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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 Physiological Condition-Dependent Changes in Ciliary GPCR Localization in the Brain(Society for Neuroscience, 2023-03-13) Brewer, Kathryn M.; Engle, Staci E.; Bansal, Ruchi; Brewer, Katlyn K.; Jasso, Kalene R.; McIntyre, Jeremy C.; Vaisse, Christian; Reiter, Jeremy F.; Berbari, Nicolas F.; Biology, School of SciencePrimary cilia are cellular appendages critical for diverse types of Signaling. They are found on most cell types, including cells throughout the CNS. Cilia preferentially localize certain G-protein-coupled receptors (GPCRs) and are critical for mediating the signaling of these receptors. Several of these neuronal GPCRs have recognized roles in feeding behavior and energy homeostasis. Cell and model systems, such as Caenorhabditis elegans and Chlamydomonas, have implicated both dynamic GPCR cilia localization and cilia length and shape changes as key for signaling. It is unclear whether mammalian ciliary GPCRs use similar mechanisms in vivo and under what conditions these processes may occur. Here, we assess two neuronal cilia GPCRs, melanin-concentrating hormone receptor 1 (MCHR1) and neuropeptide-Y receptor 2 (NPY2R), as mammalian model ciliary receptors in the mouse brain. We test the hypothesis that dynamic localization to cilia occurs under physiological conditions associated with these GPCR functions. Both receptors are involved in feeding behaviors, and MCHR1 is also associated with sleep and reward. Cilia were analyzed with a computer-assisted approach allowing for unbiased and high-throughput analysis. We measured cilia frequency, length, and receptor occupancy. We observed changes in ciliary length, receptor occupancy, and cilia frequency under different conditions for one receptor but not another and in specific brain regions. These data suggest that dynamic cilia localization of GPCRs depends on properties of individual receptors and cells where they are expressed. A better understanding of subcellular localization dynamics of ciliary GPCRs could reveal unknown molecular mechanisms regulating behaviors like feeding.Item Bardet–Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia(National Academy of Science, 2008) Berbari, Nicolas F.; Lewis, Jacqueline S.; Bishop, Georgia A.; Askwith, Candice C.; Mykytyn, Kirk; Biology, School of SciencePrimary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet–Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity.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 Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors(The American Society for Cell Biology, 2008) Berbari, Nicolas F.; Johnson, Andrew D.; Lewis, Jacqueline S.; Askwith, Candice C.; Mykytyn, Kirk; Biology, School of SciencePrimary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.Item Hippocampal and Cortical Primary Cilia Are Required for Aversive Memory in Mice(Public Library of Science, 2014-09-03) Berbari, Nicolas F.; Malarkey, Erik B.; Yazdi, S.M. Zaki R.; McNair, Andrew D.; Kippe, Jordyn M.; Croyle, Mandy J.; Kraft, Timothy W.; Yoder, Bradley K.; Biology, School of ScienceIt has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia.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 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.