Neuroblastoma (NB), which is a subtype of neural-crest-derived malignancy, is the most common extracranial solid tumor occurring in childhood. Despite extensive research, the underlying developmental ...origin of NB remains unclear. Using single-cell RNA sequencing, we generate transcriptomes of adrenal NB from 160,910 cells of 16 patients and transcriptomes of putative developmental cells of origin of NB from 12,103 cells of early human embryos and fetal adrenal glands at relatively late development stages. We find that most adrenal NB tumor cells transcriptionally mirror noradrenergic chromaffin cells. Malignant states also recapitulate the proliferation/differentiation status of chromaffin cells in the process of normal development. Our findings provide insight into developmental trajectories and cellular states underlying human initiation and progression of NB.
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•The repertoire of normal developing neural-crest-related cells defines the features of NB•NB has a predominant chromaffin-cell-like phenotype•Chromaffin cell differentiation state is highly predictive of patient outcome•MYCN amplification is robustly linked with enhanced EMT NCC-like phenotype
Dong et al. provide a detailed phenotypic landscape of human neuroblastoma (NB) using single-cell RNA sequencing and show that the proliferation/differentiation status of developing chromaffin cells is highly associated with the clinical heterogeneity of NB.
Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion ...pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180–720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.
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•Visualizing 0–490 nm pore opening, expansion, constriction, and closure in live cell•Competition between expansion and constriction generates diverse pore dynamics•Actin/tension expand the pore, and calcium/dynamin constrict the pore up to 490 nm•Establishment of a dynamic-pore theory controlling fusion and fission efficiency
The missing live-cell evidence proving the fusion pore hypothesis reveals metastable pores that are two orders of magnitude larger than previously thought and can constrict and close instantly or slowly.
Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- ...and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.
The neuroanatomical basis behind acupuncture practice is still poorly understood. Here, we used intersectional genetic strategy to ablate NPY+ noradrenergic neurons and/or adrenal chromaffin cells. ...Using endotoxin-induced systemic inflammation as a model, we found that electroacupuncture stimulation (ES) drives sympathetic pathways in somatotopy- and intensity-dependent manners. Low-intensity ES at hindlimb regions drives the vagal-adrenal axis, producing anti-inflammatory effects that depend on NPY+ adrenal chromaffin cells. High-intensity ES at the abdomen activates NPY+ splenic noradrenergic neurons via the spinal-sympathetic axis; these neurons engage incoherent feedforward regulatory loops via activation of distinct adrenergic receptors (ARs), and their ES-evoked activation produces either anti- or pro-inflammatory effects due to disease-state-dependent changes in AR profiles. The revelation of somatotopic organization and intensity dependency in driving distinct autonomic pathways could form a road map for optimizing stimulation parameters to improve both efficacy and safety in using acupuncture as a therapeutic modality.
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•Intersectional genetic manipulation of NPY+ sympathetic cells•Electroacupuncture stimulation (ES) drives distinct sympathetic pathways•ES operates in somatotopy- and intensity-dependent manners•NPY+ noradrenergic neurons bidirectionally modulate systemic inflammation
Liu et al. reveals a neuroanatomical basis for acupuncture practice, showing that electroacupuncture stimulation can drive distinct autonomic pathways and modulate systemic inflammation in somatotopy-, stimulation-intensity-, and disease-state-dependent manners.
