Purpose of review

Recent findings

Summary

Neurodevelopmental disorders (NDDs) are a broad range of conditions characterized by abnormal development of the nervous system during “critical periods” of brain growth and neuronal migration leading to lifelong impairments that are severe and often debilitating. The clinical presentation of NDDs includes a wide range of complex symptoms such as intellectual disabilities, developmental delay, recurring, uncontrollable seizures, lack of communication, poor gross and fine motor abilities, and autism spectrum disorder. There are no cures nor corrective therapies currently available and traditional medicines for symptom alleviation are minimal. To understand the intricacies of the neurobiology underlying a variety of NDDs with similar phenotypes, and potentially identify overlapping common mechanisms, while simultaneously addressing the grand unmet clinical need of efficacious therapeutics, we used and, in some cases, generated, novel preclinical genetic mouse models to identify disease-relevant translational outcome measures. Mouse models provide a valuable system to closely investigate symptoms analogous to human disease phenotypes. With the growing knowledge in genetics, NDD-relevant mouse models (knockout, knockin, humanized, etc.) with high construct validity have been generated to study the causal role of specific genes and how the loss of function contributes to NDD etiology and symptom manifestation. The research herein investigates mouse models of four distinct genetic NDDs and defines robust phenotypes that can be employed to evaluate novel therapeutic approaches. Chapter 1 investigates a novel mouse model harboring a deletion of the regulatory region 1b of the Scn1a gene which encodes for Nav1.1 sodium channel. Haploinsufficiency of SCN1A is associated with epilepsy, including Dravet Syndrome. We discovered that both heterozygous and homozygous deletion of the regulatory element led to behavioral and seizure deficits as well as electroencephalogram phenotypes, such as elevated spontaneous spiking events and spike trains, indicating that the 1b deletion model provides strong evidence to investigate the epilepsy risk gene Scn1a and highlights the importance of functional studies following modulation of regulatory elements in haploinsufficiency-related disorders. Chapter 2 focuses on CDKL5 Deficiency Disorder (CDD), a rare X-linked NDD caused by the deficiency of the gene CDKL5 (cyclin-dependent kinase-like 5), which is essential for normal brain development. Previous studies focused on male subjects from two unique mouse models to avoid any potential confounds of variable CDKL5 expression that results from X chromosome mosaicism in females; however, about 90% of individuals living with CDD are females. We performed comprehensive characterization in both sexes of the two mouse models to reproduce earlier studies in males and extend to the translationally relevant population, females. We discovered clinically relevant phenotypes including hyperactivity, learning and memory deficits, and susceptibility to seizures and abnormal EEG that will allow investigators to advance preclinical testing of therapeutics candidates for CDD in females. Chapter 3 expands on the characterization of the Snord116 deletion model of the NDD Prader-Willi Syndrome, which is caused by a loss of paternal genes on chromosome 15q11-q13a. SNORD116 is a paternally expressed imprinted gene cluster that encodes multiple copies of small nucleolar RNA. We focused on an understudied and key behavioral domain of cognition, using a heterozygous deletion of Snord116 and identified learning and memory impairments, highlighting a broader role of SNORD116, beyond the numerous previous reports focused hyperphagic and metabolic phenotypes. Finally, chapter 4 utilizes the rigorous methodologies we implemented in the earlier chapters spanning a range of behavioral domains, seizures, and electroencephalographic outcome measures to investigate a novel cell-based gene therapy for Angelman Syndrome (AS). AS is caused by the deficiency of the gene UBE3A (ubiquitin protein ligase E3A). To evaluate in vivo efficacy of our approach, we generated an immunodeficient AS mouse model capable of accepting human hematopoietic stem cells for engraftment and multilineage hematopoiesis. Upon delivery of functional UBE3A via transplantation and engraftment of CD34+ cells transduced with a Ube3a lentivector, we demonstrated rescue of AS deficits in the immunodeficient AS mouse model across two developmental time points. Currently, this study is in IND-enabling studies after a thorough review of the pre-IND package submitted to the FDA. Taken together, this research advances our understanding of NDDs and highlights the importance of neurodevelopmental behavior research in identifying clinically relevant phenotypes to meet the ultimate goal of therapeutic rescue.

