STING controls nociception via type I interferon signalling in sensory neurons

Nature

2021 Jan 13

Donnelly, CR;Jiang, C;Andriessen, AS;Wang, K;Wang, Z;Ding, H;Zhao, J;Luo, X;Lee, MS;Lei, YL;Maixner, W;Ko, MC;Ji, RR;
PMID: 33442058 | DOI: 10.1038/s41586-020-03151-1

The innate immune regulator STING is a critical sensor of self- and pathogen-derived DNA. DNA sensing by STING leads to the induction of type-I interferons (IFN-I) and other cytokines, which promote immune-cell-mediated eradication of pathogens and neoplastic cells1,2. STING is also a robust driver of antitumour immunity, which has led to the development of STING activators and small-molecule agonists as adjuvants for cancer immunotherapy3. Pain, transmitted by peripheral nociceptive sensory neurons (nociceptors), also aids in host defence by alerting organisms to the presence of potentially damaging stimuli, including pathogens and cancer cells4,5. Here we demonstrate that STING is a critical regulator of nociception through IFN-I signalling in peripheral nociceptors. We show that mice lacking STING or IFN-I signalling exhibit hypersensitivity to nociceptive stimuli and heightened nociceptor excitability. Conversely, intrathecal activation of STING produces robust antinociception in mice and non-human primates. STING-mediated antinociception is governed by IFN-Is, which rapidly suppress excitability of mouse, monkey and human nociceptors. Our findings establish the STING-IFN-I signalling axis as a critical regulator of physiological nociception and a promising new target for treating chronic pain.

Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis

Cell

2021 Jan 07

Yu, M;Ma, L;Yuan, Y;Ye, X;Montagne, A;He, J;Ho, TV;Wu, Y;Zhao, Z;Sta Maria, N;Jacobs, R;Urata, M;Wang, H;Zlokovic, BV;Chen, JF;Chai, Y;
PMID: 33417861 | DOI: 10.1016/j.cell.2020.11.037

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Graded BMP signaling within intestinal crypt architecture directs self-organization of the Wnt-secreting stem cell niche

Cell stem cell

2023 Apr 06

Kraiczy, J;McCarthy, N;Malagola, E;Tie, G;Madha, S;Boffelli, D;Wagner, DE;Wang, TC;Shivdasani, RA;
PMID: 37028407 | DOI: 10.1016/j.stem.2023.03.004

Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81+ PDGFRAlo trophocytes capably sustain ISC functions ex vivo. Here, we show that mRNA and chromatin profiles of abundant CD81- PDGFRAlo mouse stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands. Mesenchymal expression of key ISC-supportive factors extends along a spatial and molecular continuum from trophocytes into peri-cryptal CD81- CD55hi cells, which mimic trophocyte activity in organoid co-cultures. Graded expression of essential niche factors is not cell-autonomous but dictated by the distance from bone morphogenetic protein (BMP)-secreting PDGFRAhi myofibroblast aggregates. BMP signaling inhibits ISC-trophic genes in PDGFRAlo cells near high crypt tiers; that suppression is relieved in stromal cells near and below the crypt base, including trophocytes. Cell distances thus underlie a self-organized and polar ISC niche.

Emergence of CRISPR/Cas9-mediated bioimaging: A new dawn of in-situ detection

Biosensors & bioelectronics

2023 Jul 15

Tian, M;Zhang, R;Li, J;
PMID: 37086563 | DOI: 10.1016/j.bios.2023.115302

In-situ detection provides deep insights into the function of genes and their relationship with diseases by directly visualizing their spatiotemporal behavior. As an emerging in-situ imaging tool, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bioimaging can localize targets in living and fixed cells. CRISPR-mediated bioimaging has inherent advantages over the gold standard of fluorescent in-situ hybridization (FISH), including fast imaging, cost-effectiveness, and ease of preparation. Existing reviews have provided a detailed classification and overview of the principles of CRISPR-mediated bioimaging. However, the exploitation of potential clinical applicability of this bioimaging technique is still limited. Therefore, analyzing the potential value of CRISPR-mediated in-situ imaging is of great significance to the development of bioimaging. In this review, we initially discuss the available CRISPR-mediated imaging systems from the following aspects: summary of imaging substances, the design and optimization of bioimaging strategies, and factors influencing CRISPR-mediated in-situ detection. Subsequently, we highlight the potential of CRISPR-mediated bioimaging for application in biomedical research and clinical practice. Furthermore, we outline the current bottlenecks and future perspectives of CRISPR-based bioimaging. We believe that this review will facilitate the potential integration of bioimaging-related research with current clinical workflow.