Pre-blockade of the sarco-endoplasmic reticulum (ER) calcium ATPase (SERCA) with irreversible thapsigargin depresses exocytosis in adrenal bovine chromaffin cells (BCCs). Distinct expression of ...voltage-dependent Ca
2+
-channel subtypes and of the Ca
2+
-induced Ca
2+
release (CICR) mechanism in BCCs versus mouse chromaffin cells (MCCs) has been described. We present a parallel study on the effects of the acute SERCA blockade with reversible cyclopizonic acid (CPA), to repeated pulsing with acetylcholine (ACh) at short (15 s) and long intervals (60 s) at 37 °C, allowing the monitoring of the initial size of a ready-release vesicle pool (RRP) and its depletion and recovery in subsequent stimuli. We found (i) strong depression of exocytosis upon ACh pulsing at 15-s intervals and slower depression at 60-s intervals in both cell types; (ii) facilitation of exocytosis upon acute SERCA inhibition, with back to depression upon CPA washout in MCCs; (iii) blockade of exocytosis upon acute SERCA inhibition and pronounced rebound of exocytosis upon CPA washout in BCCs; (iv) basal Ca
2+
c
elevation upon stimulation with ACh at short intervals (but not at long intervals) in both cell types; and (v) augmentation of basal Ca
2+
c
and inhibition of peak Ca
2+
c
amplitude upon CPA treatment in both cell types, with milder effects upon stimulation at 60-s intervals. These results are compatible with the view that while in MCCs the uptake of Ca
2+
via SERCA contributes to the mitigation of physiological ACh triggered secretion, in BCCs the uptake of Ca
2+
into the ER facilitates such responses likely potentiating a Ca
2+
-induced Ca
2+
release mechanism. These drastic differences in the regulation of ACh-triggered secretion at 37 °C may help to understand different patterns of the regulation of exocytosis by the circulation of Ca
2+
at a functional ER Ca
2+
store.
RNA velocity of single cells La Manno, Gioele; Soldatov, Ruslan; Zeisel, Amit ...
Nature (London),
08/2018, Letnik:
560, Številka:
7719
Journal Article
Recenzirano
Odprti dostop
RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput
. However, this ...approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.
From a pathogenic perspective, Huntington's disease (HD) is being considered as a synaptopathy. As such, alterations in brain neurotransmitter release occur. As the activity of the sympathoadrenal ...axis is centrally controlled, deficits in the exocytotic release of catecholamine release may also occur. In fact, in chromaffin cells (CCs) of the adrenal medulla of the R6/1 model of HD, decrease of secretion and altered kinetics of the exocytotic fusion pore have been reported. Those alterations could be linked to mitochondrial deficits occurring in peripheral CCs, similar to those described in brain mitochondria. Here we have inquired about alterations in mitochondrial structure and function and their impact on exocytosis and calcium channel currents (ICa). We have monitored various parameters linked to those events, in wild type (WT) and the R6/1 mouse model of HD at a pre-disease stage (2 months age, 2 m), and when motor deficits are present (7 months age, 7 m). In isolated CCs from 7 m and in the adrenal medulla of R6/1 mice, we found the following alterations (with respect 7 m WT mice): (i) augmented fragmented mitochondria and oxidative stress with increased oxidized glutathione; (ii) decreased basal and maximal respiration; (iii) diminution of ATP cell levels; (iv) mitochondrial depolarization; (v) drastic decrease of catecholamine release with poorer potentiation by protonophore FCCP; (vi) decreased ICa inhibition by FCCP; and (vii) lesser potentiation by BayK8644 of ICa and smaller prolongation of current deactivation. Of note was the fact several of these alterations were already manifested in CCs from 2 m R6/1 mice at pre-disease stages. Based on those results, a plausible hypothesis can be raised in the sense that altered mitochondrial function seems to be an early primary event in HD pathogenesis. This is in line with an increasing number of mitochondrial, metabolic, and inflammatory alterations being recently reported in various HD peripheral tissues.
•Here, an investigation is presented on altered mitochondrial function and its impact on calcium and exocytosis in chromaffin cells from R6/1 mice.•Augmented mitochondrial depolarization and oxidative stress, with impaired respiration and ATP synthesis, were found, were found in R6/1 mice.•Drastic diminution of catecholamine release, and poorer potentiation of secretion by FCCP, were observed in R6/1 mice.•Lesser enhancing effects of BayK8644 on ICa, were noted in R6/1 mice.•Mitochondrial alterations were visible at pre-symptomatic disease stages, suggesting the early initiation of HD pathogenesis.
