Benjamin Deneen - US grants

DESCRIPTION (provided by applicant): Gene expression profiling of neural progenitor populations revealed that Nuclear Factor I (NFI) family genes are specifically expressed in glial progenitors in the developing spinal cord. Preliminary data suggests that NFI genes could be required for the generation of astrocytes in vivo. The goals of this study are to assess whether NFI genes are indeed required for the generation of astrocytes and to elucidate mechanisms that govern the ability of NFI genes to promote astrocytes. To examine whether NFI genes are required for the generation of astrocytes I will utilize both gain-and-loss of function analysis in the embryonic chick spinal cord and I will analyze astrocyte development in NFI mutant mice. Olig2 has been shown to suppress the generation of astrocytes, thus I will assess whether Olig2 can suppress the ability of NFI genes to promote astrocytes using in vitro culture systems and ectopic expression in the embryonic chick. If there is a antagonistic relationship between NFI genes and Olig2 I will investigate whether any biochemical relationship exists between these proteins. Lastly, given that Olig2 can both suppress astrocytes and oligodendrocytes, I will determine whether NFI gene function is epistatic to Olig2 in regard to the suppression of astrocytes and the promotion of oligodendrocytes, or whether NFI can uncouple these roles of Olig2. These experiments will rely on gain-and loss of function analysis in ovo, in vitro, and in mutant mice.

DESCRIPTION (provided by applicant): Glial cells comprise approximately 90% of the cellular constituency of the adult central nervous system (CNS) and support a vast array of physiological roles essential to CNS function. Yet, the molecular processes that control the initiation of gliogenesis from multipotent neural stem cells in vivo remain poorly understood. Thus, the overriding goal of this proposal is to elucidate the mechanisms that govern the initiation of gliogenesis from neural stem cells. We recently demonstrated that nuclear factor I (NFI) genes control the generation of glial cells in the embryonic spinal cord and are induced in neural stem cell populations coincident with the onset of gliogenesis in vivo. These properties make the NFI genes an ideal starting point from which to investigate the genetic regulatory programs that induce and maintain the early stages of gliogenesis. We, therefore, hypothesize that dissection of both the upstream and downstream events associated with NFI gene regulation will provide novel insights into the molecular control of gliogenesis. Specific Aims 1 and 2 of this proposal are based on our discovery of two distinct regulators of NFI gene expression in the embryonic spinal cord. Using enhancer screening of the NFIA promoter we have identified a highly conserved enhancer element (e123) that recapitulates the spatial and temporal patterns of NFIA induction when introduced into the embryonic chick spinal cord. Thus, in Aim 1 we propose to exploit e123 as a tool to identify a core set of transcription factors that control NFIA induction. We have also found that bone morphogenic protein (BMP) signaling controls NFI gene expression in the embryonic spinal cord in a manner that is independent of the e123 enhancer studied in Aim 1. Therefore, in Aim 2 we plan to identify BMP-responsive elements in the NFIA promoter, define the specific role of BMP signaling in the regulation of NFI gene expression, and to establish that BMP signaling does indeed operate independently of the transcriptional control mechanisms that regulate e123 induction. Finally, Aim 3 is a logical extension of temporal profiling studies of gene expression in neural stem cells in which we identified a cohort of genes upregulated after NFI gene induction in the embryonic spinal cord. Preliminary studies indicate that four of these genes are sufficient to restore gliogenesis in the absence of NFIA, suggesting that they function downstream of NFI genes. We will use gain- and loss-of function approaches in vivo to discover whether and how these genes promote gliogenesis and function downstream of NFI genes during the initiation of gliogenesis. Upon completion of these studies, we expect to have a much more comprehensive map of molecular processes, both upstream and downstream of NFI genes that control the initiation of gliogenesis during CNS development. The resultant insights into the signals that specify commitment to the glial lineage should lift understanding of glial cell specification in the embryonic spinal cord from the speculative realm to a point where clinical applications can begin to be considered. PUBLIC HEALTH RELEVANCE: This project focuses on the molecular processes that control the generation of glial cells. Glial cells have been implicated in a vast array of cancers and degenerative diseases of the nervous system and understanding the developmental processes that control their generation is a key to developing new therapeutic approaches to these disorders. This proposal is centered around a gene family that controls the generation of glial cells and is also expressed in astrocytomas and contributes to their formation. Thus, the studies herein are directly applicable to the understanding and treatment of astrocytomas.

