2008 — 2011 |
Reiter, Lawrence T |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Proteomics in Drosophila to Identify Autism Candidate Substrates of Ube3a @ University of Tennessee Health Sci Ctr
[unreadable] DESCRIPTION (provided by applicant): Autism spectrum disorders (ASD) affect ~60 in 10,000 children but in only ~10% of these individuals is autism associated with a recognized cause. Understanding the molecular pathways dysregulated in Angelman syndrome (AS), a rare and severe developmental disorder related to autism, may provide key insights leading to identification of autism susceptibility genes and pathways. Approximately 3% of all autism cases result from maternal duplications of the region containing the AS gene UBE3A. Variants in genes encoding protein targets of the ubiquitin ligase UBE3A may, therefore, confer a genetic susceptibility to autism or even cause an ASD phenotype. Here we describe a proteomics strategy utilizing the powerful genetic model organism Drosophila melanogaster to identify protein targets of human UBE3A and fly Dube3a. UBE3A will be over-expressed in the brains of flies using the GAL4/UAS system in order to increase or decrease the levels of UBE3A/Dube3a protein targets. We will then identify these targets by Rotofor-assisted proteomic profiling and mass spectrometry. Potential targets will be validated though genetic interactions in the eye and neuromuscular junction, binding assays in 293T cells and immunohistochemistry in the brains of both Ube3a-deficient and over-expressing mice. We anticipate that these studies will provide us with new autism candidate genes for future validation in families that demonstrate heritable autism risk. To this end, we propose the following specific aims: Specific Aim 1: To identify potential UBE3A and Dube3a regulated proteins using proteomic profiling in Drosophila head extracts. Completion of this aim will result in a collection of potential Dube3a and UBE3A regulated proteins for subsequent validation and autism genetic studies. Specific Aim 2: To validate physical, biochemical and genetic interactions between potential targets and Dube3a. This aim will test the hypothesis that the proteins detected in Aim #1 are regulated directly or indirectly by Dube3a. Specific Aim 3: To determine if UBE3A regulated protein expression patterns are altered in the brains of Ube3a deficient and over-expression mice. This aim will demonstrate a link between Ube3a target proteins and altered brain function in the mouse models of Angelman syndrome and proximal 15q duplication autism. PUBLIC HEALTH RELEVANCE: The primary goal of this research is to identify proteins regulated by UBE3A and to investigate the possibility that these proteins are also dysregulated or mutated in some cases of autism. Understanding how increased levels of UBE3A result in an autism phenotype at the molecular level will help us better identify and treat the underlying liability in idopathic autism. [unreadable] [unreadable] [unreadable]
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0.988 |
2012 — 2013 |
Reiter, Lawrence T |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Tooth Pulp as a Source For Neuronal Precursor Cells to Study Neurogenetic Disorde @ University of Tennessee Health Sci Ctr
DESCRIPTION (provided by applicant): The study of neurogenetic disorders like Angelman, Rett and fragile X syndromes as well as autism spectrum disorders has depended largely on the analysis of gene/protein expression in non-neuronal biospecimens like lymphoblast and fibroblast cell lines. Although some analysis has been possible in post-mortem human brain tissue for neurogenetic syndromes, this tissue is often of variable quality and in limited amounts. Recently there has been an initiative to induce pluripotent stem cells (iPSCs) from patient fibroblasts and differentiate these iPSCs into various neuronal lineages. There are several problems with this approach: 1) fibroblasts must be obtained though a fairly invasive skin biopsy which leaves a scar and causes undue pain and distress in developmentally disabled or autistic children; 3) induction of fibroblasts into stem cells (reprogramming) and then into neuronal lineages is a laborious task that may not maintain epigenetic marks on the DNA that are essential to proper gene regulation in the native neuronal tissues; and 4) viral vectors which are themselves immunogenic are used to reprogram iPSCs limiting the downstream aplication of these neurons for therapeutic transplantations. Here we propose to use dental pulps from exfoliated primary teeth (EPT), as a source of stem cells for the study of a variety of neurogenetic syndromes. Dental pulp stem cells differentiate into functional neurons in vitro and in vivo. Dental pulps from EPT are also an easily obtainable source of cranial neural crest (CNC) cells. The majority of the cells in the peripheral nervous system, including neurons, are derived from the neural crest. Based on the extensive collaborative sum of our expertise in genetics, dental pulp stem cells and neurophysiology, we feel that this novel approach will provide a fresh perspective to the way we study expresion changes, epigenetics and neurophysiology in an ex-vivo model system for human neurogenetic disorders. ! PUBLIC HEALTH RELEVANCE: The primary goal of this proposal is to develop a method to investigate the neurons of patients with neurogenetic disease that uses shed teeth (primary teeth or puled teeth). If we can determine that tooth samples provide the right type of cells in th dental pulp to make neurons in culture we will have a new way to look at the gene expression and physiology of these patient-derived neurons. Having these cultures could lead to new insights into the mechanisms of human neurogenetic disease and even normal neuronal development and function.
