NPY derived from AGRP neurons controls feeding via Y1 and energy expenditure and food foraging behaviour via Y2 signalling

Molecular metabolism

2022 Feb 12

Qi, Y;Lee, NJ;Ip, CK;Enriquez, R;Tasan, R;Zhang, L;Herzog, H;
PMID: 35167990 | DOI: 10.1016/j.molmet.2022.101455

Aguti-related protein (AGRP) neurons in the arcuate nucleus of the hypothalamus (ARC), which co-express neuropeptide Y (NPY), are key regulators of feeding and energy homeostasis. However, the precise role NPY has within these neurons and the specific pathways that it control are still unclear. In this article, we aimed to determine what aspects of feeding behaviour and energy homeostasis are controlled by NPY originating from AGRP neurons and which Y-receptor pathways are utilised to fulfil this function.Novel conditional Agrpcre/+;Npylox/lox knockout mice were generated and comprehensively phenotyped, both under standard chow as well as high-fat-diet conditions. Designer receptor exclusively activated by designer drugs (DREADD) technology was used to assess the altered responses on feeding and energy homeostasis control in the absence of NPY in these neurons. Rescue experiments utilising Npy1r- and Npy2r-selective NPY ligands were performed to assess which component of the energy homeostasis control is dependent by which specific Y-receptor pathway.We show that the specific deletion of Npy only in AGRP neurons leads to a paradoxical mild obese phenotype associated with reduced locomotion and energy expenditure and increased feeding and Respiratory Quotient (RQ) that remain elevated under a positive energy balance. The activation of Npy-deficient AGRP neurons via DREADD's is still able to drive feeding, yet with a delayed onset. Additionally, Clozapine-N-oxide (CNO) treatment reduces locomotion without impacting on energy expenditure. Rescue experiments re-introducing Npy1r- and Npy2r-selective NPY ligands revealed that the increased feeding and RQ are mostly driven by Npy1r, whereas energy expenditure and locomotion are controlled by Npy2r signalling.Together, these results demonstrate that NPY originating from AGRP neurons is not only critical to initiate but also for continuously driving feeding, and we for the first time identify which Y-receptor controls which pathway.

HPV RNA CISH score identifies two prognostic groups in a p16 positive oropharyngeal squamous cell carcinoma population

Modern Pathology

2018 Jun 20

Augustin J, Mandavit M, Outh-Gauer S, Grard O, Gasne C, Lépine C, Mirghani H, Hans S, Bonfils P, Denize T, Bruneval P, Bishop JA, Fontugne J, Péré H, Tartour E, Badoual C.
PMID: - | DOI: 10.1038/s41379-018-0090-y

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH “low” and RNA CISH “high”. Negative HPV RNA CISH cases were scored as RNA CISH “low”. This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.

Diagnosis of HPV driven oropharyngeal cancers: Comparing p16 based algorithms with the RNAscope HPV-test

Oral Oncology

2016 Oct 15

Mirghani H, Casiraghi O, Guerlain J, Amen F, He MX, Ma XJ, Luo Y, Mourareau C, Drusch F, Lakdhar AB, Melkane A, St Guily L, Badoual C, Scoazec JY, Borget I, Aupérin A, Dalstein V, Vielh P.
PMID: - | DOI: http://dx.doi.org/10.1016/j.oraloncology.2016.10.009

Abstract

Background

Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status.

Methods

To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2).

Results

105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%.

The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07–0.51, p = 0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08–0.66, p = 0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1–0.65, p = 0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13–0.81, p = 0.02).

Conclusions

Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Spindle Cell Carcinomas of the Head and Neck Rarely Harbor Transcriptionally-Active Human Papillomavirus.

Head and neck pathology, ;7(3):250–257.

Watson RF, Chernock RD, Wang X, Liu W, Ma XJ, Luo Y, Wang H, El-Mofty SK, Lewis JS Jr (2013).
PMID: 23536041 | DOI: 10.1007/s12105-013-0438-z.

