FUS-ALS hiPSC-derived astrocytes impair human motor units through both gain-of-toxicity and loss-of-support mechanisms.

BACKGROUND: Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points. METHODS: In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes from FUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings. FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems. RESULTS: We observed a dysregulation of astrocyte homeostasis, which resulted in a FUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/ß-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs. CONCLUSIONS: Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms.

CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.

Unraveling the Molecular Basis of the Dystrophic Process in Limb-Girdle Muscular Dystrophy LGMD-R12 by Differential Gene Expression Profiles in Diseased and Healthy Muscles.

Limb-girdle muscular dystrophy R12 (LGMD-R12) is caused by two mutations in anoctamin-5 (ANO5). Our aim was to identify genes and pathways that underlie LGMD-R12 and explain differences in the molecular predisposition and susceptibility between three thigh muscles that are severely (semimembranosus), moderately (vastus lateralis) or mildly (rectus femoris) affected in this disease. We performed transcriptomics on these three muscles in 16 male LGMD-R12 patients and 15 age-matched male controls. Our results showed that LGMD-R12 dystrophic muscle is associated with the expression of genes indicative of fibroblast and adipocyte replacement, such as fibroadipogenic progenitors and immune cell infiltration, while muscle protein synthesis and metabolism were downregulated. Muscle degeneration was associated with an increase in genes involved in muscle injury and inflammation, and muscle repair/regeneration. Baseline differences between muscles in healthy individuals indicated that muscles that are the most affected by LGMD-R12 have the lowest expression of transcription factor networks involved in muscle (re)generation and satellite stem cell activation. Instead, they show relative high levels of fetal/embryonic myosins, all together indicating that muscles differ in their baseline regenerative potential. To conclude, we profiled the gene expression landscape in LGMD-R12, identified baseline differences in expression levels between differently affected muscles and characterized disease-associated changes.

Single-cell Transcriptomics Uncover a Novel Role of Myeloid Cells and T-lymphocytes in the Fibrotic Microenvironment in Peyronie's Disease.

BACKGROUND: Peyronie's disease (PD) is an acquired fibrotic disease affecting the penile tunica albuginea that can lead to curvature and deformities, shortening, and erectile dysfunction. Although immunological mechanisms have been suggested for the pathophysiology of PD, these have not been investigated using single-cell transcriptomics. OBJECTIVE: To investigate the immunological signature of plaques from PD patients using immunohistochemistry (IHC) and single-cell RNA sequencing (scRNA-Seq). DESIGN, SETTING, AND PARTICIPANTS: Tunica albuginea biopsy was performed in patients undergoing penile surgery for either PD (n = 12) or plication or penile cancer (control, n = 6). The inclusion criteria for PD patients were stable chronic disease (≥12 mo in duration) and no previous penile surgery or intralesional injection therapy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: IHC was performed on surgical samples from ten patients with PD and five control subjects. An additional two PD and one control sample were used for scRNA-Seq (droplet-based; 10X Genomics). Cell clusters were visualised using heatmaps and t-distributed stochastic neighbour embedding plots (BioTuring v2.7.5). RESULTS AND LIMITATIONS: IHC revealed the presence of myeloid dendritic cells (DCs; CD68+, TLR4+, CD206+), cytotoxic T lymphocytes (CTLs; CD3+, CD8+), and B lymphocytes (CD20+) in PD plaques, which were absent in controls. scRNA-Seq yielded results for 3312 PD and 5658 control cells. Cell clusters contained fibroblasts (COL1A2+), myofibroblasts (COL1A2+, ACTA2+), smooth muscle cells (ACTA2+, DES+), endothelial cells (VWF+), myeloid cells (CD14+), T lymphocytes (CD3D+), and neutrophils (ALPL+). Myeloid cell subclustering showed infiltration of monocyte-derived cells; control tissue contained classical DCs and resident macrophages. Lymphocyte subclustering revealed mucosal-associated invariant T (MAIT) cells and CTLs in PD. Differential gene expression suggests an increase in inflammatory and immune responses in chronic PD. The study is limited by the small scRNA-seq sample size (n = 3) for IHC, mitigated by a larger cohort of historic paraffin-embedded samples (n = 15), which showed largely parallel findings. Owing to tissue stiffness and extracellular matrix adhesion, our single-cell yield was lower for PD than for the control sample. CONCLUSIONS: Our data suggest that even in the chronic PD stage (painless and stable curvature) there is a sustained inflammatory reaction. While vascularisation and collagen production are elevated, the inflammation is driven by specialised monocyte-derived CTL and MAIT cells. These findings could uncover new avenues for medical treatment of PD. PATIENT SUMMARY: We looked at the role of the immune system in patients suffering from Peyronie's disease, a condition causing shortening and curvature of the penis. We found that even in a stable, chronic stage of the disease, there is activation of the immune system. Our results suggest that there is potential for novel treatments for this condition.

