Novel variants in the stem cell niche factor WNT2B define the disease phenotype as a congenital enteropathy with ocular dysgenesis

European journal of human genetics : EJHG

2021 Feb 01

Zhang, YJ;Jimenez, L;Azova, S;Kremen, J;Chan, YM;Elhusseiny, AM;Saeed, H;Goldsmith, J;Al-Ibraheemi, A;O'Connell, AE;Kovbasnjuk, O;Rodan, L;Agrawal, PB;Thiagarajah, JR;
PMID: 33526876 | DOI: 10.1038/s41431-021-00812-1

WNT2B is a member of the Wnt family, a group of signal transduction proteins involved in embryologic development and stem cell renewal and maintenance. We recently reported homozygous nonsense variants in WNT2B in three individuals with severe, neonatal-onset diarrhea, and intestinal failure. Here we present a fourth case, from a separate family, with neonatal diarrhea associated with novel compound heterozygous WNT2B variants. One of the two variants was a frameshift variant (c.423del [p.Phe141fs]), while the other was a missense change (c.722 G > A [p.G241D]) that we predict through homology modeling to be deleterious, disrupting post-translational acylation. This patient presented as a neonate with severe diet-induced (osmotic) diarrhea and growth failure resulting in dependence on parenteral nutrition. Her gastrointestinal histology revealed abnormal cellular architecture particularly in the stomach and colon, including oxyntic atrophy, abnormal distribution of enteroendocrine cells, and a paucity of colonic crypt glands. In addition to her gastrointestinal findings, she had bilateral corneal clouding and atypical genital development later identified as a testicular 46,XX difference/disorder of sexual development. Upon review of the previously reported cases, two others also had anterior segment ocular anomalies though none had atypical genital development. This growing case series suggests that variants in WNT2B are associated with an oculo-intestinal (and possibly gonadal) syndrome, due to the protein's putative involvement in multiple developmental and stem cell maintenance pathways.

Serotonin receptor 4 in the hippocampus modulates mood and anxiety

Molecular psychiatry

2021 Jan 13

Karayol, R;Medrihan, L;Warner-Schmidt, JL;Fait, BW;Rao, MN;Holzner, EB;Greengard, P;Heintz, N;Schmidt, EF;
PMID: 33441982 | DOI: 10.1038/s41380-020-00994-y

Serotonin receptor 4 (5-HT4R) plays an important role in regulating mood, anxiety, and cognition, and drugs that activate this receptor have fast-acting antidepressant (AD)-like effects in preclinical models. However, 5-HT4R is widely expressed throughout the central nervous system (CNS) and periphery, making it difficult to pinpoint the cell types and circuits underlying its effects. Therefore, we generated a Cre-dependent 5-HT4R knockout mouse line to dissect the function of 5-HT4R in specific brain regions and cell types. We show that the loss of functional 5-HT4R specifically from excitatory neurons of hippocampus led to robust AD-like behavioral responses and an elevation in baseline anxiety. 5-HT4R was necessary to maintain the proper excitability of dentate gyrus (DG) granule cells and cell type-specific molecular profiling revealed a dysregulation of genes necessary for normal neural function and plasticity in cells lacking 5-HT4R. These adaptations were accompanied by an increase in the number of immature neurons in ventral, but not dorsal, dentate gyrus, indicating a broad impact of 5-HT4R loss on the local cellular environment. This study is the first to use conditional genetic targeting to demonstrate a direct role for hippocampal 5-HT4R signaling in modulating mood and anxiety. Our findings also underscore the need for cell type-based approaches to elucidate the complex action of neuromodulatory systems on distinct neural circuits.

LncRNA DANCR represses Doxorubicin-induced apoptosis through stabilizing MALAT1 expression in colorectal cancer cells

Cell death & disease

2021 Jan 06

Xiong, M;Wu, M;Dan Peng, ;Huang, W;Chen, Z;Ke, H;Chen, Z;Song, W;Zhao, Y;Xiang, AP;Zhong, X;
PMID: 33414433 | DOI: 10.1038/s41419-020-03318-8

Long non-coding RNA (lncRNA) DANCR has been reported to participate in key processes such as stem cell differentiation and tumorigenesis. In a high throughput screening for lncRNAs involved in Doxorubicin-induced apoptosis, we found DANCR was suppressed by Doxorubicin and it acted as an important repressor of apoptosis in colorectal cancer. Further studies demonstrated that DANCR promoted the oncogenic lncRNA MALAT1 expression via enhancing the RNA stability of MALAT1 to suppress apoptosis. MALAT1 could efficiently mediate the suppressive function of DANCR on apoptosis. Mechanistic studies found the RNA-binding protein QK served as an interacting partner of both DANCR and MALAT1, and the protein level of QK was subjected to the regulation by DANCR. Furthermore, QK was able to modulate the RNA stability of MALAT1, and the interaction between QK and MALAT1 was controlled by DANCR. In addition, QK could mediate the function of DANCR in regulating the expression of MALAT1 and suppressing apoptosis. These results revealed DANCR played a critical role in Doxorubicin-induced apoptosis in colorectal cancer cells, which was achieved by the interaction between DANCR and QK to enhance the expression of MALAT1.

