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Biology subjects

Pang, S.

Publications and source records attributed to Pang, S..

At least 19 recordsLinked to original sources

Oncostatin M-driven leak in human endothelial cells with preserved tight junctions

Blood vessels must preserve blood volume while permitting exchange of nutrients. Endothelial cells line the lumen of all blood vessels and form the intercellular junctions that govern vascular permeability. In acute critical illness the barrier fails, fluid escapes into tissues, and the resulting loss of volume and impaired nutrient exchange cause organ failure. Several inflammatory cytokines are known to disrupt junctions in cultured cells, yet therapies targeting these models have failed. How blood vessels respond to critical illness has remained largely elusive because endothelial cells are generally inaccessible. Here we profile endothelial cells recovered directly from critically ill children to show that oncostatin M drives a previously unrecognized mode of vascular leak. Oncostatin M increases permeability by forming narrow, nanoscale channels within the overlapping borders of adjacent cells, around intact tight junctions. These channels permit passage of small and intermediate sized solutes. Their formation depends on non-canonical signaling in which the kinase JAK1 activates two required parallel pathways (via ERK1 and AKT1) that converge on the transcription factor c-Fos. Inhibiting JAK1, either parallel pathway or depleting c-Fos, prevents oncostatin M-induced loss of barrier function. These results identify a permeability mechanism distinct from the junction disassembly driven by the cytokines that have dominated this field and implicate kinases already targeted by approved drugs as potential points of intervention. More broadly, vascular barriers are transiently loosened during immune-cell trafficking and metastasis, raising the possibility that diverse forms of vascular permeability arise through regulated nanoscale remodeling rather than junctional breakdown.

cell biology↗

The female larval fly CNS connectome: first draft

We release an enhanced FIB-SEM volume and associated neuron, mitochondria, synapse and glia automated segmentations for the complete, gap- and artifact-free central nervous system of the first instar female larval Drosophila melanogaster. Segmentation data is largely unproofread besides select neurons in the premotor and proprioceptive system and select subvolumes where synapses have been manually annotated, serving as training and evaluation data. The dataset represents the first complete central nervous system of the larval fly, and now awaits substantial improvement of machine vision approaches that effectively leverage domain knowledge for the automated yet data-guided proofreading, or substantial and costly human proofreading. All the data and methods are publicly available for further improvement and analysis.

neuroscience↗

miR-34/449 miRNAs regulate choroid plexus ciliogenesis to control cerebrospinal fluid production

A developmental increase in cerebrospinal fluid (CSF) production during development is essential for neuronal growth and ventricular expansion. A key regulator of CSF production is the specialized sensory multicilia of the choroid plexus (ChP), which mediate non-canonical Sonic hedgehog (Shh) signaling to suppress water channel and ion transporter expression, thereby limiting CSF production. ChP multicilia progressively shortens during development, attenuating Shh signaling and promoting CSF production. Here, we identify miR-34/449 miRNAs as essential regulators of ChP multiciliogenesis. Whereas mutations in canonical ciliogenesis genes elevate CSF production and contribute to hydrocephaly, deletion of miR-34/449 reduces CSF volume and causes microcephaly. Loss of miR-34/449 miRNAs causes excessive basal body amplification, defective basal body docking, and failure of developmental multiciliary shortening. Consequently, miR-34/449-deficient ChP cilia remain abnormally long and fail to attenuate Shh signaling, resulting in sustained repression of water channel and ion transporter expression and reduced CSF production. Mechanistically, miR-34/449 miRNAs directly target Gmnc, a master transcriptional regulator of multiciliogenesis, to restrain basal body amplification and promote basal body docking. Together, our findings identify miR-34/449 miRNAs as critical regulators of ChP multiciliogenesis and establish the developmental remodeling of ChP multicilia as a mechanism to couple Shh signaling dynamics to developmental control of CSF production.

developmental biology↗

Combining brainwide activity imaging and electron microscopy reveals novel nociceptive circuits

