Apical Signal Research Articles

Integrative evaluation and experimental validation of the immune-modulating potential of dysregulated extracellular matrix genes in high-grade serous ovarian cancer prognosis

BackgroundHigh-grade serous ovarian cancer (HGSOC) is a challenging malignancy characterized by complex interactions between tumor cells and the surrounding microenvironment. Understanding the immune landscape of HGSOC, particularly the role of the extracellular matrix (ECM), is crucial for improving prognosis and guiding therapeutic interventions.Methods and resultsUsing univariate Cox regression analysis, we identified 71 ECM genes associated with prognosis in seven HGSOC populations. The ECMscore signature, consisting of 14 genes, was validated using Cox proportional hazards regression with a lasso penalty. Cox regression analyses demonstrated that ECMscore is an excellent indicator for prognostic classification in prevalent malignancies, including HGSOC. Moreover, patients with higher ECMscores exhibited more active stromal and carcinogenic activation pathways, including apical surface signaling, Notch signaling, apical junctions, Wnt signaling, epithelial-mesenchymal transition, TGF-beta signaling, and angiogenesis. In contrast, patients with relatively low ECMscores showed more active immune-related pathways, such as interferon alpha response, interferon-gamma response, and inflammatory response. The relationship between the ECMscore and genomic anomalies was further examined. Additionally, the correlation between ECMscore and immune microenvironment components and signals in HGSOC was examined in greater detail. Moreover, the expression of MGP, COL8A2, and PAPPA and its correlation with FAP were validated using qRT-PCR on samples from HGSOC. The utility of ECMscore in predicting the prospective clinical success of immunotherapy and its potential in guiding the selection of chemotherapeutic agents were also explored. Similar results were obtained from pan-cancer research.ConclusionThe comprehensive evaluation of the ECM may help identify immune activation and assist patients in HGSOC and even pan-cancer in receiving proper therapy.

Learning cortical hierarchies with temporal Hebbian updates.

A key driver of mammalian intelligence is the ability to represent incoming sensory information across multiple abstraction levels. For example, in the visual ventral stream, incoming signals are first represented as low-level edge filters and then transformed into high-level object representations. Similar hierarchical structures routinely emerge in artificial neural networks (ANNs) trained for object recognition tasks, suggesting that similar structures may underlie biological neural networks. However, the classical ANN training algorithm, backpropagation, is considered biologically implausible, and thus alternative biologically plausible training methods have been developed such as Equilibrium Propagation, Deep Feedback Control, Supervised Predictive Coding, and Dendritic Error Backpropagation. Several of those models propose that local errors are calculated for each neuron by comparing apical and somatic activities. Notwithstanding, from a neuroscience perspective, it is not clear how a neuron could compare compartmental signals. Here, we propose a solution to this problem in that we let the apical feedback signal change the postsynaptic firing rate and combine this with a differential Hebbian update, a rate-based version of classical spiking time-dependent plasticity (STDP). We prove that weight updates of this form minimize two alternative loss functions that we prove to be equivalent to the error-based losses used in machine learning: the inference latency and the amount of top-down feedback necessary. Moreover, we show that the use of differential Hebbian updates works similarly well in other feedback-based deep learning frameworks such as Predictive Coding or Equilibrium Propagation. Finally, our work removes a key requirement of biologically plausible models for deep learning and proposes a learning mechanism that would explain how temporal Hebbian learning rules can implement supervised hierarchical learning.

ATG9A‐mediated turnover of p62 condensates requires ubiquitin and occurs independently of the LC3‐lipidation machinery

Autophagy is a tightly controlled cellular recycling process that requires a host of autophagy machinery to form a double membraned vesicle called the autophagosome. This process is most understood in the context of stress‐induced autophagy, with little known about autophagosome biogenesis in basal (nutrient replete) conditions. To understand the regulation of basal autophagy, our work has focused on the poorly understood protein ATG9A, a multi‐pass transmembrane lipid scramblase that is essential for basal autophagy. To broadly understand the role ATG9A plays in basal autophagy, we utilized a quantitative proteome‐level MS/MS approach to measure how ATG9A affects protein flux. We show that loss of ATG9A in basal conditions impairs the degradation of autophagy adaptors, particularly p62/SQSTM1. Using a panel of ATG knock‐out cells, we demonstrate that the lipid transferase proteins ATG2A, ATG2B, and ATG9A promote the basal autophagic turnover of p62 and TAX1BP1 over other autophagy adaptors and do so independently of the LC3‐lipidation machinery. Furthermore, we demonstrate that ATG2A and ATG9A lipid transferase activity regulates the rate of p62 condensate degradation. Finally, we show in CRISPR knock‐in cell lines that ubiquitin is required for recruiting ATG9A to p62 condensates. Taken together, our data suggest that the lipid transferase activity of ATG9A and ATG2A is vital to basal autophagic regulation of protein homeostasis, and that ubiquitination is an apical signal that initiates recruitment of ATG9A to p62 condensates.