Intracranial delivery of AAV9 gene therapy partially prevents retinal degeneration and visual deficits in CLN6-Batten disease mice

Molecular Therapy - Methods & Clinical Development

2021 Jan 01

White, K;Nelvagal, H;Poole, T;Lu, B;Johnson, T;Davis, S;Pratt, M;Brudvig, J;Assis, A;Meyer, K;Kaspar, B;Cooper, J;Wang, S;Weimer, J;
| DOI: 10.1016/j.omtm.2020.12.014

Batten disease is a family of rare, fatal, neuropediatric diseases presenting with memory/learning decline, blindness, and loss of motor function. Recently, we reported the use of an AAV9 mediated gene therapy that prevents disease progression in a mouse model of CLN6-Batten disease (Cln6nclf), restoring lifespans in treated animals. Despite the success of our viral-mediated gene therapy, the dosing strategy was optimized for delivery to the brain parenchyma and may limit the therapeutic potential to other disease relevant tissues, such as the eye. Here, we examine whether cerebrospinal fluid (CSF) delivery of scAAV9.CB.CLN6 is sufficient to ameliorate visual deficits in Cln6nclf mice. We show that intracerebroventricular (ICV) delivery of scAAV9.CB.CLN6 completely prevents hallmark Batten disease pathology in the visual processing centers of the brain, preserving neurons of the superior colliculus, thalamus, and cerebral cortex. Importantly, ICV delivered scAAV9.CB.CLN6 also expresses in many cells throughout the central retina, preserving many photoreceptors typically lost in Cln6nclf mice. Lastly, scAAV9.CB.CLN6 treatment partially preserved visual acuity in Cln6nclf mice as measured by optokinetic response. Taken together, we report the first instance of CSF delivered viral gene reaching and rescuing pathology in both the brain parenchyma and retinal neurons, thereby partially slowing visual deterioration.

Endodermal Wnt signaling Is required for tracheal cartilage formation

Dev Biol. 2015 Jun 17.

Snowball J, Ambalavanan M, Whitsett J, Sinner D.
PMID: 26093309 | DOI: 10.1016/j.ydbio.2015.06.009.

Tracheobronchomalacia is a common congenital defect in which the walls of the trachea and bronchi lack of adequate cartilage required for support of the airways. Deletion of Wls, a cargo receptor mediating Wnt ligand secretion, in the embryonic endoderm using ShhCre mice inhibited formation of tracheal-bronchial cartilaginous rings. The normal dorsal-ventral patterning of tracheal mesenchyme was lost. Smooth muscle cells, identified by Acta2 staining, were aberrantly located in ventral mesenchyme of the trachea, normally the region of Sox9 expression in cartilage progenitors. Wnt/β-catenin activity, indicated by Axin2 LacZ reporter, was decreased in tracheal mesenchyme of Wlsf/f;ShhCre/+ embryos. Proliferation of chondroblasts was decreased and reciprocally, proliferation of smooth muscle cells was increased in Wlsf/f;ShhCre/+ tracheal tissue. Expression of Tbx4, Tbx5, Msx1 and Msx2, known to mediate cartilage and muscle patterning, were decreased in tracheal mesenchyme of Wlsf/f;ShhCre/+ embryos. Ex vivo studies demonstrated that Wnt7b and Wnt5a, expressed by the epithelium of developing trachea, and active Wnt/β-catenin signaling are required for tracheal chondrogenesis before formation of mesenchymal condensations. In conclusion, Wnt ligands produced by the tracheal epithelium pattern the tracheal mesenchyme via modulation of gene expression and cell proliferation required for proper tracheal cartilage and smooth muscle differentiation.

Histological assessment of intestinal injury by ionizing radiation

Methods in Cell Biology

2023 May 15

Kuo, H;Daniel, A;Driver, L;Lee, C;Kirsch, D;
| DOI: 10.1016/bs.mcb.2023.03.001

Given the potential risk of radiological terrorism and disasters, it is essential to develop plans to prepare for such events. In these hazardous scenarios, radiation-induced gastrointestinal (GI) syndrome is one of the many manifestations that may happen after the organism is exposed to a lethal dose of ionizing radiation. Therefore, it is critical to better understand how the intestinal tissues initiate and orchestrate regeneration following severe radiation injury. In this chapter, we aimed to provide several key considerations for researchers who utilize histological assessment to study radiation-induced intestinal injury. Rigor and reproducibility are critical in experimental design and can be achieved by maintaining proper radiation administration, maintaining consistency in sample collection, and selecting and using appropriate controls. We also provided technical details of histological preparation of the intestines with tips on dissecting, cleaning, fixing, and preserving. Step-by-step descriptions of both bundling and Swiss rolling are provided with discussion on how to choose between the two approaches. In the following section, we detailed several histological assessment methods and then provided suggestions on how to use histological assessment to study cellular dynamics in the small intestines. Finally, we touched on some non-histological assessments. We hope that the information provided in this chapter will contribute to the research society of radiation-induced intestinal injury with an ultimate goal of promoting the development of radiation countermeasures against the GI acute radiation syndrome.

