Single-cell RNA sequencing reveals time- and sex-specific responses of mouse spinal cord microglia to peripheral nerve injury and links ApoE to chronic pain

Nature communications

2022 Feb 11

Tansley, S;Uttam, S;Ureña Guzmán, A;Yaqubi, M;Pacis, A;Parisien, M;Deamond, H;Wong, C;Rabau, O;Brown, N;Haglund, L;Ouellet, J;Santaguida, C;Ribeiro-da-Silva, A;Tahmasebi, S;Prager-Khoutorsky, M;Ragoussis, J;Zhang, J;Salter, MW;Diatchenko, L;Healy, LM;Mogil, JS;Khoutorsky, A;
PMID: 35149686 | DOI: 10.1038/s41467-022-28473-8

Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unclear whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. Using single-cell RNA-sequencing, we show that peripheral nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in male as compared to female mice. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following peripheral nerve injury. Apolipoprotein E (Apoe) is the top upregulated gene in spinal cord microglia at chronic time points after peripheral nerve injury in mice. Furthermore, polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell RNA sequencing analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a link between ApoE and chronic pain in humans.

Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce T cell paralysis

Cell reports

2022 Sep 20

Keenan, BP;McCarthy, EE;Ilano, A;Yang, H;Zhang, L;Allaire, K;Fan, Z;Li, T;Lee, DS;Sun, Y;Cheung, A;Luong, D;Chang, H;Chen, B;Marquez, J;Sheldon, B;Kelley, RK;Ye, CJ;Fong, L;
PMID: 36130508 | DOI: 10.1016/j.celrep.2022.111384

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n = 9) and matched healthy donors (n = 8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14CTX can directly suppress CD4+ T cells and induce SOCS3 expression in CD4+ T cells, rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce T cell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance.

Pharmacological intervention of the FGF-PTH axis as a potential therapeutic for craniofacial ciliopathies

Disease models & mechanisms

2022 Jul 12

Paese, CLB;Chang, CF;Kristeková, D;Brugmann, SA;
PMID: 35818799 | DOI: 10.1242/dmm.049611

Ciliopathies represent a disease class characterized by a broad range of phenotypes including polycystic kidneys and skeletal anomalies. Ciliopathic skeletal phenotypes are among the most common and most difficult to treat due to a poor understanding of the pathological mechanisms leading to disease. Using an avian model (talpid2) for a human ciliopathy with both kidney and skeletal anomalies (Orofaciodigital syndrome 14), we identified disruptions in the FGF23-PTH axis that resulted in reduced calcium uptake in the developing mandible and subsequent micrognathia. While pharmacological intervention with the FDA-approved pan-FGFR inhibitor AZD4547 alone rescued expression of the FGF target Sprouty2, it did not significantly rescue micrognathia. In contrast, treatment with a cocktail of AZD4547 and Teriparatide acetate, a PTH agonist and FDA-approved treatment for osteoporosis, resulted in a molecular, cellular, and phenotypic rescue of ciliopathic micrognathia in talpid2 mutants. Together, these data provide novel insight into pathological molecular mechanisms associated with ciliopathic skeletal phenotypes and a potential therapeutic strategy for a pleiotropic disease class with limited to no treatment options.

Single cell atlas of spinal cord injury in mice reveals a pro-regenerative signature in spinocerebellar neurons

Nature communications

2022 Sep 26

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.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Am J Respir Crit Care Med. 2018 Dec 15.

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.

Huntington disease oligodendrocyte maturation deficits revealed by single-nucleus RNAseq are rescued by thiamine-biotin supplementation

Nature communications

2022 Dec 21

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.

Signalling by senescent melanocytes hyperactivates hair growth

Nature

2023 Jun 01

Wang, X;Ramos, R;Phan, AQ;Yamaga, K;Flesher, JL;Jiang, S;Oh, JW;Jin, S;Jahid, S;Kuan, CH;Nguyen, TK;Liang, HY;Shettigar, NU;Hou, R;Tran, KH;Nguyen, A;Vu, KN;Phung, JL;Ingal, JP;Levitt, KM;Cao, X;Liu, Y;Deng, Z;Taguchi, N;Scarfone, VM;Wang, G;Paolilli, KN;Wang, X;Guerrero-Juarez, CF;Davis, RT;Greenberg, EN;Ruiz-Vega, R;Vasudeva, P;Murad, R;Widyastuti, LHP;Lee, HL;McElwee, KJ;Gadeau, AP;Lawson, DA;Andersen, B;Mortazavi, A;Yu, Z;Nie, Q;Kunisada, T;Karin, M;Tuckermann, J;Esko, JD;Ganesan, AK;Li, J;Plikus, MV;
PMID: 37344645 | DOI: 10.1038/s41586-023-06172-8

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

	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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