Inhibition of SGK1 ameliorates ventriculomegaly in a genetic rat model via regulation of TRPV4 in the choroid plexus

Hydrocephalus is a clinical manifestation of cerebrospinal fluid (CSF) accumulation, leading to ventriculomegaly and increased intracranial pressure. Diuretics and other pharmacotherapies have failed to treat hydrocephalus in the human population; therefore, treatment options remain exclusively surgical. The need to develop pharmacotherapies for the treatment of hydrocephalus remains an unmet clinical need and paramount to this need is the study of brain fluid regulation. Previous studies in our laboratory identified the transient receptor potential vanilloid 4 (TRPV4) channel as a potential regulatory hub protein in the choroid plexus responsible, in part, for the regulation of CSF production. Antagonists of the TRPV4 channel were shown to ameliorate hydrocephalus in a genetic rat model. The current studies highlight a novel drug target: serum‐ and glucocorticoid‐induced kinase 1 (SGK1) in the choroid plexus. A specific small molecular weight, membrane permeant inhibitor of SGK1, SI113, has shown promising results in cancer and metabolic research in recent years. In other tissues, SGK1 has been shown to phosphorylate and activate TRPV4 at the membrane, indicating SGK1 as an important upstream regulatory kinase. SI113 blocked TRPV4‐mediated transepithelial ion flux and barrier conductance increases in a cell culture model of human choroid plexus epithelia (HIBCPP line), and ameliorated ventriculomegaly in the genetic rat model. In hydrocephalic animals, there is an increased apical membrane signal in the choroid plexus epithelial cells via immunofluorescent localization of p‐SGK1 and TRPV4, but not of pan‐SGK1. This is not accompanied by a significant increase in either protein or transcript abundance as measured by western blotting or qPCR, respectively. The change in localization is, however, attenuated by treatment with SI113. Animals treated with SI113 show no change in amounts of TRPV4 or total SGK1, but do show significantly reduced levels of p‐SGK1. Thus, phosphorylation of SGK1 appears to be crucial for the activation of TRPV4 which is, in turn, a channel protein that is activated during the development of hydrocephalus. Like Trpv4‐/‐mice, Sgk1‐/‐mice do not show overt phenotypes, indicating pharmacological antagonism of the kinase would have minimal off‐target effects. Overall, these data provide a strong preclinical basis for the use of SGK1 inhibitors in the treatment of hydrocephalus.

Kidney Injury Stimulates Apical Translocation of NHERF1

BackgroundNHERF1 (Na+‐hydrogen exchanger regulatory factor isoform 1), recognized as a scaffold protein that regulates proximal tubule phosphate transport, also has a role in regulation of cell metabolism and survival. Our laboratory previously demonstrated more severe cisplatin‐induced acute kidney injury (AKI) in the global NHERF1 knock out (KO) mouse compared to the cisplatin‐treated wild type (WT) mouse. In the present study, we tested the hypothesis that kidney injury results in decreased NHERF1 expression by examining NHERF1 expression in two models of AKI (cisplatin and bilateral renal ischemia‐reperfusion) and two models of chronic kidney disease or CKD (adenine diet and Col4a3‐/‐) by immunofluorescent microscopy and western blot analysis.MethodsCisplatin‐induced AKI:Two‐month‐old male and female C57BL/6J WT mice received a single intraperitoneal injection of saline (vehicle) or 20 mg/kg cisplatin. Vehicle‐ and cisplatin‐treated mice were euthanized after 72 hours. Bilateral renal ischemia‐reperfusion injury AKI: Two‐month‐old male and female C57Bl/6J WT mice underwent bilateral renal artery clamp for 30 minutes followed by release. Sham and IRI surgery treated mice were euthanized at 2, 4, and 14 days post‐surgery. Adenine‐induced CKD: Two‐month‐old male and female C57Bl/6J WT mice received either regular or adenine‐supplemented chow for 20 weeks followed by euthanasia. Col4a3‐/‐ CKD: Two‐month‐old male and female C57Bl/6J heterozygous and homozygous KO mice for the collagenase type IV alpha 3 chain were followed for up to 7 months followed by euthanasia. In all four mouse models, the kidneys were removed, de‐capsulated, immersed in 3.7% formaldehyde in phosphate‐buffered saline for 24 hours, then transferred to 70% ethanol for paraffin embedding and immunofluorescent microscopy. Western blots of kidney lysates from each kidney injury model were electrophoresed in 4‐12% Bis‐Tris gradient gels and immunoblotted for NHERF1.ResultsImmunofluorescent microscopy revealed increased staining for NHERF1 at or near the apical membrane of proximal tubule epithelial cells in all four mouse injury models. For the IRI model, longitudinal immunohistochemistry demonstrated a progressive increase in the apical NHERF1 signal from baseline through day 4 post‐surgery followed by a decrease to baseline levels by day 14. Western blot analysis of lysates from all four kidney injury models showed no change in total expression of NHERF1.ConclusionsWe conclude that kidney injury results in translocation of NHERF1 to the apical membrane. These findings suggest that NHERF1 plays a significant role in the response to kidney injury, though at this time, neither the mechanism for translocation nor the specific role of NHERF1 in the response to injury is clear.