TMEM59 ablation leads to loss of olfactory sensory neurons and impairs olfactory functions via interaction with inflammation

Brain, behavior, and immunity

2023 Apr 13

Ma, Z;Li, W;Zhuang, L;Wen, T;Wang, P;Yu, H;Liu, Y;Yu, Y;
PMID: 37061103 | DOI: 10.1016/j.bbi.2023.04.005

The olfactory epithelium undergoes constant neurogenesis throughout life in mammals. Several factors including key signaling pathways and inflammatory microenvironment regulate the maintenance and regeneration of the olfactory epithelium. In this study, we identify TMEM59 (also known as DCF1) as a critical regulator to the epithelial maintenance and regeneration. Single-cell RNA-Seq data show downregulation of TMEM59 in multiple epithelial cell lineages with aging. Ablation of TMEM59 leads to apparent alteration at the transcriptional level, including genes associated with olfactory transduction and inflammatory/immune response. These differentially expressed genes are key components belonging to several signaling pathways, such as NF-κB, chemokine, etc. TMEM59 deletion impairs olfactory functions, attenuates proliferation, causes loss of both mature and immature olfactory sensory neurons, and promotes infiltration of inflammatory cells, macrophages, microglia cells and neutrophils into the olfactory epithelium and lamina propria. TMEM59 deletion deteriorates regeneration of the olfactory epithelium after injury, with significant reduction in the number of proliferative cells, immature and mature sensory neurons, accompanied by the increasing number of inflammatory cells and macrophages. Anti-inflammation by dexamethasone recovers neuronal generation and olfactory functions in the TMEM59-KO animals, suggesting the correlation between TMEM59 and inflammation in regulating the epithelial maintenance. Collectively, TMEM59 regulates olfactory functions, as well as neuronal generation in the olfactory epithelium via interaction with inflammation, suggesting a potential role in therapy against olfactory dysfunction associated with inflamm-aging.

Cytotoxic CD4+ T cells eliminate senescent cells by targeting cytomegalovirus antigen

Cell

2023 Mar 30

Hasegawa, T;Oka, T;Son, HG;Oliver-García, VS;Azin, M;Eisenhaure, TM;Lieb, DJ;Hacohen, N;Demehri, S;
PMID: 37001502 | DOI: 10.1016/j.cell.2023.02.033

Senescent cell accumulation has been implicated in the pathogenesis of aging-associated diseases, including cancer. The mechanism that prevents the accumulation of senescent cells in aging human organs is unclear. Here, we demonstrate that a virus-immune axis controls the senescent fibroblast accumulation in the human skin. Senescent fibroblasts increased in old skin compared with young skin. However, they did not increase with advancing age in the elderly. Increased CXCL9 and cytotoxic CD4+ T cells (CD4 CTLs) recruitment were significantly associated with reduced senescent fibroblasts in the old skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), becoming direct CD4 CTL targets. Skin-resident CD4 CTLs eliminated HCMV-gB+ senescent fibroblasts in an HLA-II-dependent manner, and HCMV-gB activated CD4 CTLs from the human skin. Collectively, our findings demonstrate HCMV reactivation in senescent cells, which CD4 CTLs can directly eliminate through the recognition of the HCMV-gB antigen.

A circular RNA, circPTPN14, increases MYC transcription by interacting with FUBP1 and exacerbates renal fibrosis

Cellular and molecular life sciences : CMLS

2022 Nov 17

Nie, W;Li, M;Liu, B;Zhang, Y;Wang, Y;Wang, J;Jin, L;Ni, A;Xiao, L;Shen, XZ;Chen, J;Lin, W;Han, F;
PMID: 36394649 | DOI: 10.1007/s00018-022-04603-9

