Benjamin D. Philpot - US grants

DESCRIPTION (provided by applicant): The responses of neurons in the visual cortex can undergo experience-dependent changes, particularly during early life. Abnormal experience during this "critical period" can permanently impair vision (e.g. amblyopia) due to improper cortical wiring. This demonstrates the importance of experience-dependent synaptic plasticity. Activation of NMDA-type glutamate receptors (NMDARs) is required for many forms of experience-dependent plasticity. While NMDARs are traditionally thought to exert their influences postsynaptically, a surprising recent finding is that NMDARs are also expressed presynaptically early in development. These presynaptic receptors are involved in regulating neurotransmitter release and long-term depression (LTD) of synaptic strength. The relative contribution of pre- and postsynaptic NMDARs during animal development remains unknown, yet this information is crucial to understanding how fundamental mechanisms of synaptic transmission and plasticity change at the onset of the critical period. Our data demonstrate, for the first time, an abrupt loss of presynaptic NMDAR function that coincides with the onset of the critical period for visual cortical plasticity. Here we will examine the central hypothesis that presynaptic NMDARs are involved in the induction of LTD before the onset of the critical period, but their experience- dependent loss triggers a new LTD induction mechanism to emerge. We will use electrophysiological and anatomical approaches in mice to address three crucial questions. (1) What allows presynaptic NMDARs to function, and what underlies their developmental loss? (2) Does experience modify the function of presynaptic NMDARs in an age-dependent mariner? (3) How do presynaptic NMDARs contribute to synaptic transmission and plasticity? Because LTD of excitatory synapses is one mechanism by which visual responsiveness may be lost due to aberrant visual experience, a particularly important goal is to elucidate the involvement of presynaptic NMDARs in LTD and how this might differ during the critical period. By demonstrating that both pre- and postsynaptic NMDARs contribute to the expression of synaptic plasticity, and that their relative roles shift over development, our findings are expected to define a novel, and perhaps general, property of synaptic plasticity in emerging cortical circuits. Relevance to public health: Amblyopia is the most common form of visual impairment during childhood and, if left untreated, is permanent. The weakening of synaptic inputs driven by the deprived eye is thought to underlie the visual deficits. Accordingly, knowledge of the fundamental mechanisms of synaptic weakening will lead to rational strategies for preventing the deleterious consequences of visual deprivation and will increase our understanding of normal visual cortical development.

Neurons in the brain analyze the sensory world by making appropriate neural connections during development despite an ever-changing environment. This process occurs during a sensitive period of early brain development and requires specialized receptors found at the synapses (connections) between neurons. The NMDA receptor is one such critical receptor. Although this receptor has traditionally been thought to function only at postsynaptic sites, recent evidence suggests an importance for NMDARs at presynaptic sites. Currently, the role of presynaptic NMDA receptors is poorly understood. The goal of this project is to determine how presynaptic NMDA receptors regulate neurotransmitter release and why this regulation is important for the development of appropriate neural connections. The proposed studies will specifically test the central hypothesis that plasticity mediated by presynaptic NMDA receptors is required for the normal development of early sensory functions within the brain. By demonstrating that presynaptic NMDA receptors contribute to early forms of neural plasticity, and that their contributions may shift with development, the findings from this proposal are expected to define a novel property of synaptic plasticity in emerging cortical circuits. Moreover, a fundamental understanding of the unique differences between presynaptic and postsynaptic NMDA receptors will aid future pharmacological manipulations that target NMDA receptors for the treatment of neurological disorders. The goals of this proposal will be met through promoting the involvement of underrepresented minorities in this intensive neuroscience research project.

