ItemCharacterizing Femoral Structure of the Ts66Yah Mouse Model of Down Syndrome(2023-08) Sloan, Kourtney; Roper, Randall J.; Li, Jiliang; McNulty, Margaret A.; Picard, Christine J.Down syndrome (DS) is caused by the partial or complete trisomy of human chromosome 21 (Hsa21) and can result in skeletal deficits, including lower bone mineral density (BMD) and increased risk of fracture and osteoporosis or osteopenia earlier than the general population. Mouse models of DS have been developed to understand the genetic mechanisms resulting in these phenotypes, but models differ due to the complex genetic nature of DS and differing genome structures between humans and mice. Ts65Dn mice have been a popular model of DS as they contain ~50% of Hsa21 orthologous genes on a freely segregating minichromosome, but there is speculation that the phenotypes are exaggerated by non-Hsa21 orthologous trisomic genes also present. To address this issue, the Ts66Yah mouse model was developed to remove the non-Hsa21 orthologous trisomic genes. In this study, male and female Ts66Yah mouse femurs were evaluated during bone accrual and peak bone mass to investigate structural differences using micro-computed tomography. Additionally, the role of trisomic Dyrk1a, a Hsa21 gene previously linked to bone deficits in Ts65Dn mice, was evaluated through genetic and pharmacological means in Ts66Yah femurs at postnatal day 36. Ts66Yah mice were found to have little or no trabecular deficits at any age evaluated, but sex-dependent cortical deficits were present at all ages investigated. Reducing Dyrk1a copy number in Ts66Yah mice significantly improved cortical deficits but did not return cortical bone to euploid levels. Pharmacological treatment with DYRK1A inhibitor L21 was confounded by multiple variables, making it difficult to draw conclusions about DYRK1A inhibition in this manner. Overall, these results indicate trabecular deficits associated with Ts65Dn mice may be due to the non-Hsa21 orthologous trisomic genes, and more Hsa21 orthologous trisomic genes are necessary to produce trabecular deficits in DS model mice. As more mouse models of DS are developed, multiple models need to be assessed to accurately define DS-associated phenotypes and test potential treatments. ItemExploring the Effects of Ancestry on Inference and Identity Using Bioinformatics(2023-08) Herrick, Noah; Walsh, Susan; Picard, Christine; Wilson, Jeremy; Balakrishnan, Lata; Roper, RandallAncestry is a complex and layered concept, but it must be operationalized for its objective use in genetic studies. Critical decisions in research analyses, clinical practice, and forensic investigations are based on genetic ancestry inference. For example, in genetic association studies for clinical and applied research, investigators may need to isolate one population of interest from a worldwide dataset to avoid false positive results, or in human identification, ancestry inferences can help reveal the identity of unknown DNA evidence by narrowing down a suspect list. Many studies seek to improve ancestry inference for these reasons. The research presented here offers valuable resources for exploring and improving genetic ancestry inference and intelligence toward identity. First, analyses with ‘big data’ in genomics is a resource-intensive task that requires optimization. Therefore, this research introduces a suite of automated Snakemake workflows, Iliad, that was developed to give the research community an easy-to-learn, hands-off computational tool for genomic data processing of multiple data formats. Iliad can be installed and run on a Google Cloud Platform remote server instance in less than 20 minutes when using the provided installation code in the ReadTheDocs documentation. The workflows support raw data processing from various genetic data types including microarray, sequence, and compressed alignment data, as well as performing micro-workflows on variant call format (VCF) files to merge data or lift over variant positions. When compared to a similar workflow, Iliad completed processing one sample’s raw paired-end sequence reads to a human-legible VCF file in 7.6 hours which was three-times faster than the other workflow. This suite of workflows is paramount towards building reference population panels from human whole-genome sequence (WGS) data which is useful in many research studies including imputation, ancestry estimation, and ancestry informative marker (AIM) discovery. Second, there are persistent challenges in