Activated microglia drive demyelination via CSF1R signaling

Glia

2021 Feb 23

Marzan, DE;Brügger-Verdon, V;West, BL;Liddelow, S;Samanta, J;Salzer, JL;
PMID: 33620118 | DOI: 10.1002/glia.23980

Microgliosis is a prominent pathological feature in many neurological diseases including multiple sclerosis (MS), a progressive auto-immune demyelinating disorder. The precise role of microglia, parenchymal central nervous system (CNS) macrophages, during demyelination, and the relative contributions of peripheral macrophages are incompletely understood. Classical markers used to identify microglia do not reliably discriminate between microglia and peripheral macrophages, confounding analyses. Here, we use a genetic fate mapping strategy to identify microglia as predominant responders and key effectors of demyelination in the cuprizone (CUP) model. Colony-stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor (M-CSF) - a secreted cytokine that regulates microglia development and survival-is upregulated in demyelinated white matter lesions. Depletion of microglia with the CSF1R inhibitor PLX3397 greatly abrogates the demyelination, loss of oligodendrocytes, and reactive astrocytosis that results from CUP treatment. Electron microscopy (EM) and serial block face imaging show myelin sheaths remain intact in CUP treated mice depleted of microglia. However, these CUP-damaged myelin sheaths are lost and robustly phagocytosed upon-repopulation of microglia. Direct injection of CSF1 into CNS white matter induces focal microgliosis and demyelination indicating active CSF1 signaling can promote demyelination. Finally, mice defective in adopting a toxic astrocyte phenotype that is driven by microglia nevertheless demyelinate normally upon CUP treatment implicating microglia rather than astrocytes as the primary drivers of CUP-mediated demyelination. Together, these studies indicate activated microglia are required for and can drive demyelination directly and implicate CSF1 signaling in these events.

Disruption of DNA polymerase ζ engages an innate immune response

Cell reports

2021 Feb 23

Martin, SK;Tomida, J;Wood, RD;
PMID: 33626348 | DOI: 10.1016/j.celrep.2021.108775

In mammalian cells, specialized DNA polymerase ζ (pol ζ) contributes to genomic stability during normal DNA replication. Disruption of the catalytic subunit Rev3l is toxic and results in constitutive chromosome damage, including micronuclei. As manifestations of this genomic stress are unknown, we examined the transcriptome of pol ζ-defective cells by RNA sequencing (RNA-seq). Expression of 1,117 transcripts is altered by ≥4-fold in Rev3l-disrupted cells, with a pattern consistent with an induction of an innate immune response. Increased expression of interferon-stimulated genes at the mRNA and protein levels in pol ζ-defective cells is driven by the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-signaling partner stimulator of interferon genes (STING) pathway. Expression of key interferon-stimulated chemokines is elevated in basal epithelial mouse skin cells with a disruption of Rev3l. These results indicate that the disruption of pol ζ may simultaneously increase sensitivity to genotoxins and potentially engage parts of the innate immune response, which could add an additional benefit to targeting pol ζ in cancer therapies.

FGF8 and BMP2 mediated dynamic regulation of dental mesenchyme proliferation and differentiation via Lhx8/Suv39h1 complex

Journal of cellular and molecular medicine

2021 Feb 13

Zhou, C;Chen, D;Ren, J;Huang, D;Li, R;Luo, H;Guan, C;Cao, Y;Wang, W;
PMID: 33580754 | DOI: 10.1111/jcmm.16351

The homeobox gene, LIM-homeobox 8 (Lhx8), has previously been identified as an essential transcription factor for dental mesenchymal development. However, how Lhx8 itself is regulated and regulates odontogenesis remains poorly understood. In this study, we employed an RNAscope assay to detect the co-expression pattern of Lhx8 and Suv39h1 in the dental mesenchyme, which coincided with the dynamic expression profiles of the early epithelium signal of Fibroblast Growth Factor 8 (FGF8) and the later mesenchymal signal Bone Morphogenetic Protein 2 (BMP2). Moreover, FGF8 activated Lhx8, whereas BMP2 repressed Lhx8 expression at the transcriptional level. The high expression of Lhx8 in the early dental mesenchyme maintained the cell fate in an undifferentiated status by interacting with Suv39h1, a histone-lysine N-methyltransferase constitutively expressed in the dental mesenchyme. Further in the ex vivo organ culture model, the knockdown of Suv39h1 significantly blocked the function of Lhx8 and FGF8. Mechanistically, Lhx8/Suv39h1 recognized the odontoblast differentiation-related genes and repressed gene expression via methylating H3K9 on their promoters. Taken together, our data here suggest that Lhx8/Suv39h1 complex is inversely regulated by epithelium-mesenchymal signals, balancing the differentiation and proliferation of dental mesenchyme via H3K9 methylation.

