Rett Syndrome Disorders and Angelman Syndrome as Genetic Models of Autism Spectrum Disorders
Julie Davidson – Vanderbilt University
$25,000 –  RDCRN Training Grant

This investigator will use selective phenotype study techniques to study children 3-10 years of age with AS, Rett syndrome ( RTT), and MeCP2 duplications, as genetic models of autism spectrum disorder (ASD).  It is hoped that 15 individuals from each group will be studied.  Dr. Davidson plans to use a combination of clinical assessment and passive psycho-physiological measures, including eye-tracking and event-related potentials (ERP).  ERP offers a non-invasive method of assessing the functioning of social brain circuitry and social relatedness, and does not require an overt response from the patient.   By contrasting these groups of patients, they will begin to tease apart genetic contributions to ASD symptoms and, for RTT and MeCP2 duplications in particular, the possible contributions of gene dosage to ASD features and developmental course.

Preclinical testing of a candidate Angelman syndrome therapeutic
Benjamin Philpot, PhD – University of North Carolina at Chapel Hill
$200,000 – 2-years

This grant represents a continuation of funding for this investigator’s laboratory which was instrumental in identifying that a class of topoisomerase inhibitors can unsilence the paternal UBE3A gene.  The investigator now plans to extend this work by additional experiments on topoisomerase inhibitors in the animal mouse model to determine how persistent the silencing effect is.  They will also determine drug administration parameters by evaluating intracerebral, intrathecal and IV administration of topoisomerase inhibitor agents.  They will also study tissue distribution, drug toxicity and pharmacokinetic data in the mouse model.  Finally, the rescue effects of the inhibitor medications will be studied by brain slice physiology, in vivo neuro-plasticity studies and by studies of the behavioral recovery/rescue using customary behavioral tests.  These additional studies are hoped to lay the groundwork for development of an Angelman syndrome therapeutic agent that may be available for subsequent human trials, provided issues of toxicity and efficacy are established in the mouse model.

Molecular mechanisms and biomarkers of a candidate Angelman syndrome therapeutic
Mark Zylka, PhD – University of North Carolina at Chapel Hill
$200,000 – 2-years

Dr. Zylka’s lab will expand on the work previously funded by ASF that led to the identification of a family of topoisomerase inhibitors that can unsilence the paternal UBE3A gene.  In order to learn more about this topoisomerase-linked phenomenon, this lab plans to conduct further experiments to understand the mechanisms whereby topoisomerase regulates UBE3A expression.  They will use RNA interference to knock down topoisomerase proteins in the mouse and then evaluate subsequent UBE3A expression.  They will analyze UBE3A antisense transcripts to determine how they are biomarkers for the effects of topoisomerase inhibitor actions.  They also plan to determine antisense and sense transcript levels in active and in silenced neurons and they will evaluate genome wide expression of other known imprinted genes to see if they are also affected by these inhibitors. Knowing more about how topoisomerase inhibitors mechanistically regulate Ube3a expression could identify biomarkers that might then be used as clinical indicators of drug efficacy.  Work by this investigator raises the exciting possibility that small molecule compounds could be used as an Angelman syndrome therapeutic.

The role of antisense RNA Ube3a-ATS in Ube3a imprinting and Angelman syndrome
Arthur Beaudet, MD, PhD – Baylor College of Medicine, Houston, TX

Angelman syndrome is caused by deficiency of UBE3A. Unlike ordinary autosomal genes, it is subject to genomic imprinting with expression only from maternal chromosome. It is unknown how such imprinted status is established, since no differential DNA methylation was found to be associated with the UBE3A locus. On the other hand, an antisense non-coding RNA named UBE3A-ATS overlapping UBE3A locus was identified with mono-allelic expression from paternal chromosome. Extensive negative association between Ube3a-ATS and Ube3a was reported from both mice and human studies. In this proposal, we hypothesize that Ube3a-ATS directly mediates paternal Ube3a silencing by inhibiting its transcription elongation. To test this, we have generated a mouse model with truncated Ube3a-ATS expression. Studying of this mouse model will reveal the role of Ube3a-ATS in Ube3a imprinting and Angelman syndrome. Binding of transcription initiation complex to Ube3a promoter was found to be equal on both paternal and maternal chromosomes. To test if paternal Ube3a silencing is caused by failure of transcription elongation, nascent RNAs immunoprecipitated (RNA-IP) with RNA polymerase II will be analyzed. Finally, profiles of histone modifications of Ube3a will be studied by ChIP (chromatin-immunoprecipitation)-chip, to further expand our knowledge about Ube3a imprinting. Overall, we propose to study the role of antisense RNA Ube3a-ATS in Ube3a imprinting and their interaction using mouse models. UBE3A-ATS may be developed as new therapeutic target for inactivation in Angelman syndrome to reactivate silenced paternal UBE3A, and our research will provide unique insight into the relevant regulatory mechanisms.

