Small-world connectivity dictates collective endothelial cell signaling.

Every blood vessel is lined by a single layer of highly specialized, yet adaptable and multifunctional endothelial cells. These cells, the endothelium, control vascular contractility, hemostasis, and inflammation and regulate the exchange of oxygen, nutrients, and waste products between circulating blood and tissue. To control each function, the endothelium processes endlessly arriving requests from multiple sources using separate clusters of cells specialized to detect specific stimuli. A well-developed but poorly understood communication system operates between cells to integrate multiple lines of information and coordinate endothelial responses. Here, the nature of the communication network has been addressed using single-cell Ca2+ imaging across thousands of endothelial cells in intact blood vessels. Cell activities were cross-correlated and compared to a stochastic model to determine network connections. Highly correlated Ca2+ activities occurred in scattered cell clusters, and network communication links between them exhibited unexpectedly short path lengths. The number of connections between cells (degree distribution) followed a power-law relationship revealing a scale-free network topology. The path length and degree distribution revealed an endothelial network with a "small-world" configuration. The small-world configuration confers particularly dynamic endothelial properties including high signal-propagation speed, stability, and a high degree of synchronizability. Local activation of small clusters of cells revealed that the short path lengths and rapid signal transmission were achieved by shortcuts via connecting extensions to nonlocal cells. These findings reveal that the endothelial network design is effective for local and global efficiency in the interaction of the cells and rapid and robust communication between endothelial cells in order to efficiently control cardiovascular activity.

Disrupted Cacna1c gene expression perturbs spontaneous Ca2+ activity causing abnormal brain development and increased anxiety.

The L-type voltage-gated Ca2+ channel gene CACNA1C is a risk gene for various psychiatric conditions, including schizophrenia and bipolar disorder. However, the cellular mechanism by which CACNA1C contributes to psychiatric disorders has not been elucidated. Here, we report that the embryonic deletion of Cacna1c in neurons destined for the cerebral cortex using an Emx1-Cre strategy disturbs spontaneous Ca2+ activity and causes abnormal brain development and anxiety. By combining computational modeling with electrophysiological membrane potential manipulation, we found that neural network activity was driven by intrinsic spontaneous Ca2+ activity in distinct progenitor cells expressing marginally increased levels of voltage-gated Ca2+ channels. MRI examination of the Cacna1c knockout mouse brains revealed volumetric differences in the neocortex, hippocampus, and periaqueductal gray. These results suggest that Cacna1c acts as a molecular switch and that its disruption during embryogenesis can perturb Ca2+ handling and neural development, which may increase susceptibility to psychiatric disease.

Anatomy of the complete mouse eye vasculature explored by light-sheet fluorescence microscopy exposes subvascular-specific remodeling in development and pathology.

Eye development and function rely on precise establishment, regression and maintenance of its many sub-vasculatures. These crucial vascular properties have been extensively investigated in eye development and disease utilizing genetic and experimental mouse models. However, due to technical limitations, individual studies have often restricted their focus to one specific sub-vasculature. Here, we apply a workflow that allows for visualization of complete vasculatures of mouse eyes of various developmental stages. Through tissue depigmentation, immunostaining, clearing and light-sheet fluorescence microscopy (LSFM) entire vasculatures of the retina, vitreous (hyaloids) and uvea were simultaneously imaged at high resolution. In silico dissection provided detailed information on their 3D architecture and interconnections. By this method we describe successive remodeling of the postnatal iris vasculature, involving sprouting and pruning, following its disconnection from the embryonic feeding hyaloid vasculature. In addition, we demonstrate examples of conventional and LSFM-mediated analysis of choroidal neovascularization after laser-induced wounding, showing added value of the presented workflow in analysis of modelled eye disease. These advancements in visualization and analysis of the respective eye vasculatures in development and complex eye disease open for novel observations of their functional interplay at a whole-organ level.

Consultation on UTUC II Stockholm 2022: diagnostic and prognostic methods-what's around the corner?