ancestry inference for individuals of the Middle East, especially with the use of AIMs. This research demonstrates a population genomics study pertaining to the Middle East, novel population data from Lebanon (n=190), and an unsupervised genetic clustering approach with WGS data from the 1000 Genomes Project and Human Genome Diversity Project. These efforts for AIM discovery identified two single nucleotide polymorphisms (SNPs) based on their high allelic frequency differences between the Middle East and populations in Eurasia, namely Europe and South/Central Asia. These candidate AIMs were evaluated with the most current and comprehensive AIM panel to date, the VISAGE Enhanced Tool (ET), using an external validation set of Middle Eastern WGS data (n=137). Instead of relying on pre-defined biogeographic ancestry labels to confirm the accuracy of validation sample ancestry inference, this research produced a deep, unsupervised ADMIXTURE analysis on 3,469 worldwide WGS samples with nearly 2 million independent SNPs (r2 < 0.1) which provided a genetic “ground truth”. This resulted in 136/137 validation samples as Middle East and provided valuable insights toward reference samples with varying co-ancestries that ultimately affects the classification of admixed individuals. Novel deep learning methods, specifically variational autoencoders, were introduced for visualizing one hundred percent of the genetic variance found using these AIMS in an alternative method to PCA and presents distinct population clusters in a robust ancestry space that remains static for the projection of unknown samples to aid in ancestry inference and human identification. Third, this research delves into a craniofacial study that makes improvements toward key intelligence information about physical identity by exploring the relationship between dentition and facial morphology with an advanced phenotyping approach paired with robust dental parameters used in clinical practice. Cone-beam computed tomography (CBCT) imagery was used to analyze the hard and soft tissue of the face at the same time. Low-to-moderate partial correlations were observed in several comparisons of dentition and soft tissue segments. These results included partial correlations of: i) inter-molar width and soft tissue segments nearest the nasal aperture, the lower maxillary sinuses, and a portion of the upper cheek, and ii) of lower incisor inclination and soft tissue segments overlapping the mentolabial fold. These results indicate that helpful intelligence information, potentially leading towards identity in forensic investigations, may be present where hard tissue structures are manifested in an observable way as a soft tissue phenotype. This research was a valuable preliminary study that paves the way towards the addition of facial hard tissue structures in combination with external soft tissue phenotypes to advance fundamental facial genetic research. Thus, CBCT scans greatly add to the current facial imagery landscape available for craniofacial research and provide hard and soft tissue data, each with measurable morphological variation among individuals. When paired with genetic association studies and functional biological experiments, this will ultimately lead to a greater understanding of the intricate coordination that takes place in facial morphogenesis, and in turn, guide clinical orthodontists to better treatment modalities with an emphasis on personalized medicine. Lastly, it aids intelligence methodologies when applied within the field of forensic anthropology. ItemExamining Postnatal Retinal Thickness and Retinal Ganglion Cell Count in the Ts65Dn Mouse Model of Down Syndrome(2023-05) Folz, Andrew; Roper, Randall; Goodlett, Charles; Belecky-Adams, TeriDown syndrome (DS) is a genetic condition caused by the triplication of human chromosome 21 and presents with many phenotypes including decreased brain size, hypocellularity in the brain, and assorted ocular phenotypes. Some of the ocular phenotypes seen are increased risk of cataracts, accommodation difficulties, increased risk of refractive errors, and increased retinal thickness. The Ts65Dn mouse model of DS is a classically used mouse model as it presents a number of phenotypes also seen in those with DS. Some of these phenotypes include decreased brain volume, abnormal synaptic plasticity, and ocular phenotypes. These ocular phenotypes include decreased visual acuity, cataracts, and increased retinal thickness. The Ts65Dn mouse model is trisomic for Dyrk1a, a