Fibrosis is a relentlessly progressive and irreversible cause of organ damage, as in chronic kidney disease (CKD), but its underlying mechanisms remain elusive. We found that a circular RNA, circPTPN14, is highly expressed in human kidneys with biopsy-proved chronic interstitial fibrosis, mouse kidneys subjected to ischemia/reperfusion (IR) or unilateral ureteral obstruction (UUO), and TGFβ1-stimulated renal tubule epithelial cells (TECs). The intrarenal injection of circPTPN14 shRNA alleviated the progression of fibrosis in kidneys subjected to IR or UUO. Knockdown of circPTPN14 in TECs inhibited TGFβ1-induced expression of profibrotic genes, whereas overexpressing circPTPN14 increased the profibrotic effect of TGFβ1. The profibrotic action of circPTPN14 was ascribed to an increase in MYC transcription. The binding of circPTPN14 to the KH3 and KH4 domains of far upstream element (FUSE) binding protein 1 (FUBP1) enhanced the interaction between FUBP1 and FUSE domain, which was required for the initiation of MYC transcription. In human kidneys (n = 30) with biopsy-proved chronic interstitial fibrosis, the expression of circPTPN14 positively correlated with MYC expression. Taken together these studies show a novel mechanism in the pathogenesis of renal fibrosis, mediated by circPTPN14, which can be a target in the diagnosis and treatment of CKD.

Single-cell RNA sequencing for traumatic spinal cord injury

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2022 Dec 01

Cao, Y;Zhu, S;Yu, B;Yao, C;
PMID: 36374259 | DOI: 10.1096/fj.202200943R

Traumatic spinal cord injury (tSCI) is a severe injury of the central nervous system (CNS) with complicated pathological microenvironment that results in hemorrhage, inflammation, and scar formation. The microenvironment of the injured spinal cord comprises heterogeneous neurons, glial cells, inflammatory cells, and stroma-related cells. Increasing evidence has indicated that the altered cellular and molecular microenvironment following tSCI is a key factor impeding functional recovery. Single-cell RNA sequencing (scRNA-seq) has provided deep insights into the dynamic cellular and molecular changes in the microenvironment by comprehensively characterizing the diversity of spinal cord cell types. Specifically, scRNA-seq enables the exploration of the molecular mechanisms underlying tSCI by elucidating intercellular communication in spinal cord samples between normal and injury conditions at a single-cell resolution. Here, we first described the pathological and physiological processes after tSCI and gave a brief introduction of the scRNA-seq technology. We then focused on the recent scRNA-seq researches in tSCI, which characterized diverse cell-type populations and specific cell-cell interactions in tSCI. In addition, we also highlighted some potential directions for the research of scRNA-seq in tSCI in the future.

Anatomical Methods to Study the Suprachiasmatic Nucleus

Methods in molecular biology (Clifton, N.J.)

2022 May 24

Bittman, EL;
PMID: 35610428 | DOI: 10.1007/978-1-0716-2249-0_13

The mammalian suprachiasmatic nucleus (SCN) functions as a master circadian pacemaker. In order to examine mechanisms by which it keeps time, entrains to periodic environmental signals (zeitgebers), and regulates subordinate oscillators elsewhere in the brain and in the periphery, a variety of molecular methods have been applied. Multiple label immunocytochemistry and in situ hybridization provide anatomical insights that complement physiological approaches (such as ex vivo electrophysiology and luminometry) widely used to study the SCN.The anatomical methods require interpretation of data gathered from groups of individual animals sacrificed at different time points. This imposes constraints on the design of the experiments that aim to observe changes that occur with circadian phase in free-running conditions. It is essential in such experiments to account for differences in the periods of the subjects. Nevertheless, it is possible to resolve intracellular colocalization and regional expression of functionally important transcripts and/or their peptide products that serve as neuromodulators or neurotransmitters. Armed with these tools and others, understanding of the mechanisms by which the hypothalamic pacemaker regulates circadian function is progressing apace.

Cytotoxic CD4 + T Cells Eliminate Senescent Cells by Targeting Commensal Cytomegalovirus Antigen

SSRN Electronic Journal

2022 May 27

Hasegawa, T;Oka, T;Son, H;Azin, M;Eisenhaure, T;Lieb, D;Hacohen, N;Demehri, S;
| DOI: 10.2139/ssrn.4102631

Senescent cell accumulation has been implicated in the pathogenesis of aging-associated diseases including cancer. The mechanism that prevents the accumulation of senescent cells in aging human organs is unclear. Here, we demonstrate that a commensal virus-immune axis controls the senescent fibroblast accumulation in the human skin. Senescent fibroblasts increased in old compared with young skin. However, they did not increase with advancing age in elderly. Increased CXCL9 and cytotoxic CD4+ T cell (CD4 CTL) recruitment were significantly associated with reduced senescent fibroblasts in the old skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), becoming direct CD4 CTL targets. Skin-resident CD4 CTL eliminated HCMV-gB+ senescent fibroblasts in an HLA-II-dependent manner and HCMV-gB activated CD4 CTL from the human skin. Collectively, our findings demonstrate HCMV reactivation in senescent cells, which can be directly eliminated by CD4 CTL through the recognition of the HCMV-gB antigen.

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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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