Date of Award
Doctor of Philosophy (PhD)
Lawrence T. Reiter, PhD
T.J. Hollingsworth, PhD; Jamy C. Peng, PhD; Tiffany N. Seagroves, PhD; Heather S. Smallwood, PhD
Autism; Circadian Rhythm; Dental pulp stem cells; MAGEL2; Neurogenetics; Prader-Willi syndrome
Prader-Willi syndrome (PWS) is a complex multigenic neurodevelopmental disorder resulting in hypotonia, developmental delay, hypogonadism, sleep dysfunction and childhood onset obesity affecting 1 in 10,000 to 30,000 individuals. PWS is an imprinting disorder that is caused by a loss of expression of maternally imprinted genes in the 15q11.2-q13 region including NDN, MAGEL2, SNRPN/SNURF, and a cluster of snoRNAs. The majority of cases are caused by inheriting a paternal allele deletion of this region (65-75%) and a smaller number are caused by chromosome 15 maternal uniparental disomy (UPD) (20-30%) or imprinting center defects (1-3%). Here, we used dental pulp stem cells (DPSC), differentiated to neuronal cultures, to investigate the molecular and cellular phenotypes of PWS neurons. DPSC are multipotent stem cells that accurately recapitulate the epigenetic signature of embryonic stem cells and can differentiate to a variety of cell types. Our group has collected over 40 individual PWS lines across the different genetic subgroups. In Chapter 2, we investigated the transcriptional differences in PWS subtypes. While the UPD subtype has milder classical PWS phenotypes, there is an increased risk of developing autism spectrum disorder (ASD) (30% versus 18%) and cycloid psychosis. Using RNA sequencing (RNAseq), we found a global decrease in mitochondrial transcript expression and a mitochondrial aggregate phenotype in PWS UPD +ASD neurons. In Chapter 3, we optimized a bioluminescent assay to visualize the circadian rhythms in PWS neurons. Additionally, we used this assay to analyze circadian rhythms in neurons from individuals with Schaaf-Yang syndrome (SYS), a similar syndrome resulting from mutations in MAGEL2. Using this assay, we found discordant circadian cycling in both PWS and SYS. We also identified two distinct period length phenotypes in PWS neurons that were significantly different than neurotypical control neurons. In Chapter 4, we used a novel approach to single-cell sequencing (SPLiT-Seq) to identify transcriptional differences between neuronal populations from PWS and neurotypical control subjects. In addition to finding several transcripts differentially expressed that are related to PWS phenotypes such as hypogonadism and endocytic recycling, we found a delay in neurogenesis in the PWS neurons. In Chapter 5, we focused our attention on another obesity disorder for comparison. We used RNAseq to determine the transcriptional similarities and differences between PWS, Rapid-Onset Obesity with Hypothalamic Dysregulation, Hypoventilation, and Autonomic Dysfunction (ROHHAD) and Congenital Central Hypoventilation syndrome (CCHS). ROHHAD presents with several key similarities to PWS including rapid-onset obesity and hypothalamic dysfunction. In this study, we found six transcripts differentially expressed in both PWS and ROHHAD and that these transcripts were not related to an obesity pathway or hypothalamic function. Finally, I discussed how the assays and methods described here provide a pipeline for investigating other neurogenetic disorders and how the data discussed in each of these chapters connects to possible defects caused by the spectrum of PWS genetic changes.
Victor, Anna K. (https://orcid.org/0000-0002-2242-9858), "Molecular and Cellular Investigations of Prader-Willi Syndrome" (2023). Theses and Dissertations (ETD). Paper 634. http://dx.doi.org/10.21007/etd.cghs.2023.0621.
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