Autofluorescence is a biomarker of neural stem cell activation state.

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis.

Profound seizure suppression and disease modification by targeting JAK1, a key driver of a pro-epileptogenic gene network.

Epilepsy is the 4th most prevalent neurological disorder with over 50 million cases worldwide. While a number of drugs exist to suppress seizures, approximately 1/3 of patients remain drug resistant, and no current treatments are disease modifying. Using network and systems-based approaches, we find that the histone methylase EZH2 suppresses epileptogenesis and slows disease progression, via repression of JAK1 and STAT3 signaling in hippocampal neurons. Pharmacological inhibition of JAK1 with the orally available, FDA-approved drug CP690550 (Tofacitinib) profoundly suppresses behavioral and electrographic seizures after the onset of epilepsy across preclinical rodent models of acquired epilepsy. This seizure suppression persists for weeks after drug withdrawal. Identification of an endogenous protective response to status epilepticus in the form of EZH2 induction has highlighted a critical role for the JAK1 kinase and STAT3 in both the initiation and propagation of epilepsy across preclinical rodent models and human disease. Overall, we find that STAT3 is transiently activated after insult, reactivates with spontaneous seizures, and remains targetable for disease modification in chronic epilepsy.

HAPLN1 matrikine: a bone marrow homing factor linked to poor outcomes in patients with MM.

The bone marrow (BM) microenvironment is critical for dissemination, growth, and survival of multiple myeloma (MM) cells. Homing of myeloma cells to the BM niche is a crucial step in MM dissemination, but the mechanisms involved are incompletely understood. In particular, any role of matrikines, neofunctional peptides derived from extracellular matrix proteins, remains unknown. Here, we report that a matrikine derived from hyaluronan and proteoglycan link protein 1 (HAPLN1) induces MM cell adhesion to the BM stromal components, such as fibronectin, endothelial cells, and stromal cells and, furthermore, induces their chemotactic and chemokinetic migration. In a mouse xenograft model, we show that MM cells preferentially home to HAPLN1 matrikine-conditioned BM. The transcription factor STAT1 is activated by HAPLN1 matrikine and is necessary to induce MM cell adhesion, migration, migration-related genes, and BM homing. STAT1 activation is mediated by interferon beta (IFN-ß), which is induced by NF-κB after stimulation by HAPLN1 matrikine. Finally, we also provide evidence that higher levels of HAPLN1 in BM samples correlate with poorer progression-free survival of patients with newly diagnosed MM. These data reveal that a matrikine present in the BM microenvironment acts as a chemoattractant, plays an important role in BM homing of MM cells via NF-κB-IFN-ß-STAT1 signaling, and may help identify patients with poor outcomes. This study also provides a mechanistic rationale for targeting HAPLN1 matrikine in MM therapy.

A transcriptome dataset for gonadectomy-induced changes in rat spinal cord.

Circulating sex steroid hormones are critical for neural function and development of neuroplasticity in many regions of the central nervous system. In the spinal cord, our knowledge of steroid hormone influence mostly derives from mechanistic studies of pain processing in dorsal spinal cord circuits; less is known regarding hormonal influence of ventral spinal motor function. Gonadectomy (surgical removal of the testes in males and ovaries in females) rapidly and persistently reduces circulating sex steroids in both females and males, providing a means to interrogate the role of hormones on neural function. Here we provide a next-generation RNA sequencing (RNA-seq) data set to evaluate the impact of gonadectomy on the transcriptome of ventral spinal cord tissue of adult female and male rats.

Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability.

Increased Aurora B protein expression, which is common in cancers, is expected to increase Aurora B kinase activity, yielding elevated phosphorylation of Aurora B substrates. In contrast, here we show that elevated expression of Aurora B reduces phosphorylation of six different Aurora B substrates across three species and causes defects consistent with Aurora B inhibition. Complexes of Aurora B and its binding partner INCENP autophosphorylate in trans to achieve full Aurora B activation. Increased expression of Aurora B mislocalizes INCENP, reducing the local concentration of Aurora B:INCENP complexes at the inner centromere/kinetochore. Co-expression of INCENP rescues Aurora B kinase activity and mitotic defects caused by elevated Aurora B. However, INCENP expression is not elevated in concert with Aurora B in breast cancer, and increased expression of Aurora B causes resistance rather than hypersensitivity to Aurora B inhibitors. Thus, increased Aurora B expression reduces, rather than increases, Aurora B kinase activity.

