Wolf M, Ao M, Chavez MB, Kolli TN, Thumbigere-Math V, Becker K, Chu EY, Jäger A, Somerman MJ, Foster BL.
PMID: 29533727 | DOI: 10.1177/0022034518759295
Previous studies revealed that cementum formation is tightly regulated by inorganic pyrophosphate (PPi), a mineralization inhibitor. Local PPiconcentrations are determined by regulators, including ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which increases PPiconcentrations by adenosine triphosphate hydrolysis. Orthodontic forces stimulate alveolar bone remodelling, leading to orthodontic tooth movement (OTM). To better understand how disturbed mineral metabolism and the resulting altered periodontal structures affect OTM, we employed Enpp1 mutant mice that feature reduced PPi and increased cervical cementum in a model of OTM induced by a stretched closed-coil spring ligated between the maxillary left first molar and maxillary incisors. We analyzed tooth movement, osteoclast/odontoclast response, and tooth root resorption by micro-computed tomography, histology, histomorphometry, and immunohistochemistry. Preoperatively, we noted an altered periodontium in Enpp1 mutant mice, with significantly increased periodontal ligament (PDL) volume and thickness, as well as increased PDL-bone/tooth root surface area, compared to wild-type (WT) controls. After 11 d of orthodontic treatment, Enpp1 mutant mice displayed 38% reduced tooth movement versus WT mice. Molar roots in Enpp1 mutant mice exhibited less change in PDL width in compression and tension zones compared to WT mice. Root resorption was noted in both groups with no difference in average depths, but resorption lacunae in Enpp1 mutant mice were almost entirely limited to cementum, with 150% increased cementum resorption and 92% decreased dentin resorption. Osteoclast/odontoclast cells were reduced by 64% in Enpp1 mutant mice, with a predominance of tartrate-resistant acid phosphatase (TRAP)-positive cells on root surfaces, compared to WT mice. Increased numbers of TRAP-positive cells on root surfaces were associated with robust immunolocalization of osteopontin (OPN) and receptor-activator of NF-κB ligand (RANKL). Collectively, reduced response to orthodontic forces, decreased tooth movement, and altered osteoclast/odontoclast distribution suggests Enpp1 loss of function has direct effects on clastic function/recruitment and/or indirect effects on periodontal remodeling via altered periodontal structure or tissue mineralization.
Matson, KJE;Russ, DE;Kathe, C;Hua, I;Maric, D;Ding, Y;Krynitsky, J;Pursley, R;Sathyamurthy, A;Squair, JW;Levi, BP;Courtine, G;Levine, AJ;
PMID: 36163250 | DOI: 10.1038/s41467-022-33184-1
After spinal cord injury, tissue distal to the lesion contains undamaged cells that could support or augment recovery. Targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we use single nucleus RNA sequencing to profile how each cell type in the lumbar spinal cord changes after a thoracic injury in mice. We present an atlas of these dynamic responses across dozens of cell types in the acute, subacute, and chronically injured spinal cord. Using this resource, we find rare spinal neurons that express a signature of regeneration in response to injury, including a major population that represent spinocerebellar projection neurons. We characterize these cells anatomically and observed axonal sparing, outgrowth, and remodeling in the spinal cord and cerebellum. Together, this work provides a key resource for studying cellular responses to injury and uncovers the spontaneous plasticity of spinocerebellar neurons, uncovering a potential candidate for targeted therapy.
