- Nicolas F. Berbari
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Item Artificial Intelligence Approaches to Assessing Primary Cilia(MyJove Corp., 2021-05-01) Bansal, Ruchi; Engle, Staci E.; Kamba, Tisianna K.; Brewer, Kathryn M.; Lewis, Wesley R.; Berbari, Nicolas F.; Biology, School of ScienceCilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.Item Polyciliation of GnRH Neurons in Vivo and in Vitro(Oxford University Press, 2021) Brewer, Kathryn M.; Bansal, Ruchi; Engle, Staci E.; Antonellis, Patrick J.; Cummins, Theodore R.; Berbari, Nicolas F.; Biology, School of SciencePuberty and reproduction are initiated and controlled through the hypothalamic-pituitary-gonadal (HPG) axis. A critical surge of luteinizing hormone (LH) and follicle stimulating hormone (FSH) are released from the anterior pituitary upon release of gonadotrophins from gonadotrophin releasing hormone (GnRH) neurons. Thus, GnRH neurons are key regulators of the HPG axis. GnRH neurons become active when kisspeptin (Kiss1) neuropeptides are released from neurons in the arcuate nucleus. Kiss1 binds to the Kiss1 receptor (Kiss1R), a G-protein coupled receptor (GPCR) which localizes to the primary cilia of GnRH neurons. Loss-of-function mutations of Kiss1R cause hypogonadism in mouse and human models while gain-of-function mutations are associated with precocious puberty. Interestingly, the subset of GnRH neurons that express Kiss1R are observed to be polyciliated, possessing more than one primary cilia, an uncommon property as most neurons only possess a single, primary cilium. The mechanism and conditions leading to GnRH neuron polyciliation are unknown. It is also unclear if multiple cilia impact Kiss1R or other GPCR signaling in these neurons. Here, we utilize cultured mouse primary hypothalamic neurons to begin addressing some of these questions. We have confirmed with qPCR that the ligands GnRH and Kiss1, as well as Kiss1R, are all expressed in these cultures. Surprisingly, when treated with Kiss1 and GnRH ligands we observed a small subset of polyciliated neurons compared to vehicle treated neurons. These observations mirror what is seen during sexual maturation in vivo and suggest that our model system may help elucidate fundamental questions about how ciliary localization of Kiss1r and other GPCRs participate in initiation of puberty and regulation of reproduction. Future studies will focus on the mechanisms of polyciliation and the conditions needed to induce the formation of new cilia in GnRH neurons. Investigating neuronal polyciliation could provide insights into new signaling paradigm in hypogonadism and HPG signaling.Item A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development(Wiley, 2022) Rockwell, Devan M.; O’Connor, Amber K.; Bentley-Ford, Melissa R.; Haycraft, Courtney J.; Croyle, Mandy J.; Brewer, Kathryn M.; Berbari, Nicolas F.; Kesterson, Robert A.; Yoder, Bradley K.; Biology, School of ScienceBackground: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. Results: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. Conclusion: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.Item Distribution of Prototypical Primary Cilia Markers in Subtypes of Retinal Ganglion Cells(Wiley, 2022) Kowal, Tia J.; Dhande, Onkar S.; Wang, Biao; Wang, Qing; Ning, Ke; Liu, Wendy; Berbari, Nicolas F.; Hu, Yang; Sun, Yang; Biology, School of ScienceLoss of retinal ganglion cells (RGCs) underlies several forms of retinal disease including glaucomatous optic neuropathy, a leading cause of irreversible blindness. Several rare genetic disorders associated with cilia dysfunction have retinal degeneration as a clinical hallmark. Much of the focus of ciliopathy associated blindness is on the connecting cilium of photoreceptors; however, RGCs also possess primary cilia. It is unclear what roles RGC cilia play, what proteins and signaling machinery localize to RGC cilia, or how RGC cilia are differentiated across the subtypes of RGCs. To better understand these questions, we assessed the presence or absence of a prototypical cilia marker Arl13b and a widely distributed neuronal cilia marker AC3 in different subtypes of mouse RGCs. Interestingly, not all RGC subtype cilia are the same and there are significant differences even among these standard cilia markers. Alpha-RGCs positive for osteopontin, calretinin