that negatively correlate with retrospective disease duration. We mapped molecular quantitative trait loci and found several potential ALS risk loci that may act through gene expression or splicing in the spinal cord and assign putative cell types for FNBP1, ACSL5, SH3RF1 and NFASC. Finally, we outline how common genetic variants associated with splicing of C9orf72 act as proxies for the well-known repeat expansion, and we use the same mechanism to suggest ATXN3 as a putative risk gene.

Rare variant analyses validate known ALS genes in a multi-ethnic population and identifies ANTXR2 as a candidate in PLS.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting over 30,000 people in the United States. It is characterized by the progressive decline of the nervous system that leads to the weakening of muscles which impacts physical function. Approximately, 15% of individuals diagnosed with ALS have a known genetic variant that contributes to their disease. As therapies that slow or prevent symptoms, such as antisense oligonucleotides, continue to develop, it is important to discover novel genes that could be targets for treatment. Additionally, as cohorts continue to grow, performing analyses in ALS subtypes, such as primary lateral sclerosis (PLS), becomes possible due to an increase in power. These analyses could highlight novel pathways in disease manifestation. Methods: Building on our previous discoveries using rare variant association analyses, we conducted rare variant burden testing on a substantially larger cohort of 6,970 ALS patients from a large multi-ethnic cohort as well as 166 PLS patients, and 22,524 controls. We used intolerant domain percentiles based on sub-region Residual Variation Intolerance Score (subRVIS) that have been described previously in conjunction with gene based collapsing approaches to conduct burden testing to identify genes that associate with ALS and PLS. Results: A gene based collapsing model showed significant associations with SOD1, TARDBP, and TBK1 (OR=19.18, p = 3.67 × 10-39; OR=4.73, p = 2 × 10-10; OR=2.3, p = 7.49 × 10-9, respectively). These genes have been previously associated with ALS. Additionally, a significant novel control enriched gene, ALKBH3 (p = 4.88 × 10-7), was protective for ALS in this model. An intolerant domain based collapsing model showed a significant improvement in identifying regions in TARDBP that associated with ALS (OR=10.08, p = 3.62 × 10-16). Our PLS protein truncating variant collapsing analysis demonstrated significant case enrichment in ANTXR2 (p=8.38 × 10-6). Conclusions: In a large multi-ethnic cohort of 6,970 ALS patients, rare variant burden testing validated known ALS genes and identified a novel potentially protective gene, ALKBH3. A first-ever analysis in 166 patients with PLS found a candidate association with loss-of-function mutations in ANTXR2.