Neurodevelopmental disorders (NDDs) are a large group of profound, debilitating, andlifelong conditions that impact the normal development of the central nervous system. Autism spectrum disorders, developmental delay, intellectual disability, and seizure disorders are among the clinical presentations of NDDs. Although a large population of patients live with the devastating symptoms of NDDs, no corrective therapies are currently available, stemming from the complexity of etiologies and broad range of conditions. To broaden our understanding of the mechanisms that contribute to NDDs and advance the discovery and development of therapeutics, we used preclinical mouse models to establish clinically relevant outcome measures and identify potential drug candidates. Chapter 1 introduces several strategies that address the challenges of developing therapeutics for NDDs and provides rationale for studies detailed in subsequent chapters. Chapters 2 and 3 investigate one approach proposed in the introduction: drug repurposing. Following earlier successes of repurposed drugs for other NDDs, these chapters focus on preclinical evaluation of several potential small molecule therapeutics for treating Angelman Syndrome (AS); a disorder caused by the absence of the protein UBE3A in the brain. We performed comprehensive behavioral pharmacology batteries, tailored to AS, using an AS mouse model and wildtype littermates. We treated both genotypes with one of three separate compounds, paxilline, LB-100, and lovastatin, compared to vehicle. We found that paxilline and LB-100 did not exert positive rescue effects in the motor or cognitive behavioral domains. However, lovastatin treatment of AS mice exhibited an improvement to motor ability when we assessed gait using an automated treadmill system, providing promising evidence that this treatment could have potential in the clinic. Chapter 4 examines in vitro and in vivo techniques for assessing potential drug candidates for the treatment of AS. We utilized the highlyiii objective and translational touchscreen operant chamber task for assessing cognition and the whole-body plethysmography (WBP) task for assessing pulmonary physiology and applied these methods to the AS mouse model, compared to WT, sex-matched littermates. To better inform our future behavioral pharmacology experiments, we established two in vitro assays to assess the structure and electrophysiological properties of primary neurons derived from the AS mouse model. Chapter 5 applies the lessons we learned from our research in Angelman Syndrome to another NDD, SYNGAP1-related intellectual disability (SYNGAP1-ID). SYNGAP1-ID is a disorder caused by the reduction of SynGAP1 protein in the brain, resulting in symptoms such as intellectual disability, hyperactivity, and epilepsy. We performed comprehensive behavioral characterization of a Syngap1 mutant mouse model to understand the clinically relevant phenotypes and to identify translational biomarkers. Hyperactivity, increased electroencephalographic (EEG) signal and disruptions to sleep were observed in the mutant mouse, offering clinically relevant targets for future preclinical therapeutic testing. In addition, we utilized the in vitro electrophysiological assay of primary neurons established in Chapter 4 to confirm similar hyperexcitability in both cultured neurons and recordings directly from the surface of the brain of the Syngap1 mouse model, bridging the gap between in vitro and in vivo assays of neuronal function. Taken together, this body of research increases our understanding of the complex nature of NDDs and highlights the progress toward improving preclinical biomarkers to aid in the discovery and development of therapeutics for NDDs.

Neurodevelopmental disorders (NDDs) are a broad group of conditions characterized by abnormal development of the nervous system that includes intellectual disability and autism spectrum disorders. NDDs are pervasive, lifelong, and increasing in prevalence, yet neither targeted pharmacological nor precision medicine therapies are currently available. This unmet need is in large part due to the complex and relatively unknown etiology of many NDDs. Multiple genetic loci, in combination with exposure to environmental risk factors during critical periods of development, contribute to both NDD susceptibility and symptom severity. In an effort to improve our understanding of NDD etiology, as well as help develop effective treatments, we used Sprague Dawley rats to examine the effects of two environmental risk factors and one causal genetic mutation on behaviors and neurobiological outcomes relevant to NDDs. While the mouse has predominated in recent decades as the species of choice for modeling NDDs, we sought to take advantage of the broader and more nuanced social communication repertoire of the rat. Therefore, we measured effects on a variety of behavioral domains including motor and cognition and gave particular focus to social and communication behaviors. Chapter 1 serves as an introduction to the study of social communication in rodent models of NDDs and provides detailed rationales behind many of the assays utilized in subsequent chapters. Chapters 2 and 3 investigate environmental factors that have been associated with increased risk for NDDs: air pollution and pesticides, respectively. Our findings support the hypothesis that developmental exposure to heavily trafficked roadways, or to the commonly used pesticide chlorpyrifos, increases NDD risk. Then, Chapters 4 and 5 characterize a novel genetic rat model of the NDD Angelman Syndrome, which is caused by dysfunction of the gene UBE3A (ubiquitin protein ligase E3A). We discovered that the Ube3a deletion rat model of Angelman Syndrome recapitulated many of the core behavioral and neurobiological phenotypes of the disorder, including elevated expression of positive affect signals, and thereby utilized the rat model in Chapter 6 to test the efficacy of a candidate therapeutic, insulin-like growth factor 2. Although treatment with insulin-like growth factor 2 failed to rescue the model’s deficits, the outcome measures used were robust and reproducible, indicating that the Ube3a deletion rat offers a strong preclinical model of Angelman Syndrome. Finally, Chapter 7 investigates a leading-edge “gene therapy-like” antisense oligonucleotide treatment in the rat model of Angelman Syndrome. Multiple clinical trials are currently evaluating antisense oligonucleotide therapies in humans with Angelman Syndrome and, while they have demonstrated remarkable efficacy, serious adverse reactions have been observed. By identifying an antisense oligonucleotide with molecular efficacy in the rat and delivering it via the brain, the cisterna magna, and lumbar puncture, we established a paradigm through which to elucidate the pathophysiology underlying the adverse clinical reactions. Taken together, this research advances our understanding of the complex etiology of NDDs, represents significant progress in modeling NDDs preclinically, and provides insights with direct implications to the ongoing pursuit of pharmacological treatments targeted to NDDs.