Combining mass spectrometry and genetic labeling in mice to report TRP channel expression

Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice. • We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution. • We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice

HLA-E and Its Soluble Form as Indicators of a Sex-Specific Immune Response in Patients with Oral Squamous Cell Carcinoma

The human leukocyte antigene E (HLA-E) is associated with tumorigenesis in various cancers. Immunoncology along with sex-specific aspects in cancer therapy are now in scientific focus. Therefore, immunohistochemical HLA-E expression was retrospectively analysed in a cohort of oral squamous cell carcinomas (OSCC) after surgical therapy. Then, serum concentration of HLA-E (sHLA-E) was quantified in a prospective cohort by enzyme-linked immunosorbent assay. High HLA-E expression was associated with advanced UICC stage (Spearman’s correlation: p = 0.002) and worse survival (Cox-regression: progression-free survival: hazard ratio (HR) 3.129, confidence range (CI) 1.443–6.787, p = 0.004; overall survival: HR 2.328, CI 1.071–5.060, p = 0.033). The sHLA-E concentration was significantly higher in the control group than in tumor group (Mann–Whitney U-test (MW-U): p = 0.021). Within the tumor group, women showed significantly higher sHLA-E levels than men (MW-U: p = 0.049). A closer look at the tumor group and the control group showed that gender-specific differences exist: while no differences in sHLA-E concentration were detectable between female subjects of tumor group and control group (MW-U: p = 0.916), male subjects of tumor group had a significantly lower sHLA-E concentration compared to those of control group (MW-U: p = 0.001). In summary, our results provide evidence for sex-specific differences in immune responses in OSCC. This fact should be considered regarding future immunotherapy regimens

TREM2 Is Associated with Advanced Stages and Inferior Prognosis in Oral Squamous Cell Carcinoma

Triggering receptor expressed on myeloid cells 2 (TREM2) is suggested to hamper antitumor immune response in multiple cancers. However, the role of TREM2 in oral squamous cell carcinoma (OSCC) and its expression in tumor-associated macrophages (TAMs) are unknown. In this study, TREM2 expression was analyzed in the primary tumors and corresponding lymph-node metastases of OSCC patients via immunohistochemistry on tissue microarrays. Human peripheral blood mononuclear cells (PBMCs) and single-cell suspensions of tumor and healthy adjacent tissues were analyzed for the presence of TREM2+ macrophages and TAMs using flow cytometry. The serum levels of soluble TREM2 (sTREM2) were quantified using an enzyme-linked immunosorbent assay. High TREM2 expression was associated with advanced UICC stages (Spearman’s rank correlation (SRC), p = 0.04) and significantly reduced survival rates in primary tumors (multivariate Cox regression, progression-free survival: hazard ratio (HR) of 2.548, 95% confidence interval (CI) of 1.089–5.964, p = 0.028; overall survival: HR of 2.17, 95% CI of 1.021–4.613, p = 0.044). TREM2 expression was significantly increased in the PBMCs of OSCC patients in UICC stage IV compared with healthy controls (ANOVA, p < 0.05). The serum levels of sTREM2 were higher in advanced UICC stages, but they narrowly missed significance (SRC, p = 0.059). We demonstrated that TREM2 was multi-factorially associated with advanced stages and inferior prognosis in OSCC patients and that it could serve as a prognostic biomarker in OSCC patients. Targeting TREM2 has the potential to reshape the local and systemic immune landscape for the potential enhancement of patients’ prognosis

IDO1 is highly expressed in macrophages of patients in advanced tumour stages of oral squamous cell carcinoma