Abstract/Project Summary Glial cells comprise approximately 80% of the cellular constituency of the adult central nervous system (CNS) and support a vast array of physiological roles essential to CNS function, including myelination, synapse formation, neurotransmission, and formation of the blood-brain barrier. Recent studies of glial development have documented many of the mechanisms that control the terminal differentiation and maturation of the astrocyte and oligodendrocyte sublineages. However, our knowledge of the preceding molecular processes that control the initiation of gliogenesis from multipotent neural stem cells in vivo remains rudimentary. The overriding goal of this proposal is to elucidate the molecular mechanisms that govern the initiation of gliogenesis. Recently we found that the Sox9/NFIA relationship represents a crucial regulatory node during neural stem cell commitment to the glial lineage, therefore dissection of their upstream regulatory events and downstream transcriptional networks will provide novel insight into the regulatory processes that control early gliogenesis. Our preliminary studies on the upstream regulatory events in gliogenesis suggest that Sox9 and Brn2 co-regulate NFIA through distinct enhancer elements that are brought together by Med12 mediated chromatin looping. Therefore in specific aim1 we will delineate how Brn2 regulates NFIA expression and whether Sox9/Brn2 collaboratively regulate NFIA induction and gliogenesis. In specific aim2, we will perform chromatin conformation capture (3C) to determine the three-dimensional architecture of the NFIA locus and examine whether the chromatin looping factor, Med12, regulates chromatin configuration at the NFIA locus and collaborates with Sox9/Brn2 to regulate NFIA induction. To identify key downstream events, we combined ChIP-Seq and gene expression profiling on FACS isolated, CD15+ spinal cord progenitors to dissociate the Sox9/NFIA transcriptional networks during gliogenesis. In specific aim3, we will validate and functionally analyze a set of candidate gliogenic targets identified in this screen and, in conjunction with our studies during gliogenesis, extend our ChIP-Seq analysis to earlier developmental stages to identify Sox9-specific targets in neural stem cell populations

? DESCRIPTION (provided by applicant): Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by a wide range of neurological deficits including, seizures, movement disorders, autonomic dysfunction, and marked breathing abnormalities. Nearly all cases of RTT are caused by de novo mutations in Methyl-CpG-binding protein 2 (MECP2), which functions as a global regulator of gene transcription. MeCP2 is highly expressed throughout the nervous system, and because the clinical features are associated with neuronal function, RTT has typically been assumed to be a disease of neurons. However, recent work has challenged this view, indicating that other cells within the nervous system such as astrocytes and microglia may play an important role in the pathogenesis of disease. While astrocytes directly contribute to key phenotypes associated with RTT, such as breathing and glucose sensitivity, the consequences of loss of MeCP2 on specific astrocyte sub-populations in these key regions remains completely undefined. Astrocytes have long been considered to be a uniform cell type, and in spite of recent findings indicating they perform diverse roles across the CNS, the nature of their cellular and functional heterogeneity remains shrouded in mystery. Using the brainstem and RTT as models for decoding these cellular and functional relationships, we hypothesize that MeCP2 plays a crucial role in key astrocyte subtypes within the brainstem, a brain region we have previously demonstrated to be critical in the genesis of breathing and other physiological abnormalities in RTT. To this end we have used FACS-based approaches to identify unique subpopulations of astrocytes in the adult brainstem. In specific aim 1 of this proposal, we will validate the presence of these populations in the brainstem and perform gene expression profiling on each subpopulation to decode their unique molecular signature. In specific aim 2 we will use similar FACS-based approaches to delineate astrocyte heterogeneity and their underlying molecular profiles in the brainstem of the RTT mouse. Through this work we will determine brainstem astrocyte heterogeneity and define the molecular profiles of these cells in normal and MeCP2 mutant populations, lending unprecedented insight into the nature of astrocyte heterogeneity in health and disease.