and SMI32 primarily possess AC3 positive cilia. Directionally selective RGCs that are CART positive or Trhr positive localize either Arl13b or AC3, respectively in cilia. Intrinsically photosensitive RGCs differentially localize Arl13b and AC3 based on melanopsin expression. Taken together, we characterized the localization of gold standard cilia markers in different subtypes of RGCs and conclude that cilia within RGC subtypes may be differentially organized. Future studies aimed at understanding RGC cilia function will require a fundamental ability to observe the cilia across subtypes as their signaling protein composition is elucidated. A comprehensive understanding of RGC cilia may reveal opportunities to understanding how their dysfunction leads to retinal degeneration.Item Spinophilin-dependent regulation of GluN2B-containing NMDAR-dependent calcium influx, GluN2B surface expression, and cleaved caspase expression(Wiley, 2023) Salek, Asma B.; Claeboe, Emily T.; Bansal, Ruchi; Berbari, Nicolas F.; Baucum, Anthony J., II.; Biology, School of ScienceN-methyl-d-aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor conductance and surface localization, two alterations that can modulate overall channel activity. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates the amount of calcium entering the cell and subsequent activation of calcium-dependent processes. The dendritic spine enriched protein, spinophilin, is the major synaptic protein phosphatase 1 (PP1) targeting protein. Depending on the substrate, spinophilin can act as either a PP1 targeting protein, to permit substrate dephosphorylation, or a PP1 inhibitory protein, to enhance substrate phosphorylation. Spinophilin limits NMDAR function in a PP1-dependent manner. Specifically, we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284 on GluN2B. However, how spinophilin modifies NMDAR function is unclear. Herein, we utilize a Neuro2A cell line to detail that Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs. Moreover, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression, an effect that is independent of Ser-1284 phosphorylation. In hippocampal neurons isolated from spinophilin knockout animals, there is an increase in cleaved caspase-3 levels, a marker of calcium-associated apoptosis, compared with wildtype mice. Taken together, our data demonstrate that spinophilin regulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin enhances neuronal cleaved caspase-3 expression.Item An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue(BMC, 2013-07-03) O’Connor, Amber K.; Malarkey, Erik B.; Berbari, Nicolas F.; Croyle, Mandy J.; Haycraft, Courtney J.; Bell, P. Darwin; Hohenstein, Peter; Kesterson, Robert A.; Yoder, Bradley K.; Biology, School of ScienceBackground: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. Results: To facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. Conclusions: The ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.Item Neuronal cilia in energy homeostasis(Frontiers Media, 2022-12-08) Brewer, Kathryn M.; Brewer, Katlyn K.; Richardson, Nicholas C.; Berbari, Nicolas F.; Biology, School of ScienceA subset of genetic disorders termed ciliopathies are associated with obesity. The mechanisms behind cilia dysfunction and altered energy homeostasis in these syndromes are complex and likely involve deficits in both development and adult homeostasis. Interestingly, several cilia-associated gene mutations also lead to morbid obesity. While cilia have critical and diverse functions in energy homeostasis, including their roles in centrally mediated food intake and peripheral tissues, many questions remain. Here, we briefly discuss syndromic ciliopathies and monogenic cilia signaling mutations associated with obesity. We then focus on potential ways neuronal cilia regulate energy homeostasis. We discuss the literature around cilia and leptin-melanocortin signaling and changes in ciliary G protein-coupled receptor (GPCR) signaling. We also discuss the different brain regions where cilia are implicated in energy homeostasis and the potential for cilia dysfunction in neural development to contribute to obesity. We close with a short discussion on the challenges and opportunities associated with studies looking at neuronal cilia and energy homeostasis. This review highlights how