Tang, WC;Tsao, SW;Jones, GE;Liu, X;Tsai, MH;Delecluse, HJ;Dai, W;You, C;Zhang, J;Huang, SCM;Leung, MM;Liu, T;Ching, YP;Chen, H;Lo, KW;Li, X;Tsang, CM;
PMID: 36420735 | DOI: 10.1002/path.6036
Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they were visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. This article is protected by
Lee, SH;Kim, N;Kim, M;Woo, SH;Han, I;Park, J;Kim, K;Park, KS;Kim, K;Shim, D;Park, SE;Zhang, JY;Go, DM;Kim, DY;Yoon, WK;Lee, SP;Chung, J;Kim, KW;Park, JH;Lee, SH;Lee, S;Ann, SJ;Lee, SH;Ahn, HS;Jeong, SC;Kim, TK;Oh, GT;Park, WY;Lee, HO;Choi, JH;
PMID: 36115863 | DOI: 10.1038/s41467-022-33202-2
Valvular inflammation triggered by hyperlipidemia has been considered as an important initial process of aortic valve disease; however, cellular and molecular evidence remains unclear. Here, we assess the relationship between plasma lipids and valvular inflammation, and identify association of low-density lipoprotein with increased valvular lipid and macrophage accumulation. Single-cell RNA sequencing analysis reveals the cellular heterogeneity of leukocytes, valvular interstitial cells, and valvular endothelial cells, and their phenotypic changes during hyperlipidemia leading to recruitment of monocyte-derived MHC-IIhi macrophages. Interestingly, we find activated PPARγ pathway in Cd36+ valvular endothelial cells increased in hyperlipidemic mice, and the conservation of PPARγ activation in non-calcified human aortic valves. While the PPARγ inhibition promotes inflammation, PPARγ activation using pioglitazone reduces valvular inflammation in hyperlipidemic mice. These results show that low-density lipoprotein is the main lipoprotein accumulated in the aortic valve during hyperlipidemia, leading to early-stage aortic valve disease, and PPARγ activation protects the aortic valve against inflammation.
Pathology - Research and Practice
Schwab, C;Domke, L;Rose, F;Hausser, I;Schirmacher, P;Longerich, T;
| DOI: 10.1016/j.prp.2022.154000
Pulmonary capillary microthrombosis has been proposed as a major pathogenetic factor driving severe COVID-19. Autopsy studies reported endothelialitis but it is under debate if it is caused by SARS-CoV-2 infection of endothelial cells. In this study, RNA in situ hybridization was used to detect viral RNA and to identify the infected cell types in lung tissue of 40 patients with fatal COVID-19. SARS-CoV-2 Spike protein-coding RNA showed a steadily decreasing signal abundance over a period of three weeks. Besides the original virus strain the variants of concern Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529) could also be detected by the assay. Viral RNA was mainly detected in alveolar macrophages and pulmonary epithelial cells, while only single virus-positive endothelial cells were observed even in cases with high viral load suggesting that viral infection of endothelial cells is not a key factor for the development of pulmonary capillary microthrombosis.
Low, AYT;Goldstein, N;Gaunt, JR;Huang, KP;Zainolabidin, N;Yip, AKK;Carty, JRE;Choi, JY;Miller, AM;Ho, HST;Lenherr, C;Baltar, N;Azim, E;Sessions, OM;Ch'ng, TH;Bruce, AS;Martin, LE;Halko, MA;Brady, RO;Holsen, LM;Alhadeff, AL;Chen, AI;Betley, JN;
PMID: 34789878 | DOI: 10.1038/s41586-021-04143-5
The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control1-3. Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour.
Bockmayr, M;Harnisch, K;Pohl, L;Schweizer, L;Mohme, T;Körner, M;Alawi, M;Suwala, A;Dorostkar, M;Monoranu, C;Hasselblatt, M;Wefers, A;Capper, D;Hench, J;Frank, S;Richardson, T;Tran, I;Liu, E;Snuderl, M;Engertsberger, L;Benesch, M;von Deimling, A;Obrecht, D;Mynarek, M;Rutkowski, S;Glatzel, M;Neumann, J;Schüller, U;
| DOI: 10.1093/neuonc/noac079.143
Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients’ clinical course are unknown. We assembled a cohort of 185 tumors classified as MPE based on DNA methylation from pediatric, adolescent, and adult patients. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPEB, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. Tumors in the methylation class MPE were histologically diagnosed as WHO grade I (59%), WHO grade II (37%), or WHO grade III tumors (4%). 75/77 analyzed tumors expressed HOXB13, which is a diagnostic feature not detected in other spinal ependymal tumors. Based on DNA methylation, our series split into two subtypes. MPE-A occurred in younger patients (median age 27 vs. 45 years, p=7.3e-05). They were enriched with WHO grade I tumors and associated with papillary morphology and MGMT promoter hypermethylation (all p<0.001). MPE-B included most tumors initially diagnosed as WHO grade II and cases with tanycytic morphology. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. 15/30 MPE-A could not be totally resected compared to 1/58 MPE-B (p=6.3e-08), and progression-free survival was significantly better for MPE-B (p=3.4e-06, 10-year relapse rate 33% vs. 85%). We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.