Enhanced immunoprecipitation techniques for the identification of RNA-binding protein partners: IGF2BP1 interactions in mammary epithelial cells.

RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested "exclusion-based" purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP-RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro.

TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic ß-Cell.

Transcription factor 19 (TCF19) is a gene associated with type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that Tcf19 knockdown impairs ß-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 ß-cells and human islets on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNA-seq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in viral responses, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the ß-cell and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways can provide potential targets for improving ß-cell survival.

p53 Is Not Required for High CIN to Induce Tumor Suppression.

Chromosomal instability (CIN) is a hallmark of cancer. While low levels of CIN can be tumor promoting, high levels of CIN cause cell death and tumor suppression. The widely used chemotherapeutic, paclitaxel (Taxol), exerts its anticancer effects by increasing CIN above a maximally tolerated threshold. One significant outstanding question is whether the p53 tumor suppressor is required for the cell death and tumor suppression caused by high CIN. Both p53 loss and reduction of the mitotic kinesin, centromere-associated protein-E, cause low CIN. Combining both genetic insults in the same cell leads to high CIN. Here, we test whether high CIN causes cell death and tumor suppression even in the absence p53. Despite a surprising sex-specific difference in tumor spectrum and latency in p53 heterozygous animals, these studies demonstrate that p53 is not required for high CIN to induce tumor suppression. Pharmacologic induction of high CIN results in equivalent levels of cell death due to loss of essential chromosomes in p53+/+ and p53-/- cells, further demonstrating that high CIN elicits cell death independently of p53 function. IMPLICATIONS: These results provide support for the efficacy of anticancer therapies that induce high CIN, even in tumors that lack functional p53.

Prior Hypoxia Exposure Enhances Murine Microglial Inflammatory Gene Expression in vitro Without Concomitant H3K4me3 Enrichment.

Hypoxia (Hx) is a component of multiple disorders, including stroke and sleep-disordered breathing, which often precede or are comorbid with neurodegenerative diseases. However, little is known about how hypoxia affects the ability of microglia, resident CNS macrophages, to respond to subsequent inflammatory challenges that are often present during neurodegenerative processes. We, therefore, tested the hypothesis that hypoxia would enhance or "prime" microglial pro-inflammatory gene expression in response to a later inflammatory challenge without programmatically increasing basal levels of pro-inflammatory cytokine expression. To test this, we pre-exposed immortalized N9 and primary microglia to hypoxia (1% O2) for 16 h and then challenged them with pro-inflammatory lipopolysaccharide (LPS) either immediately or 3-6 days following hypoxic exposure. We used RNA sequencing coupled with chromatin immunoprecipitation sequencing to analyze primed microglial inflammatory gene expression and modifications to histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of primed genes. We found that microglia exhibited enhanced responses to LPS 3 days and 6 days post-hypoxia. Surprisingly, however, the majority of primed genes were not enriched for H3K4me3 acutely following hypoxia exposure. Using the bioinformatics tool MAGICTRICKS and reversible pharmacological inhibition, we found that primed genes required the transcriptional activities of NF-κB. These findings provide evidence that hypoxia pre-exposure could lead to persistent and aberrant inflammatory responses in the context of CNS disorders.

Sex- and Region-Specific Differences in the Transcriptomes of Rat Microglia from the Brainstem and Cervical Spinal Cord.