Astrocytes are the most abundant cell type in the CNS that play vital roles in all facets of brain physiology. Activation of astrocytes, or reactive astrogliosis, is associated with morphological, gene expression, and functional alterations. Although changes in astrocyte dynamics a common feature in Alzheimer's disease (AD), its underlying mechanisms and functional consequences remains poorly understood. Our recent studies identified five astrocyte subpopulations that display diverse molecular and functional characteristics in the brain. Significantly, one of the subpopulations, the synaptogenic astrocytes, strongly correlated with human AD expression datasets. This is exciting because synaptic dysfunction is widely accepted as an early and causal event in AD pathogenesis. Our long term goal is to decode the astrocyte types and determine the impact of the diverse astrocyte populations in aging and AD. The objectives of this proposal are to define the cellular and molecular heterogeneity of astrocytes in the cortex and hippocampus and to ascertain the functional role of synaptogenic astrocytes during aging and in AD mouse models. We hypothesize that diverse astrocyte subpopulations in the adult brain differentially contribute to AD pathogenesis and that reduced function of synaptogenic astrocytes plays a crucial role in driving synaptic dysfunction in early AD. To test the hypotheses we will profile changes of astrocyte subpopulations and define their molecular signatures in wild-type mice and AD mouse models as a function of age and AD pathology and cross-validate these results in postmortem human samples and associated expression datasets. We will decipher the role of synaptogenic astrocytes in AD pathogenesis by genetic targeting and functional testing of selected candidates. These studies will be led by two investigators with exceptional track-record in astrocyte biology (Deneen) and AD pathophysiology (Zheng) and assisted by outstanding bioinformatics support. Overall the proposal will significantly advance our understanding of astrocyte heterogeneity in brain regions critical to AD and how early and late astrocyte dysfunction contributes to AD pathogenesis. It will also lead to the identification of novel biomarkers and therapeutic targets.

? DESCRIPTION (provided by applicant): The newborn brain is particularly sensitive to hypoxic injury (HI). Preterm HI manifests itself as periventricular leukomalacia (PVL), while in full-term infants HI presents as hypoxic ischemic encephalopathy (HIE). In newborns with moderate-severe forms of either PVL or HIE, 60-75% develop life-long neurological disabilities, resulting from extensive white matter injury (WMI), due to the loss of myelinating oligodendrocytes (OLs). This loss of OLs, coupled with their failure to regenerate, leads to impaired neuronal function, which clinically manifests as cerebral palsy (CP). In this proposal we will attack the critical problem of remyelination after HI using two distinct approaches: the differentiation of OLPs and the maintenance of axonal integrity. Our studies have identified four compounds that act on distinct pathways that contribute to the suppression of remyelination, which we will test in the neonatal brain during- and after- HI. One feature of OLPs populating white matter lesions is elevated levels of Wnt signaling, which functions to suppress regenerative myelination after WMI. Therefore, inhibition of Wnt signaling in OLPs represents a therapeutic strategy for stimulating remyelination after HI. Recently we identified Daam2 as a key proximal modulator of Wnt signaling in the developing CNS that functions through the PIP5K-PIP2 signaling axis. Leveraging this knowledge from development, we found two compounds that inhibit PIP5K activity (e.g. Sp-8-pCPT-cAMP and UNC3230) stimulate remyelination after WMI after acute hypoxia. Here we will determine whether these compounds function similarly in the neonatal brain after HI and ischemia, and whether the Daam2-PIP5K axis is expressed in OLPs in human HIE/PVL lesions. Axon integrity also plays a central role in myelination. Recently, we found that disruption of the axon initial segment (AIS) in cortical neurons blocks their eventual myelination due to loss of axonal identity. Moreover, we found that the AIS is disrupted after ischemic injury in the adult brain. Thus, we propose HI- induced loss of the AIS inhibits myelination, whereas preservation of the AIS may promote myelination after HI. Ischemic injury activates the calcium dependent protease, calpain, which proteolyzes essential AIS scaffolding proteins, resulting in the loss of axonal integrity. Calpain inhibitors (e.g. MDL28170) preserve the AIS after ischemia both in vitro and in vivo. Therefore, we will determine if maintenance of the AIS through inhibition of calpain stimulates remyelination and recovery after HI, and whether these components of the AIS are dysregulated in human HIE/PVL lesions.

