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Lowe Syndrome (LS) is a rare X-linked disorder characterized by renal dysfunction, cataracts, and several central nervous system (CNS) anomalies. The mechanisms underlying the neurological dysfunction in LS remain unclear, albeit they share some phenotypic characteristics similar to the deficiency or dysfunction of the Reelin signaling, a relevant pathway with roles in CNS development and neuronal functions. In this study, we investigated the role of OCRL1, an inositol polyphosphate 5-phosphatase encoded by the OCRL gene, mutated in LS, focusing on its impact on endosomal trafficking and receptor recycling in human neuronal cells. Specifically, we tested the effects of OCRL1 deficiency in the trafficking and signaling of ApoER2/LRP8, a receptor for the ligand Reelin. We found that loss of OCRL1 impairs ApoER2 intracellular trafficking, leading to reduced receptor expression and decreased levels at the plasma membrane. Additionally, human neurons deficient in OCRL1 showed impairments in ApoER2/Reelin-induced responses. Our findings highlight the critical role of OCRL1 in regulating ApoER2 endosomal recycling and its impact on the ApoER2/Reelin signaling pathway, providing insights into potential mechanisms underlying the neurological manifestations of LS.

Motopsin is a serine protease that plays a crucial role in synaptic functions. Loss of motopsin function causes severe intellectual disability in humans. In this study, we evaluated the role of motopsin in the neuropathological development of cognitive impairments following chemotherapy, also known as chemobrain. Motopsin knockout (KO) and wild-type (WT) mice were intravenously injected with doxorubicin (Dox) or saline four times every 8 days and were evaluated for open field, novel object recognition, and passive avoidance tests. Parvalbumin-positive neurons in the hippocampus were immunohistochemically analyzed. Dox administration significantly decreased the total distance in the open field test in both WT and motopsin KO mice without affecting the duration spent in the center square. A significant interaction between the genotype and drug treatment was detected in the recognition index (the rate to investigate a novel object) in the novel object recognition test, although Dox treatment did not affect the total investigation time. Additionally, Dox treatment significantly decreased the recognition index in WT mice, whereas it tended to increase the recognition index in motopsin KO mice. Dox treatment did not affect the latency to enter a dark compartment in either WT or motopsin KO mice in the passive avoidance test. Interestingly, Dox treatment increased the parvalbumin-positive neurons in the stratum oriens of the hippocampus CA1 region of only WT mice, not motopsin KO mice. Our data suggest that motopsin deficiency imparted partial insensitivity to Dox-induced hippocampal impairments. Alternatively, motopsin may contribute to the neuropathology of chemobrain., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Homozygous Dab1 yotari mutant mice, Dab1 yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity ( mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.

Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (Reln cKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in Reln cKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABA B receptor (GABA B R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABA B Rs in Reln cKO mice. Immunolabeling of Reln cKO cortical slices revealed a reduction in GABA B R1 and GABA B R2 surface expression at the plasma membrane and western blot of Reln cKO cortical tissue revealed decreased phosphorylation of the GABA B R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABA B R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054., (© 2020 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca 2+ /calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: K.U., A.S., S.T., and T.S. are employees of Shionogi & Co., Ltd. Other authors have declared that no competing interests exist.

Flocculation is an aggregation phenomenon of microbial cells in which they form flocs or flakes. In this study, it was found that addition of glycerol to a complex glucose medium promoted spontaneous floc formation by an Escherichia coli degP-deficient mutant strain (ΔdegP) in a dose-dependent manner. In the presence of 10% (v/v) glycerol, the amount of floc formation (quantified as floc protein) reached its maximum value (230 mg/L), five times that in its absence. 10% (v/v) glycerol was the limit concentration that does not inhibit cell growth of ΔdegP strain. Glycerol was not consumed by ΔdegP cells during floc formation. To provide media having nearly the same viscosity as that containing 10% (v/v) glycerol, carboxymethyl cellulose (CMC) or polyvinylpyrrolidone (PVP) were added to medium as viscosifying agents. Floc formation was not promoted by increasing the medium viscosity with CMC or PVP. However, addition of ethylene glycol also significantly promoted floc formation in the same manner as glycerol. Addition of short-chain polyols decreased the number of viable ΔdegP cells in the floc structure and enhanced outer membrane vesicle (OMV) production by ΔdegP cells; polyols-induced damage on the outer membrane of ΔdegP cells may contribute to the promoted floc formation., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PreP R183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Recent studies have suggested that lymphatics help to restore heart function after cardiac injury 1-6 . Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function.

