Drosophila models for human diseases have helped in advancing our knowledge on human diseases and the discovery of potential treatments. Angelman syndrome is a rare neurological disorder that results in severe mental retardation and loss of motor coordination. The disease is caused by loss-of-function mutations in the UBE3A gene encoding a HECT domain ubiquitin protein ligase. Drosophila dube3a is the fly homolog of human UBE3A and their protein products share ~55% similarity in amino acid sequence along the entire length of the proteins. My goal was to develop a Drosophila AS model that will allow us to identify the AS-associated substrate(s) of the Drosophila UBE3A homolog and ultimately, to determine why the lack of UBE3A protein causes Angelman syndrome in humans. Dube3a is present in the embryonic, larval and adult central nervous system, including the adult mushroom bodies, which is the center for learning and memory. I have generated dube3a knock-out flies and they appear normal externally, but display abnormal locomotor behaviors. Flies that overexpress wild-type dube3a in the nervous system also display locomotion defects, and these overexpression phenotypes are dependent on the presence of a conserved cysteine residue essential for HECT domain E3 enzymatic activity. Targeted overexpression of dube3a in the eye, the wing, or ubiquitously causes rough eyes, curly wings and lethality, respectively. These morphological abnormalities in the eye or wing depend on the critical catalytic cysteine of Dube3a. Overexpression of mutant dube3a carrying AS-associated point mutations does not elicit such defects, suggesting they act as loss-of-function mutants. Taken together, dube3a mutants are a candidate fly model for Angelman syndrome, and the flies that overexpress dube3a in the eye or wing are useful for genetic screens to identify the elusive UBE3A substrates relevant to Angelman syndrome.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

UBE3A is a protein with dual functions as an E3 ubiquitin ligase and as a Steroid Hormone Receptor (SHR) transcriptional coactivator. It is expressed ubiquitously in tissues and has significant importance in neurons, where it is expressed exclusively from the maternal allele. Thus, while UBE3A has a wide variety of targets in various tissues, it is especially important in the brain as UBE3A regulates several aspects of neuronal growth, function, and maintenance. Therefore deficits of Ube3a cause Angelman Syndrome (AS) and increased dosage causes Autism Spectrum Disorder (ASD), two neurological disorders. The pathological phenotype of both diseases involves behavioral dysfunctions in learning, motor skills, and sociability. Through microarray studies, our laboratory has found that Ube3a is involved in the regulation of neuronal proteins such as CBLN1, which has been found to have significant importance in parallel fiber synapse formation onto Purkinje cells. To determine how regulation of CBLN1 occurs, mutant variants of human Ube3a isoform III were then generated. E3 ligase-dead, substrate-binding defective, nonphosphorylatable mutant, and phosphor-mimetic mutants were produced and inserted into a pLVX-IRES-mCherry vector. A quantitative transcriptional analysis demonstrated that increasing wild-type (WT) UBE3A decreased Cbln1 expression. The ligase dead mutant mimicked the WT suggesting that E3 ligase activity is not required in the regulation of Cbln1. The nonphosphorylatable mutant demonstrated an increase in Cbln1 expression, which may be due to a dominant negative effect on native UBE3A causing its degradation. The phosphor-mimetic mutant had no statistical effect. This may be due to its inability to enter the nucleus and affect transcription. The substrate-binding mutant also showed no statistical effect possibly because of its inability to bind to any substrate and that may be necessary to regulate transcription. These preliminary results demonstrate that UBE3A may be regulating CBLN1 at the transcriptional level independent of its E3 ubiquitin ligase function. Future studies will be required to more precisely determine the mechanisms involved in UBE3A’s regulation of CBLN1. / 2018-06-16T00:00:00Z