The neural control system underlying breathing is sexually dimorphic with males being more vulnerable to dysfunction. Microglia also display sex differences, and their role in the architecture of brainstem respiratory rhythm circuitry and modulation of cervical spinal cord respiratory plasticity is becoming better appreciated. To further understand the molecular underpinnings of these sex differences, we performed RNA sequencing of immunomagnetically isolated microglia from brainstem and cervical spinal cord of adult male and female rats. We used various bioinformatics tools (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, STRING, MAGICTRICKS) to functionally categorize identified gene sets, as well as to pinpoint common transcriptional gene drivers that may be responsible for the observed transcriptomic differences. We found few sex differences in the microglial transcriptomes derived from the brainstem, but several hundred genes were differentially expressed by sex in cervical spinal microglia. Comparing brainstem and spinal microglia within and between sexes, we found that the major factor guiding transcriptomic differences was central nervous system (CNS) location rather than sex. We further identified key transcriptional drivers that may be responsible for the transcriptomic differences observed between sexes and CNS regions; enhancer of zeste homolog 2 emerged as the predominant driver of the differentially downregulated genes. We suggest that functional gene alterations identified in metabolism, transcription, and intercellular communication underlie critical microglial heterogeneity and sex differences in CNS regions that contribute to respiratory disorders categorized by dysfunction in neural control. These data will also serve as an important resource data base to advance our understanding of innate immune cell contributions to sex differences and the field of respiratory neural control. SIGNIFICANCE STATEMENT: The contributions of central nervous system (CNS) innate immune cells to sexually dimorphic differences in the neural circuitry controlling breathing are poorly understood. We identify key transcriptomic differences, and their transcriptional drivers, in microglia derived from the brainstem and the C3-C6 cervical spinal cord of healthy adult male and female rats. Gene alterations identified in metabolism, gene transcription, and intercellular communication likely underlie critical microglial heterogeneity and sex differences in these key CNS regions that contribute to the neural control of breathing.

CTCF loss mediates unique DNA hypermethylation landscapes in human cancers.

BACKGROUND: The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. RESULTS: Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. CONCLUSION: These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.

MAGIC: A tool for predicting transcription factors and cofactors driving gene sets using ENCODE data.

Transcriptomic profiling is an immensely powerful hypothesis generating tool. However, accurately predicting the transcription factors (TFs) and cofactors that drive transcriptomic differences between samples is challenging. A number of algorithms draw on ChIP-seq tracks to define TFs and cofactors behind gene changes. These approaches assign TFs and cofactors to genes via a binary designation of 'target', or 'non-target' followed by Fisher Exact Tests to assess enrichment of TFs and cofactors. ENCODE archives 2314 ChIP-seq tracks of 684 TFs and cofactors assayed across a 117 human cell lines under a multitude of growth and maintenance conditions. The algorithm presented herein, Mining Algorithm for GenetIc Controllers (MAGIC), uses ENCODE ChIP-seq data to look for statistical enrichment of TFs and cofactors in gene bodies and flanking regions in gene lists without an a priori binary classification of genes as targets or non-targets. When compared to other TF mining resources, MAGIC displayed favourable performance in predicting TFs and cofactors that drive gene changes in 4 settings: 1) A cell line expressing or lacking single TF, 2) Breast tumors divided along PAM50 designations 3) Whole brain samples from WT mice or mice lacking a single TF in a particular neuronal subtype 4) Single cell RNAseq analysis of neurons divided by Immediate Early Gene expression levels. In summary, MAGIC is a standalone application that produces meaningful predictions of TFs and cofactors in transcriptomic experiments.

Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways.

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.

PGC-1a integrates a metabolism and growth network linked to caloric restriction.