Objective: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice., Conclusions: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro , and that the Δ fakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla Δ fakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the Δ fakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the Δ fakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the Δ fakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in Δ fakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δ aur and Δ sspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the Δ fakA mutant during skin infection., (Copyright © 2020 American Society for Microbiology.)

The digestive enzyme chymotrypsin protects the pancreas against pancreatitis by reducing harmful trypsin activity. Genetic deficiency in chymotrypsin increases pancreatitis risk in humans and pancreatitis severity in mice. Pancreatic chymotrypsin is produced in multiple isoforms including chymotrypsin B1, B2, C and chymotrypsin-like protease (CTRL). Here we investigated the role of CTRL in cerulein-induced pancreatitis in mice. Biochemical experiments with recombinant mouse enzymes demonstrated that CTRL cleaved trypsinogens and suppressed trypsin activation. We generated a novel CTRL-deficient strain (Ctrl-KO) using CRISPR-Cas9 genome engineering. Homozygous Ctrl-KO mice expressed no detectable CTRL protein in the pancreas. Remarkably, the total chymotrypsinogen content in Ctrl-KO mice was barely reduced indicating that CTRL is a low-abundance isoform. When given cerulein, Ctrl-KO mice exhibited lower intrapancreatic chymotrypsin activation and a trend for higher trypsin activation, compared with C57BL/6N mice. Despite the altered protease activation, severity of cerulein-induced acute pancreatitis was similar in Ctrl-KO and C57BL/6N mice. We conclude that CTRL is a minor chymotrypsin isoform that plays no significant role in cerulein-induced pancreatitis in mice.

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells, via single-cell RNA-Seq and protein mass spectrometry, demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C -expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro , recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells, with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells, S1P ablation results in an increased production of granulocyte-macrophage progenitors, the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche., Competing Interests: Competing interestsDr Linda Sandell serves as the editor of the Journal of Orthopaedic Research. All other authors declare no competing interests., (© 2020. Published by The Company of Biologists Ltd.)

Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: Nonedeclared.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Inflammatory pathways contribute to disease pathogenesis; however, regulation of the underlying mechanism is not completely understood. IL-1β, a pro-inflammatory cytokine, participates in the development and progression of NAFLD. To become bioactive, IL-1β requires enzymatic processing. Mechanisms that activate IL-1β include the classical NLRP3 inflammasome-caspase-1 and the neutrophil serine proteases, neutrophil elastase, and proteinase-3. Several studies have shown that both caspase-1 and the neutrophil serine proteases are important for NAFLD development. However, it is unknown whether these pathways interact and if they have a synergistic effect in promoting NAFLD. In the present study, we developed a novel and unique mouse model by intercrossing caspase-1/11 knockout mice with neutrophil elastase/proteinase-3 double knockout mice. Subsequently, these mice were examined regarding the development of high-fat diet-induced NAFLD. Our results show that mice deficient in caspase-1, neutrophil elastase, and proteinase-3 were protected from developing diet-induced weigh gain, liver steatosis, and adipose tissue inflammation when compared with controls. We conclude that pathways that process pro-IL-1β to bioactive IL-1β play an important role in promoting the development of NAFLD and obesity-induced inflammation. Targeting these pathways could have a therapeutic potential in patients with NAFLD.

Aim: A Japanese individual with schizophrenia harboring a novel exonic deletion in RELN was recently identified by genome-wide copy-number variation analysis. Thus, the present study aimed to generate and analyze a model mouse to clarify whether Reln deficiency is associated with the pathogenesis of schizophrenia., Methods: A mouse line with a novel RELN exonic deletion (Reln-del) was established using the CRISPR/Cas9 method to elucidate the underlying molecular mechanism. Subsequently, general behavioral tests and histopathological examinations of the model mice were conducted and phenotypic analysis of the cerebellar granule cell migration was performed., Results: The phenotype of homozygous Reln-del mice was similar to that of reeler mice with cerebellar atrophy, dysplasia of the cerebral layers, and abrogated protein levels of cerebral reelin. The expression of reelin in heterozygous Reln-del mice was approximately half of that in wild-type mice. Conversely, behavioral analyses in heterozygous Reln-del mice without cerebellar atrophy or dysplasia showed abnormal social novelty in the three-chamber social interaction test. In vitro reaggregation formation and neuronal migration were severely altered in the cerebellar cultures of homozygous Reln-del mice., Conclusion: The present results in novel Reln-del mice modeled after our patient with a novel exonic deletion in RELN are expected to contribute to the development of reelin-based therapies for schizophrenia., (© 2020 The Authors Psychiatry and Clinical Neurosciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.)