<p>Ube3a is a HECT domain E3 ubiquitin ligase originally recognized for its role in degrading p53 in the presence of the human papilloma virus protein E6. Loss of maternal Ube3a expression causes Angelman syndrome, a severe neurodevelopmental disorder characterized by mental retardation, ataxia, epilepsy, lack of speech, and a unique behavioral phenotype that includes a happy demeanor and frequent laughing. However, characterization of the endogenous properties and cellular role for Ube3a has been limited. Over the last few years, an interesting cohort of Ube3a interacting partners and putative substrates were named, though the consequences of these interactions were not thoroughly investigated. These include two Golgi localized proteins - PIST and Golgin-160 - as well as several proteins that can regulate trafficking of proteins at the Golgi apparatus: Src family kinases, ubiquilin, and tuberin. Therefore, we decided to focus on whether Ube3a could regulate Golgi structure or function.</p><p>In this dissertation, I will describe a new role for Ube3a at the Golgi apparatus in the regulation of intralumenal ion homeostasis. First, I characterized the expression pattern of endogenous Ube3a and overexpressed Ube3a isoforms by immunostaining and fractionation and demonstrated that although Ube3a has diffuse nuclear/cytoplasmic localization, it also associates with membrane fractions. I also confirmed that Ube3a interacts endogenously with both PIST and Golgin-160. Next, I demonstrated that Golgi morphology is perturbed in a cell line with stable knockdown of Ube3a. I found that the Golgi apparatus in Ube3a knockdown cells is under-acidifed, and that this is the primary defect underlying the disrupted Golgi morphology. Finally, I extended these findings in vivo and examined the morphology of the Golgi apparatus in the brains of Angelman syndrome model mice. The Golgi structures in the visual cortex of these mice appeared disorganized by immunohistochemistry and individual cisternae were significantly distended by electron microscopy, consistent with a defect in ion homeostasis at the Golgi apparatus. These findings define new cellular role for Ube3a at the Golgi apparatus and provide insight into the pathogenesis of Angelman syndrome.</p> / Dissertation

Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes an E6-AP ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. Although it was previously believed that UBE3A was imprinted in a brain region-specific manner, primarily in the hippocampus and cortex, recent evidence indicates that there is a widespread knockdown of Ube3a protein throughout the AS mouse brain. As a result, it became necessary to evaluate AS human brain samples to verify the relevance and accuracy of the AS mouse model. It was determined that Ube3a is deficient throughout all major brain regions in humans with AS. The remainder of this dissertation work was focused on determining if increased UBE3A expression in the AS mouse brain would be sufficient to rescue the AS phenotype. The results show that adeno-associated virus-mediated UBE3A delivery is not effective in the AS neonatal brain. In the adult AS mouse brain, however, it increased Ube3a in the hippocampus to near wild-type levels. This was sufficient to rescue the associative fear conditioning learning deficit in the AS mouse and improve learning and memory in the Morris water maze. These studies are the first to demonstrate that increased protein production in the adult AS mouse is sufficient to improve the AS phenotype, indicating that the symptoms of AS are not necessarily embryonic developmental.

In this study, nineteen cases of suspected or clinically diagnosed Prader-Willi Syndrome (PWS) were tested for molecular deletions by in situ hybridization with two DNA probes, IR4-3R and GABRB3. Both probes are specific for sequences within the chromosome region 15q11-13, with IR4-3R located within the putative PWS region and GABRB3 in the distal area associated with Angelman Syndrome.