DESCRIPTION (provided by applicant): Angelman syndrome (AS) is a genetic disorder characterized by developmental delay, absent speech, intellectual disability, severe epilepsy, ataxia, and abnormal sleep. AS is caused by mutations in or deletion of Ube3a, an E3 ubiquitin ligase that is expressed biallelically in most tissues but is monoallelically expressed in the brain. Maternal-specific expression of Ube3a in the brain is thought to be due to production of an antisense transcript that overruns the paternal copy of Ube3a in mice and humans. Mice with maternal-specific deletions of Ube3a model many of the neurodevelopmental symptoms associated with AS, including epilepsy, learning deficits, and motor abnormalities. Using a high-throughput, unbiased screen with neurons from a Ube3a- YFP knockin mouse, we identified several small molecules that unsilence the paternal Ube3a allele at nanomolar concentrations. We hypothesize that the physiological and behavioral dysfunctions associated with Angelman syndrome can be treated by unsilencing the paternal Ube3a allele in vivo with one of these drugs. In this proposal we will: (1) Test the hypothesis that our lead compound upregulates paternal Ube3a in vivo; (2) Test the hypothesis that our lead compound can rescue physiological and behavioral deficits in Angelman syndrome model mice; (3) Test the hypothesis that genetic knockdown/out of the molecular target of our lead compound unsilences paternal Ube3a; (4) Test the hypothesis that the expression of the Ube3a-sense and Ube3a-antisense transcript levels can be used as biomarkers of drug efficacy (i.e. Ube3a unsilencing). Our research could lead to the first pharmacological treatment for Angelman syndrome (an autism spectrum disorder), and indeed for any disorder caused by mutation of an imprinted gene.

DESCRIPTION (provided by applicant): Despite the genetic heterogeneity underlying autism and neurodevelopmental syndromes with autism comorbidity, there is phenotypic convergence among these disorders, leading to the view that this may reflect a common pathological convergence in cortical circuits. A leading theory suggests that an increased ratio of excitatory to inhibitory (E/I) neurotransmission (i.e., E/I imbalance) within neocortical circuits contributesto the common phenotypic features of autism. To gain a genetic toehold for understanding E/I imbalance, we have focused on an autism disorder associated with changes in a single gene, UBE3A. Loss of UBE3A expression causes Angelman syndrome (AS), which is characterized by an absence of speech, cognitive disability, seizures, and a high comorbidity with autism. We recently demonstrated that inhibitory drive onto cortical pyramidal neurons is severely decreased in a mouse model of AS, resulting in an elevated E/I ratio. Our preliminary data led us to hypothesize that the E/I imbalance caused by loss of UBE3A protein reflects both presynaptic defects in inhibitory interneurons and postsynaptic defects in pyramidal neurons. We further hypothesize that UBE3A function is required to maintain cortical E/I balance, and therefore we predict that loss of UBE3A even in adults will increase seizure susceptibility and cognitive deficits associated with elevated E/I ratio. Furthermore, we hypothesize that reinstatement of Ube3a expression will restore cortical E/I balance and reverse some AS phenotypes. In this proposal we aim to (1) Elucidate the cellular basis of cortical E/I imbalance in AS; (2) Test the hypothesis that Ube3a expression is required throughout life to maintain cortical E/I balance and neurotypical behaviors; (3) Define treatment windows for AS phenotypes. Our research will help establish parameters for therapeutic interventions in AS and possibly other autism spectrum disorders.

Project Summary This application, ?Postdoctoral Research in Neurodevelopmental Disorders,? is a request for five years of funding for the competing continuation of an NIH National Research Service Award (NRSA) Institutional Training Grant (T32). Recent advances in behavioral science, neuroscience, and molecular genetics have led to dramatic gains in our understanding of the pathogenesis of neurodevelopmental disorders. The purpose of this training program is to develop researchers with expertise in both the biological basis and clinical manifestations of neurodevelopmental disorders. This broad-based integrated perspective will enable researchers to better relate across disciplines, and will maximize the potential for transformative advances in understanding the pathogenesis of these disorders and in developing new and more effective approaches to their treatment. Ultimately, this training will prepare researchers to interface across disciplines and will increase the number of well-rounded researchers, significantly advancing the health-related research needs of the nation (the primary objective of the T32 program). The proposed postdoctoral training plan provides a variety of opportunities including: (1) mentored research training in specific methods, disorders, and underlying pathogenetic mechanisms; (2) a range of didactic experiences (including courses, seminars, and lectures) that integrate the study of clinical disorders, normal developmental processes, mechanisms of disease, and research methods; and, (3) clinical exposures to complement previous basic science and basic science exposure to complement clinical experiences. Twenty Ph.D. or M.D. level trainees (four new trainees per year) will participate in this two-year training program, over the five-year period of this proposal. This proposal addresses the critical need for focused training opportunities beyond formal graduate programs by using the existing interdisciplinary research and administrative structure of the Carolina Institute for Developmental Disabilities (CIDD). All faculty mentors are members of the CIDD, which offers a comprehensive program for services, education, and research in developmental disabilities. Together with the excellent clinical and basic science resources of the University of North Carolina (UNC), the CIDD provides an ideal environment for integrating training in biological and behavioral research in neurodevelopmental disorders.