To understand how brains work, it is necessary to connect neural activity to synaptic-resolution circuit architecture. Recent advances in light-sheet microscopy (LSM) enable whole-brain, cellular-resolution imaging of activity of all neuronal cell bodies, however, most neurons from such datasets cannot be identified. In most organisms, neurons are identifiable based on their projections (and not based on their cell body position) which, when densely labelled, cannot be resolved using LSM. Here, we present a novel methodology to overcome this by combining whole-brain activity imaging with subsequent volume electron microscopy imaging of the same brain to visualise neuronal projections and identify neurons with interesting activity. We used this approach to identify brain neurons that process input from multisensory (nociceptive and mechanosensory) Basin interneurons that trigger vigorous escape in Drosophila larvae in response to threatening somatosensory stimuli. After whole-brain imaging of neuronal activity during Basin activation, we imaged the same brain with an enhanced focused ion-beam electron microscope (eFIB-SEM). We registered the functional and anatomical volumes and reconstructed (in the eFIB-SEM volume) the projections of neurons that responded to Basin activation to determine their developmental lineage and identity. This revealed a distributed network for processing threatening somatosensory stimuli that trigger vigorous escape spanning 25 distinct lineages and many distinct brain areas, and included direct brain targets of Basin neurons that integrate somatosensory information with other modalities, as well as brain output neurons (descending neurons [DN]) that likely contribute to action-selection. Our workflow provides a powerful framework for mapping neuronal activity onto structure across an entire brain, yielding novel insights into the distributed central processing of noxious stimuli.

neuroscience↗

Cross-species comparative connectomics reveals the evolution of an olfactory circuit

Animal behavioural diversity ultimately stems from variation in neural circuitry, yet how central neural circuits evolve remains poorly understood. Studies of neural circuit evolution often focus on a few elements within a network. However, addressing fundamental questions in evolutionary neuroscience, such as whether some elements are more evolvable than others, requires a more global and unbiased approach. Here, we used synapse-level comparative connectomics to examine how an entire olfactory circuit evolves. We compared the full antennal lobe connectome of the larvae of two closely related Drosophila species, D. melanogaster and D. erecta, which differ in their ecological niches and odour-driven behaviours. We found that evolutionary change is unevenly distributed across the network. Some features, including neuron types, neuron numbers and interneuron-to-interneuron connectivity, are highly conserved. These conserved elements delineate a core circuit blueprint presumably required for fundamental olfactory processing. Superimposed on this scaffold, we find rewiring changes that mirror each species ecologies, including a systematic shift in the excitation-to-inhibition balance in the feedforward pathways. We further show that some neurons have changed more than others, and that even within individual neurons some synaptic elements remain conserved while others display major species-specific changes, suggesting evolutionary hot-spots within the circuit. Our findings reveal constrained and adaptable elements within olfactory networks, and establish a framework for identifying general principles in the evolution of neural circuits underlying behaviour.

neuroscience↗

Lipid nanoparticle-mediated CRISPR/Cas9 delivery enables efficient trabecular meshwork gene editing in mice

Lipid nanoparticles (LNPs) have emerged as a transformative platform for mRNA delivery, enabling vaccines and gene editing with transient expression and high cargo capacity. However, their potential for ocular gene editing remains underexplored. In this study, we assessed the transduction efficiency, inflammatory response, and gene editing capability of LNP-encapsulated mRNA in murine eyes. Intravitreal delivery of LNPs achieved targeted mRNA expression in the trabecular meshwork (TM) with superior specificity and efficiency compared to adenoviral or adeno-associated viral vectors, while inducing minimal microglial activation in the retina. Using LNPs co-encapsulating SpCas9 mRNA and sgRNA, we demonstrated efficient CRISPR-mediated knockout (KO) of Matrix Gla Protein (Mgp), a key inhibitor of TM calcification. Mgp-KO eyes exhibited sustained intraocular pressure (IOP) elevation and anterior chamber deepening with normal anterior chamber angle, recapitulating key features of primary open-angle glaucoma (POAG). Chronic IOP elevation led to reactive Muller gliosis and ganglion cell complex thinning, reflecting retinal stress and progressive neurodegeneration. Our findings establish LNP-CRISPR as a safe and efficient system for TM-targeted gene editing, with broad applicability in glaucoma pathogenesis modelling and therapeutic discovery.

