A CreER Mouse to Study Melanin Concentrating Hormone Signaling in the Developing Brain

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

PhD

dissertationMammalian cells obtain cholesterol via endocytic uptake of cholesterol-rich lipoproteins and de novo synthesis through a multistep pathway. However, excess cholesterol is deleterious and cellular cholesterol levels must be strictly regulated. This regulation involves a complex set of homeostatic processes. Understanding the mechanisms involved in this regulation has implications for heart disease as well as developmental disorders. We have used a novel system for studying the effects of cholesterol synthesis inhibitors on development using zebrafish. We find that progesterone inhibits cholesterol synthesis in vitro and causes developmental abnormalities in embryos that can be prevented by the addition of water-soluble cholesterol. These findings demonstrate that cholesterol is necessary for proper development in zebrafish. Previous studies have shown that treating pregnant rats with AY-9944, a pharmacologic inhibitor of the final enzyme in the cholesterol biosynthesis pathway, causes birth defects. We find that AY-9944 confers cholesterol auxotrophy in Chinese hamster ovary (CHO) cells. We used this inhibitor in an expression cloning approach and isolated two genes that overcome AY-9944-induced cholesterol auxotrophy, when overexpressed. The first gene we isolated from this screen is subunit 3 of the COP9 signalosome sgn3. We show that this gene is most highly expressed in heart and skeletal muscle, maps to human chromosome 17p11.1-p11.2, and is deleted in all patients with the developmental disorder Smith-Magenis syndrome (SMS). These observations suggest a role for SGN3 and the COP9 signalosome in cholesterol metabolism as well as SMS. The second gene we isolated is HDL-binding protein (HBP), which is also known as vigilin. We show that cells overexpressing HBP/vigilin display no difference in cholesterol metabolism, but have an increase in fatty acid synthesis. Furthermore, we find that HBP/vigilin transcription is regulated by the nuclear hormone receptor PPAR?. These findings support a function for HBP/vigilin in fatty acid synthesis and raise the possibility that it plays a role in the adipogenic pathway

Heteromerization of ciliary G protein-coupled receptors in the mouse brain.

Nearly every cell type in the mammalian body projects from its cell surface a primary cilium that provides important sensory and signaling functions. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and adenylyl cyclase type 3, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling. Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin-concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling

Mchr1 localizes to neuronal cilia throughout the mouse brain.

<p>(A–L) Representative images of multiple brain regions in 5–6 week old WT mice showing colabeling for Mchr1 (green) and AC3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for AC3 reveals numerous primary cilia present throughout the CA1 region of the hippocampus (B), amygdala (E), piriform cortex (H), and fasciolar gyrus (K). Labeling for Mchr1 (A, D, G, & J) reveals abundant Mchr1 ciliary localization in each region. Merged images (C, F, I, L) showing colocalization between Mchr1 and AC3. Scale bars represent 10 µm.</p

Distribution of Mchr1 positive cilia in the mouse brain.

<p>The relative number of Mchr1-positive cilia in each brain region, normalized to AC3-positive cilia, is designated by: +, sparse distribution of Mchr1-positive cilia; ++, moderate distribution of Mchr1-positive cilia; +++, extensive distribution of Mchr1-positive cilia; and ++++, highest detection of Mchr1-positive cilia.</p

Mchr1 and Sstr3 colocalize in a subset of piriform cortical neuronal cilia.

<p>(A–F) Representative image of the piriform cortex from an adult WT mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the piriform cortex. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D–F) reveals a subset of neuronal cilia that are positive for both Mchr1 and Sstr3 (arrowhead). (G) Quantification of day 7 WT piriform cortical neurons colabeled for Mchr1 and AC3 or Sstr3 and AC3 reveals that Mchr1 localizes to 55.62±3.63% (n = 186) of AC3-positive cilia and Sstr3 localizes to 14.03±1.71% (n = 159) of AC3-positive cilia. (H) Graphical representation of the quantification of day 7 WT piriform cortical neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 23.36±5.11% (n = 86) of Mchr1-positive cilia. Scale bars represent 10 µm.</p

Mchr1 and Sstr3 proteins interact.

<p>Sstr3 was co-expressed with myc-tagged glyceraldehyde-3-phosphate dehydrogenase (Gapdh-myc) or myc-tagged Mchr1 (Mchr1-myc) in HEK293T cells. (A) Cell extracts were immunoprecipitated (IP) with an anti-myc antibody. Immunoprecipitates were analyzed by western blotting (IB) with an anti-Sstr3 antibody (left). Note that Sstr3 is immunoprecipitated with Mchr1, as indicated by the 45 kDa band, but not Gapdh. (B) In the reverse experiment, cell extracts were immunoprecipitated (IP) with an anti-Sstr3 antibody and immunoprecipitates were analyzed by western blotting (IB) with an anti-myc antibody (left). Note that Mchr1 is immunoprecipitated with Sstr3, as indicated by the 37 kDa band, but not Gapdh. The input, confirming expression of each protein, is also shown (right). Sstr3 appears as a 45 kDa band, which agrees with its predicted size, and 75 kDa and 150 kDa bands, which may represent higher order oligomers. Gapdh and Mchr1 are observed as 39 kDa and 37 kDa bands, respectively. The expression pattern of GPCRs often results in a multi-band pattern due to various oligomeric combinations and post translational modifications.</p

Mchr1 and Sstr3 colocalize in a subset of hypothalamic neuronal cilia.

<p>(A–F) Representative image of the hypothalamus from an adult WT mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the hypothalamus. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D–F) reveals a subset of neuronal cilia that are positive for both Mchr1 and Sstr3 (arrowhead). (G) Quantification of day 7 WT hypothalamic neurons colabeled for Mchr1 and AC3 or Sstr3 and AC3 reveals that Mchr1localizes to 63.42±4.59% (n = 108) of AC3-positive cilia and Sstr3 localizes to 33.34±5.24% (n = 129) of AC3-positive cilia. (H) Graphical representation of the quantification of day 7 WT piriform cortex neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 20.36±4.39% (n = 85) of Mchr1-positive cilia. Scale bars represent 10 µm.</p

Mchr1 and Sstr3 interact in mouse hippocampal lysate.

<p>Membrane protein enriched cell lysate from the hippocampus of 5 week old adult WT mice was immunoprecipitated (IP) with a goat anti-Mchr1 antibody or goat IgG, as a negative control. Immunoprecipitates were analyzed by western blotting (IB) with a rabbit anti-Sstr3 antibody. Note that Sstr3 is immunoprecipitated with Mchr1 but not with the IgG negative control. The input probed with anti-Sstr3 (left) or anti-Mchr1 (right), confirms the expression of Sstr3 and Mchr1. The ∼55 kDa bands in the IP lanes may be IgG heavy chain that is detected due to cross-reactivity of the secondary antibody.</p

Mchr1 and Sstr3 interact in live cells.

<p>HEK293T cells were transiently co-transfected with constructs encoding CFP-tagged Mchr1 and YFP-tagged Sstr3. Mchr1-CFP and Sstr3-YFP colocalize at the plasma membrane and in intracellular compartments (A–C). Robust FRET signals are observed between Mchr1-CFP and Sstr3-YFP localizing in intracellular compartments and moderate FRET signals are observed between Mchr1-CFP and Sstr3-YFP localizing in the cell membrane (D). The fluorescence intensity profile along the red-dotted line within the FRET_corrected image is shown (E). The intensity scale for CFP (blue) and FRET_corrected (red) are on the left. The intensity scale for YFP (green) is on the right. AU = Arbitrary Unit.</p