PROJECT SUMMARY White matter pathway deficits are common in neurodevelopmental disorders, including Angelman syndrome (AS). The few imaging studies performed to date suggest that AS individuals have generalized white matter deficits, including loss of volume and possibly delayed myelination. However, white matter deficits in AS remain poorly defined, making it difficult to link them to behavioral phenotypes and, consequently, to establish their value as therapeutic biomarkers. Accordingly, a major unmet need is to elucidate the anatomical and pathophysiological basis for white matter deficits in AS, and to test whether prevention or reversal of these deficits leads to improvement in core behavioral domains. Preliminary data from children with AS show reductions in white matter volume and integrity of white matter fiber tracts, as well as strong relationships between these WM deficits and motor delays. Our preliminary data in AS model mice demonstrate that they exhibit white matter pathway impairments that recapitulate those found in AS individuals. The white matter deficits in the AS model mice are quantifiable at all levels of analysis, from macroscopic magnetic resonance imaging (MRI) coupled with diffusion tensor imaging (DTI) to ultrastructural electron microscopy (EM). Here we will leverage the experimental tractability of AS model mice and our unique access to AS individuals through the UNC Angelman Syndrome Clinic (the first AS clinic established in the United States) to reveal the precise anatomical and pathophysiological underpinnings of white matter deficits in AS. Our research constitutes a novel effort to directly link white matter deficits in a human neurodevelopmental disorder with its mouse model, and will test our data-driven central hypothesis that white matter pathway impairments and associated AS phenotypes arise from delayed myelination and a loss of large-diameter axons that can be prevented by reinstatement of UBE3A expression in neurons. To achieve our goals, we aim to (1) Delineate white matter pathway deficits and test the hypothesis that they are a biomarker for motor phenotypes in children with AS, (2) Define the developmental trajectory and underlying anatomical basis for white matter deficits in AS model mice, and (3) Model the efficacy of early versus late therapeutic intervention toward the normalization of white matter deficits and motor outcomes in AS. Through our research, we seek to inform treatment strategies for AS and to establish WM integrity as a novel endpoint for upcoming AS clinical trials.

PROJECT SUMMARY White matter (WM) pathway deficits are common in neurodevelopmental disorders, including Angelman syndrome (AS). The few imaging studies performed to date suggest that AS individuals have loss of WM volume and possibly delayed myelination. However, these abnormalities remain poorly defined, making it difficult to link them to behavioral phenotypes and, consequently, to establish their value as therapeutic biomarkers. Accordingly, a major unmet need is to elucidate the anatomical and pathophysiological basis of abnormal WM development in AS, and to test whether prevention or reversal of WM deficits leads to improvement in core behavioral domains. Our preliminary light and electron microscopy studies of AS model mice suggest that impairments in axon growth precipitate delays in myelination and culminate in lifelong deficits in axon caliber and WM volume. Our preliminary magnetic resonance imaging (MRI) coupled with diffusion tensor imaging (DTI) data from children with AS demonstrate a conserved deficit in WM volume and indicate a similar delay in myelination. Importantly, we find in motor systems that the degree of WM insult strongly correlates with the severity of motor dysfunction in AS patients. Here we will leverage the experimental tractability of AS model mice and our unique access to AS individuals through the UNC Angelman Syndrome Clinic (the first AS clinic established in the United States) to reveal the developmental basis of WM deficits in AS. Specifically, we will test our data-driven central hypothesis that WM pathway abnormalities and associated AS phenotypes arise from deficits in the radial growth of axons and associated delays in myelination, which can be prevented by reinstatement of UBE3A expression in neurons. To achieve our goals, we aim to (1) Define the developmental trajectory and underlying anatomical basis for WM deficits in AS model mice, (2) Establish neuroimaging correlates of these deficits and test the hypothesis that they are a biomarker for motor phenotypes in children with AS, and (3) Model the efficacy of early versus late therapeutic intervention toward the normalization of WM development and motor outcomes in AS. Through our research, we seek to inform treatment strategies for AS and to establish WM integrity as a novel outcome measure for upcoming AS clinical trials.

