Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

Nature neuroscience

2022 Sep 01

Caldwell, ALM;Sancho, L;Deng, J;Bosworth, A;Miglietta, A;Diedrich, JK;Shokhirev, MN;Allen, NJ;
PMID: 36042312 | DOI: 10.1038/s41593-022-01150-1

Astrocytes negatively impact neuronal development in many models of neurodevelopmental disorders (NDs); however, how they do this, and if mechanisms are shared across disorders, is not known. In this study, we developed a cell culture system to ask how astrocyte protein secretion and gene expression change in three mouse models of genetic NDs (Rett, Fragile X and Down syndromes). ND astrocytes increase release of Igfbp2, a secreted inhibitor of insulin-like growth factor (IGF). IGF rescues neuronal deficits in many NDs, and we found that blocking Igfbp2 partially rescues inhibitory effects of Rett syndrome astrocytes, suggesting that increased astrocyte Igfbp2 contributes to decreased IGF signaling in NDs. We identified that increased BMP signaling is upstream of protein secretion changes, including Igfbp2, and blocking BMP signaling in Fragile X and Rett syndrome astrocytes reverses inhibitory effects on neurite outgrowth. This work provides a resource of astrocyte-secreted proteins in health and ND models and identifies novel targets for intervention in diverse NDs.

Single-cell RNA-sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification

Nature communications

2022 Jul 05

Petitpré, C;Faure, L;Uhl, P;Fontanet, P;Filova, I;Pavlinkova, G;Adameyko, I;Hadjab, S;Lallemend, F;
PMID: 35790771 | DOI: 10.1038/s41467-022-31580-1

Different types of spiral ganglion neurons (SGNs) are essential for auditory perception by transmitting complex auditory information from hair cells (HCs) to the brain. Here, we use deep, single cell transcriptomics to study the molecular mechanisms that govern their identity and organization in mice. We identify a core set of temporally patterned genes and gene regulatory networks that may contribute to the diversification of SGNs through sequential binary decisions and demonstrate a role for NEUROD1 in driving specification of a Ic-SGN phenotype. We also find that each trajectory of the decision tree is defined by initial co-expression of alternative subtype molecular controls followed by gradual shifts toward cell fate resolution. Finally, analysis of both developing SGN and HC types reveals cell-cell signaling potentially playing a role in the differentiation of SGNs. Our results indicate that SGN identities are drafted prior to birth and reveal molecular principles that shape their differentiation and will facilitate studies of their development, physiology, and dysfunction.

Accuracy benchmark of the GeneMind GenoLab M sequencing platform for WGS and WES analysis

BMC genomics

2022 Jul 22

Li, C;Fan, X;Guo, X;Liu, Y;Wang, M;Zhao, XC;Wu, P;Yan, Q;Sun, L;
PMID: 35869426 | DOI: 10.1186/s12864-022-08775-3

GenoLab M is a recently developed next-generation sequencing (NGS) platform from GeneMind Biosciences. To establish the performance of GenoLab M, we present the first report to benchmark and compare the WGS and WES sequencing data of the GenoLab M sequencer to NovaSeq 6000 and NextSeq 550 platform in various types of analysis. For WGS, thirty-fold sequencing from Illumina NovaSeq platform and processed by GATK pipeline is currently considered as the golden standard. Thus this dataset is generated as a benchmark reference in this study.GenoLab M showed an average of 94.62% of Q20 percentage for base quality, while the NovaSeq was slightly higher at 96.97%. However, GenoLab M outperformed NovaSeq or NextSeq at a duplication rate, suggesting more usable data after deduplication. For WGS short variant calling, GenoLab M showed significant accuracy improvement over the same depth dataset from NovaSeq, and reached similar accuracy to NovaSeq 33X dataset with 22x depth. For 100X WES, the F-score and Precision in GenoLab M were higher than NovaSeq or NextSeq, especially for InDel calling.GenoLab M is a promising NGS platform for high-performance WGS and WES applications. For WGS, 22X depth in the GenoLab M sequencing platform offers a cost-effective alternative to the current mainstream 33X depth on Illumina.