Assessment of eIF2α phosphorylation during immunogenic cell death.

Immunogenic cell death (ICD) is a modality of cellular demise that when it is induced by certain anticancer treatments can ignite an adaptive anticancer immune response. ICD is characterized by the emission of a specific set of danger-associated molecular patterns (DAMPs) including calreticulin exposure at the plasma membrane, ATP liberation, HMGB1 exodus and type-I IFN release. The apical signaling triggering the appearance of these hallmarks involves the phosphorylation on serine 51 of the α-subunit of eukaryotic initiation factor 2 (EIF2), a key protein in the orchestration of endoplasmic reticulum (ER) stress responses. EIF2α can be phosphorylated by a family of four EIF2A kinases: EIF2AK1-4 (best known as heme regulated inhibitor, HRI, protein kinase R, PKR, protein kinase R-like endoplasmic reticulum kinase, PERK, and general control non-derepressible 2, GCN2), that each respond to a specific type of cellular stress. Here, we describe different techniques to investigate the biochemical pathways leading to eIF2α phosphorylation in the context of ICD.

Interference of immunogenic chemotherapy by artificially controlled calreticulin secretion from tumor cells.

The exposure of calreticulin (CALR) on the cell surface of apoptotic cancer cells is an important "eat-me" signal that stimulates the engulfment by antigen presenting cells (APCs). When cells are exposed to immunogenic cell death (ICD) inducers, CALR translocates from the lumen of the endoplasmic reticulum (ER) to the cell surface, where it serves as a ligand for LDL-receptor-related protein 1 (LRP1, also known as CD91) expressed by dendritic cells (DCs). Surface-exposed CALR facilitates tumor antigen transfer to DCs and in turn antigen cross-presentation to cytotoxic T cells, altogether culminating in the activation of adaptive immune responses. Consistent with its role as an apical signaling event in anticancer immunity, blocking or neutralizing CALR abolishes the immune-dependent anticancer efficacy of a variety of ICD inducing anticancer agents. Recently we showed that saturating CALR receptors on DCs with abundant recombinant CALR protein, or soluble CALR secreted from cancer cells decreases the potency of ICD-mediated antitumor immune responses. Here we detail how to harness an artificially inducible release of soluble CALR from engineered cancer cells, which can blind DCs from recognizing immunogenic cancer cells, resulting in reduced anticancer immunity. This system offers precise control over the release of immunosuppressive soluble CALR, thus yielding a useful tool for the validation of ICD-inducing immunotherapies.

Signaling Pathway Genetic Variations in Apical Periodontitis

A better understanding of gene expression and variation in signaling pathways in apical periodontitis has resulted in identifying the host response and new therapeutic targets for inflammatory diseases. Inflammatory mediators can reduce cell signaling, interfering with transcription factor activation and inflammatory gene expression. In apical periodontitis (AP), genetic studies focusing on the host response and signaling pathways can establish a link between specific possible genes and the disease. The purpose of this review is to provide information on host response and genetic variations in major apical periodontitis signaling pathways.

