Quantitative Spatial Analysis of Neuroligin-3 mRNA Expression in the Enteric Nervous System Reveals a Potential Role in Neuronal-Glial Synapses and Reduced Expression in Nlgn3R451C Mice

Biomolecules

2023 Jun 30

Herath, M;Cho, E;Marklund, U;Franks, A;Bornstein, J;Hill-Yardin, E;
| DOI: 10.3390/biom13071063

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers

Scientific reports

2021 Sep 07

Lee, HJ;Myung, JK;Kim, HS;Lee, DH;Go, HS;Choi, JH;Koh, HM;Lee, SJ;Jang, B;
PMID: 34493772 | DOI: 10.1038/s41598-021-97351-y

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

Effect of sex and autism spectrum disorder on oxytocin receptor binding and mRNA expression in the dopaminergic pars compacta of the human substantia nigra

Philosophical transactions of the Royal Society of London. Series B, Biological sciences

2022 Aug 29

Frehner, SS;Dooley, KT;Palumbo, MC;Smith, AL;Goodman, MM;Bales, KL;Freeman, SM;
PMID: 35858098 | DOI: 10.1098/rstb.2021.0118

Oxytocin is an endogenous neuropeptide hormone that influences social behaviour and bonding in mammals. Variations in oxytocin receptor (OXTR) expression may play a role in the social deficits seen in autism spectrum disorder. Previous studies from our laboratory found a dense population of OXTR in the human substantia nigra (SN), a basal ganglia structure in the midbrain that is important in both movement and reward pathways. Here, we explore whether differences in OXTR can be identified in the dopaminergic SN pars compacta of individuals with autism. Postmortem human brain tissue specimens were processed for OXTR autoradiography from four groups: males with autism, females with autism, typically developing (TD) males and TD females. We found that females with autism had significantly lower levels of OXTR than the other groups. To examine potential gene expression differences, we performed in situ hybridization in adjacent slides to visualize and quantify OXTR mRNA as well as mRNA for tyrosine hydroxylase. We found no differences in mRNA levels for either gene across the four groups. These results suggest that a dysregulation in local OXTR protein translation or increased OXTR internalization/recycling may contribute to the differences in social symptoms seen in females with autism. This article is part of the theme issue 'Interplays between oxytocin and other neuromodulators in shaping complex social behaviours'.

Intraneuronal β-Amyloid Accumulation: Aging HIV-1 Human and HIV-1 Transgenic Rat Brain

Viruses

2022 Jun 10

Li, H;McLaurin, KA;Mactutus, CF;Likins, B;Huang, W;Chang, SL;Booze, RM;
PMID: 35746739 | DOI: 10.3390/v14061268

The prevalence of HIV-1 associated neurocognitive disorders (HAND) is significantly greater in older, relative to younger, HIV-1 seropositive individuals; the neural pathogenesis of HAND in older HIV-1 seropositive individuals, however, remains elusive. To address this knowledge gap, abnormal protein aggregates (i.e., β-amyloid) were investigated in the brains of aging (>12 months of age) HIV-1 transgenic (Tg) rats. In aging HIV-1 Tg rats, double immunohistochemistry staining revealed abnormal intraneuronal β-amyloid accumulation in the prefrontal cortex (PFC) and hippocampus, relative to F344/N control rats. Notably, in HIV-1 Tg animals, increased β-amyloid accumulation occurred in the absence of any genotypic changes in amyloid precursor protein (APP). Furthermore, no clear amyloid plaque deposition was observed in HIV-1 Tg animals. Critically, β-amyloid was co-localized with neurons in the cortex and hippocampus, supporting a potential mechanism underlying synaptic dysfunction in the HIV-1 Tg rat. Consistent with these neuropathological findings, HIV-1 Tg rats exhibited prominent alterations in the progression of temporal processing relative to control animals; temporal processing relies, at least in part, on the integrity of the PFC and hippocampus. In addition, in post-mortem HIV-1 seropositive individuals with HAND, intraneuronal β-amyloid accumulation was observed in the dorsolateral PFC and hippocampal dentate gyrus. Consistent with observations in the HIV-1 Tg rat, no amyloid plaques were found in these post-mortem HIV-1 seropositive individuals with HAND. Collectively, intraneuronal β-amyloid aggregation observed in the PFC and hippocampus of HIV-1 Tg rats supports a potential factor underlying HIV-1 associated synaptodendritic damage. Further, the HIV-1 Tg rat provides a biological system to model HAND in older HIV-1 seropositive individuals.