EphB2-dependent prefrontal cortex activation promotes long-range social approach and partner responsiveness

Proceedings of the National Academy of Sciences of the United States of America

2023 Feb 28

He, LN;Chen, S;Yang, Q;Wu, Z;Lao, ZK;Tang, CF;Song, JJ;Liu, XD;Lu, J;Xu, XH;Chen, JJ;Xu, TL;Sun, S;Xu, NJ;
PMID: 36802416 | DOI: 10.1073/pnas.2219952120

Social behavior starts with dynamic approach prior to the final consummation. The flexible processes ensure mutual feedback across social brains to transmit signals. However, how the brain responds to the initial social stimuli precisely to elicit timed behaviors remains elusive. Here, by using real-time calcium recording, we identify the abnormalities of EphB2 mutant with autism-associated Q858X mutation in processing long-range approach and accurate activity of prefrontal cortex (dmPFC). The EphB2-dependent dmPFC activation precedes the behavioral onset and is actively associated with subsequent social action with the partner. Furthermore, we find that partner dmPFC activity is responsive coordinately to the approaching WT mouse rather than Q858X mutant mouse, and the social defects caused by the mutation are rescued by synchro-optogenetic activation in dmPFC of paired social partners. These results thus reveal that EphB2 sustains neuronal activation in the dmPFC that is essential for the proactive modulation of social approach to initial social interaction.

Developmental diversity and unique sensitivity to injury of lung endothelial subtypes during postnatal growth

iScience

2023 Jan 01

Zanini, F;Che, X;Knutsen, C;Liu, M;Suresh, N;Domingo-Gonzalez, R;Dou, S;Zhang, D;Pryhuber, G;Jones, R;Quake, S;Cornfield, D;Alvira, C;
| DOI: 10.1016/j.isci.2023.106097

At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2), contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury which impairs angiogenesis, induced both common and unique endothelial gene signatures, dysregulated capillary EC cross-talk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.

Pooled genetic screens with image-based profiling

Molecular systems biology

2022 Nov 01

Walton, RT;Singh, A;Blainey, PC;
PMID: 36366905 | DOI: 10.15252/msb.202110768

Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype-phenotype relationships.

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Journal of visualized experiments : JoVE

2022 Oct 20

Kerloch, T;Lepko, T;Shkura, K;Guillemot, F;Gillotin, S;
PMID: 36342175 | DOI: 10.3791/64369

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Implementing a multi-colour genetic marker analysis technique for embryology education

Anatomia, histologia, embryologia

2022 Sep 30

Yahya, I;Omer, EAM;Gellisch, M;Brand-Saberi, B;Morosan-Puopolo, G;
PMID: 36177714 | DOI: 10.1111/ahe.12868

Embryology belongs to the basic sciences and is usually an integral part of the anatomy. The subject is traditionally taught by visual inspection of embryonic tissue slides stained with Haematoxylin and Eosin (H&E) to expose the dynamics of tissue histology as development proceeds. While combining in situ hybridization for gene expression analysis and immunostaining for protein expression analysis is an established technique for embryology research, the implementation of this tool in embryology teaching has not been described. The present study was conducted to assess the use of an online multi-colour gene expression analysis technique, alongside histological sections and diagrams, to improve students' understanding of embryology. The participants of this study were bachelor's students of Veterinary Medicine at the University of Khartoum. The method was also evaluated by distributing questionnaire items to Veterinary students via Google forms; subsequently, their responses were analysed qualitatively. The majority of students stated that the new technique was beneficial for their learning of embryology. The multi-colour images proved a more effective means for learning embryology than the traditional H&E image. Results from the students strengthen the belief in applying the multi-colour technique for better embryology course learning.