Angelman syndrome (AS) is a rare genetic disorder affecting 1/10,000 to 1/20,000 births. This disorder arises through the genetic disruption of the maternal UBE3A allele, which when coupled with epigenetic silencing of the paternal allele UBE3A allele, gives rise to an absence of UBE3A protein in the central nervous system. Clinical manifestations of the syndrome vary in severity and include poor motor function, deficits in language and severe intellectual impairments. Previous research in the Angelman syndrome mouse model revealed abnormalities in dendritic spine density and morphology of hippocampal pyramidal cells. As seen in humans with AS, mice show abnormal behavioral characteristics that include deficits in motor coordination and ability as well as hippocampal dependent associative fear conditioning. Physiologically, these animals exhibit severe deficits in long-term potentiation (LTP) when compared to wildtype littermates. In an attempt to reduce the time from laboratory study to human translation, we began to search a small molecule library for established compounds with the ability to improve the behavioral and physiological defects normally associated with the AS mouse. One compound, minocycline, was found to normalize the density of dendritic spines in the hippocampus as well as recover the associative memory of AS mice. Moreover, a significant increase in LTP after theta-burst stimulation was also observed in area CA1 hippocampal pyramidal neurons of AS mice treated with minocycline when compared to saline vehicle control mice. These results suggest treatment with minocycline improves synaptic function and learning and memory of AS mice and may provide similar improvements to patients with Angelman syndrome. Twenty-five participants ages 4-12 were enrolled in a clinical trial examining the safety and tolerability of minocycline in children with Angelman syndrome. Patients were evaluated at 3 time points, baseline (T1), after 8 weeks of treatment (T2) and again 8 weeks after the drug was discontinued (T3). Each evaluation was identical and included laboratory testing, EEG recording and neuropsychological examination. Results of the study showed minocycline was safe and well tolerated with only minor adverse effects reported. While no change was observed in EEG recordings, significant increases in the mean clinical global impressions severity scale score were observed after treatment with minocycline. Moreover, participants showed significant improvement in the raw scores of the communication and self-care domains of the Bayley Scale of Infant and Toddler Development, 3rd Edition. These results show for the first time, a therapeutic with the ability to improve the characteristic behaviors of Angelman syndrome. Unfortunately, currently available neuropsychological measures were found to be insensitive to the behavioral phenotype of AS. The primary outcome measure, the Bayley Scale of Infant and Toddler Development, 3rd Edition relies on verbal communication and for the examinee to perform specific tasks on command. These testing methods are not compatible with this patient population and resulted in raw scores outside of 2 standard deviations from the mean. The inability of the participants to perform on these exams led us to develop a novel outcome measure; one that relies on observation rather than verbal communication. 9 children with AS and 7 healthy children were enrolled in an observational study in which 30 minutes of free play activity was video recorded. Using behavioral coding software, 3 independent raters quantified stereotypical AS behaviors as well as communication methods. Speech attempts were categorized into five difference types of vocalizations and revealed children with AS used less advance forms of vocalization consisting mostly of phonation control. Phonetic inventories show mostly front or back vowel usage suggesting little tongue movement occurs during speech production. These results suggest deficits in verbal ability may be related to a childhood apraxia of speech. Impairments in balance and motor coordination have been associated with AS. In an attempt to measure gross motor function, spatiotemporal gait parameters were collected using an electronic walkway and gait analysis software. Results show the gait of children with AS most resembles that of patients with ataxia but without cognitive impairment. In an attempt to develop a single quantitative measure able to describe the severity of gait-related disability, statistical methods were used to create a gait index for patients with AS. The results of this study provides AS researchers with the tools necessary to accurately measure changes in behavior and gait during the clinical evaluation of potential therapeutics

The neuronal synapse is a specialized cell-cell junction that mediates communication between neurons. The formation of a synapse requires the coordinated activity of signaling molecules that can either promote or restrict synapse number and function. Tight regulation of these signaling molecules are critical to ensure that synapses form in the correct number, time and place during brain development. A number of molecular mechanisms that promote synapse formation have been elucidated, but specific mechanisms that restrict synapse formation are less well understood. The findings presented within this dissertation focus on how a specific Rho guanine nucleotide exchange factor (GEF) Ephexin5 functions to restrict early synaptic development and how perturbations in Ephexin5 signaling may lead to human neurodevelopmental disease.

