No / In this study, we have demonstrated the presence of melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptor (MCHR1) transcripts in human skin. Sequence analysis confirmed that the transcripts of both genes were identical to those previously found in human brain. In culture, endothelial cells showed pro-MCH expression whereas no signal was found in keratinocytes, melanocytes, and fibroblasts. MCHR1 expression was restricted to melanocytes and melanoma cells. Stimulation of cultured human melanocytes with MCH reduced the ¿-MSH-induced increase in cAMP production. Furthermore, the melanogenic actions of ¿-MSH were inhibited by MCH. We propose that the MCH/MCHR1 signalling system is present in human skin and may have a role with the melanocortins in regulating the melanocyte.

Introdução: O Hormônio Concentrador de Melanina (MCH) apresenta a maior expressão de seu RNA mensageiro (RNAm) no período final da lactação (19º dia) em áreas inusitadas no sistema nervoso central (SNC) de roedor, como a área pré-óptica medial (MPOA). Após esse período não encontramos mais o RNAm e a proteína na MPOA, e há descrições de flutuações no nível sérico do MCH, o que sugere uma possível função do MCH no período final da lactação. Foi descrita a presença de MCH em diversos órgãos como: pulmão, tiróide, baço, trato gastrintestinal e em destaque no SNC que é responsável pela expressão de 98% do peptídeo. Foram descritos dois receptores: MCHR1 e MCHR2. No entanto, não há descrição da presença do MCHR1 em tecido mamário, embora evidências sustentem uma possível relação do MCH e o período de lactação. Objetivos: 1) investigar a presença do MCHR1 em tecido mamário de ratas lactantes e ratas em diestro; 2) seqüenciar o receptor encontrado em tecido mamário para compará-lo com o receptor expresso no SNC e, 3) estabelecer o tipo celular reativo ao MCHR1 em tecido mamário de ratas lactantes e ratas em diestro. Material e Métodos: tecido mamário e do SNC de ratas Long-Evans lactantes e não-lactantes foram submetidos às técnicas de hibridização in situ, RT-PCR e RT-qPCR (com tecidos controles periféricos). Cortes do SNC de ratas Long-Evans (19º dia de lactação) foram submetidos à técnica RNAscope. Sequenciamento gênico foi realizado em amostras de tecidos mamários e hipocampo de rata lactante. Tecidos mamários de ratas lactantes foram submetidos às técnicas de imuno-histoquímica (fosfatase alcalina e imunofluorescência indireta) e western blot. Resultados: 1) pela técnica de hibridização in situ pudemos descrever a presença do Mchr1 em tecido mamário e do SNC (12º, 19º dias de lactação e diestro); em tecido mamário de ratas lactantes o Mchr1 se encontra em células na epiderme, derme, estroma e parênquima, e em ratas não-lactantes apenas na epiderme e derme. A técnica RNAscope confirmou a presença do Mchr1 no SNC de rata lactante. Por RT-PCR e RT-qPCR confirmou-se a presença do Mchr1 em tecidos mamários e controles de ratas lactantes e não-lactantes, com aumento da expressão gênica no hipocampo de ratas no 19º dia lactação. Por western blot, o MCHR1 encontra-se aumentado em hipocampo de ratas lactantes (12º dia); 2) o sequenciamento gênico identificou 100% de identidade da sequência do Mchr1 do tecido mamário (com pele e sem pele) com a do SNC; 3) células MCHR1-ir foram identificadas na epiderme, derme, estroma e parênquima de glândulas mamárias (ratas lactantes). Conclusão: podemos inferir, de forma inédita, que o Mchr1 é expresso no tecido mamário de ratas lactantes e nãolactantes, apresenta 100% de identidade com a sequência do SNC, a expressão varia por setor mamário, e é mais expresso no hipocampo (19º dia lactação) indicando possível neurogênese hipocampal no final da lactação e, que o MCHR1 está mais presente no tecido mamário de ratas lactantes no 12º dia lactação. Todos esses resultados sugerem um possível envolvimento do MCH no controle da lactação / Introduction: The melanin-concentrating hormone (MCH) mRNA shows the higher expression during lactation final period (around 19th day) in novel sites of central nervous system (CNS) of rodents, such as the ventral part of medial preoptic area (MPOAv). Thereafter, mRNA and protein are not found in the MPOAv and, there are seric alterations of MCH suggesting a possible function in the lactation final period. MCH is present peripheral tissues: lung, thyroid, spleen, gastrointestinal tract and the CNS is responsible for 98% of expression. The MCH has two receptors: MCHR1 and MCHR2. However, there are no descriptions of MCHR1 expression in the mammary glands of non-lactating or lactating dams albeit evidence support a possible relationship between MCH and lactation. Objective: 1) investigate the presence of MCHR1 in mammary tissue, 2) sequencing the receptor present in the mammary tissue of female rats to verify the homology to compare with central Mchr1 and 3) identify the cellular type that express the mRNA of Mchr1 and, the protein MCHR1 and. Material and Methods: mammary gland and brain tissue of Long-Evans rats (lactating and no-lactating) were submitted to in situ hibridization, RT-PCR and qRT-PCR (with peripheral control tissues). Sample of CNS Long-Evans rats (19th day lactation) was submitted to RNAscope technique. Sequencing was performed in mammary gland tissue and hippocampus of lactating rat. Mammary gland tissue of lactating rats was submitted to immunohistochemistry (alcaline phosphatase and indirect immunofluorescence) and western blot. Results: 1) Mchr1 was detected by in situ hibridization in mammary gland and CNS tissue (12th, 19th days of lactation and diestrus phase). In mammary gland of lactating rats Mchr1 was found in cells of epidermis, dermis, stroma and parenchyma, and in no-lactating rats only in the epidermis and dermis. RNAscope confirmed the presence of Mchr1 in the CNS of lactating rats. By RT-PCR and qRT-PCR, Mchr1 was detected in mammary gland and controls tissues of lactating and no-lactating rats, with higher expression in hippocampus on the 19th day lactating rats. By western blot, MCHR1 is increased in hippocampus of lactating rats (12th day); 2) the gene sequencing confirmed 100% identity os Mchr1 sequence of mammary tissue (with and without skin) compared to CNS; 3) MCHR1-ir cells were detected in epidermis, dermis, stroma and parenchyma (lactating rats). Conclusion: we can infer, in an unprecedented manner, the Mchr1 is expressed in mammary tissue of lactating and non-lactating rats, presents 100% identity with the sequence of the CNS, expression varies by mammary sector, and is best expressed in the hippocampus (19th day lactation) indicating possible hippocampal neurogenesis at the end of lactation and the MCHR1 is more present in the breast tissue of lactating rats on the 12th day lactation. All these results suggest a possible involvement of MCH in the control of lactation