Disruption of nuclear architecture as a cause of COVID-19 induced anosmia

bioRxiv : the preprint server for biology

2021 Feb 09

Zazhytska, M;Kodra, A;Hoagland, DA;Fullard, JF;Shayya, H;Omer, A;Firestein, S;Gong, Q;Canoll, PD;Goldman, JE;Roussos, P;tenOever, BR;Overdevest, JB;Lomvardas, S;
PMID: 33594368 | DOI: 10.1101/2021.02.09.430314

Olfaction relies on a coordinated partnership between odorant flow and neuronal communication. Disruption in our ability to detect odors, or anosmia, has emerged as a hallmark symptom of infection with SARS-CoV-2, yet the mechanism behind this abrupt sensory deficit remains elusive. Here, using molecular evaluation of human olfactory epithelium (OE) from subjects succumbing to COVID-19 and a hamster model of SARS-CoV-2 infection, we discovered widespread downregulation of olfactory receptors (ORs) as well as key components of their signaling pathway. OR downregulation likely represents a non-cell autonomous effect, since SARS-CoV-2 detection in OSNs is extremely rare both in human and hamster OEs. A likely explanation for the reduction of OR transcription is the striking reorganization of nuclear architecture observed in the OSN lineage, which disrupts multi-chromosomal compartments regulating OR expression in humans and hamsters. Our experiments uncover a novel molecular mechanism by which a virus with a very selective tropism can elicit persistent transcriptional changes in cells that evade it, contributing to the severity of COVID-19.

TCR-engineered T cells targeting E7 for patients with metastatic HPV-associated epithelial cancers

Nature medicine

2021 Feb 08

Nagarsheth, NB;Norberg, SM;Sinkoe, AL;Adhikary, S;Meyer, TJ;Lack, JB;Warner, AC;Schweitzer, C;Doran, SL;Korrapati, S;Stevanović, S;Trimble, CL;Kanakry, JA;Bagheri, MH;Ferraro, E;Astrow, SH;Bot, A;Faquin, WC;Stroncek, D;Gkitsas, N;Highfill, S;Hinrichs, CS;
PMID: 33558725 | DOI: 10.1038/s41591-020-01225-1

Genetically engineered T cell therapy can induce remarkable tumor responses in hematologic malignancies. However, it is not known if this type of therapy can be applied effectively to epithelial cancers, which account for 80-90% of human malignancies. We have conducted a first-in-human, phase 1 clinical trial of T cells engineered with a T cell receptor targeting HPV-16 E7 for the treatment of metastatic human papilloma virus-associated epithelial cancers (NCT02858310). The primary endpoint was maximum tolerated dose. Cell dose was not limited by toxicity with a maximum dose of 1 × 1011 engineered T cells administered. Tumor responses following treatment were evaluated using RECIST (Response Evaluation Criteria in Solid Tumors) guidelines. Robust tumor regression was observed with objective clinical responses in 6 of 12 patients, including 4 of 8 patients with anti-PD-1 refractory disease. Responses included extensive regression of bulky tumors and complete regression of most tumors in some patients. Genomic studies, which included intra-patient tumors with dichotomous treatment responses, revealed resistance mechanisms from defects in critical components of the antigen presentation and interferon response pathways. These findings demonstrate that engineered T cells can mediate regression of common carcinomas, and they reveal immune editing as a constraint on the curative potential of cellular therapy and possibly other immunotherapies in advanced epithelial cancer.

