Nicolas F. Berbari

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

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Hedgehog Signaling Regulates Apical Actin Morphology
(Office of the Vice Chancellor for Research, 2016-04-08) Anderson, Matthew; Hege, Melissa; Berbari, Nicolas; Perrin, Benjamin
Stereocilia are highly patterned actin based cell protrusions found on the apical surface of auditory hair cells. They are formed mainly from bundled filamentous actin and its associated actin cross-linking proteins. Interestingly, stereocilia develop around another cell appendage, the microtubule based kinocilium, which is the primary cilium for a hair cell. Primary cilia are found on most somatic cells and play a significant role in the regulation and proper transduction of the Hedgehog (Hh) pathway. In the current study, we are testing the hypothesis that Hh pathway activity can alter actin bundling and elongation. In support of this idea, ectopic activation or repression of Hh signaling changed the morphology of stereocilia in vivo. To further test our hypothesis, we used a CL4 porcine kidney epithelial cell culture system stably expressing the actin crosslinking protein ESPN fused to green fluorescent protein. These cells serve as an in vitro model of apical actin protrusions similar to mature stereocilia in vivo. We manipulated Hh signaling in these cells using both a genetic and a pharmacological approach. In the pharmacological approach, CL4 cells were treated with the hedgehog agonist (Purmophamine) and antagonist (Cyclopamine), at varying concentrations for 48 hours. Genetically, the Hh pathway was ectopically activated by overexpressing the transcription factor Gli1, Gli2, Gli3, and SmoA1 repressed by expressing Gli3R. Immunofluorescent (IF) and scanning electron microscopy (SEM) revealed that CL4 cells dramatically altered the apical actin structures under these conditions. In particular, activating Gli transcription decreased apical actin-based structures while antagonizing activity resulted in more actinbased protrusions. This data strongly supports the hypothesis that the Hh signaling pathway can regulate the actin cytoskeleton.
Understanding Cilia Function on POMC Neurons in Appetite and Satiety
(Office of the Vice Chancellor for Research, 2016-04-08) Boyle, Julianne; Whitehouse, Logan; Engle, Staci; Bansal, Ruchi; Berbari, Nicolas F.
Over one-third of adults in the United States are obese. Obese individuals are at an increased risk for cardiovascular diseases, type 2 diabetes, cancer, and other health conditions, resulting in premature death. Interestingly, cilia have been linked to controlling satiety in both mice and humans, and individuals with dysfunctional cilia are often obese. Cilia are cellular appendages composed of microtubules and can be motile or immotile. Primary (immotile) cilia function as sensors for important signaling pathways. The loss of cilia, specifically from hypothalamic proopiomelanocortin (POMC) expressing cells, disrupts satiety, leading to overeating and obesity. While it is known that cilia loss in POMC cells in the hypothalamus causes obesity, the age or developmental stage at which cilia loss is important for this phenotype remains unclear. The aim of this research is to determine the time point critical for proper cilia function on POMC neurons to maintain normal feeding behaviors. To do this, we utilize an inducible POMC-CreER mouse model. This model allows us to disrupt cilia formation and maintenance at specific stages of life. We take a multifaceted approach to analyze the impact of cilia loss by measuring longterm body weight and feeding behavior in adult mice, studying changes in embryonic development, as well as analyzing physiological changes in cultured primary neurons. These studies will contribute to a better understanding of the role of cilia in satiety signaling which will help lead to the development of effective treatments for weight related diseases.
Use of an Episodic Food Intake Monitoring System to Evaluate Feeding Behavior in Mice
(Office of the Vice Chancellor for Research, 2016-04-08) Engle, Staci E.; Bansal, Ruchi; Berbari, Nicolas F.
The measurement of food consumption in laboratory animals is critical to studies in metabolism and obesity. Unfortunately, feeding behavior is very sensitive to the environment. Many factors such as the change of cages, diet, and human interactions can introduce undesired experimental variation. Here we describe our experiences with a commercially available episodic food intake monitoring system, the BioDAQ Monitor. This system is designed to quantitatively record feeding behavior in mice. It continuously monitors the weight of the food and uses this information to determine bout length and size. Bouts that occur soon after one another can then be defined as meals. When an animal jostles the food hopper while eating, the weight of the hopper fluctuates and eating is considered to be in progress. Once the hopper weight has been stable for a specified time, that period of feeding is considered to be concluded. The system also has the capability to assess either food or liquid choice paradigms and to directly measure the administration of orally available drugs in either the feed or the water. In addition to these functions, the system uses an environment monitor to record temperature, humidity and lighting of the room every five minutes. Here we present data showing measurements taken in hyperphagic mutant mice, altered feeding paradigms, and under different drug and protein hormone treatments. Future studies using this system will continue to focus on the hyperphagia associated obesity phenotype observed in mice upon conditional disruption of primary cilia.
Testing Therapeutic Candidates in a Mouse Model of Polycystic Kidney Disease
(Office of the Vice Chancellor for Research, 2016-04-08) McConkey, Shannon; Yang, Jenny; Bacallao, Robert; Berbari, Nicolas F.
