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.
By analyzing gene expression data in glioblastoma in combination with matched microRNA profiles, we have uncovered a posttranscriptional regulation layer of surprising magnitude, comprising more than ...248,000 microRNA (miR)-mediated interactions. These include ∼7,000 genes whose transcripts act as miR “sponges” and 148 genes that act through alternative, nonsponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype implementation, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. siRNA silencing of 13 miR-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3′UTR-dependent manner and to increase tumor cell growth rates. Thus, miR-mediated interactions provide a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.
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► Computational inference reveals a network of miR-mediated interactions in glioblastoma ► These interactions regulate thousands of genes, including oncogenes and tumor suppressors ► These interactions regulate miR targets without affecting miR expression ► These interactions enable crosstalk between established oncogenic pathways
Computational inference uncovers a massive RNA-RNA interaction network in glioblastoma, which is shown to regulate established tumor drivers, such as PTEN, by modulating microRNA availability.
DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense ...loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration.
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•TBK1 kinase activity regulates disease progression in an ALS SOD1 mouse model•Loss of TBK1 in motor neurons increases SOD1 aggregation and accelerates disease onset•Loss of TBK1 activity in all cell types accelerates disease onset but extends survival•Loss of TBK1 activity in all cell types reduces the IFN response in microglia
DNA sequence variants in the TBK1 gene associate with familial and sporadic ALS. Gerbino et al. show that partial or complete loss-of-function TBK1 mutations alone do not induce neurodegeneration in mice. However, they profoundly affect disease onset and progression in the SOD1 ALS mouse model.
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases ...somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.
Two metrics, a rise in serum creatinine concentration and a decrease in urine output, are considered tantamount to the injury of the kidney tubule and the epithelial cells thereof (AKI). Yet neither ...criterion emphasizes the etiology or the pathogenetic heterogeneity of acute decreases in kidney excretory function. In fact, whether decreased excretory function due to contraction of the extracellular fluid volume (vAKI) or due to intrinsic kidney injury (iAKI) actually share pathogenesis and should be aggregated in the same diagnostic group remains an open question. To examine this possibility, we created mouse models of iAKI and vAKI that induced a similar increase in serum creatinine concentration. Using laser microdissection to isolate specific domains of the kidney, followed by RNA sequencing, we found that thousands of genes responded specifically to iAKI or to vAKI, but very few responded to both stimuli. In fact, the activated gene sets comprised different, functionally unrelated signal transduction pathways and were expressed in different regions of the kidney. Moreover, we identified distinctive gene expression patterns in human urine as potential biomarkers of either iAKI or vAKI, but not both. Hence, iAKI and vAKI are biologically unrelated, suggesting that molecular analysis should clarify our current definitions of acute changes in kidney excretory function.
Non-alcoholic fatty liver disease (NAFLD) affects about 24% of the world's population. Progression of early stages of NAFLD can lead to the more advanced form non-alcoholic steatohepatitis (NASH), ...and ultimately to cirrhosis or liver cancer. The current gold standard for diagnosis and assessment of NAFLD/NASH is liver biopsy followed by microscopic analysis by a pathologist. The Kleiner score is frequently used for a semi-quantitative assessment of disease progression. In this scoring system the features of active injury (steatosis, inflammation, and ballooning) and a separated fibrosis score are quantified. The procedure is time consuming for pathologists, scores have limited resolution and are subject to variation. We developed an automated deep learning method that provides full reproducibility and higher resolution. The system was established with 296 human liver biopsies and tested on 171 human liver biopsies with pathologist ground truth scores. The method is inspired by the way pathologist's analyze liver biopsies. First, the biopsies are analyzed microscopically for the relevant histopathological features. Subsequently, histopathological features are aggregated to a per-biopsy score. Scores are in the identical numeric range as the pathologist's ballooning, inflammation, steatosis, and fibrosis scores, but on a continuous scale. Resulting scores followed a pathologist's ground truth (quadratic weighted Cohen's κ on the test set: for steatosis 0.66, for inflammation 0.24, for ballooning 0.43, for fibrosis 0.62, and for the NAFLD activity score (NAS) 0.52. Mean absolute errors on a test set: for steatosis 0.29, for inflammation 0.53, for ballooning 0.61, for fibrosis 0.78, and for the NAS 0.77).
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-β signaling pathways.
Tumor heterogeneity is a major obstacle for finding effective treatment of Glioblastoma (GBM). Based on global expression analysis, GBM can be classified into distinct subtypes: Proneural, Neural, ...Classical and Mesenchymal. The signatures of these different tumor subtypes may reflect the phenotypes of cells giving rise to them. However, the experimental evidence connecting any specific subtype of GBM to particular cells of origin is lacking. In addition, it is unclear how different genetic alterations interact with cells of origin in determining tumor heterogeneity. This issue cannot be addressed by studying end-stage human tumors.
To address this issue, we used retroviruses to deliver transforming genetic lesions to glial progenitors in adult mouse brain. We compared the resulting tumors to human GBM. We found that different initiating genetic lesions gave rise to tumors with different growth rates. However all mouse tumors closely resembled the human Proneural GBM. Comparative analysis of these mouse tumors allowed us to identify a set of genes whose expression in humans with Proneural GBM correlates with survival.
This study offers insights into the relationship between adult glial progenitors and Proneural GBM, and allows us to identify molecular alterations that lead to more aggressive tumor growth. In addition, we present a new preclinical model that can be used to test treatments directed at a specific type of GBM in future studies.
Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) represent rare but serious adverse drug reactions (ADRs). Both are characterized by distinctive blistering lesions and significant ...mortality rates. While there is evidence for strong drug-specific genetic predisposition related to HLA alleles, recent genome wide association studies (GWAS) on European and Asian populations have failed to identify genetic susceptibility alleles that are common across multiple drugs. We hypothesize that this is a consequence of the low to moderate effect size of individual genetic risk factors. To test this hypothesis we developed Pointer, a new algorithm that assesses the aggregate effect of multiple low risk variants on a pathway using a gene set enrichment approach. A key advantage of our method is the capability to associate SNPs with genes by exploiting physical proximity as well as by using expression quantitative trait loci (eQTLs) that capture information about both cis- and trans-acting regulatory effects. We control for known bias-inducing aspects of enrichment based analyses, such as: 1) gene length, 2) gene set size, 3) presence of biologically related genes within the same linkage disequilibrium (LD) region, and, 4) genes shared among multiple gene sets. We applied this approach to publicly available SJS/TEN genome-wide genotype data and identified the ABC transporter and Proteasome pathways as potentially implicated in the genetic susceptibility of non-drug-specific SJS/TEN. We demonstrated that the innovative SNP-to-gene mapping phase of the method was essential in detecting the significant enrichment for those pathways. Analysis of an independent gene expression dataset provides supportive functional evidence for the involvement of Proteasome pathways in SJS/TEN cutaneous lesions. These results suggest that Pointer provides a useful framework for the integrative analysis of pharmacogenetic GWAS data, by increasing the power to detect aggregate effects of multiple low risk variants. The software is available for download at https://sourceforge.net/projects/pointergsa/.
Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified ...sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-β pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment.
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