PROJECT SUMMARY UBE3A is an E3 ubiquitin ligase that targets itself and other substrates for proteasomal degradation. In the developing brain, neuronal progenitors and immature neurons biallelically express Ube3a, but as neurons mature, Ube3a expression becomes restricted to the maternally-inherited allele. Mutations that elevate maternal or paternal Ube3a are linked to autism risk, but precisely how UBE3A excess impairs neurodevelopment is unclear. Recently, we found that phosphorylation of threonine 485 (T485) inhibits UBE3A ubiquitin ligase activity. UBE3A T485 phosphorylation initiates embryonically and peaks at birth, suggesting that phosphorylation might protectively limit UBE3A activity during early cortical development. Additionally, we found that an autism-linked de novo mutation in UBE3A (T485A) disrupts this phosphorylation site, effectively locking UBE3A always-on. We engineered a mouse that precisely models this human UBE3A T485A mutation, allowing us to evaluate how this novel gain-of-function mutation affects brain and behavioral phenotypes when inherited maternally or paternally. In preliminary studies, we found that cortical thickness and brain weight were significantly increased at birth in all three Ube3a T485A genotypes (paternal, maternal, homozygous). Mutations in other autism-linked genes increase brain weight to a similar extent. These findings suggest a novel and previously unrecognized prenatal function for UBE3A in brain development. All three Ube3a T485A mutant genotypes also had behavioral phenotypes consistent with neurodevelopmental disorders. Since little is known about how UBE3A impairs brain function at any age, we performed unbiased proteomics to identify brain-relevant substrates. Our preliminary proteomics data link UBE3A directly to the proteasome, a structure that can influence the cell cycle and signaling pathways important for brain development. These and other data lead us to hypothesize that UBE3A T485A alters the balance of cell proliferation and differentiation during brain development, in part by impairing proteasome function, and contributes to autism-associated phenotypes later in life. The experiments in this proposal will rigorously demonstrate that (1) UBE3A T485A alters the balance of progenitor proliferation and differentiation in the cerebral cortex, (2) parent-of-origin inheritance of Ube3a T485A influences autism-related brain and behavioral phenotypes, and (3) UBE3A T485A interacts with the proteasome and impairs proteasome function in the brain. Unbiased proteomics experiments will identify brain-relevant substrates of UBE3A, and broaden our understanding of which molecular pathways are affected by gain-of-function mutations that enhance UBE3A activity.

PROJECT SUMMARY Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder characterized by loss of speech, EEG abnormalities, seizures, motor impairments, and severe intellectual disabilities. Unfortunately, there are no treatments for its core symptoms. PTHS is caused by haploinsufficiency of TCF4, a transcription factor that regulates hundreds of genes, making it nearly impossible to therapeutically address the full phenotypic spectrum of the disorder by targeting downstream molecular pathways. Ideally, PTHS would be treated at its roots, by augmenting the expression of the intact TCF4 gene copy to normalize gene expression levels. We hypothesize that small molecules capable of upregulating TCF4 expression during early postnatal development, and perhaps into adulthood, will correct PTHS phenotypes. To develop an informed therapeutic intervention strategy and to identify TCF4 activators for eventual clinical trials, we will complete three Aims: (1) establish the biodistribution of TCF4 to guide therapeutic delivery, (2) assess phenotypic rescue with early- or late-onset normalization of TCF4, and (3) identify approaches to increase TCF4 levels. We have developed and validated powerful tools to facilitate each of these Aims, which are integral to guiding the clinical development of genetic normalization treatments for PTHS.