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.

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.

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.

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 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.

Peroxisome proliferator-activated receptor-γ coactivator-1α mediates neuroprotection against excitotoxic brain injury in transgenic mice: role of mitochondria and X-linked inhibitor of apoptosis protein.

Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a transcriptional coactivator involved in the regulation of mitochondrial biogenesis and cell defense. The functions of PGC-1α in physiology of brain mitochondria are, however, not fully understood. To address this we have studied wild-type and transgenic mice with a two-fold overexpression of PGC-1α in brain neurons. Data showed that the relative number and basal respiration of brain mitochondria were increased in PGC-1α transgenic mice compared with wild-type mitochondria. These changes occurred concomitantly with altered levels of proteins involved in oxidative phosphorylation (OXPHOS) as studied by proteomic analyses and immunoblottings. Cultured hippocampal neurons from PGC-1α transgenic mice were more resistant to cell degeneration induced by the glutamate receptor agonist kainic acid. In vivo kainic acid induced excitotoxic cell death in the hippocampus at 48 h in wild-type mice but significantly less so in PGC-1α transgenic mice. However, at later time points cell degeneration was also evident in the transgenic mouse hippocampus, indicating that PGC-1α overexpression can induce a delay in cell death. Immunoblotting showed that X-linked inhibitor of apoptosis protein (XIAP) was increased in PGC-1α transgenic hippocampus with no significant changes in Bcl-2 or Bcl-X. Collectively, these results show that PGC-1α overexpression contributes to enhanced neuronal viability by stimulating mitochondria number and respiration and increasing levels of OXPHOS proteins and the anti-apoptotic protein XIAP.

Tissue plasminogen activator contributes to alterations of neuronal migration and activity-dependent responses in fragile X mice.

Fragile X syndrome (FXS) is the most common inherited neurodevelopmental disorder with intellectual disability. Here, we show that the expression of tissue plasminogen activator (tPA) is increased in glial cells differentiated from neural progenitors of Fmr1 knock-out mice, a mouse model for FXS, and that tPA is involved in the altered migration and differentiation of these progenitors lacking FMR1 protein (FMRP). When tPA function is blocked with an antibody, enhanced migration of doublecortin-immunoreactive neurons in 1 d differentiated FMRP-deficient neurospheres is normalized. In time-lapse imaging, blocking the tPA function promotes early glial differentiation and reduces the velocity of nuclear movement of FMRP-deficient radial glia. In addition, we show that enhanced intracellular Ca(2+) responses to depolarization with potassium are prevented by the treatment with the tPA-neutralizing antibody in FMRP-deficient cells during early neural progenitor differentiation. Alterations of the tPA expression in the embryonic, postnatal, and adult brain of Fmr1 knock-out mice suggest an important role for tPA in the abnormal neuronal differentiation and plasticity in FXS. Altogether, the results indicate that tPA may prove to be an interesting potential target for pharmacological intervention in FXS.

Effect of glutamate receptor antagonists on migrating neural progenitor cells.

Neurotransmitters such as glutamate are potential regulators of neurogenesis. Interference with defined glutamate receptor subtypes affects proliferation, migration and differentiation of neural progenitor cells. The cellular targets for the actions of different glutamate receptor ligands are less well known. In this study we have combined calcium imaging, measurement of membrane potential, time-lapse imaging and immunocytochemistry to obtain a spatial overview of migrating mouse embryonic neural progenitor cell-derived cells responding to glutamate receptor agonists and antagonists. Responses via metabotropic glutamate receptor 5 correlated with radial glial cells and dominated in the inner migration zones close to the neurosphere. Block of metabotropic glutamate receptor 5 resulted in shorter radial glial processes, a transient increase in neuron-like cells emerging from the neurosphere and increased motility of neuron-like cells. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors are present on the majority of migrating neuronal cells, which with time accumulate at the outer edge of the migration zone. Blocking these receptors leads to an enhanced extension of radial glial processes and a reduced motility of neuron-like cells. Our results indicate that functional glutamate receptors have profound effects on the motility of neural progenitor cells. The main target for metabotropic glutamate receptor 5 appears to be radial glial cells while AMPA/kainate receptors are mainly expressed in newborn neuronal cells and regulate the migratory progress of these cells. The results suggest that both metabotropic glutamate receptor 5 and AMPA/kainate receptors are of importance for the guidance of migrating embryonic progenitor cells.

Glycosylphosphatidylinositol (GPI)-anchoring of mamba toxins enables cell-restricted receptor silencing.