PURPOSE: To map current literature and provide an overview of upcoming future diagnostic and prognostic methods for upper tract urothelial carcinoma (UTUC), including translational medical science. METHODS: A scoping review approach was applied to search the literature. Based on the published literature, and the experts own experience and opinions consensus was reached through discussions at the meeting Consultation on UTUC II in Stockholm, September 2022. RESULTS: The gene mutational profile of UTUC correlates with stage, grade, prognosis, and response to different therapeutic strategies. Analysis of pathway proteins downstream of known pathogenic mutations might be an alternative approach. Liquid biopsies of cell-free DNA may detect UTUC with a higher sensitivity and specificity than urinary cytology. Extracellular vesicles from tumour cells can be detected in urine and may be used to identify the location of the urothelial carcinoma in the urinary tract. 3D microscopy of UTUC samples may add information in the analysis of tumour stage. Chemokines and chemokine receptors were linked to overall survival and responsiveness to neoadjuvant chemotherapy in muscle-invasive bladder cancer, which is potentially also of interest in UTUC. CONCLUSION: Current diagnostic methods for UTUC have shortcomings, especially concerning prognostication, which is important for personalized treatment decisions. There are several upcoming methods that may be of interest for UTUC. Most have been studied for urothelial carcinoma of the bladder, and it is important to keep in mind that UTUC is a different entity and not all methods are adaptable or applicable to UTUC.

Altered interplay between endoplasmic reticulum and mitochondria in Charcot-Marie-Tooth type 2A neuropathy.

Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot-Marie-Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum-mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.

Glyphosate-based herbicide induces long-lasting impairment in neuronal and glial differentiation.

Glyphosate-based herbicides (GBH) are among the most sold pesticides in the world. There are several formulations based on the active ingredient glyphosate (GLY) used along with other chemicals to improve the absorption and penetration in plants. The final composition of commercial GBH may modify GLY toxicological profile, potentially enhancing its neurotoxic properties. The developing nervous system is particularly susceptible to insults occurring during the early phases of development, and exposure to chemicals in this period may lead to persistent impairments on neurogenesis and differentiation. The aim of this study was to evaluate the long-lasting effects of a sub-cytotoxic concentration, 2.5 parts per million of GBH and GLY, on the differentiation of human neuroepithelial stem cells (NES) derived from induced pluripotent stem cells (iPSC). We treated NES cells with each compound and evaluated the effects on key cellular processes, such as proliferation and differentiation in daughter cells never directly exposed to the toxicants. We found that GBH induced a more immature neuronal profile associated to increased PAX6, NESTIN and DCX expression, and a shift in the differentiation process toward glial cell fate at the expense of mature neurons, as shown by an increase in the glial markers GFAP, GLT1, GLAST and a decrease in MAP2. Such alterations were associated to dysregulation of key genes critically involved in neurogenesis, including PAX6, HES1, HES5, and DDK1. Altogether, the data indicate that subtoxic concentrations of GBH, but not of GLY, induce long-lasting impairments on the differentiation potential of NES cells.

Notch activation in the mouse mammary luminal lineage leads to ductal hyperplasia and altered partitioning of luminal cell subtypes.

Hyperactivated Notch signalling has been implicated in breast cancer, but how elevated levels of Notch signalling contribute to mammary dysplasia and tumorigenesis is not fully understood. In this study, we express an activated form of Notch1 in the mouse mammary luminal lineage and analyse the consequences for tumour formation and the transcriptomic landscape in the luminal lineage. Simultaneous conditional activation of a Notch1 intracellular domain (Notch1 ICD) and EGFP in the luminal lineage was achieved by removal of a stop cassette by CRE-recombinase expression from the whey acidic protein (WAP) promoter. Mice in which Notch1 ICD was activated in the luminal lineage (WAP-CRE;R26-N1ICD mice) exhibit ductal hyperplasia after lactation with an increase in branching frequency and in the number of side-branch ends in the ductal tree. A subset of the mice developed mammary tumours and the majority of the tumour cells expressed EGFP (as a proxy for Notch1 ICD), indicating that the tumours originate from the Notch1 ICD-expressing cells. Single-cell transcriptome analysis of the EGFP-positive mammary cells identified six subtypes of luminal cells. The same six subtypes were found in control mice (WAP-CRE;R26-tdTomato mice expressing the tdTomato reporter from WAP-CRE-mediated activation), but the proportion of cells in the various subtypes differed between the WAP-CRE;R26-N1ICD and control WAP-CRE;R26-tdTomato mice. In conclusion, we show that Notch1 ICD expression in the luminal lineage produces a ductal hyperplasia and branching phenotype accompanied by altered luminal cell subtype partitioning.

Retraction: Ghali MGZ, et al. Mechanisms underlying the generation of autonomorespiratory coupling amongst the respiratory central pattern generator, sympathetic oscillators, and cardiovagal premotoneurons. Journal of Integrative Neuroscience. 2020; 19: 521-560.

No abstract present.