Data Processing and Germline Variant Calling with the Sentieon Pipeline

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

2022 Jun 25

Aldana, R;Freed, D;
PMID: 35751805 | DOI: 10.1007/978-1-0716-2293-3_1

Public and private genomic sequencing initiatives generate ever-increasing amounts of genomic data creating a need for improved solutions for genomics data processing (Stephens et al.PLoS Biol 13:e1002195, 2015). The Sentieon Genomics software enables rapid and accurate analysis of next-generation sequence data. In this work, we present a typical use of the Sentieon Genomics software for germline variant calling. The Sentieon germline variant calling pipeline produces more accurate results than other tools on third-party benchmarks (Katherine et al. Front Genet 10:736, 2019; Shen et al. bioRxiv, 885517, 2019) in one tenth the time of comparable pipelines. Parts of this guide come from the official Sentieon Genomics software manual in https://support.sentieon.com/manual (Sentieon. Sentieon Genomics software manual, n.d.) and from the official Sentieon Genomics software application notes in https://support.sentieon.com/appnotes (Sentieon. Sentieon Genomics software application notes, n.d.) and are republished with permission. For additional details and advanced usage instructions of the Sentieon tools, refer to the software manual.

Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates

Nature biotechnology

2022 Jun 02

Brown, KM;Nair, JK;Janas, MM;Anglero-Rodriguez, YI;Dang, LTH;Peng, H;Theile, CS;Castellanos-Rizaldos, E;Brown, C;Foster, D;Kurz, J;Allen, J;Maganti, R;Li, J;Matsuda, S;Stricos, M;Chickering, T;Jung, M;Wassarman, K;Rollins, J;Woods, L;Kelin, A;Guenther, DC;Mobley, MW;Petrulis, J;McDougall, R;Racie, T;Bombardier, J;Cha, D;Agarwal, S;Johnson, L;Jiang, Y;Lentini, S;Gilbert, J;Nguyen, T;Chigas, S;LeBlanc, S;Poreci, U;Kasper, A;Rogers, AB;Chong, S;Davis, W;Sutherland, JE;Castoreno, A;Milstein, S;Schlegel, MK;Zlatev, I;Charisse, K;Keating, M;Manoharan, M;Fitzgerald, K;Wu, JT;Maier, MA;Jadhav, V;
PMID: 35654979 | DOI: 10.1038/s41587-022-01334-x

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

Mapping the cardiac vascular niche in heart failure

Nature communications

2022 May 31

Peisker, F;Halder, M;Nagai, J;Ziegler, S;Kaesler, N;Hoeft, K;Li, R;Bindels, EMJ;Kuppe, C;Moellmann, J;Lehrke, M;Stoppe, C;Schaub, MT;Schneider, RK;Costa, I;Kramann, R;
PMID: 35641541 | DOI: 10.1038/s41467-022-30682-0

The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling.

PD-1 and ICOS co-expression identifies tumor-reactive CD4 T cells in human solid tumors

The Journal of clinical investigation

2022 Apr 19

Duhen, R;Fesneau, O;Samson, KA;Frye, AK;Beymer, M;Rajamanickam, V;Ross, D;Tran, E;Bernard, B;Weinberg, AD;Duhen, T;
PMID: 35439168 | DOI: 10.1172/JCI156821

CD4 T helper (Th) cells play a key role in orchestrating immune responses, but the identity of the CD4 Th cells involved in the anti-tumor immune response remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and colorectal cancers and identified a subset of CD4 Th cells distinct from FOXP3+ regulatory T cells that co-express PD-1 and ICOS. These tumor-infiltrating CD4 Th cells (CD4 Th TIL) have a tissue-resident memory phenotype, are present in MHC class II-rich areas and proliferate in the tumor suggesting local antigen recognition. The T-cell receptor repertoire of the PD-1+ICOS+ CD4 Th TIL is oligoclonal, with T-cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4 Th TIL were shown to recognize both tumor-associated antigens and tumor-specific neoantigens. Our findings provide an approach for isolating tumor-reactive CD4 Th TIL directly ex vivo that will help define their role in the anti-tumor immune response and potentially improve future adoptive T-cell therapy approaches.