Deleterious changes in energy metabolism have been linked to aging and disease vulnerability, while activation of mitochondrial pathways has been linked to delayed aging by caloric restriction (CR). The basis for these associations is poorly understood, and the scope of impact of mitochondrial activation on cellular function has yet to be defined. Here, we show that mitochondrial regulator PGC-1a is induced by CR in multiple tissues, and at the cellular level, CR-like activation of PGC-1a impacts a network that integrates mitochondrial status with metabolism and growth parameters. Transcriptional profiling reveals that diverse functions, including immune pathways, growth, structure, and macromolecule homeostasis, are responsive to PGC-1a. Mechanistically, these changes in gene expression were linked to chromatin remodeling and RNA processing. Metabolic changes implicated in the transcriptional data were confirmed functionally including shifts in NAD metabolism, lipid metabolism, and membrane lipid composition. Delayed cellular proliferation, altered cytoskeleton, and attenuated growth signaling through post-transcriptional and post-translational mechanisms were also identified as outcomes of PGC-1a-directed mitochondrial activation. Furthermore, in vivo in tissues from a genetically heterogeneous mouse population, endogenous PGC-1a expression was correlated with this same metabolism and growth network. These data show that small changes in metabolism have broad consequences that arguably would profoundly alter cell function. We suggest that this PGC-1a sensitive network may be the basis for the association between mitochondrial function and aging where small deficiencies precipitate loss of function across a spectrum of cellular activities.

Histone deacetylase inhibitors restore normal hippocampal synaptic plasticity and seizure threshold in a mouse model of Tuberous Sclerosis Complex.

Abnormal synaptic plasticity has been implicated in several neurological disorders including epilepsy, dementia and Autism Spectrum Disorder (ASD). Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder that manifests with seizures, autism, and cognitive deficits. The abnormal intracellular signaling underlying TSC has been the focus of many studies. However, nothing is known about the role of histone modifications in contributing to the neurological manifestations in TSC. Dynamic regulation of chromatin structure via post translational modification of histone tails has been implicated in learning, memory and synaptic plasticity. Histone acetylation and associated gene activation plays a key role in plasticity and so we asked whether histone acetylation might be dysregulated in TSC. In this study, we report a general reduction in hippocampal histone H3 acetylation levels in a mouse model of TSC2. Pharmacological inhibition of Histone Deacetylase (HDAC) activity restores histone H3 acetylation levels and ameliorates the aberrant plasticity in TSC2+/- mice. We describe a novel seizure phenotype in TSC2+/- mice that is also normalized with HDAC inhibitors (HDACis). The results from this study suggest an unanticipated role for chromatin modification in TSC and may inform novel therapeutic strategies for TSC patients.

A systems approach identifies Enhancer of Zeste Homolog 2 (EZH2) as a protective factor in epilepsy.

Complex neurological conditions can give rise to large scale transcriptomic changes that drive disease progression. It is likely that alterations in one or a few transcription factors or cofactors underlie these transcriptomic alterations. Identifying the driving transcription factors/cofactors is a non-trivial problem and a limiting step in the understanding of neurological disorders. Epilepsy has a prevalence of 1% and is the fourth most common neurological disorder. While a number of anti-seizure drugs exist to treat seizures symptomatically, none is curative or preventive. This reflects a lack of understanding of disease progression. We used a novel systems approach to mine transcriptome profiles of rodent and human epileptic brain samples to identify regulators of transcriptional networks in the epileptic brain. We find that Enhancer of Zeste Homolog 2 (EZH2) regulates differentially expressed genes in epilepsy across multiple rodent models of acquired epilepsy. EZH2 undergoes a prolonged upregulation in the epileptic brain. A transient inhibition of EZH2 immediately after status epilepticus (SE) robustly increases spontaneous seizure burden weeks later. This suggests that EZH2 upregulation is a protective. These findings are the first to characterize a role for EZH2 in opposing epileptogenesis and debut a bioinformatic approach to identify nuclear drivers of complex transcriptional changes in disease.

Altered circadian rhythms and oscillation of clock genes and sirtuin 1 in a model of sudden unexpected death in epilepsy.