Whole-Mount Multicolor Fluorescent Labeling by In Situ Hybridization in Astyanax mexicanus Embryos and Larvae

Neuromethods

2023 Jan 01

Blin, M;Rétaux, S;Torres-Paz, J;
| DOI: 10.1007/978-1-0716-2875-1_13

Gene expression analyses by molecular histology are a crucial step in understanding gene function in any model organism. In the teleost _Astyanax mexicanus_, here we describe in detail the method we have developed to perform double fluorescent in situ hybridization on whole-mount samples. As an illustration, in the result section, we present an analysis of the expression patterns of four mRNAs expressed in the hypothalamus of the surface and cave morphs of _A. mexicanus_ at 3.5 days postfertilization, three neuropeptides (_npy_, _pomca_, _agrp_) and one transcription factor (_isl1_). Confocal imaging after fluorescent in situ hybridization allows counting cells in distinct but closely related hypothalamic areas. The step-by-step protocol and the comprehensive table of reagents presented here will allow researchers to analyze gene expression in different structures and at various stages, from embryos to older larvae.

Single cell RNA sequencing reveals the role of Myelin regulatory factor (MYRF) in regulating melanogenesis and cell structure during retinal pigment epithelial development.

Investigative Ophthalmology & Visual Science

2022 Jan 01

Prasov, L;Brinkmeier, ML;Wang, SQ;

RESULTS : Cell clustering revealed that Myrf deficiency altered cell type distributions with reductions in RPE cells at all timepoints. Cell cycle dynamics were stable, consistent with increased cell death in mutants. There was also a compensatory increase in retinal progenitor (RPC) population at P0, without alteration in overall cell cycle dynamics. Differential gene expression analysis and PANTHER gene ontology-term analysis revealed down regulation of key pathways in mutant RPE cells, including melanosome biogenesis, cytoskeleton, and extracellular matrix. EM analysis and immunofluorescence staining of RPE flatmounts confirmed structural defects in RPE and disorganization of photoreceptor outer segments, loss of melanosomes, and alterations in novel structural proteins in the apical RPE. Compensatory upgregulation of _Prss56_, another gene implicated in nanophthalmos, was found in the RPC population.

Interleukin-6 Stromal Expression is Correlated with Epithelial-Mesenchymal Transition at Tumor Budding in Colorectal Cancer

International journal of surgical pathology

2023 Jun 12

Uehara, T;Sato, K;Iwaya, M;Asaka, S;Nakajima, T;Nagaya, T;Kitazawa, M;Ota, H;
PMID: 37306249 | DOI: 10.1177/10668969231177705

Background. Tumor budding is a poor prognostic factor in colorectal adenocarcinoma, but the underlying mechanism remains unclear. Interleukin-6 (IL6) is one of the main cytokines produced by cancer-associated fibroblasts. IL6 is linked with cancer progression and poor prognosis by activating cancer cells and modifying the cancer microenvironment. However, little is known about the expression of IL6 in tumor budding and its association with tumor budding in colorectal adenocarcinoma. Methods. The clinicopathological and prognostic significance of IL6 in tumor budding was examined using a tissue microarray consisting of 36 patient samples of tumor budding in colorectal adenocarcinoma. IL6 mRNA was detected by RNAscope. Patients were stratified into negative and positive IL6 expression groups. Results. IL6 expression was overwhelmingly observed in cancer stroma but was negligible in cancer cells. Tumor budding grade was higher in the IL6-positive group in cancer stroma than in the IL6-negative group (P = .0161), while the IL6-positive group significantly exhibited the epithelial-mesenchymal transition phenotype compared with the IL6-negative group in cancer stroma (P = .0301). There was no significant difference in overall survival between colorectal adenocarcinoma patients in the IL6-positive and -negative groups in cancer stroma. Conclusion. Tumor budding may be affected by IL6 expression, and IL6 expression in cancer stroma at tumor budding may be an important prognostic marker.

Caspase 6/NR4A1/SOX9 signaling axis regulates hepatic inflammation and pyroptosis in ischemia-stressed fatty liver

Cell death discovery

2023 Mar 28

Sheng, M;Weng, Y;Cao, Y;Zhang, C;Lin, Y;Yu, W;
PMID: 36977670 | DOI: 10.1038/s41420-023-01396-z