Project Summary Large-scale national and international cancer sequencing programs are generating a compendium of tumor- associated genomic alterations to prioritize the most promising therapeutic targets for drug development. These efforts have uncovered a staggering level of genome complexity in cancer. Although much is known about the function and clinical impact of recurrent aberrations in well-known cancer genes, less is known about which and how the more abundant, low-frequency mutations contribute to tumor progression. Effective translation of tumor genomic datasets into cancer therapeutics will require new experimental systems to inform the functional activity of targets in the relevant biological context encompassing inter- and intra-tumoral heterogeneity. To address these needs, we propose a CTD2 Center that will provide the research community high-throughput informatic and experimental approaches to characterize and validate pathogenic ?driver? mutations and fusion genes as well as identify molecular markers that meaningfully predict responses or resistance to anticancer therapies. We will pursue the following Specific Aims: In Aim 1 we will implement an algorithmic framework for identifying driver mutations with high sensitivity and specificity. We will focus our algorithm development, training and testing efforts on predicting oncogenic, gain-of-function mutation drivers of glioblastoma multiforme (GBM), pancreatic ductal adenocarcinoma (PDAC) and epithelial ovarian cancer (EOC). These computational approaches will be amenable to the analysis of all cancer types. We will next engineer ~1,500 selected mutations and ~400 fusion genes into expression vectors along with cohorts of personalized, patient-defined coding mutations. In Aim 2 we will enter mutant alleles and fusion genes into GBM, PDAC and EOC context-specific, in vivo functional screens that take into account the importance of genetic context, tumor microenvironment and heterogeneity in the selection of single and combinatorial drivers of tumorigenesis. In Aim 3 we will determine the consequences of intra-tumoral heterogeneity on tumor sensitivity and resistance to therapeutic agents using DNA-barcoded, human patient-derived xenograft models that recapitulate the heterogeneity of cancer. We will determine the extent to which single targeted agents and their rational combinations alter tumor population dynamics. We will also leverage Aim 1 informatics and functional characterizations in Aim 2 and 4 to characterize ?persistor? populations to identify aberrations associated with drug resistance. In Aim 4 we will use high-throughput functional proteomics, innovative protein- protein interaction assays and informer drug library screening studies to elucidate underlying mechanisms and therapeutic liabilities engendered by validated drivers. The foundational platform implemented in our CTD2 Center will provide a validated pipeline for the rapid characterization of gain-of-function aberrations that can be industrialized across tumor lineages to guide clinical management of cancer patients.

Glioblastoma cells trigger pharmacoresistant seizures that may promote tumor growth and diminish the quality of remaining life. To define the relationship between growth of glial tumors and their neuronal microenvironment, and to identify genomic biomarkers and mechanisms that may point to better prognosis and treatment of drug resistant epilepsy in brain cancer, we are analyzing a new generation of genetically defined CRISPR/in utero electroporation inborn glioblastoma (GBM) tumor models engineered in mice. The molecular pathophysiology of glioblastoma cells and surrounding neurons and untransformed astrocytes will be compared at serial stages of tumor development in three genetic mouse strains: wild type, seizure prone, and seizure resistant. Preliminary data reveal that epileptiform EEG spiking is a very early and reliable preclinical signature of GBM expansion preceding other neurological deficits in these mice, followed by rapidly progressive seizures and death within weeks. Transcriptomic analysis of cortical astrocytes reveals the expansion of a subgroup enriched in pro-synaptogenic genes that may drive hyperexcitability, a novel mechanism of epileptogenesis. In Specific Aim 1 we will systematically define the earliest appearance of cortical hyperexcitability in wild type mice with a prototypical GBM and correlate its progression with in vivo and neuropathological imaging of invasive tumor cell location, in vitro electrophysiology, and molecular markers of key epilepsy pathogenic cascades in peritumoral neurons, including impaired glutamate reuptake, altered GABA gated-chloride gradients, and synaptic densities. In Specific Aim 2 we will correlate these findings with detailed FACS-sorted transcriptomic profiles of both transformed and wild type astrocytes in the peritumoral region to test the novel hypothesis that peritumoral hyperexcitability is driven in part by astrocytic subtypes that disrupt synaptic E/I homeostasis. In Specific Aim 3, we will use this benchmark approach in WT brain to compare growth, electrophysiological and molecular pathological profiles of the same tumor generated in a hyperexcitable brain bearing a single gene deletion (Kcna1) that dramatically lowers the threshold for seizures and shortens lifespan, and in a monogenic deletion strain (MapT/tau) that raises cortical seizure threshold and prolongs life, in order to examine the contribution of host neuronal excitability to tumor expansion. Our approach sets the stage to broadly explore the developmental biology of personalized tumor/host interactions in mice engineered with novel human tumor mutations in specified glial cell lineages.

