Murat Gunel, MD - US grants

? DESCRIPTION (provided by applicant): Autism Spectrum Disorders (ASDs) are a group of related neurodevelopmental syndromes defined by social communication deficits and by restricted and repetitive behaviors. The public health burden is enormous, with an estimated cost of $35 billion in the U.S alone. Behavioral approaches are currently the mainstay of treatment; options for somatic therapies remain extremely limited. A new generation of more effective treatments will require a far deeper understanding of the pathobiology of ASD. In addition, while there has been significant recent progress in clarifying the genomic architecture of autism, only a small number of the hundreds of genes and genomic regions thought to be involved in ASD have so far been identified. The current proposal focuses on discovering additional rare recessive mutations leading to autism via the study of consanguineous families from Egypt and Turkey. The project leverages a long-standing collaboration between the Gunel and State labs, which have independently and collectively demonstrated the productivity of homozygosity mapping and whole exome sequencing for a range of developmental disorders including autism. Our long-term goal is to make use of genetics to identify therapeutic targets in ASD while contributing to translating such findings to clinical practice. Our hypothesis is that th discovery of additional rare recessive, highly penetrant ASD mutations in consanguineous families will advance the understanding of molecular mechanisms; that these will show overlap with the emerging picture of the genetic architecture and biology of idiopathic ASD in outbred populations, and that, combined, these advances will lay the foundation for the development of novel, rational, and more efficacious treatments. Therefore, we focused on 3 specific aims: 1) To expand our current cohort of consanguineous ASD families with an additional 250 carefully diagnosed ASD kindreds from Egypt and Turkey, 2) To identify novel, rare, highly penetrant genetic variants that contribute to ASD by employing homozygosity mapping and whole-exome sequencing in 384 ASD subjects and parents; and 3) To search for clustering of these variants among unrelated families as well as to evaluate the overlap in risk loci for inbred versus outbred ASD populations, cross- referencing findings from these Middle Eastern families with data from the Simons Simplex Collection (SSC), which we have been studying for the past 5 years and to evaluate the identified homozygous variants with reference to ASD-associated developmental co-expression networks using high confidence ASD genes discovered in outbred families. Overall this proposal is aimed at advancing the understanding of the genetics and biology of ASD in the interests of identifying novel approaches to diagnosis, and therapeutic development.

? DESCRIPTION (provided by applicant): This is a renewal application for the Yale Center for Mendelian Genomics. The biology linking Mendelian mutations to traits has transformed our understanding of every organ system, identifying therapeutic targets, and allowing preclinical diagnosis and mitigation of disease risk. We know the consequence of mutation of fewer than 3,000 genes. With ~19,000 protein-coding genes, the vast majority of which are conserved across phylogeny, even allowing for 30% lethality, there are doubtless thousands of Mendelian loci awaiting discovery. The full utility of clinical sequencing will not be realized without bette understanding of the consequence of mutation of every gene. The advent of robust exome and genome sequencing allows unprecedented opportunity for discovery of new Mendelian trait loci. In the current cycle, by sequencing more than 7000 exomes from investigators world-wide we have identified 180 new Mendelian trait loci with high confidence, 35 phenotypic expansions, and hundreds more that are likely new trait loci across a range of traits and genetic mechanisms, including de novo mutations, incomplete penetrance, and complex rare recessive traits. Several new loci have immediate therapeutic implications. These results underscore that many new trait loci remain to be described and solved, motivating efforts to complete the human `knock out' map. We now propose, by building upon the current studies and through reduction in high quality exome cost to $330, to identify at least another 500 trait loci via the sequencing of more than 20,000 samples, advancing the understanding of genomes, health and disease.