Validation of Arc and Ephexin5 as Novel Therapeutic Targets for the Treatment of Angelman Syndrome
Michael Greenberg, PhD – President and Fellows of Harvard College, Harvard Medical School
$200,000 – 2-years

While it has been appreciated for some time that loss-of-function mutations in the UBE3A gene are the primary causative factor for Angelman syndrome (AS), the precise mechanisms by which the loss of Ube3A in the nervous system gives rise to the spectrum of cognitive and behavioral features characteristic of this disorder remain unclear. This gap in knowledge has significantly limited the scope of clearly defined therapeutic strategies for AS. Moreover, therapeutic interventions based on the reactivation of mutated genes such as UBE3A have historically proven extremely challenging to implement. Thus, identification of the molecular mechanisms by which the loss of Ube3A function gives rise to the neurophysiological and cognitive dysfunction associated with AS will provide important insight into AS etiology and open new potential therapeutic avenues to combat AS.  Dr. Greenberg’s lab has recently identified two neuronal proteins, termed Arc and Ephexin5, that act as downstream targets of Ube3A. In the absence of Ube3A activity, elevated levels of these two proteins accumulate in neurons, and reduction of the levels of these factors in Ube3A-deficient neuronal cultures is sufficient to reverse specific cellular deficits associated with AS. Building on these findings, they plan to determine the extent to which elevated Arc or Ephexin5 expression contributes to the neurophysiological and cognitive dysfunction associated with AS in the context of a well-characterized mouse model of the disorder. Implication of one or more these factors in specific aspects of AS pathology will provide new opportunities for therapeutic intervention

Pathophysiology in a human stem cell model of Angelman syndrome
Eric Levine, PhD – University of Connecticut Health Center, Farmington, CT
$120,000 – 2-years

The discovery of genomic reprogramming of human skin cells into induced pluripotent stem cells (iPSCs) provides a novel way to model human diseases with complex genetics. By reprogramming skin cells obtained from patient samples, cell lines can be isolated that have the potential to develop into functional brain cells. We have recently succeeded in reprogramming skin samples from AS patients, as well as age-matched control subjects, into iPSCs, and then differentiated the iPSCs into functional brain cells that maintain the specific genetic profile that is seen in AS patients. The proposed research will use these patient-specific cell lines to explore the underlying physiological and morphological defects in the brain cells of AS patients. Establishment of a human cell culture model of AS provides a unique opportunity to understand the cellular mechanisms that underlie the behavioral and developmental deficits observed in AS patients. This model may also be valuable for identifying novel targets for drug discovery and for screening potential therapeutics aimed at ameliorating and/or curing the seizures, movement disorders, and language and cognitive impairments in Angelman syndrome.

An inducible mouse model for Angelman Syndrome: Follow up
Ype Elgersma, PhD – Erasmus University Medical Center, Rotterdam, Netherlands
$92,144 – 1-year

With previous support from the ASF, Dr. Elgersma engineered a UBE3A- inducible mouse in which the UBE3A gene (responsible for causing AS) is non-functional when the mouse is born.  However, by a medication injection at any desired age, this inducible mouse model could now restore UBE3A gene function in every cell, including the brain.  Dr. Elgersma, under this grant funding will now study how to restore gene function at various ages and will assess the phenotype of these genetically cured mice using various behavioral and electrophysiological assays. This research work will address the issue as to what extent the UBE3A gene is involved in brain development, and to what extent one can reverse the symptoms.  Although not offering a specific therapeutic intervention for patients, this work may provide pivotal proof of principle that a putative mechanism-based treatment is still effective after the onset of the neurological symptoms. This knowledge is essential for future trials, aimed at alleviating the symptoms of AS. In addition, this project will give us valuable information in understanding the role of UBE3A in brain development and/or in mature brain function.

Examining rescue of neurological deficits in Angelman syndrome mice by expression of the E6-AP isoforms
Scott Dindot, PhD – Texas A & M University, College Station, TX

In this study, Dr. Dindot plans to investigate whether gene therapy in the AS mouse (using lentiviruses as the vector) is a viable therapeutic option, and thus a future possibility for individuals with AS. He will also examine the function of the E6-AP (UBE3A) isoforms. He will use live animal testing of brain function and cell morphological studies in a mouse model of AS to determine if gene therapy can ameliorate the neurological deficits exhibited by these mice. This proposal will examine two unresolved questions regarding gene therapy in AS. First, he will examine whether re-introduction of UBE3A expression into the brain of post-adolescent and adult mice rescues abnormal neuronal morphology, reduces seizures, enhances learning and memory, and improves motor-coordination. Second, he will examine the efficacy of the 3 E6-AP isoforms to improve brain function in AS mice with the intent of identifying a particular isoform that can be used in gene therapy studies.