ABSTRACT Parent grant: Genetic susceptibility plays a significant role in glioma development. An individual with two or more first- and/or second-degree affected relatives has a two-fold increased risk of the disease. We were the first to suggest mutations in POT1 (Protection of Telomeres 1) as causative in familial glioma (FG). We have now established the presence of POT1 mutations in 5 different families, providing the strongest evidence of its role in glioma. However, we do not yet have direct functional evidence that loss of POT1 is causal in glioma leaving few options for carrier surveillance or potential treatment targets. We are currently able to explain the genetic basis of glioma in up to 12% of our families, using highly stringent criteria for calling a mutation deleterious and causal. In contrast, the majority of our families remain unexplained though several candidate genes have emerged as ?suspects of interest (SOIs)?. We propose a data-driven, knowledge-based, computational approach to guide candidate gene selection for functional characterization. In order to further our efforts to explain the genetic basis of FG we propose two specific aims to: Identify new gene candidates that may cause FG through WGS (Aim 1). We will identify SNVs, small indels, and structural variants in both coding and noncoding regions of the genome, intensively annotate those variants using more than 50 data sources, and we will rank these variants using multiple criteria based on their likelihood to cause disease. In addition to the 270 FG cases (from 203 FG families) with sequence data already available, we will also sequence an additional 100 cases (from 100 families) already collected in our Glioma International Case-Control Study with a reported family history using Gliogene criteria, and 200 newly recruited cases (from 100 families) with a strong family history of glioma to enhance our discovery, and 150 familial glioma tumor samples. The second aim is to functionally validate SOIs to include: A) POT1 mutations and B) newly discovered FG susceptibility genes (SOIs) from Aim 1 using a novel experimental mouse model of gliomagenesis. To determine the functional contributions of POT1 and novel mutations identified in our WGS studies, we will evaluate these genes in glioma mouse models using CRISPR gene editing technology. This study has the potential for future genetic testing in high-risk families, success will offer much needed insight on the underlying biology and etiology of glioma in both familial and sporadic cases. Supplement: The FDA?s expanded access program (EAP)?sometimes called ?compassionate use? for individual requests?is a potential option for patients with immediate, life-threatening conditions when no comparable or satisfactory options are available to gain access to experimental medicines outside of clinical trials. Yet we know little about how oncologists and patients navigate EAP requests, their understanding of federal policy, or how they manage informed consent after a request has been approved. There is no empirically-derived ethical guidance for oncologists considering experimental drugs as treatment options for their patients. We propose to develop and offer guidance to neuro-oncologists through a stepwise approach of empirical research and normative ethics. First, we will conduct individual, in-depth interviews through family-physician dyads, and ask about their experiences in discussing options of providing an experimental drug through compassionate use or expanded access. Family cohorts will include at least two living glioma cases, and will be identified through recruitment in the Discovery, Biology and Risk of Inherited Variants in Glioma (GLIOGENE) parent grant. Then, we will combine ethical principles and paradigmatic cases from the literature with the themes and cases derived from interviews to identify salient ethical areas of concern. Once identified, we will synthesize case scenarios and questions for normative discussion with investigators on the parent grant and three practicing neuro-oncologists. This exercise will be used to generate a set of clinically-relevant recommendations and decision tools for practitioners.