gene of interest in DS research. We hypothesize that there will be a genotypic and sex effect of retinal thickness and retinal ganglion cell (RGC) count at postnatal day 15 in the Ts65Dn mouse model of DS. Retinal slices were taken from male and female trisomic and euploid Ts65Dn mice at P15 and fluorescently labeled for RGCs and bipolar cells via immunohistochemistry. The retinas were measured for total retinal thickness and RNA-binding protein (RBPMS) positive cells in the RGC layer were counted. There was no genotypic or sex effect when comparing retinal thickness in trisomic mice as compared to euploid mice. There was a genotypic effect of RBPMS positive cell count in which the trisomic mice had a higher number of RBPMS positive cells than euploid mice. Increased retinal thickness along with increased RGC number have both been implicated with decreased apoptosis in the retina. In the Ts65Dn mouse model along with in individuals with DS, this could be due to an increase in DYRK1A protein levels reducing apoptosis. In future studies, determining DYRK1A’s influence in retinal thickness and RGC number could result in a treatment for overactive DYRK1A that could normalize retinal thickness and RGC number in those with DS. ItemElucidating Cellular Mechanisms Underlying Retinal Ganglion Cell Neurodegeneration in a Human Pluripotent Stem Cell-Derived Model(2022-12) Huang, Kang-Chieh; Cummins, Theodore R.; Meyer, Jason S.; Marrs, James A.; Perrin, Benjamin J.; Lasagna Reeves, Cristian A.Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration. ItemResistance to Three Common Herbicides in Chameleon Plant (Houttuynia Cordata Thunb.), a Highly Invasive Exotic Species(2022-12) Ice, David; Wang, Xianzhong; Clark, Patricia B.; Watson, JohnChameleon plant (Houttuynia cordata Thunb.) is native to Southern and Southeastern Asia. It can reproduce sexually through seeds and asexually through rhizomes and is invasive in multiple countries including the U.S. There has been much research on H. cordata as a medicinal species, and its potential as an invasive species is well documented. However, its herbicidal resistance has not previously been quantified. The objective of this study is to assess H. cordata’s resistance to herbicides. This study consisted of two rounds of tests to examine the resistance of H. cordata plants to three commonly used herbicides: SpeedZone, Weed-B-Gon, and Roundup. Two concentrations of each herbicide were used during each trial in the study: the recommended concentration and twice the recommended concentration. Herbicide treatments were applied outside the greenhouse. Herbicides were sprayed uniformly on the plants until the herbicide was dripping off the leaves. The growth of the treated plants was then monitored in the greenhouse. The herbicides generally reduced growth of the plants temporarily. However, plant extermination was not achieved. Plant samples from all herbicidal treatments regrew from rhizomes after all herbicide treatments. Results from the study showed that H. cordata could not be controlled by the recommended concentrations of herbicides commercially available for horticultural uses in the U.S. Doubling the recommended herbicide concentration was also ineffective in exterminating H. cordata plants. This research clearly showed that H. cordata has the potential to become a highly invasive species with the potential to negatively affect the ecological integrity of many communities in the U.S. ItemZebrafish Asd Discovery Models for Epileptic Mutations of Scn2a and Scn8a(2022-12) Milder, Patrick; Marrs, James A.; Baucum, Anthony J., II.; Overholser, Brian R.; Cummins, Theodore R.; Mastracci, Teresa L.Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs (ASD) and treatment methods. Therefore, a critical need exists to efficiently screen ASDs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel ASDs. Here, we present an optimized PTZ assay to improve reliability and reproducibility based on work in our laboratory. This optimized assay improves robustness in our screening of anti-seizure drugs (topiramate, lamotrigine, carbamazepine and GS967). These findings show that electroencephalogram (EEG) and calcium sensitive GFP from fusion protein (GCaMP) assays largely correlate with the behavioral findings, helping us connect physiological and behavioral responses to ASDs. Genetic epilepsy syndromes, like voltage gated sodium channel SCN2A and SCN8A pathogenic variants, are often poorly controlled by current medications. Our optimized