Single cell analysis of autism patient with bi-allelic NRXN1-alpha deletion reveals skewed fate choice in neural progenitors and impaired neuronal functionality.

We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.

Mechanisms underlying the generation of autonomorespiratory coupling amongst the respiratory central pattern generator, sympathetic oscillators, and cardiovagal premotoneurons.

The respiratory rhythm and pattern and sympathetic and parasympathetic outflows are generated by distinct, though overlapping, propriobulbar arrays of neuronal microcircuit oscillators constituting networks utilizing mutual excitatory and inhibitory neuronal interactions, residing principally within the metencephalon and myelencephalon, and modulated by synaptic influences from the cerebrum, thalamus, hypothalamus, cerebellum, and mesencephalon and ascending influences deriving from peripheral stimuli relayed by cranial nerve afferent axons. Though the respiratory and cardiovascular regulatory effector mechanisms utilize distinct generators, there exists significant overlap and interconnectivity amongst and between these oscillators and pathways, evidenced reciprocally by breathing modulation of sympathetic oscillations and sympathetic modulation of neural breathing. These coupling mechanisms are well-demonstrated coordinately in sympathetic- and respiratory-related central neuronal and efferent neurogram recordings and quantified by the findings of cross-correlation, spectra, and coherence analyses, combined with empirical interventions including lesioning and pharmacological agonist and antagonist microinjection studies, baroloading, barounloading, and hypoxic and/or hypercapnic peripheral and/or central chemoreceptor stimulation. Sympathetic and parasympathetic central neuronal and efferent neural discharge recordings evidence classic fast rhythms produced by propriobulbar neuronal networks located within the medullary division of the lateral tegmental field, coherent with cardiac sympathetic nerve discharge. These neural efferent nerve discharges coordinately evidence slow synchronous oscillations, constituted by Traube Hering (i.e., high frequency), Mayer wave (i.e., medium or low frequency), and vasogenic autorhythmicity (i.e., very low frequency) wave spectral bands. These oscillations contribute to coupling neural breathing, sympathetic oscillations, and parasympathetic cardiovagal premotoneuronal activity. The mechanisms underlying the origins of and coupling amongst, these waves remains to be unresolved.

Voltage-dependent calcium channel signaling mediates GABAA receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection, γ-aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination.

Volumetric imaging: a potential tool to stage upper tract urothelial carcinoma.

PURPOSE: To investigate whether volumetric imaging of tumor vasculature can be used to phenotypically characterize advanced upper tract urothelial carcinoma, and if this technique can distinguish aggressive invasive tumors from non-aggressive superficial ones. METHODS: In a pilot study, two TaG1 and two T3G3 formalin-fixed paraffin-embedded (FFPE) tumor samples were examined using the DIPCO pipeline (Tanaka et al. in Nature Biomed Eng 1(10):796-806. https://doi.org/10.1038/s41551-017-0139-0 , 2017). Briefly, punch biopsies of FFPE tumors were deparaffinized, cleared, immunolabeled with the vessel marker CD34 and imaged with a light-sheet microscope. Thereafter, the three-dimensional (3D) vasculature of the tumors was analyzed and characterized using a specialized image processing software. RESULTS: We found that T3G3 tumors had increased CD34 density kurtosis and skewness compared to TaG1 tumors. This suggests that analysis of the 3D vasculature can distinguish between high-grade invasive and low-grade superficial tumors. CONCLUSIONS: Volumetric imaging of tumor samples may represent novel methodology that can complement conventional histopathology. Volumetric imaging enabled us to differentiate between invasive and non-invasive upper tract urothelial carcinoma. The method is of particular interest in diagnostic work-up of patients with upper tract urothelial carcinoma as previous findings indicate that volumetric imaging of vascular patterns could be used to differentiate superficial and invasive urothelial carcinoma, irrespective of if the tumor sample was deep or superficial. However, further and more extensive studies are required before this method can be applied clinically.

Regulation of radial glial process growth by glutamate via mGluR5/TRPC3 and neuregulin/ErbB4.

Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ErbB4 causes Ca2+ mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.

Mapping of the three-dimensional lymphatic microvasculature in bladder tumours using light-sheet microscopy.