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population. IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses., (Copyright © 2019 American Society for Microbiology.)

Multiple sclerosis is a neurodegenerative disease affecting predominantly female patients between 20 and 45 years of age. We previously reported the significant contribution of mouse mast cell protease 4 (mMCP-4) in the synthesis of endothelin-1 (ET-1) in healthy mice and in a murine model of experimental autoimmune encephalomyelitis (EAE). In the current study, the cardiovascular effects of ET-1 and big endothelin-1 (big-ET-1) administered systemically or intrathecally were assessed in the early preclinical phase of EAE in telemetry instrumented/conscious mice. Chymase-specific enzymatic activity was also measured in the lung, brain, and mast cell extracts in vitro. Finally, the impact of EAE immunization was studied on the pulmonary and brain mRNA expression of different genes of the endothelin pathway, interleukin-33 (IL-33), and monitoring of immunoreactive tumor necrosis factor- α (TNF- α ). Systemically or intrathecally administered big-ET-1 triggered increases in blood pressure in conscious mice. One week post-EAE, the pressor responses to big-ET-1 were potentiated in wild-type (WT) mice but not in mMCP-4 knockout (KO) mice. EAE triggered mMCP-4-specific activity in cerebral homogenates and peritoneal mast cells. Enhanced pulmonary, but not cerebral preproendothelin-1 and IL-33 mRNA were found in KO mice and further increased 1 week post-EAE immunization, but not in WT animals. Finally, TNF- α levels were also increased in serum from mMCP-4 KO mice, but not WT, 1 week post-EAE. Our study suggests that mMCP-4 activity is enhanced both centrally and systemically in a mouse model of EAE., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

The surface protein haemagglutinin (HA) of influenza A viruses (IAV) needs to be cleaved by a host protease to become functional. Here, we investigated if IAV of the H10 subtype also requires TMPRSS2 for replication and pathogenesis in mice. We first showed in cell culture that TMPRSS2 is able to cleave H10-HA. When Tmprss2 -/- deficient mice were infected with a re-assorted virus H10-HA, they did not lose body weight and no viral replication was observed in contrast to wild-type mice. Histopathological analysis showed that inflammatory lesions in the lung of Tmprss2 -/- mice were reduced compared to wild-type mice. In addition, no viral antigen was detected in the lungs of Tmprss2 -/- mice and no evidence for HA cleavage was observed. We conclude from these studies that TMPRSS2 activity is also essential for in vivo replication and pathogenesis of H10 IAV.

Mouse mast cell protease-4 (mMCP4) is a chymase that has been implicated in cardiovascular diseases, including myocardial infarction (MI). This study tested a direct role of mMCP4 in mouse post-MI cardiac dysfunction and myocardial remodeling. Immunoblot and immunofluorescent double staining demonstrated mMCP4 expression in cardiomyocytes from the infarct zone from mouse heart at 28 day post-MI. At this time point, mMCP4-deficient Mcpt4 -/- mice showed no difference in survival from wild-type (WT) control mice, yet demonstrated smaller infarct size, improved cardiac functions, reduced macrophage content but increased T-cell accumulation in the infarct region compared with those of WT littermates. mMCP4-deficiency also reduced cardiomyocyte apoptosis and expression of TGF-β1, p-Smad2, and p-Smad3 in the infarct region, but did not affect collagen deposition or α-smooth muscle actin expression in the same area. Gelatin gel zymography and immunoblot analysis revealed reduced activities of matrix metalloproteinases and expression of cysteinyl cathepsins in the myocardium, macrophages, and T cells from Mcpt4 -/- mice. Immunoblot analysis also found reduced p-Smad2 and p-Smad3 in the myocardium from Mcpt4 -/- mice, yet fibroblasts from Mcpt4 -/- mice showed comparable levels of p-Smad2 and p-Smad3 to those of WT fibroblasts. Flow cytometry, immunoblot analysis, and immunofluorescent staining demonstrated that mMCP4-deficiency reduced the expression of proapoptotic cathepsins in cardiomyocytes and protected cardiomyocytes from H 2 O 2 -induced apoptosis. This study established a role of mMCP4 in mouse post-MI dysfunction by regulating myocardial protease expression and cardiomyocyte death without significant impact on myocardial fibrosis or survival post-MI in mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cajal-Retzius (CR) cells are early-born glutamatergic neurons that are primarily known as the early main source of the signal protein Reelin. In the reeler mutant, the absence of Reelin causes severe defects in the radial migration of neurons, resulting in abnormal cortical layering. To date, the exact morphological properties of CR-cells independent of Reelin are unknown. With this in view, we studied the ontogenesis, density, and distribution of CR-cells in reeler mice that were cross-bred with a CXCR4-EGFP reporter mouse line, thus enabling us to clearly identify CR-cells positions in the disorganized hippocampus of the reeler mouse. As evidenced by morphological analysis, differences were found regarding CR-cell distribution and density: generally, we found fewer CR-cells in the developing and adult reeler hippocampus as compared to the hippocampus of wild-type animals (WT); however, in reeler mice, CR-cells were much more closely associated to the hippocampal fissure (HF), resulting in relatively higher local CR-cell densities. This higher local cell density was accompanied by stronger immunoreactivity of the CXCR4 ligand, stroma-derived factor-1 (SDF-1) that is known to regulate CR-cell positioning. Importantly, confocal microscopy indicates an integration of CR-cells into the developing and adult hippocampal network in reeler mice, raising evidence that network integration of CR-cells might be independent of Reelin., (© 2018 Wiley Periodicals, Inc.)