Intracranial aneurysms (IA) represent a significant health issue in the US and worldwide. Their rupture leads to intracranial hemorrhage, with devastating outcomes: 30% of patients with ruptured IA die within a month of the initial event, and 50% of survivors are left with severe neurological deficits requiring long-term care. In our previous studies, we completed a series of genome-wide association studies (GWAS), which identified several IA risk loci containing candidate IA risk genes. We now propose to investigate the biological significance of select candidate genes using genome editing in human endothelial and vascular smooth muscle cell lines, and validate these findings in zebrafish and mouse, two model organisms that are ideal for genetic manipulations and analyses of brain vasculature. The proposed studies will establish the mechanisms by which modulation of gene dosage may enhance risk of aneurysm formation, testing to the hypothesis that they impact vascular homeostasis and vessel tone regulation. If successful, these studies will stimulate future research into rational and molecularly informed therapeutic approaches for IA.

? DESCRIPTION (provided by applicant): Glioblastomas are aggressive and invasive brain tumors that generally lead to death within a year of diagnosis. No cure is available. Current treatments prolong life by only a few months, often at the expense of quality of life. Here we test the general hypothesis that a novel recombinant chimeric virus will target and kill gliomas with no detectable adverse effect to the brain. Of the large number of viruses we have tested, vesicular stomatitis virus (VSV) appears to be one of the most effective for targeting and destroying brain tumors. However, VSV has the potential unwanted side effect of infecting neurons, and half of our efforts in the last few years have focused on reducing or controlling potential neurotoxicity of VSV. To avoid complications and toxicity of the VSV G-protein, particularly its binding to neurons, we have compared a number of recombinant chimeric viruses in which the VSV G-protein gene was replaced by genes coding for binding glycoproteins of non-related viruses including rabies, lymphocytic choriomeningitis, Marburg, Ebola, and Lassa viruses. Of these chimeric viruses tested, one stood out as a clear superlative safe oncolytic candidate: a chimeric virus consisting of a gene coding for the Lassa glycoprotein together with genes coding for the VSV N,P,M, and L proteins, and a GFP reporter gene which further attenuates the virus. Lassa-VSV is safe, both in rodents and primates. Of considerable importance, our direct injection of Lassa-VSV into the brains of normal mice or rats, or even into the brains of immunodeficient mice has not resulted in any detectable adverse effects, whereas injections of native or other attenuated VSVs generated neurological complications sometimes resulting in death. Equally important, in our preliminary experiments in vitro and in vivo, Lassa-VSV targets and destroys gliomas completely without damage to the host brain, and extends survival of tumor-bearing mice indefinitely. In the first set of experiments, we test the hypothess that Lassa-VSV successfully targets and kills glioblastoma cells that are transplanted into the brain, both after an intratumoral virus injection, and after intravenous inoculation. We use both glioma cell lines and primary human gliomas transplanted into immunodeficient mice. We also test syngeneic mouse glioma implanted into immunocompetent mouse host brains. Tumors are detected by expression of a red fluorescent reporter and virally infected cells are detected by expression of a reporter gene coding for green fluorescent protein. In the second Aim, we address the hypothesis that the mechanism behind the safety and selectivity of Lassa-VSV in the brain is that the virus either does not bind to receptors on neurons or normal glia, or is not internalized, whereas binding and internalization in glioma is robust. The lack of virus infection of neurons is studied by blocking or enhancing various steps in the infectious pathway coupled with reverse transcriptase quantitative PCR, and corroborated with electron microscopy, and in additional species and in human brain slices. A key hypothesis we test in Aim 3 is that Lassa-VSV initiates an attack by the systemic immune system, particularly by CD8+ T cells, on the glioma that continues even after the virus is eliminated, thereby preventing the recurrence of tumor. This is tested by infection of glioma in the brain; after the virus is eliminated, we examin the potential of newly implanted glioma to grow in the presence of the enhanced immune response. Immune targeting is complemented with adoptive transfer, CD8-T cell elimination, and immunocytochemistry to detect immune cells recruited to the infected tumor. Lassa-VSV is remarkable in that it can completely kill glioma with no detectable adverse side effects in the brain or elsewhere. If our experiments are successful, we think this virus would be a top priority candidate for clinical trials.