Approximately 1 in 500 middle aged people in the United States will be diagnosed with Polycystic Kidney Disease (PKD), an inherited genetic disorder that results in extreme cysts on the kidneys. PKD eventually leads to end-stage kidney failure and current treatments are limited to dialysis or transplantation. Thus, a pharmacological approach to prevent, delay, or slow the progression of PKD would revolutionize treatment and improve mortality. Interestingly, many proteins associated with PKD have been found in and around the primary cilia of renal epithelial cells. Cilia are small microtubule-based cellular appendages found on the surface of most cell types in the human body and are broadly classified as either “motile” or “primary” (immotile). Primary cilia are known to be mechano- and environmental sensors, and play a critical role in cell-to-cell communication. The aim of this proposed research is to use potential therapeutics identified in silico and in vitro in animal models of PKD to determine if the compound can delay or prevent cystogenesis. Here we test Sildenafil citrate (Viagra) in an animal model of rapidly progressing cyst formation for its ability to ameliorate the phenotype. Further research directed at understanding the cilia, cell-cycle, and cilia-mediated signalling activity will hopefully provide important insights into the mechanisms of renal cyst pathogenesis and lead to better approaches for therapeutic intervention for PKD.
Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease
(Plos, 2016-07-29) Lewis, Wesley R.; Malarkey, Erik B.; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C.; Porath, Jonathan D.; Birket, Susan E.; Saunier, Sophie; Antignac, Corinne; Knowles, Michael R.; Leigh, Margaret W.; Zariwala, Maimoona A.; Challa, Anil K.; Keterson, Robert A.; Drummond, Iaian A.; Parant, John M.; Rowe, Steven M.; Hildebrandt, Friedhelm; Porter, Mary E.; Yoder, Bradley K.; Berbari, Nicolas F.; Department of Biology, School of Science
Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.
Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse
(Elsevier, 2016-04-15) Pasek, Raymond C.; Malarkey, Erik; Berbari, Nicolas F.; Sharma, Neeraj; Kesterson, Robert A.; Tres, Laura L.; Kierszenbaum, Abraham L.; Yoder, Bradley K.; Department of Biology, School of Science
Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans.
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.
Cilia and Obesity
(Cold Spring Harbor Laboratory Press, 2017-07-05) Vaisse, Christian; Reiter, Jeremy F.; Berbari, Nicolas F.; Biology, School of Science
The ciliopathies Bardet-Biedl syndrome and Alström syndrome cause obesity. How ciliary dysfunction leads to obesity has remained mysterious, partly because of a lack of understanding of the physiological roles of primary cilia in the organs and pathways involved in the regulation of metabolism and energy homeostasis. Historically, the study of rare monogenetic disorders that present with obesity has informed our molecular understanding of the mechanisms involved in nonsyndromic forms of obesity. Here, we present a framework, based on genetic studies in mice and humans, of the molecular and cellular pathways underlying long-term regulation of energy homeostasis. We focus on recent progress linking these pathways to the function of the primary cilia with a particular emphasis on the roles of neuronal primary cilia in the regulation of satiety.
Ciliary gene RPGRIP1L is required for hypothalamic arcuate neuron development
(American Society for Clinical Investigation, 2019-02-07) Wang, Liheng; De Solis, Alain J.; Goffer, Yossef; Birkenbach, Kathryn E.; Engle, Staci E.; Tanis, Ross; Levenson, Jacob M.; Li, Xueting; Rausch, Richard; Purohit, Manika; Lee, Jen-Yi; Tan, Jerica; De Rosa, Maria Caterina; Doege, Claudia A.; Aaron, Holly L.; Martins, Gabriela J.; Brüning, Jens C.; Egli, Dieter; Costa, Rui; Berbari, Nicolas; Leibel, Rudolph L.; Stratigopoulos, George; Biology, School of Science
Intronic polymorphisms in the α-ketoglutarate-dependent dioxygenase gene (FTO) that are highly associated with increased body weight have been implicated in the transcriptional control of a nearby ciliary gene, retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Previous studies have shown that congenital Rpgrip1l hypomorphism in murine proopiomelanocortin (Pomc) neurons causes obesity by increasing food intake. Here, we show by congenital and adult-onset Rpgrip1l deletion in Pomc-expressing neurons that the hyperphagia and obesity are likely due to neurodevelopmental effects that are characterized by a reduction in the Pomc/Neuropeptide Y (Npy) neuronal number ratio and marked increases in arcuate hypothalamic-paraventricular hypothalamic (ARH-PVH) axonal projections. Biallelic RPGRIP1L mutations result in fewer cilia-positive human induced pluripotent stem cell-derived (iPSC-derived) neurons and blunted responses to Sonic Hedgehog (SHH). Isogenic human ARH-like embryonic stem cell-derived (ESc-derived) neurons homozygous for the obesity-risk alleles at rs8050136 or rs1421085 have decreased RPGRIP1L expression and have lower numbers of POMC neurons. RPGRIP1L overexpression increases POMC cell number. These findings suggest that apparently functional intronic polymorphisms affect hypothalamic RPGRIP1L expression and impact development of POMC neurons and their derivatives, leading to hyperphagia and increased adiposity.
MicroRNA-31 is required for astrocyte specification
(Wiley, 2018-05) Meares, Gordon P.; Rajbhandari, Rajani; Gerigk, Magda; Tien, Chih-Liang; Chang, Chenbei; Fehling, Samuel C.; Rowse, Amber; Mulhern, Kayln C.; Nair, Sindhu; Gray, G. Kenneth; Berbari, Nicolas F.; Bredel, Markus; Benveniste, Etty N.; Nozell, Susan E.; Biology, School of Science
Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis.

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