Abstract The goal of the parent grant is to understand the transcriptional mechanisms that control the physiological functions of astrocytes in the brain. The transcription factor Nuclear Factor I-A (NFIA) is the focal point of the parent grant and we propose to determine its role in maintaining astrocyte physiology and neuronal circuits across a host of brain regions. These topics are particularly relevant to the pathogenesis of Alzheimer?s Disease (AD) as patients with AD have reactive astrocytes closely associated with degenerating neurons across multiple brain regions; these observations have been recapitulated in mouse models of AD. Despite these clear links between AD pathology and astrocytes, evaluation of astrocyte phenotypes in AD typically focuses on GFAP upregulation and various, poorly defined states of reactivity. These broad molecular criteria overlook how pathological states manifest in AD disrupt normal astrocyte function and their essential interactions with neurons during the formative stages of disease and throughout progression. Therefore, the overarching goal of this supplement is to bring tools developed in the parent grant to bear on mouse models of AD in order to decipher how physiological changes in astrocytes contribute to AD pathogenesis. We have generated a set of mouse tools and developed a platform for comprehensively evaluating astrocyte physiology and contributions to established circuits that we will apply to mouse models of AD. In the first aim, we will decipher how astrocyte physiology and their interactions with neurons change across of a series of landmark timepoints in mouse models of AD. Here we will assess a battery of functional criteria including: morphology, Ca2+ activities, proximity to neurons, handling of neurotransmitters, and activity of associated neurons. In the second aim, we will use our mouse tools to evaluate the role of astrocytic NFIA in AD pathogenesis. Using these tools, we discovered that astrocytic NFIA plays an essential role in maintaining astrocyte function and regulating hippocampal circuits, a brain region that is vulnerable to AD. Moreover, NFIA is highly expressed in reactive astrocytes found in human neurological diseases. Using these observations as our premise, we will determine the expression of NFIA in reactive astrocytes in human AD, how its loss modifies pathological benchmarks of AD, and how AD modifies astrocytic NFIA function. Upon completion of this supplement these studies will reveal when physiological changes in astrocytes take root during AD pathogenesis and how NFIA contributes to these changes in astrocytes.

Summary Astrocytes are the most abundant type of glial cell in the CNS and play vital roles in all facets of brain physiology. Recent studies from our lab identified five molecularly and functionally distinct astrocyte subpopulations in the brain. One of these subpopulations is specifically labeled by the cell surface maker CD51 and is endowed with enhanced synaptogenic function, leading us to hypothesize that CD51+ astrocytes play an important role in functioning brain circuits. To determine how CD51+ astrocytes contribute to circuit function, we created a CD51-FLP mouse line that allows us selectively label and manipulate this population of astrocytes in the brain. To decipher the mechanisms that regulate CD51+ astrocytes, we found that the transcription factor Sox9 is enriched in this subpopulation. Preliminary studies in a newly generated mouse line that specifically eliminates Sox9 in astrocytes revealed that this transcription factor selectively regulates astrocyte morphology and circuit function in the hippocampus and olfactory bulb in an aging-dependent manner. Together, these new mouse lines will enable us to uncover how CD51+ astrocytes regulate brain circuits and reveal novel roles for Sox9 in controlling astrocyte diversity and function during aging. Based on the strength of these preliminary observations, we propose the following specific aims. In specific aim 1, we will define the anatomical, morphological and physiological properties of CD51+ and CD51- astrocytes in the aging brain and decipher how they contribute to circuit function in the hippocampus and olfactory bulb. In specific aim 2, we will use our astrocyte-specific Sox9 knockout mouse to decipher how it regulates astrocyte function in the aging brain and how these changes in astrocytes impact circuit function in the hippocampus and olfactory bulb. In specific aim 3 we will determine how Sox9 regulates astrocyte diversity during aging and integrate our findings with Sox9 ChIP-Seq to decipher Sox9 target gene networks.