It is known that chronic ethanol (EtOH) consumption leads to hypertension development and has been associated with deleterious effects on the cardiovascular system. Whether this condition alters ...calcium (Ca2+) signaling and exocytosis in adrenal chromaffin cells (CCs) as the case is for genetic hypertension, is unknown. We explored this question in four randomized experimental groups, male Wistar Kyoto (WKY/EtOH) and Spontaneously Hypertensive (SHR/EtOH) rats were subjected to the intake of increasing EtOH concentrations (5–20%, for 30 days) and their respective controls (WKY/Control and SHR/Control) received water. WKY/EtOH developed hypertension and cardiac hypertrophy; blood aldehyde dehydrogenase (ALDH) and H2O2 were also augmented. In comparison with WKY/Control, CCs from WKY/EtOH had the following features: (i) depolarization and higher frequency of spontaneous action potentials; (ii) decreased Ca2+ currents with slower inactivation; (iii) decreased K+ currents; (iv) augmented K+-elicited cytosolic Ca2+ transients (Ca2+c); (v) enhanced K+-elicited catecholamine release. These cardiovascular, blood and CCs changes were qualitatively similar to those undergone by SHR/Control and SHR/EtOH. The results suggest that the hypertension elicited by chronic EtOH has pathogenic features common to genetic hypertension namely, augmented Ca2+c transients and catecholamine release from their CCs.
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The hypersecretory phenotype of adrenal chromaffin cells (CCs) from early spontaneously hypertensive rats (SHRs) mainly results from enhanced Ca
2+
-induced Ca
2+
-release (CICR). A key question is ...if these abnormalities can be traced to the prehypertensive stage. Spontaneous and stimulus-induced catecholamine exocytosis, intracellular Ca
2+
signals, and dense-core granule size and density were examined in CCs from prehypertensive and hypertensive SHRs and compared with age-matched Wistar-Kyoto rats (WKY). During the prehypertensive stage, the depolarization-elicited catecholamine exocytosis was ~ 2.9-fold greater in SHR than in WKY CCs. Interestingly, in half of CCs the exocytosis was indistinguishable from WKY CCs, while it was between 3- and sixfold larger in the other half. Likewise, caffeine-induced exocytosis was ~ twofold larger in prehypertensive SHR. Accordingly, depolarization and caffeine application elicited Ca
2+
i rises ~ 1.5-fold larger in prehypertensive SHR than in WKY CCs. Ryanodine reduced the depolarization-induced secretion in prehypertensive SHR by 57%, compared to 14% in WKY CCs, suggesting a greater contribution of intracellular Ca
2+
release to exocytosis. In SHR CCs, the mean spike amplitude and charge per spike were significantly larger than in WKY CCs, regardless of age and stimulus type. This difference in granule content could explain in part the enhanced exocytosis in SHR CCs. However, electron microscopy did not reveal significant differences in granule size between SHRs and WKY rats’ adrenal medulla. Nonetheless, preSHR and hypSHR display 63% and 82% more granules than WKY, which could explain in part the enhanced catecholamine secretion. The mechanism responsible for the heterogeneous population of prehypertensive SHR CCs and the bias towards secreting more medium and large granules remains unexplained.
Endocytosis generates spherical or ellipsoid-like vesicles from the plasma membrane, which recycles vesicles that fuse with the plasma member during exocytosis in neurons and endocrine secretory ...cells. Although tension in the plasma membrane is generally considered to be an important factor in regulating endocytosis, whether membrane tension inhibits or facilitates endocytosis remains debated in the endocytosis field, and has been rarely studied for vesicular endocytosis in secretory cells. Here we report that increasing membrane tension by adjusting osmolarity inhibited both the rapid (a few seconds) and slow (tens of seconds) endocytosis in calyx-type nerve terminals containing conventional active zones and in neuroendocrine chromaffin cells. We address the mechanism of this phenomenon by computational modeling of the energy barrier that the system must overcome at the stage of membrane budding by an assembling protein coat. We show that this barrier grows with increasing tension, which may slow down or prevent membrane budding. These results suggest that in live secretory cells, membrane tension exerts inhibitory action on endocytosis.
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