L'axe MCH/MCHR1 a d'abord été étudié pour son rôle dans les comportements alimentaires. Ici, nous étudions son implication dans certains comportements liés à la consommation de drogues d'abus, telles que les psychostimulants, chez des souris génétiquement dépourvues du récepteur de type 1 (MCHR1).

Indiana University-Purdue University Indianapolis (IUPUI) / Obesity is a growing pandemic that claims close to three hundred thousand lives per year in the United States alone. Despite strong interest and investment in potential treatments, obesity remains a complex and challenging disorder. In the study of obesity, mouse models have been excellent tools that help in understanding the function of different genes that contribute to this disease of energy homeostasis. However, it was surprising when disfunction in primary cilia was found to be linked to syndromic obesity. To understand the role of primary cilia in obesity, a growing subset of GPCRs have been identified to selectively localize to the organelle. Several of which have known roles in energy homeostasis. In some examples, ciliary GPCRs appear to dynamically localize to the organelle; such is the case of GPR161 and smoothened in the hedgehog signaling pathway. Thus, we were interested to see if other GPCRs dynamically localize to the primary cilia as part of their regulation of energy homeostasis. For example, the GPCR MCHR1 selectively localizes to the cilia and is involved in energy homeostasis. Although much is known about the expression of the receptor in the brain, how its ciliary subcellular localization impacts its roles in energy homeostasis is unknown. Observing neuronal cilia in vivo remains a difficult task as some of the available tools such as tagged alleles rely on overexpression of ciliary protein which may impact function. Additionally, most of the work is done in vitro, leaving much to be discovered about neuronal cilia in vivo. In this thesis, we show that using a newly constructed reporter allele mCherryMCHR1, we can see ciliary expression of MCHR1 in the brain of developing and adult mice; more specifically in the ARC and PVN. Subsequently, using a novel Artificial intelligence analysis approach, we measured the length and composition of MCHR1 positive cilia under physiological conditions associated with MCHR1 function. Although in this work we are reporting no changes in dynamic localization of MCHR1 in the hypothalamus specifically, we are not excluding the potential for changes in other regions of the brain or under other conditions; and we are suggesting that pharmacological approaches may help highlight potential ciliary GPCR dynamic localization.

Indiana University-Purdue University Indianapolis (IUPUI) / Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.

<p>  </p> <p>Cilia are cell appendages that sense our environment and are critical in cell-to-cell communication. Dysfunction of cilia can result in several disease states including obesity. While cilia in the brain are known to be important for feeding behavior, it is unclear how they regulate energy homeostasis. Classically, cilia coordinate signaling through surface receptors called G-protein coupled receptors (GPCRs). For example, cilia mediated GPCR signaling is critical for both our senses of vision and smell. How cilia regulate the signaling of GPCRs in other areas of the body including the brain is only now emerging. To answer cell biology questions around cilia mediated GPCR signaling in neurons, we developed a system for primary neuronal cultures. We discovered that the cilia mediated hedgehog pathway influences the ability of neurons to respond to GPCR ligands. For the first time, this result highlights the role of the hedgehog pathway in neurons. We continue to explore how cilia integrate the hedgehog pathway and GPCR signaling in the central nervous system, and the potential connections to energy homeostasis. We discovered that hedgehog pathway activity in feeding centers of the brain changes based upon feeding conditions like fasting. We also learned that activating the hedgehog pathway in these brain regions is sufficient to cause obesity in mice. These novel results highlight an unrecognized role for the hedgehog pathway in the regulation of feeding behavior. Overall, this work provides a better understanding of ciliopathy associated obesity and may reveal more common mechanisms of obesity in the general population. In addition, this work implicates the hedgehog pathway in regulating behaviors and new modes of cell-cell communication within the central nervous system.</p>