Lhx6 regulates canonical Wnt signaling to control the fate of mesenchymal progenitor cells during mouse molar root patterning

PLoS genetics

2021 Feb 01

He, J;Jing, J;Feng, J;Han, X;Yuan, Y;Guo, T;Pei, F;Ma, Y;Cho, C;Ho, TV;Chai, Y;
PMID: 33596195 | DOI: 10.1371/journal.pgen.1009320

Mammalian tooth crown formation has long served as a model for investigating how patterning and morphogenesis are orchestrated during development. However, the mechanism underlying root patterning and morphogenesis remains poorly understood. In this study, we find that Lhx6 labels a subpopulation of root progenitor cells in the apical dental mesenchyme, which is closely associated with furcation development. Loss of Lhx6 leads to furcation and root number defects, indicating that Lhx6 is a key root patterning regulator. Among the multiple cellular events regulated by Lhx6 is the odontoblast fate commitment of progenitor cells, which it controls in a cell-autonomous manner. Specifically, Lhx6 loss leads to elevated expression of the Wnt antagonist Sfrp2 and down-regulation of Wnt signaling in the furcation region, while overactivation of Wnt signaling in Lhx6+ progenitor cells partially restore the furcation defects in Lhx6-/- mice. Collectively, our findings have important implications for understanding organ morphogenesis and future strategies for tooth root regeneration.

Medial septum glutamatergic neurons control wakefulness through a septo-hypothalamic circuit

Current biology : CB

2021 Jan 28

An, S;Sun, H;Wu, M;Xie, D;Hu, SW;Ding, HL;Cao, JL;
PMID: 33545041 | DOI: 10.1016/j.cub.2021.01.019

The medial septum (MS) is involved in arousal-based behaviors and modulates general anesthesia response. However, the role of MS in wakefulness control remains unknown. Here, combining double fluorescence in situ hybridization and optrode recording, we showed that MS glutamatergic neurons exhibited higher activities preferentially during wakefulness. Activating these neurons, either optogenetically or chemogenetically, strongly promoted wakefulness, mainly through the transition from non-rapid eye movement (NREM) sleep to wakefulness. In contrast, inactivation of these neurons reduced wakefulness by the transition from wakefulness to NREM sleep. Furthermore, both rabies-mediated monosynaptic retrograde and anterograde tracing showed that MS glutamatergic neurons monosynaptically innervated lateral hypothalamus (LH) glutamatergic neurons, which were also wake-active as well as wake-promoting. Activating MS-derived glutamatergic terminals in LH enhanced wakefulness, whereas silencing MS glutamatergic neurons destabilized the wake-active preference of LH glutamatergic neurons. These results reveal a vital role of MS glutamatergic neurons in wakefulness control and depict a novel septo-hypothalamic circuit for wakefulness.

Zebrafish heart regenerates after chemoptogenetic cardiomyocyte depletion

Developmental dynamics : an official publication of the American Association of Anatomists

2021 Jan 26

Missinato, MA;Zuppo, DA;Watkins, SC;Bruchez, MP;Tsang, M;
PMID: 33501711 | DOI: 10.1002/dvdy.305

Zebrafish can regenerate adult cardiac tissue following injuries from ventricular apex amputation, cryoinjury, and cardiomyocyte genetic ablation. Here, we characterize cardiac regeneration from cardiomyocyte chemoptogenetic ablation caused by localized near-infrared excited photosensitizer-mediated Reactive Oxygen Species (ROS) generation. Exposure of transgenic adult zebrafish, Tg(myl7:fapdl5-cerulean), to di-iodinated derivative of the cell- permeable Malachite Green ester fluorogen (MG-2I) and whole-body illumination with 660nm light resulted in cytotoxic damage to about 30% of cardiac tissue. After chemoptogenetic cardiomyocyte ablation, heart function was compromised, and macrophage infiltration was detected, but epicardial and endocardial activation response was much muted when compared to ventricular amputation. The spared cardiomyocytes underwent proliferation and restored the heart structure and function in 45-60 days after ablation. This cardiomyocyte ablation system did not appear to activate the epicardium and endocardium as is noted in other cardiac injury models. This approach represents a useful model to study specifically cardiomyocyte injury, proliferation and regeneration in the absence of whole organ activation. Moreover, this system can be adapted to ablate distinct cell populations in any organ system to study their function in regeneration. This article is protected by