Lentiviral Vectors for Ocular Gene Therapy

Pharmaceutics

2022 Jul 31

Arsenijevic, Y;Berger, A;Udry, F;Kostic, C;
PMID: 36015231 | DOI: 10.3390/pharmaceutics14081605

This review offers the basics of lentiviral vector technologies, their advantages and pitfalls, and an overview of their use in the field of ophthalmology. First, the description of the global challenges encountered to develop safe and efficient lentiviral recombinant vectors for clinical application is provided. The risks and the measures taken to minimize secondary effects as well as new strategies using these vectors are also discussed. This review then focuses on lentiviral vectors specifically designed for ocular therapy and goes over preclinical and clinical studies describing their safety and efficacy. A therapeutic approach using lentiviral vector-mediated gene therapy is currently being developed for many ocular diseases, e.g., aged-related macular degeneration, retinopathy of prematurity, inherited retinal dystrophies (Leber congenital amaurosis type 2, Stargardt disease, Usher syndrome), glaucoma, and corneal fibrosis or engraftment rejection. In summary, this review shows how lentiviral vectors offer an interesting alternative for gene therapy in all ocular compartments.

Repair of the Murine Tympanic Membrane Displays Hallmarks of Regeneration

The FASEB Journal

2022 May 01

Scaria, S;Frumm, S;Sheth, A;Tward, A;
| DOI: 10.1096/fasebj.2022.36.S1.R3190

RNA expression data from all timepoints of perforation were merged and analyzed, revealing 8 distinct major populations of cells and revealing time-dependent transcriptional shifts in each layer of the TM. From both cross-sectional and whole-mount views, the TM shows a rapid, proliferative response to injury by 18 hours post-injury, predominantly in the KCs. 3 days after perforation, there are large transcriptional shifts in the immune, mesenchymal, and mucosal populations. The multi-layered tissue shows a large volumetric increase by day 7 but quickly remodels and restores the original volume of the TM by day 14. At slightly longer timepoints, the radial and circular collagen patterning of the TM is also restored, creating a scar-free structure. We identified a regeneration-induced “wounded epithelial” population, characterized by a combination of distinct marker genes. A _K5Cre-ERT2;Confetti_ mouse model shows that the population migrates from known stem cell regions of the organ to the site of injury. Based on expression values and immunostaining, EGFR signaling is upregulated during regeneration, corresponding with increased expression of EGFR ligands and processing co-factors. When EGFR is deleted _in vivo_, using a _K5-CreERT2_;_Egfrfl/fl; R26mTmG/mTmG_ mouse model, TMs no longer display proliferation post-injury and cannot repair perforations.

Odorant Receptor Choice Mechanism Revealed by Analysis of a Highly Represented Odorant Receptor Transgene

SSRN Electronic Journal

2022 May 28

Makhlouf, M;D'Hulst, C;Omura, M;Rosa, A;Mina, R;Bernal-Garcia, S;Lempert, E;Saraiva, L;Feinstein, P;
| DOI: 10.2139/ssrn.4119003

In the mouse, more than 1,100 odorant receptors (ORs) are expressed in a monogenic and monoallelic fashion, referred to as singular gene expression. Using a 21bp singular-choice enhancer (x21), we radically increase representation of olfactory sensory neurons (OSNs) choosing a 5x21 enhanced OR transgene, but not overexpression of its mRNA on a per cell basis. RNA-sequencing and differential expression analysis identified 425 differentially expressed genes (DEGs). ORs make up 86% of all DEGs, of which 325 have decreased representation and 40 have increased representation. Underrepresented ORs include Class I, Class II and TAAR genes and within each of their respective olfactory bulb domains: DI, DII, and DIII (TAAR) we committedly observe multiple homogeneous glomeruli with an OR1A1-identity. The underrepresentation of endogenous, class-specific ORs across evolutionarily distinct cell types in favor of the expression of the 5x21-OR1A1 transgene argues that a common mechanism of singular gene choice is present for all OR-expressing OSNs.

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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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