Paolo Guarnieri

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.

Glioblastoma Models Reveal the Connection between Adult Glial Progenitors and the Proneural Phenotype

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

A Molecular Signature Predictive of Indolent Prostate Cancer

A three-gene panel derived from mechanistic models of cell senescence predicts outcome of low Gleason score prostate tumors. To Treat or Not to Treat...* ...That is often the question for prostate cancer patients and their caretakers. Now, Irshad et al. describe a gene signature that may guide treatment choices when prognosis is unclear. Along with other clinical and molecular parameters, pathologists use the Gleason grading system to stage prostate cancers and predict patient prognosis. A Gleason score is assigned to a cancer on the basis of its microscopic features and is directly related to tumor aggressiveness and poor prognosis. Most newly diagnosed prostate cancers with low Gleason scores require no treatment intervention and are monitored with active surveillance (indolent tumors). However, the pinpointing of tumors that are aggressive and lethal despite having low Gleason scores is a clinical challenge. In these cases, new tools are needed to answer the title question. Irshad and colleagues show that low Gleason score prostate tumors can be separated into distinct indolent and aggressive subgroups on the basis of their expression of aging and senescence genes. Using patient tissue samples and gene expression data along with computational biology techniques, including a decision tree learning model, the authors identified three genes—FGFR1, PMP22, and CDKN1A—that predicted the clinical outcome of low Gleason score prostate tumors. The prognostic power of the three-gene signature was validated in independent patient cohorts, and expression of the FGFR1, PMP22, and CDKN1A proteins in biopsy samples identified Gleason 6 patients who had failed surveillance over a 10-year period. Just as Hamlet laments in his famous soliloquy, oncologists and patients need more information about the unknown before making a decision. The new signature might aid in the choice between “bear[ing] those ills [they] have” with active surveillance or actively treating—and hopefully thwarting—aggressive tumors. *Paraphrased from the “To be, or not to be” soliloquy in Hamlet by William Shakespeare. Many newly diagnosed prostate cancers present as low Gleason score tumors that require no treatment intervention. Distinguishing the many indolent tumors from the minority of lethal ones remains a major clinical challenge. We now show that low Gleason score prostate tumors can be distinguished as indolent and aggressive subgroups on the basis of their expression of genes associated with aging and senescence. Using gene set enrichment analysis, we identified a 19-gene signature enriched in indolent prostate tumors. We then further classified this signature with a decision tree learning model to identify three genes—FGFR1, PMP22, and CDKN1A—that together accurately predicted outcome of low Gleason score tumors. Validation of this three-gene panel on independent cohorts confirmed its independent prognostic value as well as its ability to improve prognosis with currently used clinical nomograms. Furthermore, protein expression of this three-gene panel in biopsy samples distinguished Gleason 6 patients who failed surveillance over a 10-year period. We propose that this signature may be incorporated into prognostic assays for monitoring patients on active surveillance to facilitate appropriate courses of treatment.

Intricate interplay between astrocytes and motor neurons in ALS

Significance Although ALS is a motor neuron disease, processes within glial cells contribute significantly to motor neuron-specific degeneration. Using a mouse model of ALS, we identified cell autonomous and nonautonomous changes in gene expression in motor neurons cocultured with glia. We also 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 this model. We 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. 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 hSOD1G93A (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.

The testis-specific double bromodomain-containing protein BRDT forms a complex with multiple spliceosome components and is required for mRNA splicing and 3′-UTR truncation in round spermatids

Members of the BET (bromodomain and extra terminal motif) family of proteins have been shown to be chromatin-interacting regulators of transcription. We previously generated a mutation in the testis-specific mammalian BET gene Brdt (bromodomain, testis-specific) that yields protein lacking the first bromodomain (BRDTΔBD1) and observed disrupted spermiogenesis and male sterility. To determine whether BRDTΔBD1 protein results in altered transcription, we analyzed the transcriptomes of control versus BrdtΔBD1/ΔBD1 round spermatids. Over 400 genes showed statistically significant differential expression, and among the up-regulated genes, there was an enrichment of RNA splicing genes. Over 60% of these splicing genes had transcripts that lacked truncation of their 3′-untranslated region (UTR) typical of round spermatids. We selected four of these genes to characterize: Srsf2, Ddx5, Hnrnpk and Tardbp. The 3′-UTRs of Srsf2, Ddx5 and Hnrnpk mRNAs were longer in mutant round spermatids and resulted in reduced protein levels. Tardbp was transcriptionally up-regulated and a splicing shift toward the longer variant was observed. All four splicing proteins were found to complex with BRDT in control and mutant testes. We thus suggest that, along with modulating transcription, BRDT modulates gene expression as part of the splicing machinery. These modulations alter 3′-UTR processing in round spermatids; importantly, the BD1 is essential for these functions.