neuroscience↗

Multicilia dynamically transduce Shh signaling to regulate choroid plexus functions

Choroid plexus is a major site for cerebrospinal fluid (CSF) production, characterized by a multiciliated epithelial monolayer that regulates CSF production. We demonstrate that defective choroid plexus ciliogenesis or Intraflagellar transport yields neonatal hydrocephalus, at least in part, due to increased water channel Aqp1 and ion transporter Atp1a2 expression. We demonstrate choroid plexus multicilia as sensory cilia, transducing both canonical and non-canonical Shh signaling. Interestingly, it is the non-canonical Shh signaling that represses Aqp1 and Atp1a2 expression by Smo/Gi/cAMP pathway. Choroid plexus multicilia exhibit unique ciliary ultrastructure, carrying features of both primary and motile cilia. Unlike most cilia that elongate during maturation, choroid plexus ciliary length decreases during development, causing a decline of Shh signaling intensity in developing choroid plexus, a derepression of Aqp1 and Atp1a2, and ultimately, an increased CSF production. Hence, developmental dynamics of choroid plexus multicilia dampens the Shh signaling intensity to promote CSF production.

developmental biology↗

The first complete 3D reconstruction and morphofunctional mapping of an insect eye

The structure of compound eyes in arthropods has been the subject of many studies, revealing important biological principles. Until recently, these studies were constrained by the two-dimensional nature of available ultrastructural data. By taking advantage of the novel three-dimensional ultrastructural dataset obtained using volume electron microscopy (vEM), we present the first cellular-level reconstruction of the whole compound eye of an insect, the miniaturized parasitoid wasp Megaphragma viggianii. The compound eye of the female M. viggianii consists of 29 ommatidia and contains 478 cells. Despite the almost anucleate brain, all cells of the compound eye contain nuclei. As in larger insects, the dorsal rim area (DRA) of the eye in M. viggianii contains ommatidia that are believed to be specialized in polarized light detection as reflected in their corneal and retinal morphology. We report the presence of three ectopic photoreceptors. Our results offer new insights into the miniaturization of compound eyes and scaling of sensory organs in general.

zoology↗

Structural and functional dissection of the Pacinian corpuscle reveals an active role of the inner core in touch detection

Pacinian corpuscles are rapidly adapting mechanoreceptor end-organs that detect transient touch and high-frequency vibration. In the prevailing model, these properties are determined by the outer core, which acts as a mechanical filter limiting static and low-frequency stimuli from reaching the afferent terminal--the sole site of touch detection in corpuscles. Here, we determine the detailed 3D architecture of corpuscular components and reveal their contribution to touch detection. We show that the outer core is dispensable for rapid adaptation and frequency tuning. Instead, these properties arise from the inner core, composed of gap junction-coupled lamellar Schwann cells (LSCs) surrounding the afferent terminal. By acting as additional touch sensing structures, LSCs potentiate mechanosensitivity of the terminal, which detects touch via fast-inactivating ion channels. We propose a model in which Pacinian corpuscle function is mediated by an interplay between mechanosensitive LSCs and the afferent terminal in the inner core. HighlightsO_LIeFIB-SEM reveals detailed 3D architecture of the entire Pacinian (Herbst) corpuscle C_LIO_LIInner, not outer core mediates rapid adaptation and frequency tuning C_LIO_LIAfferent terminal detects touch via fast-inactivating ion channels C_LIO_LIMechanosensitive lamellar Schwann cells tune afferent terminal sensitivity to touch C_LI

neuroscience↗

Periodic ER-plasma membrane junctions support long-range Ca2+ signal integration in dendrites