neuronal cilia-mediated signaling is critical for proper energy homeostasis.Item Primary cilia enhance kisspeptin receptor signaling on gonadotropin-releasing hormone neurons(National Academy of Science, 2014) Koemeter-Cox, Andrew I.; Sherwood, Thomas W.; Green, Jill A.; Steiner, Robert A.; Berbari, Nicolas F.; Yoder, Bradley K.; Kauffman, Alexander S.; Monsma, Paula C.; Brown, Anthony; Askwith, Candice C.; Mykytyn, Kirk; Biology, School of ScienceMost central neurons in the mammalian brain possess an appendage called a primary cilium that projects from the soma into the extracellular space. The importance of these organelles is highlighted by the fact that primary cilia dysfunction is associated with numerous neuropathologies, including hyperphagia-induced obesity, hypogonadism, and learning and memory deficits. Neuronal cilia are enriched for signaling molecules, including certain G protein-coupled receptors (GPCRs), suggesting that neuronal cilia sense and respond to neuromodulators in the extracellular space. However, the impact of cilia on signaling to central neurons has never been demonstrated. Here, we show that the kisspeptin receptor (Kiss1r), a GPCR that is activated by kisspeptin to regulate the onset of puberty and adult reproductive function, is enriched in cilia projecting from mouse gonadotropin-releasing hormone (GnRH) neurons. Interestingly, GnRH neurons in adult animals are multiciliated and the percentage of GnRH neurons possessing multiple Kiss1r-positive cilia increases during postnatal development in a progression that correlates with sexual maturation. Remarkably, disruption of cilia selectively on GnRH neurons leads to a significant reduction in kisspeptin-mediated GnRH neuronal activity. To our knowledge, this result is the first demonstration of cilia disruption affecting central neuronal activity and highlights the importance of cilia for proper GPCR signaling.Item A 360-kb interchromosomal duplication of the human HYDIN locus(Elsevier, 2006) Doggett, Norman A.; Xie, Gary; Meincke, Linda J.; Sutherland, Robert D.; Mundt, Mark O.; Berbari, Nicolas F.; Davy, Brian E.; Robinson, Michael L.; Rudd, M. Katharine; Weber, James L.; Stallings, Raymond L.; Han, Cliff; Biology, School of ScienceThe HYDIN gene located in human chromosome band 16q22.2 is a large gene encompassing 423 kb of genomic DNA that has been suggested as a candidate for an autosomal recessive form of congenital hydrocephalus. We have found that the human HYDIN locus has been very recently duplicated, with a nearly identical 360-kb paralogous segment inserted on chromosome 1q21.1. The duplication, among the largest interchromosomal segmental duplications described in humans, is not accounted for in the current human genome assembly and appears to be part of a greater than 550-kb contig that must lie within 1 of the 11 sequence gaps currently remaining in 1q21.1. Both copies of the HYDIN gene are expressed in alternatively spliced transcripts. Elucidation of the role of HYDIN in human disease susceptibility will require careful discrimination among the paralogous copies.Item Loss of tumor suppressive microRNA-31 enhances TRADD/NF-κB signaling in glioblastoma(Impact Journals, 2015) Rajbhandari, Rajani; McFarland, Braden C.; Patel, Ashish; Gerigk, Magda; Gray, Kenneth; Fehling, Samuel C.; Bredel, Markus; Berbari, Nicolas F.; Kim, Hyunsoo; Marks, Margaret P.; Meares, Gordon P.; Sinha, Tanvi; Chuang, Jeffrey; Benveniste, Etty N.; Nozell, Susan E.; Biology, School of ScienceGlioblastomas (GBMs) are deadly tumors of the central nervous system. Most GBM exhibit homozygous deletions of the CDKN2A and CDKN2B tumor suppressors at 9p21.3, although loss of CDKN2A/B alone is insufficient to drive gliomagenesis. MIR31HG, which encodes microRNA-31 (miR-31), is a novel non-coding tumor suppressor positioned adjacent to CDKN2A/B at 9p21.3. We have determined that miR-31 expression is compromised in >72% of all GBM, and for patients, this predicts significantly shortened survival times independent of CDKN2A/B status. We show that miR-31 inhibits NF-κB signaling by targeting TRADD, its upstream activator. Moreover, upon reintroduction, miR-31 significantly reduces tumor burden and lengthens survival times in animal models. As such, our work identifies loss of miR-31 as a novel non-coding tumor-driving event in GBM.