Spindle cell carcinoma is an uncommon variant of squamous cell carcinoma characterized by spindled or pleomorphic cells which appear to be a true sarcoma but are actually epithelial. Some head and neck squamous cell carcinoma variants can be human papillomavirus (HPV)-related and have improved outcomes. We sought to determine if spindle cell carcinomas are associated with transcriptionally-active HPV. Cases of spindle cell carcinoma were retrieved from department files. Transcriptionally-active HPV was determined by mRNA in situ hybridization for high risk HPV E6 and E7 transcripts and by a surrogate marker, p16 immunohistochemistry, with a 50% staining cutoff. RT-PCR for high risk HPV mRNA was performed on the cases that were technical failures by in situ hybridization. Medical records and follow up information were retrieved for all patients. Of 31 cases, 5 were from the oropharynx, 12 from the oral cavity, and 14 from the larynx or hypopharynx. One purely spindled oral cavity spindle cell carcinoma was HPV positive. It was also diffusely positive for p16. Another laryngeal spindle cell carcinoma was HPV positive in both the squamous and spindle cell components, but was negative for p16. None of the five oropharyngeal spindle cell carcinomas were positive for p16 or HPV RNA. The HPV positive patients both presented at high stage (IV) and died with disease within 2 years of diagnosis. The majority of spindle cell carcinomas of the head and neck, including those arising in the oropharynx, are not related to transcriptionally active HPV. Although the number of cases is too small for any definitive conclusions, for the rare HPV positive spindle cell carcinoma cases, positive viral status does not appear to confer any prognostic benefit.

Potential clinical implications of HPV status and expressions of p53 and cyclin D1 among oropharyngeal cancer patients.

J Oral Pathol Med.

2018 Sep 06

David Lu XJ, Liu KYP, Soares RC, Thomson T, Prisman E, Wu J, Poh CF.
PMID: 30191616 | DOI: 10.1111/jop.12779

Abstract

BACKGROUND:

There is increasing evidence that high-risk human papillomavirus plays significant role in oropharyngeal cancer; however, there is lack of knowledge on the interplay between the virus and its downstream related molecules and their possible prognostic values. The objectives of the study are to better understand the interplay of the HR-HPV and its associated downstream molecules and to evaluate potential biomarkers for patient outcomes.

METHODS:

We conducted a retrospective study with available formalin-fixed, paraffin-embedded tissue from 244 oropharyngeal cancer patients that received curative radiotherapy or concurrent chemoradiotherapy from 2000 to 2008. In addition to chart review, we performed HPV DNA and RNA in situ hybridization and immunohistochemistry for p53, the retinoblastoma protein, p16, and cyclin D1 analysis. Cox-proportional hazard and Kaplan-Meier survival analysis were used to determine the prognostic markers for clinical outcomes.

RESULTS:

Patients averaged 57.3±9.4 year-old and were mostly males (76.2%) and ever-smokers (76.2%). All patients received curative radiotherapy and 44.3% received concurrent chemoradiotherapy. We detected the human papillomavirus in 77.9% of study patients. Ever-smokers, more advanced tumor stage, and receiving radiotherapy only had poorer 5-year overall survival, disease-specific survival, and loco-regional recurrence. Cases with positive human papillomavirus and p53 overexpression had poorer disease-specific survival. Cases without human papillomavirus, but cyclin D1 overexpression, was associated with poorer 5-year overall survival.

CONCLUSIONS:

Our data suggests that additional p53 and cyclin D1 testing may benefit oropharyngeal cancer patients with known human papillomavirus status.

Molecular ontology of the parabrachial nucleus

The Journal of comparative neurology

2022 Feb 08

Karthik, S;Huang, D;Delgado, Y;Laing, JJ;Peltekian, L;Iverson, GN;Grady, F;Miller, RL;McCann, CM;Fritzsch, B;Iskusnykh, IY;Chizhikov, VV;Geerling, JC;
PMID: 35134251 | DOI: 10.1002/cne.25307

Diverse neurons in the parabrachial nucleus (PB) communicate with widespread brain regions. Despite evidence linking them to a variety of homeostatic functions, it remains difficult to determine which PB neurons influence which functions because their subpopulations intermingle extensively. An improved framework for identifying these intermingled subpopulations would help advance our understanding of neural circuit functions linked to this region. Here, we present the foundation of a developmental-genetic ontology that classifies PB neurons based on their intrinsic, molecular features. By combining transcription factor labeling with Cre fate-mapping, we find that the PB is a blend of two, developmentally distinct macropopulations of glutamatergic neurons. Neurons in the first macropopulation express Lmx1b (and, to a lesser extent, Lmx1a) and are mutually exclusive with those in a second macropopulation, which derive from precursors expressing Atoh1. This second, Atoh1-derived macropopulation includes many Foxp2-expressing neurons, but Foxp2 also identifies a subset of Lmx1b-expressing neurons in the Kölliker-Fuse nucleus (KF) and a population of GABAergic neurons ventrolateral to the PB ("caudal KF"). Immediately ventral to the PB, Phox2b-expressing glutamatergic neurons (some coexpressing Lmx1b) occupy the KF, supratrigeminal nucleus, and reticular formation. We show that this molecular framework organizes subsidiary patterns of adult gene expression (including Satb2, Calca, Grp, and Pdyn) and predicts output projections to the amygdala (Lmx1b), hypothalamus (Atoh1), and hindbrain (Phox2b/Lmx1b). Using this molecular ontology to organize, interpret, and communicate PB-related information could accelerate the translation of experimental findings from animal models to human patients.