BACKGROUND: Cancers are heterogeneous and contain various types of irregular structures that can go undetected when examining them with standard two-dimensional microscopes. Studies of intricate networks of vasculature systems, e.g., the tumour lymphatic microvessels, benefit largely from three-dimensional imaging data analysis. METHODS: The new DIPCO (Diagnosing Immunolabeled Paraffin-Embedded Cleared Organs) imaging platform uses three-dimensional light-sheet microscopy and whole-mount immunolabelling of cleared samples to study proteins and micro-anatomies deep inside of tumours. RESULTS: Here, we uncovered the whole three-dimensional lymphatic microvasculature of formalin-fixed paraffin-embedded (FFPE) tumours from a cohort of 30 patients with bladder cancer. Our results revealed more heterogeneous spatial deviations in more advanced bladder tumours. We also showed that three-dimensional imaging could determine tumour stage and identify vascular or lymphatic system invasion with higher accuracy than standard two-dimensional histological diagnostic methods. There was no association between sample storage times and outcomes, demonstrating that the DIPCO pipeline could be successfully applied on old FFPE samples. CONCLUSIONS: Studying tumour samples with three-dimensional imaging could help us understand the pathological nature of cancers and provide essential information that might improve the accuracy of cancer staging.

Mapping genes for calcium signaling and their associated human genetic disorders.

MOTIVATION: Signal transduction via calcium ions (Ca2+) represents a fundamental signaling pathway in all eukaryotic cells. A large portion of the human genome encodes proteins used to assemble signaling systems that can transduce signals with diverse spatial and temporal dynamics. RESULTS: Here, we provide a map of all of the genes involved in Ca2+ signaling and link these genes to human genetic disorders. Using Gene Ontology terms and genome databases, 1805 genes were identified as regulators or targets of intracellular Ca2+ signals. Associating these 1805 genes with human genetic disorders uncovered 1470 diseases with mutated 'Ca2+ genes'. A network with scale-free properties appeared when the Ca2+ genes were mapped to their associated genetic disorders. AVAILABILITY AND IMPLEMENTATION: The Ca2+ genome database is freely available at http://cagedb.uhlenlab.org and will foster studies of gene functions and genetic disorders associated with Ca2+ signaling. CONTACT: per.uhlen@ki.se. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An ex vivo spinal cord injury model to study ependymal cells in adult mouse tissue.

Traumatic spinal cord injury is characterized by an initial cell loss that is followed by a concerted cellular response in an attempt to restore the damaged tissue. Nevertheless, little is known about the signaling mechanisms governing the cellular response to injury. Here, we have established an adult ex vivo system that exhibits multiple hallmarks of spinal cord injury and allows the study of complex processes that are difficult to address using animal models. We have characterized the ependymal cell response to injury in this model system and found that ependymal cells can become activated, proliferate, migrate out of the central canal lining and differentiate in a manner resembling the in vivo situation. Moreover, we show that these cells respond to external adenosine triphosphate and exhibit spontaneous Ca2+ activity, processes that may play a significant role in the regulation of their response to spinal cord injury. This model provides an attractive tool to deepen our understanding of the ependymal cell response after spinal cord injury, which may contribute to the development of new treatment options for spinal cord injury.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain.

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

Cxcl12/Cxcr4 signaling controls the migration and process orientation of A9-A10 dopaminergic neurons.

CXCL12/CXCR4 signaling has been reported to regulate three essential processes for the establishment of neural networks in different neuronal systems: neuronal migration, cell positioning and axon wiring. However, it is not known whether it regulates the development of A9-A10 tyrosine hydroxylase positive (TH(+)) midbrain dopaminergic (mDA) neurons. We report here that Cxcl12 is expressed in the meninges surrounding the ventral midbrain (VM), whereas CXCR4 is present in NURR1(+) mDA precursors and mDA neurons from E10.5 to E14.5. CXCR4 is activated in NURR1(+) cells as they migrate towards the meninges. Accordingly, VM meninges and CXCL12 promoted migration and neuritogenesis of TH(+) cells in VM explants in a CXCR4-dependent manner. Moreover, in vivo electroporation of Cxcl12 at E12.5 in the basal plate resulted in lateral migration, whereas expression in the midline resulted in retention of TH(+) cells in the IZ close to the midline. Analysis of Cxcr4(-/-) mice revealed the presence of VM TH(+) cells with disoriented processes in the intermediate zone (IZ) at E11.5 and marginal zone (MZ) at E14. Consistently, pharmacological blockade of CXCR4 or genetic deletion of Cxcr4 resulted in an accumulation of TH(+) cells in the lateral aspect of the IZ at E14, indicating that CXCR4 is required for the radial migration of mDA neurons in vivo. Altogether, our findings demonstrate that CXCL12/CXCR4 regulates the migration and orientation of processes in A9-A10 mDA neurons.

MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division.

The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.