Manganese is an essential metal that is required for a wide range of biological functions. Ferroportin (FPN), the only known cellular exporter of iron, has also been proposed to play a role in manganese export, but this relationship is incompletely understood. To investigate this in more detail in vivo, we examined the relative distributions of manganese and iron in TMPRSS6 deficient mice, which are characterized by constitutively high expression of the iron regulatory hormone hepcidin and, consequently, very low FPN levels in their tissues. Tmprss6-/- mice showed frank iron deficiency and reduced iron levels in most tissues, consistent with FPN playing an important role in the distribution of this metal, but manganese levels were largely unaffected. Associated studies using intestine-specific FPN knockout mice showed that loss of FPN significantly reduced the dietary absorption of iron, but had no effect on manganese intake. Taken together, our data suggest that FPN does not play a major role in Mn transport in vivo. They do not exclude a minor role for FPN in manganese homeostasis, nor the possibility that the transporter may be relevant at high Mn levels, but at physiological levels of this metal, other transport proteins appear to be more important.

The Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse β-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamB G439D We show that the folding defect of LamB G439D results in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutant degS A323E allele. The mutant DegS A323E protein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream σ E stress response signaling. Furthermore, the enhanced σ E activity of DegS A323E suppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of σ E activity. We propose that the increased basal levels of σ E signaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation. IMPORTANCE Gram-negative bacteria, such as Escherichia coli , inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σ E stress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival., (Copyright © 2019 American Society for Microbiology.)

In Escherichia coli , the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in the degS gene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of DegS's protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS's basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS's protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σ E -mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of Δ tolB Δ degP mutants stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised. IMPORTANCE The Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented here show that DegP's periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed., (Copyright © 2019 American Society for Microbiology.)

The serine protease PRSS8 has shown important physiological and pathological functions, but its roles in cancer initiation and progression are unclear. We developed and dynamically characterized a conditional knockout Prss8 fl/fl , p-Villin-Cre + mouse model. We found that genetic deficiency of the Prss8 gene caused spontaneous colitis and an inflamed rectum at an early age and caused intestinal tumors at a late age, which were linked to increased intestinal cell proliferation and migration but decreased cell differentiation. Increased PRSS8 expression inhibited cancer cell growth and metastasis in nude mice and inhibited cancer cell migration, invasion, colony formation and tumor sphere formation in vitro, but decreased PRSS8 expression facilitated malignancies in vivo and in vitro. Gene profiling on manipulated cancer cells and intestinal epithelial cells of Prss8 mouse models, gene set enrichment analysis and mechanistic studies revealed that PRSS8 targeted the Wnt/β-catenin, epithelial-mesenchymal transition, and stem cell signaling pathways, which were further supported by the results from the TCGA data mining and validated by immunohistochemical staining on colorectal cancer tissue microarrays. In conclusion, PRSS8 is a novel tumor suppressor that plays critical roles in the suppression of colorectal carcinogenesis and metastasis.