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at [~]1 m intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca2+ channels and ER Ca2+-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca2+ homeostasis, and local activation of the Ca2+/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca2+ modulatory machinery facilitating voltage-independent signal transmission and ryanodine receptor-dependent Ca2+ release at ER-PM junctions over 20 m away. Thus, interconnected ER-PM junctions support signal propagation and Ca2+ release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations. HighlightsO_LIPeriodic ER-PM junctions tile neuronal dendritic plasma membrane in rodent and fly. C_LIO_LIER-PM junctions are populated by ER tethering and Ca2+ release and influx machinery. C_LIO_LIER-PM junctions act as sites for local activation of CaMKII. C_LIO_LILocal spine activation drives Ca2+ release from RyRs at ER-PM junctions over 20 m. C_LI

neuroscience↗

Structure, interaction, and nervous connectivity of beta cell primary cilia

Primary cilia are sensory organelles present in many cell types. Based on an array of microtubules termed axoneme they form a specialized membrane compartment partaking in various signaling processes. Primary cilia of pancreatic islet beta cells play a role in autocrine and paracrine signaling and are linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of complete mouse and human beta cell cilia, revealing a disorganized 9+0 axoneme structure. Within the islet, cilia are spatially confined within deep ciliary pockets or squeezed into narrow extracellular spaces between adjacent cells. Beta and alpha cell cilia physically interact with neighboring islet cells pushing and strongly bending their plasma membranes. Furthermore, beta cells can contain multiple cilia that can meet with other islet cell cilia in the extracellular space. Additionally, beta cell cilia establish connections with islet-projecting nerves. These findings highlight the pivotal role of beta cell primary cilia in islet cell connectivity, pointing at their potential functional role in integrating islet intrinsic and extrinsic signals. These novel insights contribute to understanding their significance in health and diabetes.

cell biology↗

A rapid microglial metabolic response controls metabolism and improves memory

Chronic high-fat feeding triggers chronic metabolic dysfunction including obesity, insulin resistance, and diabetes. How high-fat intake first triggers these pathophysiological states remains unknown. Here, we identify an acute microglial metabolic response that rapidly translates intake of high-fat diet (HFD) to a surprisingly beneficial effect on metabolism and spatial / learning memory. High-fat intake rapidly increases palmitate levels in cerebrospinal fluid and triggers a wave of microglial metabolic activation characterized by mitochondrial membrane activation and fission as well as metabolic skewing towards aerobic glycolysis. These effects are detectable throughout the brain and can be detected within as little as 12 hours of HFD exposure. In vivo, microglial ablation and conditional DRP1 deletion show that the microglial metabolic response is necessary for the acute effects of HFD. 13C-tracing experiments reveal that in addition to processing via {beta}-oxidation, microglia shunt a substantial fraction of palmitate towards anaplerosis and re-release of bioenergetic carbons into the extracellular milieu in the form of lactate, glutamate, succinate, and intriguingly, the neuro-protective metabolite itaconate. Together, these data identify microglia as a critical nutrient regulatory node in the brain, metabolizing away harmful fatty acids and releasing the same carbons as alternate bioenergetic and protective substrates for surrounding cells. The data identify a surprisingly beneficial effect of short-term HFD on learning and memory. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/535373v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b9699corg.highwire.dtl.DTLVardef@1c48efcorg.highwire.dtl.DTLVardef@18757d0org.highwire.dtl.DTLVardef@962cf6_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

3D architecture and a bi-cellular mechanism of touch detection in mechanosensory corpuscle

Mechanosensory corpuscles detect transient touch and vibratory signals in the skin of vertebrates, enabling navigation, foraging, and precise manipulation of objects1. The corpuscle core comprises a terminal neurite of a mechanoreceptor afferent, the only known touch-sensing element within corpuscles, surrounded by terminal Schwann cells called lamellar cells (LCs)2-4. However, the precise corpuscular ultrastructure, and the role of LCs in touch detection are unknown. Here we used enhanced focused ion beam scanning electron microscopy and electron tomography to reveal the three-dimensional architecture of avian Meissner (Grandry) corpuscle5. We show that corpuscles contain a stack of LCs innervated by two afferents, which form large-area contacts with LCs. LCs form tether-like connections with the afferent membrane and contain dense core vesicles which release their content onto the afferent. Furthermore, by performing simultaneous electrophysiological recordings from both cell types, we show that mechanosensitive LCs use calcium influx to trigger action potential firing in the afferent and thus serve as physiological touch sensors in the skin. Our findings suggest a bi-cellular mechanism of touch detection, which comprises the afferent and LCs, likely enables corpuscles to encode the nuances of tactile stimuli.