In situ hybridization detection methods for HPV16 E6/E7 mRNA in identifying transcriptionally active HPV infection of oropharyngeal carcinoma: an updating

Human Pathology

2017 Oct 06

Volpi CC, Ciniselli CM, Gualeni AV, Plebani M, Alfieri S, Verderio P, Locati L, Perrone F, Quattronea P, Carbone A, Pilotti S, Gloghini A.
PMID: 28993274 | DOI: 10.1016/j.humpath.2017.09.011

The aim of this study is comparing two in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, i.e. the RNAscope™ 2.0 High Definition (HD) and the upgraded RNAscope™ 2.5 HD version. The RNAscope™ 2.5 HD has recently replaced the RNAscope™ 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma (OSCC) with the final goal to apply it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify OSCC that are truly driven by high risk-HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real time reverse transcriptase-polymerase chain reaction (RT-PCR), in a fraction of cases where material for HPV E6/E7 mRNA real time RT-PCR was available. Furthermore, the algorithm that associates p16 immunohistochemistry (IHC) with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 IHC with the identification of HPV DNA by ISH.

NMDAR-dependent synaptic potentiation via APPL1 signaling is required for the accessibility of a prefrontal neuronal assembly in retrieving fear extinction

Biological psychiatry

2023 Feb 24

Hua, SS;Ding, JJ;Sun, TC;Guo, C;Zhang, Y;Yu, ZH;Cao, YQ;Zhong, LH;Wu, Y;Guo, LY;Luo, JH;Cui, YH;Qiu, S;
PMID: 36842495 | DOI: 10.1016/j.biopsych.2023.02.013

The ventromedial prefrontal cortex (vmPFC) has been viewed as a locus to store and recall extinction memory. However, the synaptic and cellular mechanisms underlying this process remain elusive.We combined transgenic mice, electrophysiological recording, activity-dependent cell labeling, and chemogenetic manipulation to analyze the role of adaptor protein APPL1 in the vmPFC for fear extinction retrieval.We found that both constitutive and conditional APPL1 knockout decreases NMDA receptor (NMDAR) function in the vmPFC and impairs fear extinction retrieval. Moreover, APPL1 undergoes nuclear translocation during extinction retrieval. Blocking APPL1 nucleocytoplasmic translocation reduces NMDAR currents and disrupts extinction retrieval. We further identified a prefrontal neuronal ensemble that is both necessary and sufficient for the storage of extinction memory. Inducible APPL1 knockout in this ensemble abolishes NMDAR-dependent synaptic potentiation and disrupts extinction retrieval, while simultaneously chemogenetic activation of this ensemble rescues the impaired behaviors.Therefore, our results indicate that a prefrontal neuronal ensemble stores extinction memory, and APPL1 signaling supports these neurons to retrieve extinction memory via controlling NMDAR-dependent potentiation.

Lateral Habenula and Its Potential Roles in Pain and Related Behaviors

ACS chemical neuroscience

2022 Apr 20

Dai, D;Li, W;Chen, A;Gao, XF;Xiong, L;
PMID: 35412792 | DOI: 10.1021/acschemneuro.2c00067

The lateral habenula (LHb) is a tiny structure that acts as a hub, relaying signals from the limbic forebrain structures and basal ganglia to the brainstem modulatory area. Facilitated by updated knowledge and more precise manipulation of circuits, the progress in figuring out the neural circuits and functions of the LHb has increased dramatically over the past decade. Importantly, LHb is found to play an integrative role and has profound effects on a variety of behaviors associated with pain, including depression-like and anxiety-like behaviors, antireward or aversion, aggression, defensive behavior, and substance use disorder. Thus, LHb is a potential target for improving pain management and related disorders. In this review, we focused on the functions, related circuits, and neurotransmissions of the LHb in pain processing and related behaviors. A comprehensive understanding of the relationship between the LHb and pain will help to find new pain treatments.