Hematopoietic stem and progenitor cells (HSPCs) undergo self-renewal and differentiation to guarantee a constant supply of short-lived blood cells. Both intrinsic and extrinsic factors determine HSPC fate, but the underlying mechanisms remain elusive. Here, we report that Proteinase 3 (PR3), a serine protease mainly confined to granulocytes, is also expressed in HSPCs. PR3 deficiency intrinsically suppressed cleavage and activation of caspase-3, leading to expansion of the bone marrow (BM) HSPC population due to decreased apoptosis. PR3-deficient HSPCs outcompete the long-term reconstitution potential of wild-type counterparts. Collectively, our results establish PR3 as a physiological regulator of HSPC numbers. PR3 inhibition is a potential therapeutic target to accelerate and increase the efficiency of BM reconstitution during transplantation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Reelin is a secreted protein essential for the development and function of the mammalian brain. The receptors for Reelin, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, belong to the low-density lipoprotein receptor family, but it is not known whether Reelin is involved in the brain lipid metabolism. In the present study, we performed lipidomic analysis of the cerebral cortex of wild-type and Reelin-deficient (reeler) mice, and found that reeler mice exhibited several compositional changes in phospholipids. First, the ratio of phospholipids containing one saturated fatty acid (FA) and one docosahexaenoic acid (DHA) or arachidonic acid (ARA) decreased. Secondly, the ratio of phospholipids containing one monounsaturated FA (MUFA) and one DHA or ARA increased. Thirdly, the ratio of phospholipids containing 5,8,11-eicosatrienoic acid, or Mead acid (MA), increased. Finally, the expression of stearoyl-CoA desaturase-1 (SCD-1) increased. As the increase of MA is seen as an index of polyunsaturated FA (PUFA) deficiency, and the expression of SCD-1 is suppressed by PUFA, these results strongly suggest that the loss of Reelin leads to PUFA deficiency. Hence, MUFA and MA are synthesized in response to this deficiency, in part by inducing SCD-1 expression. This is the first report of changes of FA composition in the reeler mouse brain and provides a basis for further investigating the new role of Reelin in the development and function of the brain., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Our previous work revealed that rhomboid domain-containing protein 1 (RHBDD1) participates in the modulation of cell growth and apoptosis in colorectal cancer cells. This study aimed to investigate the function of RHBDD1 in regulating breast cancer progression and its underlying molecular basis., Methods: Immunohistochemistry was performed to evaluate RHBDD1 expression in 116 breast cancer tissue and 39 adjacent normal tissue and expression of RHBDD1, phospho-Akt (p-Akt) and cyclin-dependent kinase 2 (CDK2) in the same 84 breast cancer specimens. RHBDD1-knock-out cells were established using breast cancer cell lines. In vitro studies were carried out to estimate the function of RHBDD1 in cell proliferation, migration and invasion. Fluorescence microscopy assay and flow cytometric analysis were used to measure apoptosis and cell cycle regulation. RNA sequencing and western blot analysis were used to investigate the molecular mechanisms of RHBDD1., Results: RHBDD1 was highly up-regulated in breast cancer tissue compared with that in normal tissue and associated with pathological tumor (pT) stage, pathological tumor-node-metastasis (pTNM) stage and estrogen receptor (ER) expression. RHBDD1 up-regulation was associated with poor prognosis in several subtypes of breast cancer. Deletion of RHBDD1 promoted apoptosis and suppressed proliferation, migration and invasion in breast cancer cells. RHBDD1 deletion suppressed Akt activation and decreased CDK2 protein level via proteasome pathway, thus inhibited cell cycle progression and G1/S phase transition. Moreover, the protein level of RHBDD1, p-Akt and CDK2 was significantly positively correlated in breast cancer tissue., Conclusions: Our study reveals that RHBDD1 promotes breast cancer progression by regulating p-Akt and CDK2 protein levels, and might be a potential biomarker and prognostic indicator for breast cancer patients.

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Reelin protein (RELN), an extracellular matrix protein, plays multiple roles that range from embryonic neuronal migration to spine formation in the adult brain. Results from genetic studies have suggested that RELN is associated with the risk of psychiatric disorders, including schizophrenia (SCZ). We previously identified a novel exonic deletion of RELN in a patient with SCZ. High-resolution copy number variation analysis revealed that this deletion included exons 52 to 58, which truncated the RELN in a similar manner to the Reln Orleans mutation (Reln rl-Orl ). We examined the clinical features of this patient and confirmed a decreased serum level of RELN. To elucidate the pathophysiological role of the exonic deletion of RELN in SCZ, we conducted behavioral and neurochemical analyses using heterozygous Reln rl-Orl/+ mice. These mice exhibited abnormalities in anxiety, social behavior, and motor learning; the deficits in motor learning were ameliorated by antipsychotics. Methamphetamine-induced hyperactivity and dopamine release were significantly reduced in the Reln rl-Orl/+ mice. In addition, the levels of GABAergic markers were decreased in the brain of these mice. Taken together, our results suggest that the exonic deletion of RELN plays a pathological role, implicating functional changes in the dopaminergic and GABAergic systems, in the pathophysiology of SCZ.