Gemcitabine Recruits M2-Type Tumor-Associated Macrophages into the Stroma of Pancreatic Cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a very lethal disease that can develop therapy resistance over time. The dense stroma in PDAC plays a critical role in tumor progression and resistance. How this stroma interacts with the tumor cells and how this is influenced by chemotherapy remain poorly understood. METHODS: The backbone of this study is the parallel transcriptome analysis of human tumor and mouse stroma in two molecular and clinical representative patient-derived tumor xenografts models. Mice (8 animals per group) were treated for 4 weeks with gemcitabine or control. We studied tumor growth, RNA expression in the stroma, tumor-associated macrophages (TAMs) with immunofluorescence, and cytokines in the serum. RESULTS: A method for parallel transcriptome analysis was optimized. We found that the tumor (differentiation, gene expression) determines the infiltration of macrophages into the stroma. In aggressive PDAC (epithelial-to-mesenchymal transition high), we find more M2 polarized TAMs and the activation of cytokines and growth factors (TNFα, TGFß1, and IL6). There are increased stromal glycolysis, reduced fatty acid oxidation, and reduced mitochondrial oxidation (tricarboxylic acid cycle and oxidative phosphorylation). Treatment with gemcitabine results in a shift of innate immune cells, especially additional infiltration of protumoral M2 TAMs (P < .001) and metabolic reprogramming. CONCLUSIONS: Gemcitabine treatment of PDAC xenografts stimulates a protumoral macrophage phenotype, and this, in combination with a shift of the tumor cells to a mesenchymal phenotype that we reported previously, contributes to tumor progression and therapeutic resistance. Targeting M2-polarized TAMs may benefit PDAC patients at risk to become refractory to current anticancer regimens.

Dysregulation of Macropinocytosis Processes in Glioblastomas May Be Exploited to Increase Intracellular Anti-Cancer Drug Levels: The Example of Temozolomide.

Macropinocytosis is a clathrin-independent endocytosis of extracellular fluid that may contribute to cancer aggressiveness through nutrient supply, recycling of plasma membrane and receptors, and exosome internalization. Macropinocytosis may be notably triggered by epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), two well-known markers for glioblastoma aggressiveness. Therefore, we studied whether the expression of key actors of macropinocytosis is modified in human glioma datasets. Strong deregulation has been evidenced at the mRNA level according to the grade of the tumor, and 38 macropinocytosis-related gene signatures allowed discrimination of the glioblastoma (GBM) samples. Honokiol-induced vacuolization was then compared to vacquinol-1 and MOMIPP, two known macropinocytosis inducers. Despite high phase-contrast morphological similarities, honokiol-induced vacuoles appeared to originate from both endocytosis and ER. Also, acridine orange staining suggested differences in the macropinosomes' fate: their fusion with lysosomes appeared very limited in 3-(5-methoxy -2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP)-treated cells. Nevertheless, each of the compounds markedly increased temozolomide uptake by glioma cells, as evidenced by LC-MS. In conclusion, the observed deregulation of macropinocytosis in GBM makes them prone to respond to various compounds affecting their formation and/or intracellular fate. Considering that sustained macropinocytosis may also trigger cell death of both sensitive and resistant GBM cells, we propose to envisage macropinocytosis inducers in combination approaches to obtain dual benefits: increased drug uptake and additive/synergistic effects.