The mechanism of nonalcoholic fatty liver susceptibility to ischemia/reperfusion (IR) injury has not been fully clarified. Caspase 6 is a critical regulator in innate immunity and host defense. We aimed to characterize the specific role of Caspase 6 in IR-induced inflammatory responses in fatty livers. Human fatty liver samples were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. in mice model, we generated Caspase 6-knockout (Caspase 6KO) mice to investigate cellular and molecular mechanisms of macrophage Caspase 6 in IR-stimulated fatty livers. In human liver biopsies, Caspase 6 expression was upregulated combined with enhanced serum ALT level and severe histopathological injury in ischemic fatty livers. Moreover, Caspase 6 was mainly accumulated in macrophages but not hepatocytes. Unlike in controls, the Caspase 6-deficiency attenuated liver damage and inflammation activation. Activation of macrophage NR4A1 or SOX9 in Caspase 6-deficient livers aggravated liver inflammation. Mechanistically, macrophage NR4A1 co-localized with SOX9 in the nuclear under inflammatory conditions. Specifically, SOX9 acts as a coactivator of NR4A1 to directly target S100A9 transcription. Furthermore, macrophage S100A9 ablation dampened NEK7/NLRP3-driven inflammatory response and pyroptosis in macrophages. In conclusion, our findings identify a novel role of Caspase 6 in regulating NR4A1/SOX9 interaction in response to IR-stimulated fatty liver inflammation, and provide potential therapeutic targets for the prevention of fatty liver IR injury.

Netrin-1 regulates the balance of synaptic glutamate signaling in the adult ventral tegmental area

eLife

2023 Mar 17

Cline, MM;Juarez, B;Hunker, A;Regiarto, EG;Hariadi, B;Soden, ME;Zweifel, LS;
PMID: 36927614 | DOI: 10.7554/eLife.83760

The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding (Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, the reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Simultaneous loss of Ntn1 function in both cell types largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for Ntn1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.

Role of Eosinophils in Purinergic Receptor P2X3Expression in Mouse Sensory Neurons

B63. FROM AIRWAYS TO ALVEOLI: MECHANISTIC AND CLINICAL STUDIES

2022 May 01

Mize, E;
| DOI: 10.1164/ajrccm-conference.2022.205.1_meetingabstracts.a3282

: P2X3 purinoceptors are expressed by airway sensory neurons and are activated by extracellular ATP released during periods of cell stress. In asthma, sensitivity to ATP is increased. Airway eosinophilia, which is common in a majority of asthmatics, increases airway epithelial sensory nerve density in mice and in humans. Whether eosinophils increase neuronal P2X3 expression in asthma is unknown. Methods: P2X3 expression was quantified in sensory neurons in vagal ganglia from wild-type (WT) mice, transgenic mice with chronic pulmonary eosinophilia (NJ1726 lineage), transgenic mice with chronic systemic eosinophilia (NJ1638 lineage), and eosinophil-deficient mice (PHIL) using RNAscope in situ hybridization. Images were obtained using an ApoTome confocal microscope (40X, 1.3 N.A.). P2X3 expression was determined by measuring the percentage of P2X3 positive pixels within three, randomly assigned, non-overlapping 50x50 micron sections of vagal ganglia using ImageJ. Replicates were averaged to generate a single data point for each animal. Results: P2X3 RNA was detected in vagal ganglia tissue sections in all mouse groups. P2X3 RNA expression was present in 38.9 ± 1.2% of vagal neuron tissue analyzed in WT mice, 62.6 ± 11.9% in mice with chronic pulmonary eosinophilia, 55.7 ± 4.3% in mice with chronic systemic eosinophilia, and 56.6 ± 2.1% in eosinophil-deficient mice. Conclusions: P2X3 is highly expressed in vagal sensory neurons. Increased P2X3 expression in both the setting of chronic eosinophilia and absence of tissue eosinophils suggests eosinophils both positively and negatively regulate purinoceptor expression in sensory neurons. Eosinophil effects may contribute to increased ATP sensitivity in humans with eosinophilic asthma.

Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia

Molecular psychiatry

2022 May 26

Mätlik, K;Garton, DR;Montaño-Rodríguez, AR;Olfat, S;Eren, F;Casserly, L;Damdimopoulos, A;Panhelainen, A;Porokuokka, LL;Kopra, JJ;Turconi, G;Schweizer, N;Bereczki, E;Piehl, F;Engberg, G;Cervenka, S;Piepponen, TP;Zhang, FP;Sipilä, P;Jakobsson, J;Sellgren, CM;Erhardt, S;Andressoo, JO;
PMID: 35618883 | DOI: 10.1038/s41380-022-01554-2

Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2-3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A2AR), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A2AR with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF-A2AR crosstalk may regulate dopamine function in a therapeutically targetable manner.

Beta2-adrenoreceptor agonist clenbuterol produces transient decreases in alpha-synuclein mRNA but no long-term reduction in protein

NPJ Parkinson's disease

2022 May 24

Patterson, JR;Hirst, WD;Howe, JW;Russell, CP;Cole-Strauss, A;Kemp, CJ;Duffy, MF;Lamp, J;Umstead, A;Kubik, M;Stoll, AC;Vega, IE;Steece-Collier, K;Chen, Y;Campbell, AC;Nezich, CL;Glajch, KE;Sortwell, CE;
PMID: 35610264 | DOI: 10.1038/s41531-022-00322-x

β2-adrenoreceptor (β2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of β2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

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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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