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.

Alterations in iron status have frequently been associated with obesity and other metabolic disorders. The hormone hepcidin stands out as a key regulator in the maintenance of iron homeostasis by controlling the main iron exporter, ferroportin. Here we demonstrate that the deficiency in the hepcidin repressor matriptase-2 (Tmprss6) protects from high-fat diet-induced obesity. Tmprss6 -/- mice show a significant decrease in body fat, improved glucose tolerance and insulin sensitivity, and are protected against hepatic steatosis. Moreover, these mice exhibit a significant increase in fat lipolysis, consistent with their dramatic reduction in adiposity. Rescue experiments that block hepcidin up-regulation and restore iron levels in Tmprss6 -/- mice via anti-hemojuvelin (HJV) therapy, revert the obesity-resistant phenotype of Tmprss6 -/- mice. Overall, this study describes a role for matritpase-2 and hepcidin in obesity and highlights the relevance of iron regulation in the control of adipose tissue function.

Prolyl oligopeptidase (PREP) inhibition by small-molecule inhibitors can reduce alpha-synuclein (aSyn) aggregation, a key player in Parkinson's disease pathology. However, the significance of PREP protein for aSyn aggregation and toxicity is not known. We studied this in vivo by using PREP knock-out mice with viral vector injections of aSyn and PREP. Animal behavior was studied by locomotor activity and cylinder tests, microdialysis and HPLC were used to analyze dopamine levels, and different aSyn forms and loss of dopaminergic neurons were studied by immunostainings. Additionally, PREP knock-out cells were used to characterize the impact of PREP and aSyn on autophagy, proteasomal system and aSyn secretion. PREP knock-out animals were nonresponsive to aSyn-induced unilateral toxicity but combination of PREP and aSyn injections increased aSyn toxicity. Phosphorylated p129, proteinase K resistant aSyn levels and tyrosine hydroxylase positive cells were decreased in aSyn and PREP injected knock-out animals. These changes were accompanied by altered dopamine metabolite levels. PREP knock-out cells showed reduced response to aSyn, while cells were restored to wild-type cell levels after PREP overexpression. Taken together, our data suggests that PREP can enhance aSyn toxicity in vivo.

Alpha-synuclein is the main component of Lewy bodies, a histopathological finding of Parkinson's disease. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and accelerates its aggregation in vitro. PREP enzyme inhibitors have been shown to block the α-synuclein aggregation process in vitro and in cellular models, and also to enhance the clearance of α-synuclein aggregates in transgenic mouse models. Moreover, PREP inhibitors have induced alterations in dopamine and metabolite levels, and dopamine transporter immunoreactivity in the nigrostriatal tissue. In this study, we characterized the role of PREP in the nigrostriatal dopaminergic and GABAergic systems of wild-type C57Bl/6 and PREP knockout mice, and the effects of PREP overexpression on these systems. Extracellular concentrations of dopamine and protein levels of phosphorylated dopamine transporter were increased and dopamine reuptake was decreased in the striatum of PREP knockout mice, suggesting increased internalization of dopamine transporter from the presynaptic membrane. Furthermore, PREP overexpression increased the level of dopamine transporters in the nigrostriatal tissue but decreased phosphorylated dopamine transporters in the striatum in wild-type mice. Our results suggest that PREP regulates the function of dopamine transporter, possibly by controlling the phosphorylation and transport of dopamine transporter into the striatum or synaptic membrane.

PurposePREPL deficiency causes neonatal hypotonia, ptosis, neonatal feeding difficulties, childhood obesity, xerostomia, and growth hormone deficiency. Different recessive contiguous gene deletion syndromes involving PREPL and a variable combination of SLC3A1 (hypotonia-cystinuria syndrome), CAMKMT (atypical hypotonia-cystinuria syndrome), and PPM1B (2p21 deletion syndrome) have been described. In isolated PREPL deficiency, previously described only once, the absence of cystinuria complicates the diagnosis. Therefore, we developed a PREPL blood assay and further delineated the phenotype.MethodsClinical features of new subjects with PREPL deficiency were recorded. The presence of PREPL in lymphocytes and its reactivity with an activity-based probe were evaluated by western blot.ResultsFive subjects with isolated PREPL deficiency, three with hypotonia-cystinuria syndrome, and two with atypical hypotonia-cystinuria syndrome had nine novel alleles. Their IQs ranged from 64 to 112. Adult neuromuscular signs included ptosis, nasal dysarthria, facial weakness, and variable proximal and neck flexor weakness. Autonomic features are prevalent. PREPL protein and reactivity were absent in lymphocytes from subjects with PREPL deficiency, but normal in the clinically similar Prader-Willi syndrome.ConclusionPREPL deficiency causes neuromuscular, autonomic, cognitive, endocrine, and dysmorphic clinical features. PREPL is not deficient in Prader-Willi syndrome. The novel blood test should facilitate the confirmation of PREPL deficiency.

