Nicolas F. Berbari

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https://hdl.handle.net/1805/35564

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Cilia Signaling and Obesity
(Elsevier, 2021) Engle, Staci E.; Bansal, Ruchi; Antonellis, Patrick J.; Berbari, Nicolas F.; Biology, School of Science
An emerging number of rare genetic disorders termed ciliopathies are associated with pediatric obesity. It is becoming clear that the mechanisms associated with cilia dysfunction and obesity in these syndromes are complex. In addition to ciliopathic syndromic forms of obesity, several cilia-associated signaling 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 as well as in peripheral tissues, many questions remain. Here, we briefly discuss the syndromic ciliopathies and monoallelic cilia signaling gene mutations associated with obesity. We also describe potential ways cilia may be involved in common obesity. We discuss how neuronal cilia impact food intake potentially through leptin signaling and changes in ciliary G protein-coupled receptor (GPCR) signaling. We highlight several recent studies that have implicated the potential for cilia in peripheral tissues such as adipose and the pancreas to contribute to metabolic dysfunction. Then we discuss the potential for cilia to impact energy homeostasis through their roles in both development and adult tissue homeostasis. The studies discussed in this review highlight how a comprehensive understanding of the requirement of cilia for the regulation of diverse biological functions will contribute to our understanding of common forms of obesity.
A CreER mouse to study melanin concentrating hormone signaling in the developing brain
(Wiley, 2018-08) Engle, Staci E.; Antonellis, Patrick J.; Whitehouse, Logan S.; Bansal, Ruchi; Emond, Michelle R.; Jontes, James D.; Kesterson, Robert A.; Mykytyn, Kirk; Berbari, Nicolas F.; Biology, School of Science
The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.
A CreER Mouse to Study Melanin Concentrating Hormone Signaling in the Developing Brain
(Wiley, 2018) Engle, Staci E.; Antonellis, Patrick J.; Whitehouse, Logan S.; Bansal, Ruchi; Emond, Michelle R.; Jontes, James D.; Kesterson, Robert A.; Mykytyn, Kirk; Berbari, Nicolas F.; Biology, School of Science
The neuropeptide, melanin concentrating hormone (MCH), and its G protein‐coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1‐Cre) exists, there is a need for an inducible Mchr1‐Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1‐Cre expression pattern are recapitulated by the Mchr1‐CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1‐CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1‐CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.
Hedgehog Pathway Activation Alters Ciliary Signaling in Primary Hypothalamic Cultures
(Frontiers, 2019-06-12) Bansal, Ruchi; Engle, Staci E.; Antonellis, Patrick J.; Whitehouse, Logan S.; Baucum, Anthony J.; Cummins, Theodore R.; Reiter, Jeremy F.; Berbari, Nicolas F.; Biology, School of Science
Primary cilia dysfunction has been associated with hyperphagia and obesity in both ciliopathy patients and mouse models of cilia perturbation. Neurons throughout the brain possess these solitary cellular appendages, including in the feeding centers of the hypothalamus. Several cell biology questions associated with primary neuronal cilia signaling are challenging to address in vivo. Here we utilize primary hypothalamic neuronal cultures to study ciliary signaling in relevant cell types. Importantly, these cultures contain neuronal populations critical for appetite and satiety such as pro-opiomelanocortin (POMC) and agouti related peptide (AgRP) expressing neurons and are thus useful for studying signaling involved in feeding behavior. Correspondingly, these cultured neurons also display electrophysiological activity and respond to both local and peripheral signals that act on the hypothalamus to influence feeding behaviors, such as leptin and melanin concentrating hormone (MCH). Interestingly, we found that cilia mediated hedgehog signaling, generally associated with developmental processes, can influence ciliary GPCR signaling (Mchr1) in terminally differentiated neurons. Specifically, pharmacological activation of the hedgehog-signaling pathway using the smoothened agonist, SAG, attenuated the ability of neurons to respond to ligands (MCH) of ciliary GPCRs. Understanding how the hedgehog pathway influences cilia GPCR signaling in terminally differentiated neurons could reveal the molecular mechanisms associated with clinical features of ciliopathies, such as hyperphagia-associated obesity.
The Hedgehog Signaling Pathway is Expressed in the Adult Mouse Hypothalamus and Modulated by Fasting
(Society for Neuroscience, 2021-09-29) Antonellis, Patrick J.; Engle, Staci E.; Brewer, Kathryn M.; Berbari, Nicolas F.; Biology, School of Science
The hedgehog signaling pathway is best known for its role in developmental patterning of the neural tube and limb bud. More recently, hedgehog signaling has been recognized for its roles in growth of adult tissues and maintenance of progenitor cell niches. However, the role of hedgehog signaling in fully differentiated cells like neurons in the adult brain is less clear. In mammals, coordination of hedgehog pathway activity relies on primary cilia and patients with ciliopathies such as Bardet-Biedl and Alström syndrome exhibit clinical features clearly attributable to errant hedgehog such as polydactyly. However, these ciliopathies also present with features not clearly associated with hedgehog signaling such as hyperphagia-associated obesity. How hedgehog signaling may contribute to feeding behavior is complex and unclear, but cilia are critical for proper energy homeostasis. Here, we provide a detailed analysis of the expression of core components of the hedgehog signaling pathway in the adult mouse hypothalamus with an emphasis on feeding centers. We show that hedgehog pathway genes continue to be expressed in differentiated neurons important for the regulation of feeding behavior. Furthermore, we demonstrate for the first time that pathway activity is regulated at the transcriptional level by fasting. These data suggest that hedgehog signaling is involved in the proper functioning of brain regions that regulate feeding behavior and that hedgehog pathway dysfunction may play a role in the obesity observed in certain ciliopathies.
Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone
(Federation of American Society of Experimental Biology (FASEB), 2019-01) Lewis, Wesley R.; Bales, Katie L.; Revell, Dustin Z.; Croyle, Mandy J.; Engle, Staci E.; Song, Cheng Jack; Malarkey, Erik B.; Uytingco, Cedric R.; Shan, Dan; Antonellis, Patrick J.; Nagy, Tim R.; Kesterson, Robert A.; Mrug, Michal M.; Martens, Jeffrey R.; Berbari, Nicolas F.; Gross, Alecia K.; Yoder, Bradley K.; Biology, School of Science
The transition zone (TZ) is a domain at the base of the cilium that is involved in maintaining ciliary compartment-specific sensory and signaling activity by regulating cilia protein composition. Mutations in TZ proteins result in cilia dysfunction, often causing pleiotropic effects observed in a group of human diseases classified as ciliopathies. The purpose of this study is to describe the importance of the TZ component Meckel-Grüber syndrome 6 (Mks6) in several organ systems and tissues regarding ciliogenesis and cilia maintenance using congenital and conditional mutant mouse models. Similar to MKS, congenital loss of Mks6 is embryonic lethal, displaying cilia loss and altered cytoskeletal microtubule modifications but only in specific cell types. Conditional Mks6 mutants have a variable cystic kidney phenotype along with severe retinal degeneration with mislocalization of phototransduction cascade proteins. However, other phenotypes, such as anosmia and obesity, which are typically associated with cilia and TZ dysfunction, were not evident. These data indicate that despite Mks6 being a core TZ component, it has tissue- or cell type-specific functions important for cilia formation and cilia sensory and signaling activities.
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 Science
Puberty 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.

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