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.

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.

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

Abstract The Prader-Willi (PWS) and Angelman (AS) syndromes are clinically distinct developmental disorders which are caused by genetic defects in the imprinted domain at chromosome 15q11-q13, resulting in the loss of paternal (PWS) or maternal (AS) gene function. In this study, the genetic changes of 15q11-q13 and their possible inheritance in Finnish PWS (n=76) and AS (n=47) patients are described. The diagnosis could be confirmed by laboratory methods in all PWS and in 43 (91%) AS patients. A deletion of 15q11-q13 accounted for 76% of the PWS and 67% of the AS patients in whom a specific genetic defect had been established. The origin of deletion was always paternal in PWS and maternal in AS. In PWS, deletions of four different sizes were detected, while in AS only type I or II deletions were found. The smallest overlap of deletions/critical region detected was from locus D15S13 to locus D15S10 in PWS and from locus D15S128 to locus D15S12 in AS. A rare recurrence of del(15)(q11q13) due to maternal germ line mosaicism is described. Maternal uniparental disomy of chromosome 15 accounted for 21% of PWS patients and paternal UPD for 2% of AS patients. In PWS, most UPD cases were due to errors in maternal meiosis (87%), most commonly leading to maternal heterodisomy (MI error). In AS, a rare error in the second segregation of paternal meiosis was found. UPD was associated with advanced maternal age, the mean being 34.6 years. Imprinting defects were found in 3% of PWS (two non-IC-deletions) and 11% of AS (IC deletion in one sib pair and three non-IC-deletions) patients. In the case with IC deletion, the mutation was seen in several generations. The non-deletion cases were thought to be due to a de novo prezygotic or postzygotic error. In the non-deletion PWS cases, the maternally imprinted paternal chromosome region was shown to have been inherited from the paternal grandmother, while in AS the paternally imprinted maternal chromosome region had been inherited from either the maternal grandfather or the maternal grandmother. The region of IC involved in AS was defined to be 1.15 kb. Five (11%) AS patients with normal DNA methylation test results had a UBE3A mutation. One of the two novel missense mutations (902A→C) had been inherited from the mosaic mother, while the mutation 975T→C was a new one. De novo deletions 1930delAG and 3093delAAGA have also been described previously, suggesting that these sites may be mutation hotspots in UBE3A. Identification of different genetic aetiologies with different recurrence risks is essential for genetic counselling, and close co-operation between clinicians and the laboratory is required both for diagnosis and for the detection of possible inheritance.

The prevalence of Autism Spectrum Disorder (ASD) and its defining features has been increasingly investigated in genetic syndromes associated with intellectual disability, with syndrome specific profiles reported. The experience of catatonia and other movement disorders in people with ASD has been increasing highlighted within both research and diagnostic guidelines. However, these issues have not typically been investigated alongside other features of ASD within research into genetic syndromes. The first paper in this thesis provides a review of the literature on movement disorders in genetic syndromes associated with ASD, which focuses on the prevalence of reported movement disorders, the methods of assessment used, and the quality of research to date. An empirical study is reported in Paper 2. Within a cohort of individuals with Cornelia de Lange and Fragile X syndromes the prevalence of attenuated behaviour [autistic catatonia] is examined, based on parent/carer report, and the extent to which features of ASD predict later attenuated behaviour is investigated. Paper 3 provides a critical reflection on the first two papers as well as some wider considerations on undertaking research in this area. The results of both the literature review and the empirical study indicated that across a number of genetic syndromes (Angelman syndrome, Cornelia de Lange syndrome, Fragile X syndrome and Rett syndrome) attenuated behaviour [autistic catatonia] and/or movement disorders affect a substantial proportion of individuals. Furthermore, repetitive behaviours, one of the characteristic features of ASD, appear to predict later attenuated behaviour in Cornelia de Lange and Fragile X syndromesThe results presented in this thesis have important implications for the way services support individuals with specific genetic syndromes. Paper 1 confirms the high prevalence of movement problems in Angelman and Rett syndromes, and Paper 2 provides a new insight into movement problems in Cornelia de Lange and Fragile X syndromes. Movement disorders are reported to impact negatively on wellbeing and quality of life in people with ASD, and are likely to have a similar impact on the lives of people with genetic syndromes. Greater awareness and recognition of movement problems in CdLS and FXS is required, and although specialist services may already be aware of some of the above issues, there should be an increased emphasis on ensuring that community services are aware of the needs of individuals with genetic syndromes, including the implications of movement problems for support needs and quality of life.

