Extraction and sequencing of single nuclei from murine skeletal muscles
Santos, M;Gioftsidi, S;Backer, S;Machado, L;Relaix, F;Maire, P;Mourikis, P;
| DOI: 10.1016/j.xpro.2021.100694
Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021).
Stress-Potentiated Memory Updating as a Novel Intervention for Non-Treatment Seeking Smokers
Barnabe, A;Gamache, K;Paes de Mello de Camargo, J;Allen-Flanagan, E;Rioux, M;Pruessner, J;Leyton, M;Nader, K;
| DOI: 10.1016/j.biopsych.2021.02.301
Interventions to disrupt memory reconsolidation have held promise for the treatment of stress- and anxiety-related disorders. In the present study, we tested whether an intervention based on these principles, called memory updating, could be adapted for reward-seeking behaviors. Non-treatment seeking tobacco smokers were exposed to smoking cues and/or stress, two stimuli known to trigger smoking. It was predicted that exposure to a stress task would enhance the cues’ motivational salience and yield greater susceptibility to the memory updating procedure.
Burke, K;, ;Zhou, X;Wang, Y;Wei, H;, ;, ;, ;
| DOI: 10.21926/obm.geriatr.2104184
The supplement telomerase activator TA-65 (purified from Astragalus membranaceus) has been shown to retard cellular senescence, boost the aging immune system, and retard age-related symptoms. Lengthened telomeres retard aging, but because cancers often maintain longevity by lengthening telomeres, dietary telomerase activator might possibly increase tumorigenesis. This study investigated whether oral TA-65 effects the timing of onset and/or the incidence of skin cancers induced by UVB-irradiation and whether that possible effect is different if the oral supplementation is begun only after tumors are first detected clinically or if supplementation is begun before initiation of tumors as well as during and after the inciting UVB exposure. Three groups of ten Skh:1 hairless, nonpigmented mice exposed to UVB for twenty weeks were given (1) no supplementation, (2) TA-65 supplementation starting when the first UV-induced skin cancers were clinically observed, after which the UV exposure was terminated, and (3) TA-65 supplementation before, during, and after UV exposure (as more tumors subsequently appeared). Except for two time points when Group 3 had borderline or statistically more tumors ≥ 2mm per mouse, overall, there was no statistically significant difference in the time of onset, the incidence, or the tumor load of skin cancers with TA-65 with either timing, confirming the safety of this anti-aging supplement in this model of the most frequent human malignancy.
Muñoz-Manchado AB, Bengtsson Gonzales C, Zeisel A, Munguba H, Bekkouche B, Skene NG, Lönnerberg P, Ryge J, Harris KD, Linnarsson S, Hjerling-Leffler J.
PMID: 30134177 | DOI: 10.1016/j.celrep.2018.07.053
Striatal locally projecting neurons, or interneurons, act on nearby circuits and shape functional output to the rest of the basal ganglia. We performed single-cell RNA sequencing of striatal cells enriching for interneurons. We find seven discrete interneuron types, six of which are GABAergic. In addition to providing specific markers for the populations previously described, including those expressing Sst/Npy, Th, Npy without Sst, and Chat, we identify two small populations of cells expressing Cck with or without Vip. Surprisingly, the Pvalb-expressing cells do not constitute a discrete cluster but rather are part of a larger group of cells expressing Pthlh with a spatial gradient of Pvalb expression. Using PatchSeq, we show that Pthlh cells exhibit a continuum of electrophysiological properties correlated with expression of Pvalb. Furthermore, we find significant molecular differences that correlate with differences in electrophysiological properties between Pvalb-expressing cells of the striatum and those of the cortex.
Qi, Y;Lee, NJ;Ip, CK;Enriquez, R;Tasan, R;Zhang, L;Herzog, H;
PMID: 37201523 | DOI: 10.1016/j.cmet.2023.04.020
Neuropeptide Y (NPY) in the arcuate nucleus (ARC) is known as one of the most critical regulators of feeding. However, how NPY promotes feeding under obese conditions is unclear. Here, we show that positive energy balance, induced by high-fat diet (HFD) or in genetically obese leptin-receptor-deficient mice, leads to elevated Npy2r expression especially on proopiomelanocortin (POMC) neurons, which also alters leptin responsiveness. Circuit mapping identified a subset of ARC agouti-related peptide (Agrp)-negative NPY neurons that control these Npy2r expressing POMC neurons. Chemogenetic activation of this newly discovered circuitry strongly drives feeding, while optogenetic inhibition reduces feeding. Consistent with that, lack of Npy2r on POMC neurons leads to reduced food intake and fat mass. This suggests that under energy surplus conditions, when ARC NPY levels generally drop, high-affinity NPY2R on POMC neurons is still able to drive food intake and enhance obesity development via NPY released predominantly from Agrp-negative NPY neurons.
