Josh Bonkowsky - US grants

[unreadable] DESCRIPTION (provided by applicant): The development of the basal ganglia and the formation of their connections are complex and poorly understood processes. The basal ganglia are integral to motor function, to cognitive and language development, and to social and emotional regulation. While some genes have been identified with roles in basal ganglia neuron specification, or in diseases of the basal ganglia, how these and other genes regulate the development of basal ganglia connectivity is not known. Further, the actual process of basal ganglia axon pathfinding during development has not been characterized. My hypothesis is that connectivity of the striatum and the mesodiencephalic dopaminergic (mesDA) neurons is controlled by the pathfinding gene families of the robos, the slits, and the ephslephrins. My long-term objective is to characterize the development and genetic control of basal ganglia connectivity, using zebrafish (Danio rerio) as a model system. In order to explore the development of basal ganglia connections, this project has three specific aims: Aim 1. Develop molecular and genetic markers to visualize striatal and mesodiencephalic dopaminergic (mesDA) neurons and their axons. I am using in situ gene markers and antibodies to label these nuclei. To study their pathfinding, I have generated novel enhancer lines specific for basal ganglia neurons, including dlx(mini):gfp and foxP2-enhancerA:egfp. Aim 2. Characterize the normal axon pathfinding of the zebrafish striatum and mesDA neurons during development. I will use enhancer:gfp constructs and transgenic lines to describe the development of connectivity, and compare the pathfinding to the expression patterns of the ratios, slits, ephs, and ephrins. Aim 3. Evaluate the role of robo, slit, and ephlephrin genes in striatal and mesDA neuron pathfinding. I will test the function and effects of different members of these gene families by using a combination of mutant fish lines and morpholinos. Clinical Significance: Our results will improve our understanding of the development of basal ganglia connectivity, which is affected in neurodevelopmental and neurodegenerative disorders. Summary: The work described here will be the first description of axon pathfinding of the basal ganglia, characterizing both the development of connectivity and its genetic basis. My results will include both descriptive elements of basal ganglia development, as well as analyses of the role of specific genes. This project consists of a well-structured career development plan, extensively supported with institutional resources and an internationally known mentor, to assist me with the transition during the K award to an independent tenure-track faculty position. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Provided by the applicant) Abstract: Specific visualization and manipulation of neural circuitry has remained a vexing problem in neurobiology. Classical methods rely upon analysis in fixed tissue, preventing characterization of function or behavior. Newer methods allow genetic targeting to specific neuron types and even identify single neurons, but synaptic partners and functional circuits are not accessible by these current methods. A more general related issue is how to induce expression of a transgene in a vertebrate system when two cells make contact. A solution to these issues could have wide applicability, both for experimental studies, as well as for potentially a variety of therapeutic options. My project, Trans-Cellular Activation of Transcriptin to Analyze Dopaminergic Axon Reorganization, describes a novel strategy to analyze vertebrate circuit construction and function. It is the first genetic method for visualizing and driving expression in two cells that make contact, and offers the potential to identify and manipulate neuronal circuits in a vertebrate organism. I designed TCAT (trans-cellular activation of transcription) based on components from the conserved receptor/ligand pair of Notch/Delta. Upon ligand binding to receptor, the intracellular domain of Notch is cleaved and translocates to the nucleus. But by replacing the intracellular domain of Notch with the yeast transcriptional activator Gal4, I can specifically drive expression of transgenes at the Gal4-binding site UAS. TCAT uses the homologs LAG-2 (Delta) and LIN-12 (Notch) from the nematode C. elegans to prevent cross-reactivity with the endogenous zebrafish proteins. Specificity of labeling is provided by restricting expression of LAG-2 or LIN-12 to specific cell types. Expression from the UAS occurs when Gal4 is targeted to the nucleus, and this only occurs in the presence of ligand-receptor (cell-cell) LAG-2 to LIN-12 binding. Critically, this method is more than a means for labeling cells, but allows any inducible form of manipulation to be driven. There has been no other comparable method reported that activates transcription when two cells come into contact. Thus, TCAT will have applicability not only for use in the nervous system, but also in the study of other biological processes. In this proposal I introduce and explain TCAT, and show proof-of-principle that TCAT works in vivo, including when it is expressed by cell-type-specific enhancers. I describe how I will use it in mapping functional neural circuitry, by designing reagents to target TCAT to synapses. Finally, I outline how I will use TCAT to characterize dopamine circuit reorganization following injury, and the relevance of this for both basic and clinical neuroscience. TCAT is a significant technical innovation for mapping and manipulating circuits, but its real power is the ability it provides to create a full and functional understandig of the vertebrate CNS connectome. Public Health Relevance: The project develops a novel method to analyze and understand complex nervous systems, based on genetic approaches in the vertebrate model organism D. rerio. We apply this method to study the molecular mechanisms by which dopamine neurons alter their connections after injury, a clinically important cause of neurological and psychiatric diseases.