Purpose Strategies for Indolamine-2,3-dioxygenase 1 (IDO1) inhibition in cancer immunotherapy once produced encouraging results, but failed in clinical trials. Recent evidence indicates that immune cells in the tumour microenvironment, especially macrophages, contribute to immune dysregulation and therefore might play a critical role in drug resistance. Methods In this study, we investigated the signifcance of IDO1 expressing immune cells in primary tumours and corresponding lymph node metastases (LNMs) in oral squamous cell carcinoma (OSCC) by immunohistochemistry. The link between IDO1 and macrophages was investigated by fow cytometry in tumour tissue, healthy adjacent tissue and peripheral blood mononuclear cells (PBMCs). IDO1 activity (measured as Kynurenine/Tryptophan ratio) was assessed by ELISAs. Results High IDO1 expression in tumour-infltrating immune cells was signifcantly correlated with advanced stages [Spearman’s rank correlation (SRC), p=0.027] and reduced progression-free survival (multivariate Cox regression, p=0.034). IDO1 was signifcantly higher expressed in PBMCs of patients in advanced stages than in healthy controls (ANOVA, p<0.05) and IDO1+ macrophages were more abundant in intratumoural areas than peritumoural (t test, p<0.001). IDO1 expression in PBMCs was signifcantly correlated with IDO1 activity in serum (SRC, p<0.05). IDO1 activity was signifcantly higher in patients with LNMs (t test, p<0.01). Conclusion All in all, IDO1 expressing immune cells, especially macrophages, are more abundant in advanced stages of OSCC and are associated with reduced progression-free survival. Further investigations are needed to explore their role in local and systemic immune response. The IDO1 activity might be a suitable biomarker of metastasis in OSCC patients

Ovulation is triggered by a cyclical modulation of gonadotropes into a hyperexcitable state

Gonadotropes in the anterior pituitary gland are essential for fertility and provide a functional link between the brain and the gonads. To trigger ovulation, gonadotrope cells release massive amounts of luteinizing hormone (LH). The mechanism underlying this remains unclear. Here, we utilize a mouse model expressing a genetically encoded Ca2+ indicator exclusively in gonadotropes to dissect this mechanism in intact pituitaries. We demonstrate that female gonadotropes exclusively exhibit a state of hyperexcitability during the LH surge, resulting in spontaneous [Ca2+]i transients in these cells, which persist in the absence of any in vivo hormonal signals. L-type Ca2+ channels and transient receptor potential channel A1 (TRPA1) together with intracellular reactive oxygen species (ROS) levels ensure this state of hyperexcitability. Consistent with this, virus-assisted triple knockout of Trpa1 and L-type Ca2+ subunits in gonadotropes leads to vaginal closure in cycling females. Our data provide insight into molecular mechanisms required for ovulation and reproductive success in mammals

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis

The median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism

Subunit composition, molecular environment, and activation of native TRPC channels encoded by their interactomes.

peer reviewedIn the mammalian brain TRPC channels, a family of Ca2+-permeable cation channels, are involved in a variety of processes from neuronal growth and synapse formation to transmitter release, synaptic transmission and plasticity. The molecular appearance and operation of native TRPC channels, however, remained poorly understood. Here, we used high-resolution proteomics to show that TRPC channels in the rodent brain are macro-molecular complexes of more than 1 MDa in size that result from the co-assembly of the tetrameric channel core with an ensemble of interacting proteins (interactome). The core(s) of TRPC1-, C4-, and C5-containing channels are mostly heteromers with defined stoichiometries for each subtype, whereas TRPC3, C6, and C7 preferentially form homomers. In addition, TRPC1/C4/C5 channels may co-assemble with the metabotropic glutamate receptor mGluR1, thus guaranteeing both specificity and reliability of channel activation via the phospholipase-Ca2+ pathway. Our results unveil the subunit composition of native TRPC channels and resolve the molecular details underlying their activation

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis.

peer reviewedThe median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease

Genetic strategies to dissect hypothalamic cell populations controlling body homeostasis in mice