Xie, L;Xiong, Y;Ma, D;Shi, K;Chen, J;Yang, Q;Yan, J;
PMID: 37172583 | DOI: 10.1016/j.neuron.2023.04.016
The suprachiasmatic nucleus (SCN) can generate robust circadian behaviors in mammals under different environments, but the underlying neural mechanisms remained unclear. Here, we showed that the activities of cholecystokinin (CCK) neurons in the mouse SCN preceded the onset of behavioral activities under different photoperiods. CCK-neuron-deficient mice displayed shortened free-running periods, failed to compress their activities under a long photoperiod, and developed rapid splitting or became arrhythmic under constant light. Furthermore, unlike vasoactive intestinal polypeptide (VIP) neurons, CCK neurons are not directly light sensitive, but their activation can elicit phase advance and counter light-induced phase delay mediated by VIP neurons. Under long photoperiods, the impact of CCK neurons on SCN dominates over that of VIP neurons. Finally, we found that the slow-responding CCK neurons control the rate of recovery during jet lag. Together, our results demonstrated that SCN CCK neurons are crucial for the robustness and plasticity of the mammalian circadian clock.
Shan, D;Wang, Y;Chang, Y;Cui, H;Tao, M;Sheng, Y;Kang, H;Jia, P;Song, J;
| DOI: 10.1016/j.isci.2023.106646
Ischemia reperfusion injury (IRI), often related to surgical procedures, is one of the important causes of acute kidney injury (AKI). To decipher the dynamic process of AKI caused by IRI (with prolonged ischemia phase), we performed single-cell RNA sequencing (scRNA-seq) of clinically relevant IRI murine model with different ischemic intervals. We discovered that Slc5a2hi proximal tubular cells were susceptible to AKI and highly expressed neutral amino acid transporter gene Slc6a19, which was dramatically decreased over the time course. With the usage of mass spectrometry-based metabolomic analysis, we detected that the level of neutral amino acid isoleucine dropped off in AKI mouse plasma metabolites. And the reduction of plasma isoleucine was also verified in patients with cardiac surgery-associated acute kidney injury (CSA-AKI). The findings advanced the understanding of dynamic process of AKI and introduced reduction of isoleucine as a potential biomarker for CSA-AKI.
Royal Society open science
Kaganer, AW;Ossiboff, RJ;Keith, NI;Schuler, KL;Comizzoli, P;Hare, MP;Fleischer, RC;Gratwicke, B;Bunting, EM;
PMID: 36756057 | DOI: 10.1098/rsos.220810
Dynamic interactions between host, pathogen and host-associated microbiome dictate infection outcomes. Pathogens including Batrachochytrium dendrobatidis (Bd) threaten global biodiversity, but conservation efforts are hindered by limited understanding of amphibian host, Bd and microbiome interactions. We conducted a vaccination and infection experiment using Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) challenged with Bd to observe infection, skin microbial communities and gene expression of host skin, pathogen and microbiome throughout the experiment. Most animals survived high Bd loads regardless of their vaccination status and vaccination did not affect pathogen load, but host gene expression differed based on vaccination. Oral vaccination (exposure to killed Bd) stimulated immune gene upregulation while topically and sham-vaccinated animals did not significantly upregulate immune genes. In early infection, topically vaccinated animals upregulated immune genes but orally and sham-vaccinated animals downregulated immune genes. Bd increased pathogenicity-associated gene expression in late infection when Bd loads were highest. The microbiome was altered by Bd, but there was no correlation between anti-Bd microbe abundance or richness and pathogen burden. Our observations suggest that hellbenders initially generate a vigorous immune response to Bd, which is ineffective at controlling disease and is subsequently modulated. Interactions with antifungal skin microbiota did not influence disease progression.
Jiang, D;Burger, CA;Akhanov, V;Liang, JH;Mackin, RD;Albrecht, NE;Andrade, P;Schafer, DP;Samuel, MA;
PMID: 36379210 | DOI: 10.1016/j.immuni.2022.10.018
Microglia utilize their phagocytic activity to prune redundant synapses and refine neural circuits during precise developmental periods. However, the neuronal signals that control this phagocytic clockwork remain largely undefined. Here, we show that neuronal signal-regulatory protein alpha (SIRPα) is a permissive cue for microglial phagocytosis in the developing murine retina. Removal of neuronal, but not microglial, SIRPα reduced microglial phagocytosis, increased synpase numbers, and impaired circuit function. Conversely, prolonging neuronal SIRPα expression extended developmental microglial phagocytosis. These outcomes depended on the interaction of presynaptic SIRPα with postsynaptic CD47. Global CD47 deficiency modestly increased microglial phagocytosis, while CD47 overexpression reduced it. This effect was rescued by coexpression of neuronal SIRPα or codeletion of neuronal SIRPα and CD47. These data indicate that neuronal SIRPα regulates microglial phagocytosis by limiting microglial SIRPα access to neuronal CD47. This discovery may aid our understanding of synapse loss in neurological diseases.