Reciprocal interaction between mesenchymal stem cells and transit amplifying cells regulates tissue homeostasis

eLife

2021 Jan 22

Jing, J;Feng, J;Li, J;Zhao, H;Ho, TV;He, J;Yuan, Y;Guo, T;Du, J;Urata, M;Sharpe, P;Chai, Y;
PMID: 33480845 | DOI: 10.7554/eLife.59459

Interaction between adult stem cells and their progeny is critical for tissue homeostasis and regeneration. In multiple organs, mesenchymal stem cells (MSCs) give rise to transit amplifying cells (TACs), which then differentiate into different cell types. However, whether and how MSCs interact with TACs remains unknown. Using the adult mouse incisor as a model, we present in vivo evidence that TACs and MSCs have distinct genetic programs and engage in reciprocal signaling cross talk to maintain tissue homeostasis. Specifically, an IGF-WNT signaling cascade is involved in the feedforward from MSCs to TACs. TACs are regulated by tissue-autonomous canonical WNT signaling and can feedback to MSCs and regulate MSC maintenance via Wnt5a/Ror2-mediated non-canonical WNT signaling. Collectively, these findings highlight the importance of coordinated bidirectional signaling interaction between MSCs and TACs in instructing mesenchymal tissue homeostasis, and the mechanisms identified here have important implications for MSC-TAC interaction in other organs.

Runx2-Twist1 interaction coordinates cranial neural crest guidance of soft palate myogenesis

eLife

2021 Jan 22

Han, X;Feng, J;Guo, T;Loh, YE;Yuan, Y;Ho, TV;Cho, CK;Li, J;Jing, J;Janeckova, E;He, J;Pei, F;Bi, J;Song, B;Chai, Y;
PMID: 33482080 | DOI: 10.7554/eLife.62387

Cranial neural crest (CNC) cells give rise to bone, cartilage, tendons, and ligaments of the vertebrate craniofacial musculoskeletal complex, as well as regulate mesoderm-derived craniofacial muscle development through cell-cell interactions. Using the mouse soft palate as a model, we performed an unbiased single-cell RNA-seq analysis to investigate the heterogeneity and lineage commitment of CNC derivatives during craniofacial muscle development. We show that Runx2, a known osteogenic regulator, is expressed in the CNC-derived perimysial and progenitor populations. Loss of Runx2 in CNC-derivatives results in reduced expression of perimysial markers (Aldh1a2 and Hic1) as well as soft palate muscle defects in Osr2-Cre;Runx2fl/fl mice. We further reveal that Runx2 maintains perimysial marker expression through suppressing Twist1, and that myogenesis is restored in Osr2-Cre;Runx2fl/fl;Twist1fl/+ mice. Collectively, our findings highlight the roles of Runx2, Twist1, and their interaction in regulating the fate of CNC-derived cells as they guide craniofacial muscle development through cell-cell interactions.

Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkwormBombyx mori

Proceedings of the National Academy of Sciences

2021 Jan 05

Tsuchiya, R;Kaneshima, A;Kobayashi, M;Yamazaki, M;Takasu, Y;Sezutsu, H;Tanaka, Y;Mizoguchi, A;Shiomi, K;
| DOI: 10.1073/pnas.2020028118

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkwormBombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

Adoptive T Cell Immunotherapy for Medullary Thyroid Carcinoma Targeting GDNF Family Receptor alpha 4

Molecular Therapy - Oncolytics

2021 Jan 01

Bhoj, V;Li, L;Parvathaneni, K;Zhang, Z;Kacir, S;Arhontoulis, D;Zhou, K;McGettigan-Croce, B;Nunez-Cruz, S;Gulendran, G;Boesteanu, A;Johnson, L;Feldman, M;Radaelli, E;Mansfield, K;Nasrallah, M;Goydel, R;Peng, H;Rader, C;Milone, M;Siegel, D;
| DOI: 10.1016/j.omto.2021.01.012

Metastatic medullary thyroid cancer (MTC) is a rare, but often, aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC.

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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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