The hypothalamus, located in the diencephalon, close to the median eminence (ME) and the pituitary gland controls body homeostasis by the release of hormones and integration of periphery feedback. To orchestrate these body axes, such as the sexually dimorphic hypothalamus-pituitary-gonadal axis (hpg-axis), the hypothalamus consists of numerous cell populations, each expressing a specific repertoire of proteins. The ion channel family of transient receptor potential (TRP) channels is essential for this orchestration and is widely expressed in the body. TRP-expression patterns with cellular resolution are of great interest, especially in the hypothalamus, but detection is challenging due to multiple reasons. TRP channels are membrane-bound proteins, posttranslationally glycosylated and their expression levels are generally rather low. This makes epitope-directed antibody production and detection at the RNA level with cellular resolution diffcult, unfortunately including the hypothalamus. Reporter mice which label these cell populations with a fluorescent marker, are an elegant tool to overcome these caveats. To investigate TRP expression in the hypothalamus and to identify the individual cell type for each TRP channel, I utilized the newly generated TRPM5, TRPM6, TRPV6, TRPA1, TRPML3, TRPC2, TRPC4 and TRPC5 tGFP-reporter mice and iDISCO clearing. TRM5 is expressed in a subpopulation of tanycytes at the floor of the third ventricle, having access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations, potentially mediating bidirectional communication between the cerebrospinal fluid and the blood. In addition to tanycytes, I identified TRPM5, and also TRPC5 and TRPA1 cells in the pars tuberalis in close contact with blood vessels. Interestingly, TRPV6 is not expressed in the brain, whereas TRPM6 was found in every blood vessel of the blood brain barrier (BBB). TRPC4 was identified in pericytes, wrapping their processes around blood vessels and tGFP expression was found in fibers in the ME of TRPC2 reporter animals. These fibers are in close contact with fenestrated blood vessels in the posterior ME, indicating that these fibers terminate here. I identified the origin of these fibers in acutely TRPC2 expressing neurons in the paraventricular nucleus (PVN), projecting ventro-latterally into the ME. A subset of these neurons co-express corticotropin-releasing-hormone (CRH), but ablation or silencing of TRPC2 neurons did not have an effect on adrenocorticotropin (ACTH) levels. Activation of TRPC2 neurons resulted in increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. This shows a potential role of TRPC2 neurons in mediating hormone release in the ME. The hpg-axis is controlled by sex steroids, such as oestrogen, in a sexually dimorphic manner. The rate-limiting enzyme for oestrogen synthesis is aromatase. To gain a better understanding how oestrogen impacts sexual differentiation, I investigated aromatase expression through embryonic and prenatal development. To address this question, I capitalized on a Cre reporter mouse which labels aromatase cells with tGFP. Aromatase neurons were first visible at embryonic day 13.5 (E13.5) and dramatically increased in number by E16.5. At birth I revealed a prominent sexual dimorphism in the arcuate nucleus (Arc). Here, aromatase is only expressed in male animals. The majority of oestrogen receptor (ER)-expressing neurons were devoid of aromatase. This indicates that aromatase neurons can convert gonadal and/or brainborn testosterone to oestrogens to regulate neuron activity via ER-dependent paracrine mechanisms. To test this hypothesis we collaborated with the group of Prof. Erik Hrabovszky to perform whole-cell-patch-clamp experiments on kisspeptin neurons in the Arc. These neurons are oestrogen sensitive and are not co-expressing aromatase. Testosterone reduced kisspeptin neuron firing by ~50% and bath application of the aromatase inhibitor Letrozole or the ER-inhibitor ICI182780 entirely prevented this effect. My study provides detailed spatio-temporal information on aromatase neuron development and highlights a novel paracrine mechanism whereby aromatase neurons regulate the activity of distinct neuronal populations expressing ERs.Der Hypothalamus, lokalisiert im Diencephalon in der N¨ahe der Eminentia mediana (ME) und der Hypophyse, steuert die K¨orperhom¨oostase und die Fertilit¨at durch die Freisetzung von Hormonen und die Integration von R¨uckmeldungen aus der Peripherie. Um