OBJECTIVES: Circadian rhythms are affected in many neurological disorders. Although sleep disturbances are known in epilepsy, data on circadian rhythm disturbances in epilepsy are sparse. Here, we examined diurnal and circadian rest-activity and sleep-wake patterns in Kcna1-null mice, which exhibit spontaneous recurrent seizures and are a model of sudden unexpected death in epilepsy. Furthermore, we sought to determine whether seizures or aberrant oscillation of core clock genes and a regulator, sirtuin 1 (Sirt1), is associated with disrupted rhythms. METHODS: We used passive infrared actigraphy to assess rest-activity patterns, electroencephalography for seizure and sleep analysis, and reverse transcription polymerase chain reaction and Western blotting to evaluate expression of clock genes and Sirt1 in Kcna1-null and wild-type mice. RESULTS: Epileptic Kcna1-null animals have disrupted diurnal and circadian rest-activity patterns, tending to exhibit prolonged circadian periods. Electroencephalographic analysis confirmed disturbances in sleep architecture, with more time spent awake and less asleep. Although all epileptic mice manifested disrupted diurnal and circadian rest-activity patterns, we found no correlation between actual seizure burden and degree of sleep disruption. However, we found attenuated oscillations of several clock genes (ie, Clock, Bmal1, Per1, and Per2) and diurnal Sirt1 mRNA in the anterior hypothalamus. SIGNIFICANCE: Attenuated oscillation of several core clock genes correlates with, and may underlie, aberrant diurnal and circadian rest-activity and sleep-wake patterns observed in Kcna1-null mice. This could contribute to late complications in epilepsy, such as sudden unexpected death in epilepsy. Sirt1 may represent a useful therapeutic target for rescuing circadian clock gene rhythmicity and sleep patterns in epilepsy.

Human papillomavirus oncogenes reprogram the cervical cancer microenvironment independently of and synergistically with estrogen.

High-risk human papillomaviruses (HPVs) infect epithelial cells and are causally associated with cervical cancer, but HPV infection is not sufficient for carcinogenesis. Previously, we reported that estrogen signaling in the stromal tumor microenvironment is associated with cervical cancer maintenance and progression. We have now determined how HPV oncogenes and estrogen treatment affect genome-wide host gene expression in laser-captured regions of the cervical epithelium and stroma of untreated or estrogen-treated nontransgenic and HPV-transgenic mice. HPV oncogene expression in the cervical epithelium elicited significant gene-expression changes in the proximal stromal compartment, and estrogen treatment uniquely affected gene expression in the cervical microenvironment of HPV-transgenic mice compared with nontransgenic mice. Several potential estrogen-induced paracrine-acting factors were identified in the expression profile of the cervical tumor microenvironment. The microenvironment of estrogen-treated HPV-transgenic mice was significantly enriched for chemokine/cytokine activity and inflammatory and immune functions associated with carcinogenesis. This inflammatory signature included several proangiogenic CXCR2 receptor ligands. A subset of the same CXCR2 ligands was likewise increased in cocultures of early-passage cells from human cervical samples, with levels highest in cocultures of cervical fibroblasts and cancer-derived epithelial cells. Our studies demonstrate that high-risk HPV oncogenes profoundly reprogram the tumor microenvironment independently of and synergistically with estrogen. These observations illuminate important means by which HPVs can cause cancer through alterations in the tumor microenvironment.

Transcriptional profile of hippocampal dentate granule cells in four rat epilepsy models.

Global expression profiling of neurologic or psychiatric disorders has been confounded by variability among laboratories, animal models, tissues sampled, and experimental platforms, with the result being that few genes demonstrate consistent expression changes. We attempted to minimize these confounds by pooling dentate granule cell transcriptional profiles from 164 rats in seven laboratories, using three status epilepticus (SE) epilepsy models (pilocarpine, kainate, self-sustained SE), plus amygdala kindling. In each epilepsy model, RNA was harvested from laser-captured dentate granule cells from six rats at four time points early in the process of developing epilepsy, and data were collected from two independent laboratories in each rodent model except SSSE. Hierarchical clustering of differentially-expressed transcripts in the three SE models revealed complete separation between controls and SE rats isolated 1 day after SE. However, concordance of gene expression changes in the SE models was only 26-38% between laboratories, and 4.5% among models, validating the consortium approach. Transcripts with unusually highly variable control expression across laboratories provide a 'red herring' list for low-powered studies.