Differentiating the origin of outflow tract premature ventricular complexes using the QRS-right ventricular apex interval

Abstract Background For localization of outflow tract Premature ventricular complexes (PVCs) many ECG criteria have been proposed, however in some cases it is difficult to accurately localize the origin of PVCs using the surface ECG. Objective This study aims to study the relation between QRS-right ventricular apex interval and the origin of the outflow tract PVCs. Patients and Methods The study included 30 patients (27 female, age 37.20 ± 7.87, RVOT origin 18) referred for PVCs ablation and we measured the interval from the onset of the earliest QRS complex of premature ventricular contractions (PVCs) to the distal right ventricular apical signal, (the QRS-RVA interval) and correlated this interval with origin of outflow tract PVCs as identified by the successful ablation during the procedure. Results Compared to PVCs originating from RVOT, the QRS-RVA interval was significantly longer in PVCs originating from LVOT (67.33±7.56 for LVOT PVCs vs. 37.11±4.34 for RVOT PVCs, p < 0.001). Receiver operating characteristic (ROC) analysis showed that a QRSRVA interval ≥47 ms has a sensitivity, specificity, positive and negative predictive values of 100%, 100%, 100%, 100% respectively, for prediction of an LVOT origin of PVCs Conclusion The QRS-RVA interval is a simple and accurate criterion for differentiating the origin of outflow tract arrhythmia during electrophysiology study, A QRS-RVA interval ≥47 ms suggests an LVOT origin of PVCs.

Identification of miRNAs mediating shoot growth of grafted sweet cherry through small RNA and degradome sequencing

Sweet cherry (Prunus avium L) is an economically important fruit tree in the worldwide, in which grafting is widely used for plant propagation. Rootstock plays a vital role in shoot growth. However, thus far there have been few reports concerning the molecular mechanism of how rootstock regulates scion growth. MicroRNAs (miRNAs) are crucial small RNAs involved in multiple biological processes. The regulatory roles of miRNAs in the grafting of sweet cherry are not yet clear. In the current work, twelve libraries were constructed using the leaves and stems of sweet cherry grafted on two types of Chinese cherry (Prunus pseudocerasus) rootstocks by high-throughput sequencing. A total of 238 known miRNAs were cataloged into 61 families, and 40 novel ones were identified. Among these miRNAs, 13 and 21 miRNAs were differentially expressed in leaves and stems, respectively. The degradome results and prediction analysis of differentially expressed miRNAs and their targets indicated that miRNAs mediate the growth of scion via several aspects: the shoot apical meristem development, phytohormone synthesis and signal transduction, the absorption and balance of nutrient elements. The available evidence facilitates a better understanding of the role of miRNAs in the response of sweet cherry scion to Chinese cherry rootstock after grafting.

Synaptotagmin-Like Protein 2a Regulates Angiogenic Lumen Formation via Weibel-Palade Body Apical Secretion of Angiopoietin-2.

[Figure: see text].

Structural basis of the (in)activity of the apical DNA damage response kinases ATM, ATR and DNA-PKcs

The DNA damage response (DDR) is orchestrated by three apical signalling kinases: ATM, ATR and DNA-PKcs. Despite their central roles, structural and biochemical understanding has remained limited, mainly due to their large size. Recent advances in cryo-electron microscopy allowed for the structural analysis of these kinases, revealing their overall architecture and providing high resolution structures of their active sites. Combined with novel biochemical insights it is now possible to dissect the elements that are important for activation. In this review we discuss the recent structures of these kinases, the possible mechanisms to regulate their activity, substrate recognition and emerging insights in the elements required for kinase activation.

High and asymmetric somato-dendritic coupling of V1 layer 5 neurons independent of visual stimulation and locomotion.

Active dendrites impact sensory processing and behaviour. However, it remains unclear how active dendritic integration relates to somatic output in vivo. We imaged semi-simultaneously GCaMP6s signals in the soma, trunk and distal tuft dendrites of layer 5 pyramidal neurons in the awake mouse primary visual cortex. We found that apical tuft signals were dominated by widespread, highly correlated calcium transients throughout the tuft. While these signals were highly coupled to trunk and somatic transients, the frequency of calcium transients was found to decrease in a distance-dependent manner from soma to tuft. Ex vivo recordings suggest that low-frequency back-propagating action potentials underlie the distance-dependent loss of signals, while coupled somato-dendritic signals can be triggered by high-frequency somatic bursts or strong apical tuft depolarization. Visual stimulation and locomotion increased neuronal activity without affecting somato-dendritic coupling. High, asymmetric somato-dendritic coupling is therefore a widespread feature of layer 5 neurons activity in vivo.