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0.988 |
2017 — 2018 |
Reiter, Lawrence T |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
An in Vivo Chemical Screen For Seizure Suppression in Duplication 15q Syndrome. @ University of Tennessee Health Sci Ctr
The objective of this study is to perform a chemical screen to identify currently approved medications that can be repurposed as potential anti-epileptics in both Duplication 15q syndrome (Dup15q) and inherited epilepsy. Children with Dup15q have an extremely high incidence of seizures that often begins in the post-natal stage as infantile spasms and later in childhood progresses to epilepsy that is difficult to manage using currently available antiepileptic medications. In this proposal we use a newly developed Drosophila model of Dup15q syndrome that recapitulates the seizure phenotypes observed in humans. We will perform high-throughput screening in fly larval brains to identify compounds that can re-activate gene expression for a specific set of synapse associated genes down regulated in the Dup15q model, some of which are also associated with inherited human epilepsies. We will then attempt to suppress the seizure phenotype in our model using these compounds from our screen and also investigate the molecular consequences of this chemical suppression of seizures. These studies have broad therapeutic implications not only for pediatric disorders like Dup15q syndrome, but also for other inherited forms of epilepsy which result from de novo mutations in synaptic genes. Since the compound libraries we are using contain previously approved drugs, it will be possible to quickly translate our findings to clinical practice in off-label therapeutic intervention studies for epilepsy.
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0.988 |
2021 |
Reiter, Lawrence T Zakharenko, Stanislav S (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Ube3a in Gliopathic Seizures @ University of Tennessee Health Sci Ctr
As many as ~3 million individuals suffer from epileptic seizures in the US alone. Although some forms of epilepsy respond quite well to anti-seizure medications, a substantial portion of individuals suffering from epilepsy do not respond well to medication or dietary treatments. One way to investigate the underlying molecular and cellular pathology of seizure disorder is by studying a genetically defined syndrome where epilepsy is a prominent feature. Approximately 25-50% of individuals with Duplication 15q syndrome (Dup15q) suffer from difficult to control seizures. The prevailing hypotheses to explain seizures in Dup15q are maternal specific neuronal elevation of the ubiquitin E3 ligase UBE3A and/or duplication of a cluster of GABA receptor genes located adjacent to UBE3A at 15q11.2-q13.1. Studies using the fruit fly, Drosophila melanogaster, from our laboratory indicates that these seizures may be caused by elevated levels of UBE3A in glia, not neurons as previously proposed. The premise for this proposal is that seizures are caused by elevated levels of the UBE3A protein in glia, not neurons, providing not only a pathway to molecular mechanism for Dup15q related epilepsy, but also a paradigm shift, directly implicating glial cells in the etiology of seizures. The experiments outlined in this proposal are designed to investigate the developmental timing and molecular changes in glial cells over-expressing Dube3a and to reveal how these changes are recapitulated in a new mouse model we developed that expresses Ube3a in glial cells. Everything we learn from these studies in flies will be directly disseminated to a team of seizure experts who work at the Duplication 15q centers of excellence throughout the country. The identification of new therapeutic targets for Dup15q epilepsy may also provide new treatment options to individuals who are pharmacoresistant to current anti-epileptics.
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0.988 |