Aß-accelerated neurodegeneration caused by Alzheimer's-associated ACE variant R1279Q is rescued by angiotensin system inhibition in mice.

Recent genome-wide association studies identified the angiotensin-converting enzyme gene (ACE) as an Alzheimer's disease (AD) risk locus. However, the pathogenic mechanism by which ACE causes AD is unknown. Using whole-genome sequencing, we identified rare ACE coding variants in AD families and investigated one, ACE1 R1279Q, in knockin (KI) mice. Similar to AD, ACE1 was increased in neurons, but not microglia or astrocytes, of KI brains, which became elevated further with age. Angiotensin II (angII) and angII receptor AT1R signaling were also increased in KI brains. Autosomal dominant neurodegeneration and neuroinflammation occurred with aging in KI hippocampus, which were absent in the cortex and cerebellum. Female KI mice exhibited greater hippocampal electroencephalograph disruption and memory impairment compared to males. ACE variant effects were more pronounced in female KI mice, suggesting a mechanism for higher AD risk in women. Hippocampal neurodegeneration was completely rescued by treatment with brain-penetrant drugs that inhibit ACE1 and AT1R. Although ACE variant-induced neurodegeneration did not depend on ß-amyloid (Aß) pathology, amyloidosis in 5XFAD mice crossed to KI mice accelerated neurodegeneration and neuroinflammation, whereas Aß deposition was unchanged. KI mice had normal blood pressure and cerebrovascular functions. Our findings strongly suggest that increased ACE1/angII signaling causes aging-dependent, Aß-accelerated selective hippocampal neuron vulnerability and female susceptibility, hallmarks of AD that have hitherto been enigmatic. We conclude that repurposed brain-penetrant ACE inhibitors and AT1R blockers may protect against AD.

A cell atlas of the adult Drosophila midgut.

Studies of the adult Drosophila midgut have led to many insights in our understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. We used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing functions of genes in the midgut epithelium.

Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis.

BACKGROUND AND AIMS: Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis. APPROACH AND RESULTS: Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor ß2) and inflammation (tumor necrosis factor, interleukin 1ß). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl4 injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis. CONCLUSIONS: Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis.

ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair.

The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.

Basal-A Triple-Negative Breast Cancer Cells Selectively Rely on RNA Splicing for Survival.

Prognosis of triple-negative breast cancer (TNBC) remains poor. To identify shared and selective vulnerabilities of basal-like TNBC, the most common TNBC subtype, a directed siRNA lethality screen was performed in 7 human breast cancer cell lines, focusing on 154 previously identified dependency genes of 1 TNBC line. Thirty common dependency genes were identified, including multiple proteasome and RNA splicing genes, especially those associated with the U4/U6.U5 tri-snRNP complex (e.g., PRPF8, PRPF38A). PRPF8 or PRPF38A knockdown or the splicing modulator E7107 led to widespread intronic retention and altered splicing of transcripts involved in multiple basal-like TNBC dependencies, including protein homeostasis, mitosis, and apoptosis. E7107 treatment suppressed the growth of basal-A TNBC cell line and patient-derived basal-like TNBC xenografts at a well-tolerated dose. The antitumor response was enhanced by adding the proteasome inhibitor bortezomib. Thus, inhibiting both splicing and the proteasome might be an effective approach for treating basal-like TNBC. Mol Cancer Ther; 16(12); 2849-61. ©2017 AACR.

Silencing of the Drosophila ortholog of SOX5 leads to abnormal neuronal development and behavioral impairment.

SOX5 encodes a transcription factor that is expressed in multiple tissues including heart, lung and brain. Mutations in SOX5 have been previously found in patients with amyotrophic lateral sclerosis (ALS) and developmental delay, intellectual disability and dysmorphic features. To characterize the neuronal role of SOX5, we silenced the Drosophila ortholog of SOX5, Sox102F, by RNAi in various neuronal subtypes in Drosophila. Silencing of Sox102F led to misorientated and disorganized michrochaetes, neurons with shorter dendritic arborization (DA) and reduced complexity, diminished larval peristaltic contractions, loss of neuromuscular junction bouton structures, impaired olfactory perception, and severe neurodegeneration in brain. Silencing of SOX5 in human SH-SY5Y neuroblastoma cells resulted in a significant repression of WNT signaling activity and altered expression of WNT-related genes. Genetic association and meta-analyses of the results in several large family-based and case-control late-onset familial Alzheimer's disease (LOAD) samples of SOX5 variants revealed several variants that show significant association with AD disease status. In addition, analysis for rare and highly penetrate functional variants revealed four novel variants/mutations in SOX5, which taken together with functional prediction analysis, suggests a strong role of SOX5 causing AD in the carrier families. Collectively, these findings indicate that SOX5 is a novel candidate gene for LOAD with an important role in neuronal function. The genetic findings warrant further studies to identify and characterize SOX5 variants that confer risk for AD, ALS and intellectual disability.

HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay.

UNLABELLED: Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4(+)model, and the cultured TCM(central memory) CD4(+)model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. IMPORTANCE: Despite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration into the human genome in vivo We have developed a novel probe-based sequence enrichment assay to sequence and analyze integrated HIV. We compared HIV integration site characteristics between four cellular models and to previously described patient data sets. Significant differences were detected in the distribution of HIV integration sites between cellular models of HIV latency and compared to data sets from patient samples. The results from this study have implications for how well these cellular models of HIV infection truly reflect HIV integration in vivo and their applicability in drug discovery for novel latency-reversing agents.

An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates.

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.

edgeRun: an R package for sensitive, functionally relevant differential expression discovery using an unconditional exact test.

Next-generation sequencing platforms for measuring digital expression such as RNA-Seq are displacing traditional microarray-based methods in biological experiments. The detection of differentially expressed genes between groups of biological conditions has led to the development of numerous bioinformatics tools, but so far, few exploit the expanded dynamic range afforded by the new technologies. We present edgeRun, an R package that implements an unconditional exact test that is a more powerful version of the exact test in edgeR. This increase in power is especially pronounced for experiments with as few as two replicates per condition, for genes with low total expression and with large biological coefficient of variation. In comparison with a panel of other tools, edgeRun consistently captures functionally similar differentially expressed genes.

Sequencing of captive target transcripts identifies the network of regulated genes and functions of primate-specific miR-522.

Identifying microRNA (miRNA)-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical "seed" base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly noncanonical MREs were identified by sequencing RNase-resistant fragments. miR-522 overexpression reduced mRNA, protein levels, and luciferase activity of >70% of a random list of candidate target genes and MREs. Bioinformatic analysis suggested that miR-522 regulates cell proliferation, detachment, migration, and epithelial-mesenchymal transition. miR-522 induces G1 cell-cycle arrest and causes cells to detach without anoikis, become invasive, and express mesenchymal genes. Thus, our method provides a simple but effective technique for identifying miRNA-regulated genes and biological function.