assay relied on a fast and precise zebrafish seizure model using mRNA overexpression of hSCN2A and hSCN8A variants including: hSCN2A R1882Q and R853Q and hSCN8A R1872Q. All three pathogenic variants increased seizure activity, and the ASDs significantly decreased this seizure activity. This mRNA overexpression assay can be used to quickly evaluate seizure activity induced by pathogenic variants in voltage gated sodium channel genes and test ASDs to determine efficacy. In a separate study, we tested if the addition of the human SCN2A sodium channel could potentially rescue the loss of the zebrafish scn1Lab gene. Our GCaMP assay data indicates that this loss was successfully rescued. Cumulatively, these findings can be used to improve the screening of novel ASDs and treatments for patients with refractory epilepsy. ItemAnalyses of the development and function of stem cell derived cells in neurodegenerative diseases(2022-12) Lavekar, Sailee Sham; Meyer, Jason; Canfield, Scott; Belecky-Adams, Teri; Mastracci, Teresa; Perrin, BenjaminHuman pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases. ItemNeurabin's Influence on Striatal Dependent Behaviors(2022-08) Corey, Wesley; Cummins, Theodore; Berbari, Nicolas; Baucum, Anthony J., II.The striatum is a key brain region involved in regulating motor output and integration. The dorsal and ventral subdivisions of the striatum work in concert to mediate the reinforcing and motor behavioral outputs of the striatum. Moreover, dysfunction of these striatal regions is involved in various diseases including Parkinson’s disease and drug addiction. Therefore, understanding and characterizing biochemical and molecular changes within the striatum associated with these diseases is key in devolving novel therapeutics to treat these disease states. The main output neurons of the striatum are GABAergic, medium-spiny neurons (MSNs), and striatal functionality is mediated by neuroplastic changes in MSN activity. Within MSNs, dopaminergic receptor activation triggers a cascade of reversable phosphorylation, which is facilitated by the activation of specific protein kinases and inhibition of specific protein phosphatases. In comparison to the 350 serine/threonine protein kinases expressed within the striatum, there are only 40 major serine/threonine protein phosphatases. However, serine/threonine protein phosphatases, such as protein phosphatase 1 (PP1), gain their target specificity by interacting with phosphatase-targeting proteins. Within the striatum, the neurabins, termed neurabin and spinophilin, are the most abundant PP1 targeting proteins in dendritic spines. Spinophilin’s expression in the striatum has been strongly characterized, and spinophilin has been shown to regulate striatal-dependent motor-skill learning and amphetamine-induced locomotor sensitization. In contrast to spinophilin, neurabin’s expression within the striatum and its involvement in these striatal-dependent behaviors has not been fully probed. I found that neurabin expression in the striatum is not sex-dependent but is age-dependent. In addition to these data, I also present validation of new global, constitutive and conditional neurabin knock-out mouse lines. Finally, I present data that, unlike previous studies in spinophilin knockout mice, neurabin knockout mice have enhanced striatal-dependent motor-skill learning, but do not impact amphetamine-induced locomotor sensitization. Further characterization of neurabin’s expression in the striatum, and its role in these key striatal behaviors could provide a druggable target for therapeutics designed to address striatal dysfunction. ItemRole of Choroid Plexus TRPV4 Channel in Health and Disease(2022-08) Hochstetler, Alexandra; Blazer-Yost, Bonnie L; Berbari, Nicolas; Baucum II, AJ; Roper, Randall; Raskin, JeffreyPediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the Tmem67P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-andglucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using in vitro models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease. ItemUnderstanding the Role of Hypusine Biosynthesis in Endocrine-Exocrine Crosstalk(2022-08) Dale, Dorian J.; Mastracci, Teresa L.; Berbari, Nicolas; Balakrishnan, Lata; Spaeth, JasonTraditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (Dhps) to post- translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of Dhps in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein Eif5a is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.