Prognostic relevance of molecular subtypes and master regulators in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic cancer is poorly characterized at genetic and non-genetic levels. The current study evaluates in a large cohort of patients the prognostic relevance of molecular subtypes and key transcription factors in pancreatic ductal adenocarcinoma (PDAC). METHODS: We performed gene expression analysis of whole-tumor tissue obtained from 118 surgically resected PDAC and 13 histologically normal pancreatic tissue samples. Cox regression models were used to study the effect on survival of molecular subtypes and 16 clinicopathological prognostic factors. In order to better understand the biology of PDAC we used iRegulon to identify transcription factors (TFs) as master regulators of PDAC and its subtypes. RESULTS: We confirmed the PDAssign gene signature as classifier of PDAC in molecular subtypes with prognostic relevance. We found molecular subtypes, but not clinicopathological factors, as independent predictors of survival. Regulatory network analysis predicted that HNF1A/B are among thousand TFs the top enriched master regulators of the genes expressed in the normal pancreatic tissue compared to the PDAC regulatory network. On immunohistochemistry staining of PDAC samples, we observed low expression of HNF1B in well differentiated towards no expression in poorly differentiated PDAC samples. We predicted IRF/STAT, AP-1, and ETS-family members as key transcription factors in gene signatures downstream of mutated KRAS. CONCLUSIONS: PDAC can be classified in molecular subtypes that independently predict survival. HNF1A/B seem to be good candidates as master regulators of pancreatic differentiation, which at the protein level loses its expression in malignant ductal cells of the pancreas, suggesting its putative role as tumor suppressor in pancreatic cancer. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov under the number NCT01116791 (May 3, 2010).

p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

iRegulon: from a gene list to a gene regulatory network using large motif and track collections.

Identifying master regulators of biological processes and mapping their downstream gene networks are key challenges in systems biology. We developed a computational method, called iRegulon, to reverse-engineer the transcriptional regulatory network underlying a co-expressed gene set using cis-regulatory sequence analysis. iRegulon implements a genome-wide ranking-and-recovery approach to detect enriched transcription factor motifs and their optimal sets of direct targets. We increase the accuracy of network inference by using very large motif collections of up to ten thousand position weight matrices collected from various species, and linking these to candidate human TFs via a motif2TF procedure. We validate iRegulon on gene sets derived from ENCODE ChIP-seq data with increasing levels of noise, and we compare iRegulon with existing motif discovery methods. Next, we use iRegulon on more challenging types of gene lists, including microRNA target sets, protein-protein interaction networks, and genetic perturbation data. In particular, we over-activate p53 in breast cancer cells, followed by RNA-seq and ChIP-seq, and could identify an extensive up-regulated network controlled directly by p53. Similarly we map a repressive network with no indication of direct p53 regulation but rather an indirect effect via E2F and NFY. Finally, we generalize our computational framework to include regulatory tracks such as ChIP-seq data and show how motif and track discovery can be combined to map functional regulatory interactions among co-expressed genes. iRegulon is available as a Cytoscape plugin from http://iregulon.aertslab.org.

The transcriptional program controlled by the stem cell leukemia gene Scl/Tal1 during early embryonic hematopoietic development.

The basic helix-loop-helix transcription factor Scl/Tal1 controls the development and subsequent differentiation of hematopoietic stem cells (HSCs). However, because few Scl target genes have been validated to date, the underlying mechanisms have remained largely unknown. In this study, we have used ChIP-Seq technology (coupling chromatin immunoprecipitation with deep sequencing) to generate a genome-wide catalog of Scl-binding events in a stem/progenitor cell line, followed by validation using primary fetal liver cells and comprehensive transgenic mouse assays. Transgenic analysis provided in vivo validation of multiple new direct Scl target genes and allowed us to reconstruct an in vivo validated network consisting of 17 factors and their respective regulatory elements. By coupling ChIP-Seq in model cell lines with in vivo transgenic validation and sophisticated bioinformatic analysis, we have identified a widely applicable strategy for the reconstruction of stem cell regulatory networks in which biologic material is otherwise limiting. Moreover, in addition to revealing multiple previously unrecognized links to known HSC regulators, as well as novel links to genes not previously implicated in HSC function, comprehensive transgenic analysis of regulatory elements provided substantial new insights into the transcriptional control of several important hematopoietic regulators, including Cbfa2t3h/Eto2, Cebpe, Nfe2, Zfpm1/Fog1, Erg, Mafk, Gfi1b, and Myb.