Functional antagonism between ΔNp63α and GCM1 regulates human trophoblast stemness and differentiation

Nature communications

2022 Mar 25

Wang, LJ;Chen, CP;Lee, YS;Ng, PS;Chang, GD;Pao, YH;Lo, HF;Peng, CH;Cheong, ML;Chen, H;
PMID: 35338152 | DOI: 10.1038/s41467-022-29312-6

The combination of EGF, CHIR99021, A83-01, SB431542, VPA, and Y27632 (EGF/CASVY) facilitates the derivation of trophoblast stem (TS) cells from human blastocysts and first-trimester, but not term, cytotrophoblasts. The mechanism underlying this chemical induction of TS cells remains elusive. Here we demonstrate that the induction efficiency of cytotrophoblast is determined by functional antagonism of the placental transcription factor GCM1 and the stemness regulator ΔNp63α. ΔNp63α reduces GCM1 transcriptional activity, whereas GCM1 inhibits ΔNp63α oligomerization and autoregulation. EGF/CASVY cocktail activates ΔNp63α, thereby partially inhibiting GCM1 activity and reverting term cytotrophoblasts into stem cells. By applying hypoxia condition, we can further reduce GCM1 activity and successfully induce term cytotrophoblasts into TS cells. Consequently, we identify mitochondrial creatine kinase 1 (CKMT1) as a key GCM1 target crucial for syncytiotrophoblast differentiation and reveal decreased CKMT1 expression in preeclampsia. Our study delineates the molecular underpinnings of trophoblast stemness and differentiation and an efficient method to establish TS cells from term placentas.

Disruption of mitochondrial complex III in cap mesenchyme but not in ureteric progenitors results in defective nephrogenesis associated with amino acid deficiency

Kidney international

2022 Mar 24

Guan, N;Kobayashi, H;Ishii, K;Davidoff, O;Sha, F;Ikizler, TA;Hao, CM;Chandel, NS;Haase, VH;
PMID: 35341793 | DOI: 10.1016/j.kint.2022.02.030

Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)-expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4-mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development.

Human microglial models to study HIV infection and neuropathogenesis: a literature overview and comparative analyses

Journal of neurovirology

2022 Feb 09

Gumbs, SBH;Kübler, R;Gharu, L;Schipper, PJ;Borst, AL;Snijders, GJLJ;Ormel, PR;van Berlekom, AB;Wensing, AMJ;de Witte, LD;Nijhuis, M;
PMID: 35138593 | DOI: 10.1007/s13365-021-01049-w

HIV persistence in the CNS despite antiretroviral therapy may cause neurological disorders and poses a critical challenge for HIV cure. Understanding the pathobiology of HIV-infected microglia, the main viral CNS reservoir, is imperative. Here, we provide a comprehensive comparison of human microglial culture models: cultured primary microglia (pMG), microglial cell lines, monocyte-derived microglia (MDMi), stem cell-derived microglia (iPSC-MG), and microglia grown in 3D cerebral organoids (oMG) as potential model systems to advance HIV research on microglia. Functional characterization revealed phagocytic capabilities and responsiveness to LPS across all models. Microglial transcriptome profiles of uncultured pMG showed the highest similarity to cultured pMG and oMG, followed by iPSC-MG and then MDMi. Direct comparison of HIV infection showed a striking difference, with high levels of viral replication in cultured pMG and MDMi and relatively low levels in oMG resembling HIV infection observed in post-mortem biopsies, while the SV40 and HMC3 cell lines did not support HIV infection. Altogether, based on transcriptional similarities to uncultured pMG and susceptibility to HIV infection, MDMi may serve as a first screening tool, whereas oMG, cultured pMG, and iPSC-MG provide more representative microglial culture models for HIV research. The use of current human microglial cell lines (SV40, HMC3) is not recommended.

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences

eLife

2022 Jan 20

Lee, JY;Wing, PA;Gala, DS;Noerenberg, M;Järvelin, AI;Titlow, J;Zhuang, X;Palmalux, N;Iselin, L;Thompson, MK;Parton, RM;Prange-Barczynska, M;Wainman, A;Salguero, FJ;Bishop, T;Agranoff, D;James, W;Castello, A;McKeating, JA;Davis, I;
PMID: 35049501 | DOI: 10.7554/eLife.74153

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.

Naringenin potentiates anti-tumor immunity against oral cancer by inducing lymph node CD169-positive macrophage activation and cytotoxic T cell infiltration

Cancer immunology, immunotherapy : CII

2022 Jan 19

Kawaguchi, S;Kawahara, K;Fujiwara, Y;Ohnishi, K;Pan, C;Yano, H;Hirosue, A;Nagata, M;Hirayama, M;Sakata, J;Nakashima, H;Arita, H;Yamana, K;Gohara, S;Nagao, Y;Maeshiro, M;Iwamoto, A;Hirayama, M;Yoshida, R;Komohara, Y;Nakayama, H;
PMID: 35044489 | DOI: 10.1007/s00262-022-03149-w

The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.

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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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