Vasopressin V1a Receptor of Renal Collecting Duct Intercalated Cells Promotes Urinary Proton Secretion

Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a therapeutic strategy for retardation of chronic kidney disorders. Physiologically, V1aR signaling has been linked with acid‐base homeostasis, although the underlying mechanisms remain debatable. In this study, we tested the hypothesis that V1aR activation in type A intercalated cells (A‐ICs) induces urinary H+ secretion.We generated an anti‐V1aR antibody and verified its specificity using V1aR‐knockout tissues. Localization studies in mice, rat and human tissue, were preformed using immunofluorescence and confocal microscopy or 3D‐structured illumination microscopy (3D‐SIM). For functional studies in vivo, V1aR‐specific agonist, AO‐4‐67, was administered in AVP‐deficient Brattleboro rats and C57/BI6J mice (0.2, 2, or 10μg/kg b.w.; 1 to 4h). Urine was collected in metabolic cages or via ureteral catheter and plasma samples were taken. Isolated, microperfused mouse collecting ducts were analyzed for V1aR‐dependent luminal pH changes and cultured inner medullary collecting duct cells for proton‐secreting vH+‐ATPase regulation.Localization of V1aR in mouse, rat and human kidneys produced basolateral signal in A‐ICs and perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts throughout these species. In addition, basolateral V1aR signal was detected in macula densa cells of mouse but not rat or human kidneys. Administration of the V1aR agonist, A0‐4‐67, significantly decreased urinary pH in vasopressin‐deficient Brattleboro rats (10 μg/kg body weight for 4 hours i.p.; pH 7.38 to 6.71 after 1 hour, P<0.01) and C57/BI6J mice (2 μg/kg body weight for 1 hour i.p.; pH 7.18 to 6.78 after 20 minutes, P<0.05). Basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular Ca2+ levels in intercalated cells and decreased luminal pH (pH −5% after 3 minutes, P<0.05) suggesting V1aR‐dependent Ca2+ release and stimulation of proton secreting proteins. Basolateral treatment of inner medullary collecting duct cells with the V1aR agonist induced luminal translocation of vacuolar H+‐ATPase in A‐ICs (apical signal intensity +93%, P<0.001).In summary, our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A‐ICs, which may have clinical implications for pharmacological targeting of V1aR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synchronizing Protein Traffic to the Primary Cilium.

The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane.

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine-type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine.

The REN4 rheostat dynamically coordinates the apical and lateral domains of Arabidopsis pollen tubes

The dynamic maintenance of polar domains in the plasma membrane (PM) is critical for many fundamental processes, e.g., polar cell growth and growth guidance but remains poorly characterized. Rapid tip growth of Arabidopsis pollen tubes requires dynamic distribution of active ROP1 GTPase to the apical domain. Here, we show that clathrin-mediated endocytosis (CME) coordinates lateral REN4 with apical ROP1 signaling. REN4 interacted with but antagonized active ROP1. REN4 also interacts and co-localizes with CME components, but exhibits an opposite role to CME, which removes both REN4 and active ROP1 from the PM. Mathematical modeling shows that REN4 restrains the spatial distribution of active ROP1 and is important for the robustness of polarity control. Hence our results indicate that REN4 acts as a spatiotemporal rheostat by interacting with ROP1 to initiate their removal from the PM by CME, thereby coordinating a dynamic demarcation between apical and lateral domains during rapid tip growth.

Anisotropy of cell division and epithelial sheet bending via apical constriction shape the complex folding pattern of beetle horn primordia

Insects can dramatically change their outer morphology at molting. To prepare for this drastic transformation, insects generate new external organs as folded primordia under the old cuticle. At molting, these folded primordia are physically extended to form their final outer shape in a very short time. Beetle horns are a typical example. Horn primordia are derived from a flat head epithelial sheet, on which deep furrows are densely added to construct the complex folded structure. Because the 3D structure of the pupa horn is coded in the complex furrow pattern, it is indispensable to know how and where the furrows are set. Here, we studied the mechanism of furrow formation using dachsous (ds) gene knocked down beetles that have shorter and fatter adult horns. The global shape of the beetle horn primordia is mushroom like, with dense local furrows across its surface. Knockdown of ds by RNAi changed the global shape of the primordia, causing the stalk region become apparently thicker. The direction of cell division is biased in wildtype horns to make the stalk shape thin and tall. However, in ds knocked down beetles, it became random, resulting in the short and thick stalk shape. On the other hand, a fine and dense local furrow was not significantly affected by the ds knockdown. In developing wildtype horn primordia, we observed that, before the local furrow is formed, the apical constriction signal emerged at the position of the future furrow, suggesting the pre-pattern for the fine furrow pattern. According to the results, we propose that development of complex horn primordia can be roughly divided to two distinct processes, 1) development of global primordia shape by anisotropic cell division, and 2) local furrow formation via actin-myosin dependent apical constriction of specific cells.