Reciprocal changes in UBE3A gene dosage cause two neurodevelopmental disorders. Maternally inherited deletions of UBE3A cause Angelman syndrome, characterized by intellectual disability, motor defects, seizures, and a pathognomonic increased social motivation. Whereas maternally inherited triplications of UBE3A as in Idic15 Autism underlie decreased sociability and increased repetitive restrictive behaviors of this disorder. Identifying the cellular compartments and neuronal subtypes where excess and loss of Ube3a impair behavior is essential to understanding and potentially treating the disorders. In Chapter 2, we show that mouse models of maternal loss of UBE3A (Ube3a-mKO, Angelman syndrome) and triplication of UBE3A (Ube3a-2x, Idic15) have opposite effects on social behavioral and cortical gene expression. Social preference and social vocalizations are reduced in Ube3a-2x and increased in Ube3a-mKO mimicking the human phenotypes. Using a nuclear localizing signal tagged Ube3a transgenic mouse (Ube3a-NLS), we show that Ube3a acts in the nucleus to impair social behavior and cortical gene expression. Many of the genes reciprocally regulated by nuclear Ube3a are part of an Autism protein-interaction network. In Chapter 3, we demonstrate Ube3a-2x mice have increased aggression, an important Autism comorbidity. In contrast, maternal loss of Ube3a reduces aggression, consistent with the gregarious, amiable nature of individuals with Angelman syndrome. We then mapped the loci where increased Ube3a increases aggression. Increased aggression was observed when Ube3a was targeted to glutamatergic and vasopressinergic but not to GABAergic or oxytocinergic neurons. In Chapter 4, we show that in mice, social behavior is downregulated by social experience via Ube3a. In wild-type mice, altering their social environment strongly regulates their social behavior: individual housing causes hypersocial whereas group housing causes hyposocial behavior. In Ube3a-2x animals, group housing caused an excessive downregulation of social behavior whereas single housing fully rescued their social behavior deficits. By contrast, in Ube3a-mKO animals, the suppressive effects of group housing on social experience was largely blocked, suggesting Ube3a is required for this process. In summary, this thesis characterizes the role of UBE3A gene dosage in regulating social and aggression behaviors and identifies the subcellular compartment and neuronal subtypes where changes in Ube3a gene dosage disturb the homeostasis of these behaviors.

abstract: Angelman syndrome (AS) is a neurodevelopmental disorder characterized by developmental delays, intellectual disabilities, impaired language and speech, and movement defects. Most AS cases are caused by dysfunction of a maternally-expressed E3 ubiquitin ligase (UBE3A, also known as E6 associated protein, E6-AP) in neurons. Currently, the mechanism on how loss-of-function of the enzyme influences the nervous system development remains unknown. We hypothesize that impaired metabolism of proteins, most likely those related to E6-AP substrates, may alter the developmental trajectory of neuronal structures including dendrites, spines and synaptic proteins, which leads to disrupted activity/experience-dependent synaptic plasticity and maturation. To test this hypothesis, we conducted a detailed investigation on neuronal morphology and electrophysiological properties in the prefrontal cortex (PFC) layer 5 (L5) corticostriatal pyramidal neurons (target neurons). We found smaller soma size in the maternal Ube3a deficient mice (m-/p+; 'AS' mice) at postnatal 17-19 (P17-19), P28-35 and older than 70 days (>P70), and decreased basal dendritic processes at P28-35. Surprisingly, both excitatory and inhibitory miniature postsynaptic currents (mEPSCs and mIPSCs) decreased on these neurons. These neurons also exhibited abnormalities in the local neural circuits, short-term synaptic plasticity and AMPA/NMDA ratio: the excitatory inputs from L2/3 and L5A, and inhibitory inputs from L5 significantly reduced in AS mice from P17-19; Both the release probability (Pr) and readily-releasable vesicle (RRV) pool replenishment of presynaptic neurons of the target neurons were disrupted at P17-19 and P28-35, and the change of RRV pool replenishment maintained through adulthood (>P70). The AMPA/NMDA ratio showed abnormality in the L5 corticostriatal neurons of PFC in AS mice older than P28-35, during which it decreased significantly compared to that of age-matched WT littermates. Western Blot analysis revealed that the expression level of a key regulator of the cytoskeleton system, Rho family small GTPase cell division control protein 42 homolog (cdc42), reduced significantly in the PFC of AS mice at P28-35.These impairments of synaptic transmission and short-term synaptic plasticity may account for the impaired neuronal morphology and synaptic deficits observed in the PFC target neurons, and contribute to the phenotypes in AS model mice. The present work reveals for the first time that the E6-AP deficiency influences brain function in both brain region-specific and age-dependent ways, demonstrates the functional impairment at the neural circuit level, and reveals that the presynaptic mechanisms are disrupted in AS model. These novel findings shed light on our understanding of the AS pathogenesis and inform potential novel therapeutic explorations. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2017