Reprogramming of the microRNA transcriptome mediates resistance to rapamycin.

Background: The role of miRNAs in the cellular response to mTOR inhibitors has never been tested. Results: Rapamycin resistance is associated with up-regulation of oncogenic miRNAs and down-regulation of tumor suppressor miRNAs. Conclusion: miRNAs influence the cellular response to mTOR inhibitors. Significance: miRNAs are potential markers and novel targets for cancer therapy involving mTOR inhibitors. The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation that is often deregulated in cancer. Inhibitors of mTOR, including rapamycin and its analogues, are being evaluated as antitumor agents. For their promise to be fulfilled, it is of paramount importance to identify the mechanisms of resistance and develop novel therapies to overcome it. Given the emerging role of microRNAs (miRNAs) in tumorigenesis, we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Long-term rapamycin treatment showed extensive reprogramming of miRNA expression, characterized by up-regulation of miR-17–92 and related clusters and down-regulation of tumor suppressor miRNAs. Inhibition of members of the miR-17–92 clusters or delivery of tumor suppressor miRNAs restored sensitivity to rapamycin. This study identifies miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. It also identifies potential markers to assess the efficacy of treatment and provides novel therapeutic targets to treat rapamycin-resistant tumors.

The Transcriptional Regulatory Network of Proneural Glioma Determines the Genetic Alterations Selected During Tumor Progression

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression.

Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS

Significance Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that leads to death of spinal motor neurons and paralysis. Genetic studies of ALS patients have identified mutations in autophagy pathway genes including p62/SQSTM1, OPTN, TBK1, VCP, and C9ORF72. However, the mechanisms by which these mutations cause ALS are not understood. Here we investigated the role of autophagy in a mouse model of ALS by specifically disrupting the critical autophagy gene Atg7 in motor neurons. We found that inhibition of autophagy in motor neurons accelerated disease onset but prolonged lifespan. This increase in longevity was associated with a reduction in glial inflammation and interneuron pathology, indicating a non–cell-autonomous role for motor neuron autophagy in disease pathogenesis. Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non–cell-autonomous manner to promote disease progression.

Unique Transcriptional Programs Identify Subtypes of AKI.

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.

Inhibition of cyclo-oxygenase 2 reduces tumor metastasis and inflammatory signaling during blockade of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) blockade is an effective therapy for human cancer, yet virtually all neoplasms resume primary tumor growth or metastasize during therapy. Mechanisms of progression have been proposed to include genes that control vascular remodeling and are elicited by hypoperfusion, such as the inducible enzyme cyclooxygenase-2 (COX-2). We have previously shown that COX-2 inhibition by the celecoxib analog SC236 attenuates perivascular stromal cell recruitment and tumor growth. We therefore examined the effect of combined SC236 and VEGF blockade, using the metastasizing orthotopic SKNEP1 model of pediatric cancer. Combined treatment perturbed tumor vessel remodeling and macrophage recruitment, but did not further limit primary tumor growth as compared to VEGF blockade alone. However, combining SC236 and VEGF inhibition significantly reduced the incidence of lung metastasis, suggesting a distinct effect on prometastatic mechanisms. We found that SC236 limited tumor cell viability and migration in vitro, with effects enhanced by hypoxia, but did not change tumor proliferation or matrix metalloproteinase expression in vivo. Gene set expression analysis (GSEA) indicated that the addition of SC236 to VEGF inhibition significantly reduced expression of gene sets linked to macrophage mobilization. Perivascular recruitment of macrophages induced by VEGF blockade was disrupted in tumors treated with combined VEGF- and COX-2-inhibition. Collectively, these findings suggest that during VEGF blockade COX-2 may restrict metastasis by limiting both prometastatic behaviors in individual tumor cells and mobilization of macrophages to the tumor vasculature.