Broeckel, RM;Feldmann, F;McNally, KL;Chiramel, AI;Sturdevant, GL;Leung, JM;Hanley, PW;Lovaglio, J;Rosenke, R;Scott, DP;Saturday, G;Bouamr, F;Rasmussen, AL;Robertson, SJ;Best, SM;
PMID: 34855915 | DOI: 10.1371/journal.ppat.1009678
Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.
Am J Respir Crit Care Med. 2018 Dec 15.
Reyfman PA, Walter JM, Joshi N, Anekalla KR, McQuattie-Pimentel AC, Chiu S, Fernandez R, Akbarpour M, Chen CI, Ren Z, Verma R, Abdala-Valencia H, Nam K, Chi M, Han S, Gonzalez-Gonzalez FJ, Soberanes S, Watanabe S, Williams KJN, Flozak AS, Nicholson TT, Morgan VK, Winter DR, Hinchcliff M, Hrusch CL, Guzy RD, Bonham CA, Sperling AI, Bag R, Hamanaka RB, Mutlu GM, Yeldandi AV, Marshall SA, Shilatifard A, Amaral LAN, Perlman H, Sznajder JI, Argento AC, Gillespie CT, Dematte J, Jain M, Singer BD, Ridge KM, Lam AP, Bharat A, Bhorade SM, Gottardi CJ, Budinger GRS, Misharin AV.
PMID: 30554520 | DOI: 10.1164/rccm.201712-2410OC
Abstract RATIONALE: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. OBJECTIVES: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells or other cell types in lung tissue from subjects with pulmonary fibrosis compared with controls. METHODS: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data in using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. MEASUREMENTS AND MAIN RESULTS: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to non-overlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. CONCLUSIONS: We generated a single cell atlas of pulmonary fibrosis. Using this atlas we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
He, S;Bhatt, R;Brown, C;Brown, EA;Buhr, DL;Chantranuvatana, K;Danaher, P;Dunaway, D;Garrison, RG;Geiss, G;Gregory, MT;Hoang, ML;Khafizov, R;Killingbeck, EE;Kim, D;Kim, TK;Kim, Y;Klock, A;Korukonda, M;Kutchma, A;Lewis, ZR;Liang, Y;Nelson, JS;Ong, GT;Perillo, EP;Phan, JC;Phan-Everson, T;Piazza, E;Rane, T;Reitz, Z;Rhodes, M;Rosenbloom, A;Ross, D;Sato, H;Wardhani, AW;Williams-Wietzikoski, CA;Wu, L;Beechem, JM;
PMID: 36203011 | DOI: 10.1038/s41587-022-01483-z
Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Lovatt, D;Tamburino, A;Krasowska-Zoladek, A;Sanoja, R;Li, L;Peterson, V;Wang, X;Uslaner, J;
PMID: 36261573 | DOI: 10.1038/s42003-022-03970-0
Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.
Lim, RG;Al-Dalahmah, O;Wu, J;Gold, MP;Reidling, JC;Tang, G;Adam, M;Dansu, DK;Park, HJ;Casaccia, P;Miramontes, R;Reyes-Ortiz, AM;Lau, A;Hickman, RA;Khan, F;Paryani, F;Tang, A;Ofori, K;Miyoshi, E;Michael, N;McClure, N;Flowers, XE;Vonsattel, JP;Davidson, S;Menon, V;Swarup, V;Fraenkel, E;Goldman, JE;Thompson, LM;
PMID: 36543778 | DOI: 10.1038/s41467-022-35388-x
The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.