PROJECT SUMMARY / ABSTRACT Meningiomas are the most common primary brain tumors. While typically of benign histology, they can be associated with significant neurological morbidity and have the potential for malignant transformation. In our previous studies, we completed genomic analysis of over 700 meningiomas using whole exome and targeted next-generation sequencing, identifying driver mutations in 12 genes and establishing mutually exclusive molecular subgroups. We now propose to investigate the molecular mechanisms underlying the formation of TRAF7-dependent meningiomas (which represent up to one quarter of all meningiomas), undertake molecular genomic analyses to identify somatic coding and non-coding or genomic events in TRAF7 tumors that progress to higher grades. We also propose to test and validate candidate drug efficacy using primary cultures from surgically resected meningiomas carrying TRAF7 aberrations, as well as mouse models of meningioma that we have established.

Title: Zona incerta GABA neurons modulate energy homeostasis Abstract. Obesity, which often leads to secondary health complications including heart disease, diabetes, stroke, cancer, and early death, has become a major health concern in the US. Here we test the general hypothesis that neurons in the rostromedial zona incerta (ZI), and particularly inhibitory GABA neurons, play an unexpectedly profound orexigenic role in increasing food intake and body weight. Most of the work done on the neuronal regulation of energy homeostasis has previously focused on neurons in other brain regions. The first Aim examines the structural substrates for interaction between ZI GABA axons and their postsynaptic targets. Using confocal scanning laser microscopy and dual immunolabel electron microscopy coupled with cre recombinase-dependent AAV and rabies virus tracers, we test the hypothesis that ZI axons project to a number of sites, including the paraventricular thalamus (PVT) and hypothalamic ventromedial nucleus (VMH) where direct synaptic connections are made with excitatory neurons. We use multiple transgenic mouse lines expressing Cre recombinase under control of various neuron-selective promoters including mice that express Cre in GABA neurons driven by a vGAT promoter. These will be coupled with intracerebral microinjections of AAV viral vectors containing floxed GFP or tdTomato reporter genes to study ZI GABA neuron efferent and afferent axon projections. We will also employ injection of a Cre recombinase-dependent Brainbow-type pseudorabies virus into the ZI; after retrograde axonal transport this PRV expresses a red reporter in wild-type cells, but in Cre-expressing GABA cells, reporter expression changes to yellow or cyan, helping define the cell of interest. Aim 2 tests the hypothesis that ZI GABA cells respond to long distance signals of energy homeostasis including ghrelin and leptin, and also to axonally released neuropeptide modulators of food intake. Whole cell recording allows us to test different mechanisms of action on ZI GABA cells produced by neuromodulator signals from other neurons involved in energy homeostasis. C-fos expression will be examined after food deprivation to test the hypothesis that ZI GABA neurons are more active during reduced food availability. Optogenetics is used in brain slices to test the hypothesis that release of transmitter from ZI GABA axons will exert similar inhibitory effects on PVT and VMH neurons; neighboring ZI dopamine cells are also tested. In Aim 3, we examine the role that rostromedial ZI GABA neurons play in ongoing energy homeostasis by cell silencing (using Gi-DREADDs and caspase) to test the hypothesis that body weight and food intake is reduced. Optogenetic activation with ChR2 variant ChIEF will test the hypothesis that stimulation of ZI GABA axons in different terminal zones will each enhance food intake and body weight. We also test the hypothesis that ZI GABA neuron activation provides a positive emotional valence. Together, these experiments examine converging structural, electrophysiological, and behavioral analyses, focusing on the role of the GABA ZI neurons in energy homeostasis. With the growing levels of obesity in this country approaching 30% of the adult population, and the associated health complications, identifying and understanding the brain cells that control and sense energy homeostasis will help to identify novel approaches to reducing the trend toward obesity.