neuroscience↗

Three-dimensional reconstructions of mechanosensory end organs suggest a unifying mechanism underlying dynamic, light touch

Specialized mechanosensory end organs within mammalian skin--hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles--enable our perception of light, dynamic touch1. In each of these end organs, fast-conducting mechanically sensitive neurons, called A{beta} low-threshold mechanoreceptors (A{beta} LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating A{beta} LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli1-6. How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo27-15 and A{beta} RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by A{beta} RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the A{beta} RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for A{beta} RA-LTMR activation in which axon protrusions anchor A{beta} RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.

neuroscience↗

TEAD1 is crucial for myelination, Remak bundles and functional regeneration of peripheral nerves

Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin (Grove et al., 2017; Grove, Lee, Zhao, & Son, 2020). Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

neuroscience↗

Co-targeting B-RAF and PTEN enables sensory axons to regenerate across and beyond the spinal cord injury

Primary sensory axons in adult mammals fail to regenerate after spinal cord injury (SCI), in part due to insufficient intrinsic growth potential. Robustly boosting their growth potential continues to be a challenge. Previously, we showed that constitutive activation of B-RAF (rapidly accelerated fibrosarcoma kinase) markedly promotes axon regeneration after dorsal root and optic nerve injuries. The regrowth is further augmented by supplemental deletion of PTEN (phosphatase and tensin homolog). Here, we examined whether concurrent B-RAF activation and PTEN deletion promotes dorsal column axon regeneration after SCI. Remarkably, genetically targeting B-RAF and PTEN selectively in DRG neurons of adult mice enables many DC axons to enter, cross, and grow beyond the lesion site after SCI; some axons reach ~2 mm rostral to the lesion by 3 weeks post-injury. Co-targeting B-RAF and PTEN promotes more robust DC regeneration than a pre-conditioning lesion, which additively enhances the regeneration triggered by B-RAF/PTEN. We also found that post-injury targeting of B-RAF and PTEN enhances DC axon regeneration. These results demonstrate that co-targeting B-RAF and PTEN effectively enhances the intrinsic growth potential of DC axons after SCI and therefore may help to develop a novel strategy to promote robust long-distance regeneration of primary sensory axons.

neuroscience↗

A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility

Chemical synapses between axons and dendrites mediate much of the brains intercellular communication. Here we describe a new kind of synapse - the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam - scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

cell biology↗

Transcriptome-wide association study of coronary artery disease identifies novel susceptibility genes

Transcriptome-wide association studies (TWAS) explore genetic variants affecting gene expression for association with a trait. Here we studied coronary artery disease (CAD) using this approach by first determining genotype-regulated expression levels in nine CAD relevant tissues by EpiXcan in two genetics-of-gene-expression panels, the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) and the Genotype-Tissue Expression (GTEx). Based on these data we next imputed gene expression in respective nine tissues from individual level genotype data on 37,997 CAD cases and 42,854 controls for a subsequent gene-trait association analysis. Transcriptome-wide significant association (P < 3.85e-6) was observed for 114 genes, which by genetic means were differentially expressed predominately in arterial, liver, and fat tissues. Of these, 96 resided within previously identified GWAS risk loci and 18 were novel (CAND1, EGFLAM, EZR, FAM114A1, FOCAD, GAS8, HOMER3, KPTN, MGP, NLRC4, RGS19, SDCCAG3, STX4, TSPAN11, TXNRD3, UFL1, WASF1, and WWP2). Gene set analyses showed that TWAS genes were strongly enriched in CAD-related pathways and risk traits. Associations with CAD or related traits were also observed for damaging mutations in 67 of these TWAS genes (11 novel) in whole-exome sequencing data of UK Biobank. Association studies in human genotype data of UK Biobank and expression-trait association statistics of atherosclerosis mouse models suggested that newly identified genes predominantly affect lipid metabolism, a classic risk factor for CAD. Finally, CRISPR/Cas9-based gene knockdown of RGS19 and KPTN in a human hepatocyte cell line resulted in reduced secretion of APOB100 and lipids in the cell culture medium. Taken together, our TWAS approach was able to i) prioritize genes at known GWAS risk loci and ii) identify novel genes which are associated with CAD.

genetics↗