diese K¨orperachsen, wie die sexuell dimorphe Hypothalamus- Hypophysen-Gonaden-Achse (HPG-Achse), zu koordinieren, besteht der Hypothalamus aus zahlreichen Zelltypen, die jeweils ein spezifisches Repertoire an Proteinen exprimieren. Die Ionenkanalfamilie der Transient- Receptor-Potential (TRP)-Kan¨ale ist essentiell f¨ur diese Koordinierung und ist in zahlreichen Organen exprimiert. Die TRP-Expressionsmuster im Gesamtorganismus, vor allem aber im Hypothalamus, sind von großem Interesse, aber zuverl¨assige Daten mit zellul¨arer Aufl¨osung fehlen aus unterschiedlichen Gr¨unden. TRP-Kan¨ale sind in der Membran lokalisiert, werden posttranslational glykosyliert und zeigen in der Regel ein eher niedriges Expressionsniveau. Dies erschwert die epitopgerichtete Antik¨orperproduktion und den Nachweis auf RNAEbene, gerade auch im Hypothalamus. Reporterm¨ause, die diese Zellpopulationen mit einem Fluoreszenzfarbsto markieren, k¨onnen genutzt werden, um diese Schwierigkeiten zu umgehen. Um die TRP-Expression im Hypothalamus zu untersuchen und f¨ur jeden TRP-Kanal die spezifischen Zelltypen zu identifizieren, habe ich die neu generierten TRPM5- TRPM6-, TRPV6-, TRPA1-, TRPML3-, TRPC2-, TRPC4- und TRPC5- GFP Reporterm¨ause und iDISCO-Clearing verwendet. Eine Subpopulation von Tanyzyten im dritten Ventrikel exprimiert TRPM5 und stehen potenziell im bidirektionalen Austausch mit dem Blutkreislauf ¨uber ihre Fasern welche in Richtung der fenestrierten Endothelzellen in der ME projizieren. Zus¨atzlich zu den Tanyzyten identifizierte ich TRPM5 sowie TRPC5- und TRPA1 Zellen in der Pars tuberalis in engem Kontakt mit Blutgef¨aßen. Interessanterweise wird TRPV6 nicht im Gehirn exprimiert, w¨ahrend TRPM6 in jedem Blutgef¨aß der Blut- Hirn-Schranke zu finden ist. TRPC4 wurde in Perizyten identifiziert, welche ihre Fasern um Blutgef¨aße wickeln, und GFP-Expression wurde in Fasern in der ME von TRPC2-Reporter Tieren gefunden. Diese Fasern stehen in engem Kontakt mit fenestrierten Blutgef¨aßen im posterioren Teil der ME, was darauf hindeutet, dass diese Fasern hier terminieren. Diese Fasern haben ihren Urpsrung in akut TRPC2 exprimierenden Neuronen im paraventrikul¨aren Nukleus (PVN), die ventro-lateral in die ME projizieren. Eine kleine Subpopulation dieser Neurone exprimiert ebenfalls Corticotropin-Freisetzendes-Hormon (CFH), aber Ablation oder synaptisches silencing von TRPC2 Neuronen hatte keinen Einfluss auf den Adrenocorticotropin (ACTH)-Spiegel. Die Aktivierung von TRPC2 Neuronen f¨uhrte zu erh¨ohten luteinisierendem Hormon (LH) und follikelstimulierendem Hormon (FSH) Spiegeln. Dies deutet auf eine m¨ogliche Rolle der TRPC2 Neuronen bei der Regulation der Hormonfreisetzung in der ME hin. Die HPG-Achse wird durch Sexualhormone, wie z. B. O¨ strogen, in sexuell dimorpher Weise gesteuert. Das geschwindigkeitsbestimmende Enzym fu¨r die O¨ strogensynthese ist Aromatase. Um zu verstehen, wie O¨ strogen die sexuelle Di erenzierung beeinflusst, habe ich die Aromatase- Expression w¨ahrend der embryonalen und pr¨anatalen Entwicklung untersucht. Hierzu nutzte ich ebenfalls eine Cre-Reportermaus, die die Aromatasezellen mit GFP markiert. Aromataseneurone waren erstmals am Embryonaltag 13,5 (E13,5) sichtbar und nahmen bis E16,5 in ihrer Anzahl dramatisch zu. Bei der Geburt zeigte sich ein ausgepr¨agter sexueller Dimorphismus im Nucleus arcuatus (Arc). Hier wird Aromatase nur in m¨annlichen Tieren exprimiert. Die Mehrheit der O¨ strogenrezeptor (O¨ R) exprimierenden Neurone ist Aromatase negativ. Dies deutet darauf hin, dass Aromataseneurone entweder in den Gonaden oder im Gehirn synthetisiertes Testosteron in O¨ strogen umwandeln ko¨nnen, um die Neuronenaktivita¨t u¨ber O¨ R-abha¨ngige parakrine Mechanismen zu regulieren. Um diese Hypothese zu testen, haben wir in Zusammenarbeit mit der Gruppe von Prof. Erik Hrabovszky wholecell-Patch-Clamp Experimente an Kisspeptin Neuronen im Arc durchgef¨uhrt. Diese Neurone sind ¨ostrogen-sensitiv und Aromatase-negativ. Testosteron reduzierte die Erregbarkeit der Kisspeptin- Neuronen um 50% und die Applikation des Aromatase-Inhibitors Letrozol oder des O¨ R-Inhibitors ICI182780 verhinderte diesen E ekt vollst¨andig. Meine Ergebnisse liefern detaillierte r¨aumlich-zeitliche Informationen ¨uber die Entwicklung von Aromatase-Neuronen. Außerdem heben sie einen neuartigen parakrinen Mechanismus hervor, durch den Aromataseneurone die Aktivita¨t verschiedener neuronaler Populationen, die O¨ Rs exprimieren, regulieren