Chen, J;Katada, Y;Okimura, K;Yamaguchi, T;Guh, YJ;Nakayama, T;Maruyama, M;Furukawa, Y;Nakane, Y;Yamamoto, N;Sato, Y;Ando, H;Sugimura, A;Tabata, K;Sato, A;Yoshimura, T;
PMID: 36306789 | DOI: 10.1016/j.cub.2022.09.062
Many organisms living along the coastlines synchronize their reproduction with the lunar cycle. At the time of spring tide, thousands of grass puffers (Takifugu alboplumbeus) aggregate and vigorously tremble their bodies at the water's edge to spawn. To understand the mechanisms underlying this spectacular semilunar beach spawning, we collected the hypothalamus and pituitary from male grass puffers every week for 2 months. RNA sequencing (RNA-seq) analysis identified 125 semilunar genes, including genes crucial for reproduction (e.g., gonadotropin-releasing hormone 1 [gnrh1], luteinizing hormone β subunit [lhb]) and receptors for pheromone prostaglandin E (PGE). PGE2 is secreted into the seawater during the spawning, and its administration activates olfactory sensory neurons and triggers trembling behavior of surrounding individuals. These results suggest that PGE2 synchronizes lunar-regulated beach-spawning behavior in grass puffers. To further explore the mechanism that regulates the lunar-synchronized transcription of semilunar genes, we searched for semilunar transcription factors. Spatial transcriptomics and multiplex fluorescent in situ hybridization showed co-localization of the semilunar transcription factor CCAAT/enhancer-binding protein δ (cebpd) and gnrh1, and cebpd induced the promoter activity of gnrh1. Taken together, our study demonstrates semilunar genes that mediate lunar-synchronized beach-spawning behavior. VIDEO ABSTRACT.
Zhang, Z;Tian, X;Lu, J;Boit, K;Ablaeva, J;Tolibzoda Zakusilo, F;Emmrich, S;Firsanov, D;Rydkina, E;Biashad, S;Lu, Q;Tyshkovsky, A;Gladyshev, V;Horvath, S;Seluanov, A;Gorbunova, V;
| DOI: 10.2139/ssrn.4185135
Production of abundant high molecular weight hyaluronic acid (HMW-HA) contributes to cancer resistance and possibly longevity of the longest-lived rodent, the naked mole-rat. To study whether the benefits of HMW-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHAS2). nmrHAS2 mice showed lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHAS2 mice shifted towards that of longer-lived species. The most striking change observed in nmrHAS2 mice was attenuated inflammation across multiple tissues. HMW-HA reduced inflammation via several pathways including direct immunoregulatory effect on immune cells, protection from oxidative stress, and improved gut barrier function during aging. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new avenues for using HMW-HA to improve lifespan and healthspan.
Otero-Garcia, M;Mahajani, SU;Wakhloo, D;Tang, W;Xue, YQ;Morabito, S;Pan, J;Oberhauser, J;Madira, AE;Shakouri, T;Deng, Y;Allison, T;He, Z;Lowry, WE;Kawaguchi, R;Swarup, V;Cobos, I;
PMID: 35882228 | DOI: 10.1016/j.neuron.2022.06.021
Tau aggregation in neurofibrillary tangles (NFTs) is closely associated with neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the molecular signatures that distinguish between aggregation-prone and aggregation-resistant cell states are unknown. We developed methods for the high-throughput isolation and transcriptome profiling of single somas with NFTs from the human AD brain, quantified the susceptibility of 20 neocortical subtypes for NFT formation and death, and identified both shared and cell-type-specific signatures. NFT-bearing neurons shared a marked upregulation of synaptic transmission-related genes, including a core set of 63 genes enriched for synaptic vesicle cycling. Oxidative phosphorylation and mitochondrial dysfunction were highly cell-type dependent. Apoptosis was only modestly enriched, and the susceptibilities of NFT-bearing and NFT-free neurons for death were highly similar. Our analysis suggests that NFTs represent cell-type-specific responses to stress and synaptic dysfunction. We provide a resource for biomarker discovery and the investigation of tau-dependent and tau-independent mechanisms of neurodegeneration.