Pancreatic ductal adenocarcinomas (PDAs) are desmoplastic and can undergo epithelial-to-mesenchymal transition to confer metastasis and chemoresistance. Studies have demonstrated that phenotypically and functionally distinct stromal cell populations exist in PDAs. Fibroblast activation protein-expressing (FAP-expressing) cells act to enhance PDA progression, while α-smooth muscle actin myofibroblasts can restrain PDA. Thus, identification of precise molecular targets that mediate the protumorigenic activity of FAP+ cells will guide development of therapy for PDA. Herein, we demonstrate that FAP overexpression in the tumor microenvironment correlates with poor overall and disease-free survival of PDA patients. Genetic deletion of FAP delayed onset of primary tumor and prolonged survival of mice in the KPC mouse model of PDA. While genetic deletion of FAP did not affect primary tumor weight in advanced disease, FAP deficiency increased tumor necrosis and impeded metastasis to multiple organs. Lineage-tracing studies unexpectedly showed that FAP is not only expressed by stromal cells, but can also be detected in a subset of CD90+ mesenchymal PDA cells, representing up to 20% of total intratumoral FAP+ cells. These data suggest that FAP may regulate PDA progression and metastasis in cell-autonomous and/or non-cell-autonomous fashions. Together, these data support pursuing FAP as a therapeutic target in PDA.

Half of all prostate cancers are caused by the TMPRSS2-ERG gene-fusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells. Recent genomic landscape studies of such cancers have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF. Here we show these ERF mutations cause decreased protein stability and mostly occur in tumours without ERG upregulation. ERF loss recapitulates the morphological and phenotypic features of ERG gain in normal mouse prostate cells, including expansion of the androgen receptor transcriptional repertoire, and ERF has tumour suppressor activity in the same genetic background of Pten loss that yields oncogenic activity by ERG. In the more common scenario of ERG upregulation, chromatin immunoprecipitation followed by sequencing indicates that ERG inhibits the ability of ERF to bind DNA at consensus ETS sites both in normal and in cancerous prostate cells. Consistent with a competition model, ERF overexpression blocks ERG-dependent tumour growth, and ERF loss rescues TMPRSS2-ERG-positive prostate cancer cells from ERG dependency. Collectively, these data provide evidence that the oncogenicity of ERG is mediated, in part, by competition with ERF and they raise the larger question of whether other gain-of-function oncogenic transcription factors might also inactivate endogenous tumour suppressors.

Background and Purpose: Angiotensin II (Ang II), whose generation largely depends on angiotensin-converting enzyme (ACE) activity, mediates most of the renin-angiotensin-system (RAS) effects. Elastase-2 (ELA-2), a chymotrypsin-serine protease elastase family member 2A, alternatively generates Ang II in rat arteries. Myocardial infarction (MI) leads to intense RAS activation, but mechanisms involved in Ang II-generation in resistance arteries are unknown. We hypothesized that ELA-2 contributes to vascular Ang II generation and cardiac damage in mice subjected to MI., Experimental Approach: Concentration-effect curves to Ang I and Ang II were performed in mesenteric resistance arteries from male wild type (WT) and ELA-2 knockout (ELA-2KO) mice subjected to left anterior descending coronary artery ligation (MI)., Key Results: MI size was similar in WT and ELA-2KO mice. Ejection fraction and fractional shortening after MI similarly decreased in both strains. However, MI decreased stroke volume and cardiac output in WT, but not in ELA-2KO mice. Ang I-induced contractions increased in WT mice subjected to MI (MI-WT) compared with sham-WT mice. No differences were observed in Ang I reactivity between arteries from ELA-2KO and ELA-2KO subjected to MI (MI-ELA-2KO). Ang I contractions increased in arteries from MI-WT versus MI-ELA-2KO mice. Chymostatin attenuated Ang I-induced vascular contractions in WT mice, but did not affect Ang I responses in ELA-2KO arteries., Conclusions and Implications: These results provide the first evidence that ELA-2 contributes to increased Ang II formation in resistance arteries and modulates cardiac function after MI, implicating ELA-2 as a key player in ACE-independent dysregulation of the RAS., (© 2017 The British Pharmacological Society.)