Muscarinic toxins (MTs) are snake venom peptides found to selectively target specific subtypes of G-protein-coupled receptors. In here, we have attached a glycosylphosphatidylinositol (GPI) tail to three different toxin molecules and evaluated their receptor-blocking effects in a heterologous expression system. MT7-GPI remained anchored to the cell surface and selectively inhibited M(1) muscarinic receptor signaling expressed in the same cell. To further demonstrate the utility of the GPI tail, we generated MT3- and MTα-like gene sequences and fused these to the signal sequence for GPI attachment. Functional assessment of these membrane-anchored toxins on coexpressed target receptors indicated a prominent antagonistic effect. In ligand binding experiments the GPI-anchored toxins were found to exhibit similar selection profiles among receptor subtypes as the soluble toxins. The results indicate that GPI attachment of MTs and related receptor toxins could be used to assess the role of receptor subtypes in specific organs or even cells in vivo by transgenic approaches.

Re-Analysis of Single-Nucleus Transcriptomics Reveals Diverse Dorsal Root Ganglia Macrophage Responses Following Peripheral Nerve Injury.

An increasing amount of evidence points to an important role of macrophages in peripheral nerve injury (PNI) and associated pain. Peripheral nerve macrophages facilitate the regeneration, while dorsal root ganglia (DRG) macrophages might propagate the injury after a PNI. These differences might be explained by various in vivo models of PNIs or non-uniform methodologies to phenotype the macrophages. Unbiased methods to phenotype macrophages using single whole cell or nucleus transcriptomics have been rarely applied on PNIs outside the nerves themselves. Here, we compare the effects of the transection or crush of the sciatic nerve and spinal nerve transection on the DRG macrophage phenotypes utilizing a publicly available single-nucleus transcriptomic DRG dataset. Our results demonstrate that unique and time-dependent DRG macrophage gene expression profiles were produced by the three PNI models with particular macrophage clusters being enriched that were dependent on the severity of the neuronal injury score. PNI associated DRG macrophages were not purely anti- or pro-inflammatory. These results suggest that various functions of DRG macrophage subtypes are carefully orchestrated upon a PNI. These findings open a new avenue for studying the DRG macrophage subtypes in PNIs and encourage further unbiased phenotyping efforts to better understand their relevance in neuropathic pain.

GIT1 protects against breast cancer growth through negative regulation of Notch.

Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.

BDNF and TrkB in neuronal differentiation of Fmr1-knockout mouse.

Fragile X syndrome (FXS) is a common cause of inherited mental retardation and the best characterized form of autistic spectrum disorders. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP), which leads to abnormalities in the differentiation of neural progenitor cells (NPCs) and in the development of dendritic spines and neuronal circuits. Brain-derived neurotrophic factor (BDNF) and its TrkB receptors play a central role in neuronal maturation and plasticity. We studied BDNF/TrkB actions in the absence of FMRP and show that an increase in catalytic TrkB expression in undifferentiated NPCs of Fmr1-knockout (KO) mice, a mouse model for FXS, is associated with changes in the differentiation and migration of neurons expressing TrkB in neurosphere cultures and in the developing cortex. Aberrant intracellular calcium responses to BDNF and ATP in subpopulations of differentiating NPCs combined with changes in the expression of BDNF and TrkB suggest cell subtype-specific alterations during early neuronal maturation in the absence of FMRP. Furthermore, we show that dendritic targeting of Bdnf mRNA was increased under basal conditions and further enhanced in cortical layer V and hippocampal CA1 neurons of Fmr1-KO mice by pilocarpine-induced neuronal activity represented by convulsive seizures, suggesting that BDNF/TrkB-mediated feedback mechanisms for strengthening the synapses were compromised in the absence of FMRP. Pilocarpine-induced seizures caused an accumulation of Bdnf mRNA transcripts in the most proximal segments of dendrites in cortical but not in hippocampal neurons of Fmr1-KO mice. In addition, BDNF protein levels were increased in the hippocampus but reduced in the cortex of Fmr1-KO mice in line with regional differences of synaptic plasticity in the brain of Fmr1-KO mice. Altogether, the present data suggest that alterations in the BDNF/TrkB signaling modulate brain development and impair synaptic plasticity in FXS.

Neurotransmitters and Endothelins Acting on Radial Glial G-Protein-Coupled Receptors Are, Through Proteolytic NRG/ErbB4 Activation, Able to Modify the Migratory Behavior of Neocortical Cells and Mediate Bipolar-to-Multipolar Transition.

Cell-cell communication plays a central role in the guidance of migrating neurons during the development of the cerebral cortex. Neuregulins (NRGs) are essential mediators for migration and maintenance of the radial glial scaffold. We show, in this study that soluble NRG reduces neuronal motility, causes transition of bipolar cells to multipolar ones, and induces neuronal mitosis. Blocking the NRG receptor, ErbB4, results in reduction of neuron-neuron and neuron-radial glial contacts and causes an increase in neuronal motility. Blocking the radial glial metabotropic glutamate receptor 5 (mGluR5), the nonselective cation channel transient receptor potential 3 (TRPC3), or matrix metalloproteinases (MMPs) results in similar effects as ErbB4 blockade. Soluble NRG counteract the changes in motility pattern. Stimulation of other radial glial G-protein-coupled receptors (GPCRs), such as muscarinic acetylcholine receptors or endothelin receptors counteract all the effect of mGluR5 blockade, but not that of ErbB4, TRPC3, and MMP blockade. The results indicate that neurotransmitters and endothelins acting on radial glial GPCRs are, through proteolytic NRG/ErbB4 activation, able to modify the migratory behavior of neurons.

Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

Radiation Triggers a Dynamic Sequence of Transient Microglial Alterations in Juvenile Brain.

Cranial irradiation (IR), an effective tool to treat malignant brain tumors, triggers a chronic pro-inflammatory microglial response, at least in the adult brain. Using single-cell and bulk RNA sequencing, combined with histology, we show that the microglial response in the juvenile mouse hippocampus is rapid but returns toward normal within 1 week. The response is characterized by a series of temporally distinct homeostasis-, sensome-, and inflammation-related molecular signatures. We find that a single microglial cell simultaneously upregulates transcripts associated with pro- and anti-inflammatory microglial phenotypes. Finally, we show that juvenile and adult irradiated microglia are already transcriptionally distinct in the early phase after IR. Our results indicate that microglia are involved in the initial stages but may not be responsible for driving long-term inflammation in the juvenile brain.

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells.

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression.

The T-type Ca2+ Channel Cav3.2 Regulates Differentiation of Neural Progenitor Cells during Cortical Development via Caspase-3.

Here we report that the low-voltage-dependent T-type calcium (Ca2+) channel Cav3.2, encoded by the CACNA1H gene, regulates neuronal differentiation during early embryonic brain development through activating caspase-3. At the onset of neuronal differentiation, neural progenitor cells exhibited spontaneous Ca2+ activity. This activity strongly correlated with the upregulation of CACNA1H mRNA. Cells exhibiting robust spontaneous Ca2+ signaling had increased caspase-3 activity unrelated to apoptosis. Inhibition of Cav3.2 by drugs or viral CACNA1H knock down resulted in decreased caspase-3 activity followed by suppressed neurogenesis. In contrast, when CACNA1H was overexpressed, increased neurogenesis was detected. Cortical slices from Cacna1h knockout mice showed decreased spontaneous Ca2+ activity, a significantly lower protein level of cleaved caspase-3, and microanatomical abnormalities in the subventricular/ventricular and cortical plate zones when compared to their respective embryonic controls. In summary, we demonstrate a novel relationship between Cav3.2 and caspase-3 signaling that affects neurogenesis in the developing brain.

BCG-induced cytokine release in bladder cancer cells is regulated by Ca2+ signaling.

Bacillus Calmette-Guérin (BCG) is widely used in the clinic to effectively treat superficial urinary bladder cancer. However, a significant proportion of patients who fail to respond to BCG risk cystectomy or death. Though more than 3 million cancer treatments with BCG occur annually, surprisingly little is known about the initial signaling cascades activated by BCG. Here, we report that BCG induces a rapid intracellular Ca2+ (calcium ion) signal in bladder cancer cells that is essential for activating the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and for synthesizing and secreting proinflammatory cytokines, including interleukin 8 (IL-8). A similar Ca2+ response was observed when cells were exposed to the supernatant of BCG. Studying cellular molecular mechanisms involved in the BCG signaling event, we found pivotal roles for phospholipase C and the Toll-like receptor 4. Further assessment revealed that this signaling pathway induces synthesis of IL-8, whereas exocytosis appeared to be controlled by global Ca2+ signaling. These results shed new light on the molecular mechanisms underlying BCG treatment of bladder cancer, which can help in improving therapeutic efficacy and reducing adverse side effects.

Endocannabinoid Signaling in Embryonic Neuronal Motility and Cell-Cell Contact - Role of mGluR5 and TRPC3 Channels.

Cell-cell communication plays a central role in the guidance of migrating neuronal precursor cells during the development of the cerebral cortex. Endocannabinoids (eCBs) have previously been shown to be one of the central factors regulating neuronal migration. In this study the effects of eCBs on different parameters, expected to affect embryonic cortical neuronal motility have been analyzed in neurosphere-derived neuroblasts using time-lapse microscopy. Increased endogenous production of the endocannabinoid 2-arachidonyl glycerol (2-AG) causes bursts of neuroblast motility. The neuroblasts move longer distances and show a low frequency of turning, and the number of neuron-neuron contacts are reduced. Similar changes occur interfering with the function of the metabotropic glutamate receptor 5 (mGluR5) or its transducer canonical transient receptor potential channel 3 (TRPC3) or the neuregulin receptor ErbB4. Blocking of 2-AG production reverses these effects. The data suggest that eCB-regulated neuronal motility is controlled by mGluR5/TRPC3 activity possibly via NRG/ErbB4 signaling.