DESCRIPTION (provided by applicant): In spite of unparalleled opportunities to assess novel therapies for both common and rare neurological disorders, investigators involved in multicenter clinical trials face an increasing array of challenges in efficiently designing, powering and performing such trials. The Network of Excellence in Neuroscience Clinical Trials (NEXT) is intended to help address this increasingly complex environment and facilitate an efficient robust mechanism for multicenter neuroscience trials via the creation of centralized infrastructure supporting a nationalized network of high quality sites and coordinating centers. Based at the University of Utah, the Utah Regional Network for Excellence in Neuroscience Clinical Trials (UR-NEXT) will provide an ideal mechanism for coordinating recruitment and access throughout a 5 state region in the Intermountain West. The coordinated efforts of a leadership team with a track record of collaboration and experience in both pediatric and adult clinical studies and trial and an experienced clinical trials manager will ensure the performance of high quality neuroscience trials across a broad range of diseases. The UR-NEXT will be centered at the University of Utah, where longstanding and close partnerships exist between the Clinical Neuroscience Center, the only integrated neuroscience center in a 5 state region, the Salt Lake City Veteran's Administration Medical Center, the western anchor for the Rocky Mountain Region, and Primary Children's Medical Center (PCMC), the leading pediatric referral center for the region. UR-NEXT will additionally benefit from an established network of referring community neurologists with interest in clinical trials, the Western Intermountain Neurological Organization (WINO), which has members ranging from southern Utah to Montana, and Nevada to western Colorado. The Neurology Department has sections in each major subspecialty and substantial expertise in the management and performance of both adult and pediatric multicenter clinical trials. The UR-NEXT will benefit from the Center for Clinical and Translational Science as well as numerous other collaborative resources. Overall, the network has primary access to a population of approximately 8 million covered lives, making it the ideal choice for a NEXT site for this region.

? DESCRIPTION (provided by applicant): Each year 500,000 infants are born prematurely in the U.S. Premature infants have a 3-times higher risk for developing an autism spectrum disorder (ASD), and the prevalence of ASDs approaches 25% in the very most prematurely-born infants. Premature infants have been shown to experience chronic hypoxia during a period of development when axon connections are forming; and the extent of hypoxic exposure for premature infants correlates with the risk for ASDs. Ex-premature infants who develop ASDs lack conspicuous brain abnormalities, but have evidence of decreased brain serotonin (5-HT) and alterations in connectivity. Our work addresses the gap in understanding the mechanism by which hypoxia disrupts axon connections. We investigate a novel role for 5- HT: sensing a developmental perturbation (hypoxia), and in response altering neural circuitry. Work from our lab has shown that hypoxia disrupts axon pathfinding in vertebrates (Stevenson et al. 2012). Hypoxia can disrupt 5-HT signaling, and recent in vitro evidence demonstrates that 5-HT can modulate axon guidance. Our hypothesis is that developmental hypoxia disrupts axon pathfinding acting through serotonin. We have developed a powerful platform to test our hypothesis. We use studies in zebrafish, combining the relevancy of vertebrate CNS structures and genes, with rapidity and efficiency for testing molecular mechanisms. We have generated unique and novel fluorescent reporter and expression/misexpression lines; and capability to generate large numbers of animals for sufficient statistical power. Our experiments combine knock- down, knock-out, and misexpression of genes; pharmacological manipulations; and an established hypoxia model. Our preliminary data shows that 5-HT2 receptors (htr2) are expressed in commissural foxP2 neurons, and that pharmacological blockade of htr2 receptors causes midline axon pathfinding errors. We use knock-down of htr2 in the foxP2 neurons to confirm the role of 5-HT on axon guidance in vivo (Aim 1). We genetically ablate the 5-HT source neurons (raphe nucleus) to demonstrate that the pathfinding is regulated by 5-HT; and we test whether 5-HT's effect is mediated by the guidance receptor ephrinB2a which is expressed in foxP2 neurons. In Aim 2 we will study whether 5-HT is a sensor for developmental hypoxia to regulate circuit and behavior development. 