Establishment of Microvillus Inclusion Disease (MVID) human models using small intestinal enteroids and embryonic stem cells

Microvillus Inclusion Disease (MVID) is a severe genetic disease resulting from loss‐of‐function mutations in the apical recycling associated actin motor, myosin Vb (Myo5B). MVID affects newborns (early‐stage) or infants (late‐stage), resulting in uncontrolled secretory diarrhea, and death. The diagnostic and pathognomonic features are found in enterocytes of the small intestine, specifically in the duodenum. Our laboratory investigates how apical ion transporter trafficking and signaling in the intestine results in MVID diarrhea. Treatment is unavailable due to limited understanding of MVID pathogenesis, and, access to human MVID intestinal tissues for research is not feasible due to the severity of illness in affected children. Therefore, generating appropriate research models is critical to understanding the disease and development of therapies.Our laboratory generated cultured intestinal cell models lacking Myo5B (lentivirus shRNA knockdown) for MVID studies (Kravtsov DV et al. 2016). Although cancer‐derived cell lines remain valuable, superior cutting‐edge technologies and models, such as enteroids/organoids and CRISPR/Cas 9 gene editing, are now available. Since MVID is an epithelial disease resulting in ion transporter defects in the proximal small intestine, we hypothesized that successful development of a human small intestinal model of MVID will provide a superior model for studies of MVID pathogenesis and treatment. Our goal is to develop complementary human intestinal models of MVID by impeding Myo5B function in both human duodenal enteroids (hDEs) and human embryonic stem cells (hESCs) that will be differentiated into small intestinal organoids.First, Myo5B will be silenced in 2D hDEs using lentiviral transfection as before. Immunoblot analysis will confirm Myo5B knockdown and enteroids will be characterized for expression, location, and function of ion transporters and proteins involved in MVID pathogenesis. A complementary MVID model will employ H9 hESCs for gene editing of the Myo5B single nucleotide polymorphism, G1125A, using CRISPR/Cas 9 technology. Clones will be sequenced to confirm successful gene editing of Myo5B. Once confirmed, H9 hESCs will be differentiated into small intestine and cultured into 3D intestinal organoids and characterized for MVID.To date, we have cultured undifferentiated (immature) and differentiated (mature) hDEs replicating crypt and villus enterocytes of the duodenum. For H9 hESC studies, the donor DNA guide, forward and reverse single guide RNAs were designed (http://crispr.mit.edu). Guide RNAs and donor DNA were transfected into H9 hESCs and clones sequenced to confirm Myo5B G1125A gene editing. H9 Myo5B G1125A cells will be differentiated into small intestine using an established protocol by McCracken KW et al. 2013. Successful establishment of these models will facilitate studies of ion transporter trafficking and signaling, and screening of compounds for treatment of MVID using physiologically relevant human intestinal models.Support or Funding InformationFunding: R01 DK077065This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Golgi Lipid Signaling Pathway Controls Apical Golgi Distribution and Cell Polarity during Neurogenesis

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) stimulate PtdIns-4-P synthesis and signaling in eukaryotic cells, but to what biological outcomes such signaling circuits are coupled remains unclear. Herein, we show that two highly related StART-like PITPs, PITPNA and PITPNB, act in a redundant fashion to support development of the embryonic mammalian neocortex. PITPNA/PITPNB do so by driving PtdIns-4-P-dependent recruitment of GOLPH3, and likely ceramide transferprotein (CERT), to Golgi membranes with GOLPH3 recruitment serving to promote MYO18A- and F-actin-directed loading of the Golgi network to apical processes of neural stem cells (NSCs). We propose the primary role for PITP/PtdIns-4-P/GOLPH3/CERT signaling in NSC Golgi is not in regulating bulk membrane trafficking but in optimizing apically directed membrane trafficking and/or apical membrane signaling during neurogenesis.