A neural circuit for excessive feeding driven by environmental context in mice

Nature neuroscience

2021 Jun 24

Mohammad, H;Senol, E;Graf, M;Lee, CY;Li, Q;Liu, Q;Yeo, XY;Wang, M;Laskaratos, A;Xu, F;Luo, SX;Jung, S;Augustine, GJ;Fu, Y;
PMID: 34168339 | DOI: 10.1038/s41593-021-00875-9

Despite notable genetic influences, obesity mainly results from the overconsumption of food, which arises from the interplay of physiological, cognitive and environmental factors. In patients with obesity, eating is determined more by external cues than by internal physiological needs. However, how environmental context drives non-homeostatic feeding is elusive. Here, we identify a population of somatostatin (TNSST) neurons in the mouse hypothalamic tuberal nucleus that are preferentially activated by palatable food. Activation of TNSST neurons enabled a context to drive non-homeostatic feeding in sated mice and required inputs from the subiculum. Pairing a context with palatable food greatly potentiated synaptic transmission between the subiculum and TNSST neurons and drove non-homeostatic feeding that could be selectively suppressed by inhibiting TNSST neurons or the subiculum but not other major orexigenic neurons. These results reveal how palatable food, through a specific hypothalamic circuit, empowers environmental context to drive non-homeostatic feeding.

Defining the better algorithm for the accurate identification of HPV status among oropharyngeal squamous-cell carcinoma. Results from a pilot study

WCRJ 2015; 2 (1): e497

Gloghini A, Volpi CC, Gualeni AV, Cortellazzi B, Perrone F, Pilotti S.
PMID: //www.wcrj.net/wp-content/uploads/2015/04/WCRJ-2015-2-1-e497-Gloghini-WCRJ.pdf

Abstract: Background: The recognition of tumor infection by human papilloma virus (HPV) in oropharyngeal squamous-cell carcinoma (OSCC) is emerging as a valid biomarker to more accurate selection of patients for specific treatment, surveillance and tumor staging. To this aim, the HPV detection strategy in OSCC must dissect between HPV that is acting as a driver of malignant transformation, and transcriptionally silent virus involved in productive infection. The aim of this study is to define the better method for the accurate identification of HPV status among OSCC. Patients and Methods: Thirty-six patients were selected for HPV status assessment combining different methods, such as immunohistochemistry (IHC) for p16, in-situ hybridization (ISH) for high risk (HR)-HPV DNA and HR-HPV E6/E7 mRNA along with real-time PCR of HPV16 E6/E7 mRNA. All these cases were originally classified as HPV negative by DNA-based ISH but p16 positive by the IHC. Results: Twenty-six cases showed concordance between methods; whereas, nine cases resulted negative for HPV E6/E7 mRNA RT-PCR but positive for HPV E6/E7 mRNA ISH. Conclusion: By considering that the bright field HPV E6/E7 mRNA ISH could be more sensitive than mRNA-based real-time RT-PCR, and that it provides the precise identification of transcriptionally active HPV infected cells, a randomized analysis to validate the robustness of this preliminary assay will be undertaken.

Fast neurotransmitter identity of MCH neurons: Do contents depend on context?

Frontiers in neuroendocrinology

2023 May 05

Beekly, BG;Rupp, A;Burgess, CR;Elias, CF;
PMID: 37149229 | DOI: 10.1016/j.yfrne.2023.101069

Hypothalamic melanin-concentrating hormone (MCH) neurons participate in many fundamental neuroendocrine processes. While some of their effects can be attributed to MCH itself, others appear to depend on co-released neurotransmitters. Historically, the subject of fast neurotransmitter co-release from MCH neurons has been contentious, with data to support MCH neurons releasing GABA, glutamate, both, and neither. Rather than assuming a position in that debate, this review considers the evidence for all sides and presents an alternative explanation: neurochemical identity, including classical neurotransmitter content, is subject to change. With an emphasis on the variability of experimental details, we posit that MCH neurons may release GABA and/or glutamate at different points according to environmental and contextual factors. Through the lens of the MCH system, we offer evidence that the field of neuroendocrinology would benefit from a more nuanced and dynamic interpretation of neurotransmitter identity.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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