5-HT circuits are known to be involved in anxiety-related behaviors dysregulated in ASDs, and our preliminary work has found that hypoxia causes a persistent decrease in 5-HT expression. We will characterize 5- HT's role in hypoxia axon pathfinding errors by rescuing pathfinding using the 5-HT reuptake inhibitor fluoxetine. To determine if 5-HT circuitry changes modulate behavior, we will compare anxiety behavior (thigmotaxis/wall- hugging) in control or experimental animals exposed to hypoxia or to 5-HT pharmacological blockade; to hypoxia animals treated with fluoxetine; or to animals with 2-photon ablation of foxP2 axons. Our approach provides important mechanistic insights into the effects of hypoxia accompanying prematurity, and its involvement in disruptions of CNS connectivity associated with autism spectrum disorders.

Project Summary/Abstract Vanishing white matter disease (VWMD) is a common inherited leukodystrophy affecting almost ~1:15,000 live births. VWMD imposes tremendous and often lethal burdens on patients. New treatments are needed. Our objective is to generate and validate a novel small vertebrate model (zebrafish) for vanishing white matter disease (VWMD). Mutations in five different subunits of the eukaryotic initiation factor EIF2B (1-5) are known to cause VWMD, but discovery of treatments for VWMD has been limited by slow disease progression in mouse models, difficulty in large-scale screening, and high costs. Zebrafish (Danio rerio) offers key benefits to circumvent these issues: myelin development begins in the first 3 days after fertilization; there is conservation of genes for myelin development; and low costs and small size facilitate screening not possible in other systems. We have four specific aims for the R21 and R33 phases of this project. In the R21 phase we will first, generate zebrafish mutants for vanishing white matter disease, using CRISPR/Cas9 targeting of exons 1 and 2 of eif2B5, and obtaining an eif2B2 splice-site mutant. Second, we will validate zebrafish VWMD molecular and biochemical phenotypes. We will perform immunohistochemistry for myelin, oligodendrocytes, and axonal integrity; assays of larval motor (swimming) behavior; and survival curves. For progression to the R33 phase, Go/No-Go milestones will be the generation and raising of mutants in eif2B2 and eif2B5; demonstration with PCR and sequencing of the genomic mutations; that RNA transcript and protein product are lost or diminished; that oligodendrocyte and myelin development is impaired; and that survival and motor ability are affected. In the R33 phase we will first, characterize zebrafish VWMD mutant phenotypes compared to human VWMD. We will test whether the zebrafish mutants have hallmark features of human VWMD, including increased mortality; inducible myelin loss; myelin changes on MRI; impaired somatic growth; and increased CSF glycine levels. Next, we will test whether zebrafish VWMD disease pathology is rescued by expression of the human gene, demonstrating conservation of the genetic and biochemical pathophysiology. Second, we will determine zebrafish VWMD phenotype range and disease and scale parameters. We will examine ability to generate sufficient numbers of animals for drug screening. We will test whether swimming behavior or a fluorescent-myelin GFP reporter in the mutants could be used for screening, and determine the signal-to-noise ratio; throughput capacity for screening; and expected effect size in comparison to a genetic rescue. In summary, the work described in this proposal will establish and validate a small vertebrate model for VWMD. This work is carried out in vivo and utilizes state-of-the-art techniques. This proposal addresses a significant unmet need, uses unique reagents, and offers significant potential for a therapeutics screening pipeline.