Angelman Syndrome (AS) is a severe neurodevelopmental disorder that affects 1:12000 newborns. It is characterized by mental retardation, delayed major motor and cognitive milestones, seizures, absence of speech and excessive laughter. The majority of AS cases arise from deletions or mutations of UBE3A gene located on the chromosome 15q11-13. UBE3A codes for E3-ubiquitin ligase that target specific proteins for degradation. To date, a wide variety of Ube3a substrates has been identified. The accumulation of Ube3a-dependent proteins and their effect on the multitude of signal transduction pathways are` considered the main cause of the AS pathology. While the majority of research has been directed towards target identifications, the overall role of Ube3a in activity-dependent synaptic plasticity has been greatly overlooked. The present work is designed to fill some of these knowledge gaps. Chapter 2 is focused on the activity-dependent aspect of Ube3a expression following neuronal stimulation in vivo and in vitro. We examined total Ube3a expression followed by KCl depolarization in neuronal primary culture. By utilizing a subcellular fractionation technique, we were able to determine which cellular pools are responsive to the depolarization. Next, a fear conditioning paradigm (FC) was used to activate neurons in the paternal Ube3a-YFP reporter mouse brain. This mouse model allowed us to resolve spatial and temporal alterations of the maternal and the paternal Ube3a in hippocampus and cortex followed by FC. In accordance to KCl depolarization results, we observed alterations in Ube3a protein but at later time points. Furthermore, we investigated if the absence of activity-dependent Ube3a changes has any effect on learning and memory kinase activation. We utilized KCl and FC to determine synaptic activity-induced ERK 1/2 phosphorylation in acute hippocampal slices and in CA1 area of hippocampus of wild type (Ube3a m+/p+) and Ube3a deficient mice (Ube3a m-/p+). We demonstrated that Ube3a loss leads to impaired activity-dependent ERK 1/2 phosphorylation. It has been established that Ube3a m-/p+ mice have a profound deficit in LTP, implying the importance of this ligase in excitatory synaptic transmission. The abnormal LTP could be partially explained by an aberrant CaMKII function, decreased activity-dependent ERK 1/2 phosphorylation and reduced phosphatase activity. These proteins have also been implicated in another form of synaptic plasticity such as long-term depression (LTD). Chapter 3, we investigated the contribution of Ube3a to NMDAR - dependent and - independent LTD. Our data showed that Ube3a m-/p+ P21-30 animals exhibit the impairments in both forms of LTD. Next, we focused on elucidating molecular mechanism underlying the reduced mGluR1/5-LTD. We discovered that mGluR1/5 kinase activation such as ERK, mTOR and p38 is not affected by Ube3a loss. In concordance with previous work, we detected increased Arc expression together with abnormal AMPAR distribution in the Ube3a m-/p+ hippocampus. Surprisingly, the mGluR1/5 induced GluR2 trafficking was normal. Our findings infer that elevated Arc levels together with the increased internalization of AMPAR may result in compromised basal state of the synapses leading to a more depression-like state in Ube3a m-/p+ mice. Evidence points that loss of Ube3a produces alterations in a variety of activity-dependent signal transduction cascades that may ultimately result in impaired synaptic plasticity and cognition. Similar to AS, abnormal molecular and behavioral phenotypes have already been observed in other mouse models of human mental retardation such as Fragile X Mental Retardation Syndrome (FXS). Chapter 4 is set to explore if any correlation can be found in between these neurodevelopmental disorders. Analysis of crude synaptoneurosomes of adult Fmr1 KO mice revealed a significant reduction in Ube3a protein. Additionally, a blunted translation of Ube3a in response to mGluR1/5 stimulation was observed. However, we didn't find any evidence of direct interaction between Ube3a mRNA and Fragile X Mental Retardation Protein (FMRP). To examine if some of the pathology seen in Fmr1 KO mice is due to Ube3a downregulation, we performed a rescue experiment by increasing overall levels of Ube3a in hippocampus of FRMP deficient mice. An exhaustive battery of behavioral testing indicated that alterations of Ube3a expression impacted only associative fear conditioning. In summary, the present work has attempted to answer some of the fundamental questions about Ube3a and its role in synaptic plasticity. We have demonstrated that Ube3a expression is modulated by synaptic activation and its activity-dependent alterations are essential for normal brain functioning. Additionally, our data suggest that Ube3a is not only significant for the synaptic excitation but also crucial for the synaptic depression. Finally, our findings indicate that the alteration of Ube3a expression may contribute to the cognitive phenotypes in other neurodevelopmental disorders such as FXS suggesting an advantage of exploring Ube3a function outside the AS research.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 141 pages. Includes Vita. Includes bibliographical references.