We discovered a hypomorphic reelin (Reln) mutant with abnormal cortical lamination and no cerebellar hypoplasia. This mutant, Reln CTRdel , carries a chemically induced splice-site mutation that truncates the C-terminal region (CTR) domain of RELN protein and displays remarkably distinct phenotypes from reeler The mutant does not have an inverted cortex, but cortical neurons overmigrate and invade the marginal zone, which are characteristics similar to a phenotype seen in the cerebral cortex of Vldlr null mice. The dentate gyrus shows a novel phenotype: the infrapyramidal blade is absent, while the suprapyramidal blade is present and laminated. Genetic epistasis analysis showed that Reln CTRdel /Apoer2 null double homozygotes have phenotypes akin to those of reeler mutants, while Reln CTRdel /Vldlr null mice do not. Given that the receptor double knock-out mice resemble reeler mutants, we infer that Reln CTRdel /Apoer2 null double homozygotes have both receptor pathways disrupted. This suggests that CTR-truncation disrupts an interaction with VLDLR (very low-density lipoprotein receptor), while the APOER2 signaling pathway remains active, which accounts for the hypomorphic phenotype in Reln CTRdel mice. A RELN-binding assay confirms that CTR truncation significantly decreases RELN binding to VLDLR, but not to APOER2. Together, the in vitro and in vivo results demonstrate that the CTR domain confers receptor-binding specificity of RELN., Significance Statement: Reelin signaling is important for brain development and is associated with human type II lissencephaly. Reln mutations in mice and humans are usually associated with cerebellar hypoplasia. A new Reln mutant with a truncation of the C-terminal region (CTR) domain shows that Reln mutation can cause abnormal phenotypes in the cortex and hippocampus without cerebellar hypoplasia. Genetic analysis suggested that CTR truncation disrupts an interaction with the RELN receptor VLDLR (very low-density lipoprotein receptor); this was confirmed by a RELN-binding assay. This result provides a mechanistic explanation for the hypomorphic phenotype of the CTR-deletion mutant, and further suggests that Reln mutations may cause more subtle forms of human brain malformation than classic lissencephalies., (Copyright © 2017 the authors 0270-6474/17/370960-12$15.00/0.)

Essentials Factor VII-activating protease (FSAP) is a plasma protease involved in vascular processes. Neointima formation was investigated after vascular injury in FSAP -/- mice. The neointimal lesion size and the accumulation of macrophages were increased in FSAP -/- mice. This was due to an increased activity of the chemokine (C-C motif) ligand 2 (CCL2)., Summary: Background Factor VII-activating protease (FSAP) is a multifunctional circulating plasma serine protease involved in thrombosis and vascular remodeling processes. The Marburg I single-nucleotide polymorphism (MI-SNP) in the FSAP-coding gene is characterized by low proteolytic activity, and is associated with increased rates of stroke and carotid stenosis in humans. Objectives To determine whether neointima formation after vascular injury is increased in FSAP -/- mice. Methods and Results The neointimal lesion size and the proliferation of vascular smooth muscle cells (VSMCs) were significantly enhanced in FSAP -/- mice as compared with C57BL/6 control mice after wire-induced injury of the femoral artery. Accumulation of leukocytes and macrophages was increased within the lesions of FSAP -/- mice at day 3 and day 14. Quantitative zymography demonstrated enhanced activity of gelatinases/matrix metalloproteinase (MMP)-2 and MMP-9 within the neointimal lesions of FSAP -/- mice, and immunohistochemistry showed particular costaining of MMP-9 with accumulating leukocytes. Using intravital microscopy, we observed that FSAP deficiency promoted the intravascular adherence and the subsequent transmigration of leukocytes in vivo in response to chemokine ligand 2 (CCL2). CCL2 expression was increased in FSAP -/- monocytes but not in the vessel wall. There was no difference in the expression of platelet-derived growth factor (PDGF-BB). Conclusions FSAP deficiency causes an increase in CCL2 expression and CCL2-mediated infiltration of leukocytes into the injured vessel, thereby promoting SMC proliferation and migration by the activation of leukocyte-derived gelatinases. These results provide a possible explanation for the observed association of the loss-of-function MI-SNP with vascular proliferative diseases., (© 2016 International Society on Thrombosis and Haemostasis.)

Prolyl oligopeptidase (PREP) has been implicated in neurodegeneration and neuroinflammation and has been considered a drug target to enhance memory in dementia. However, the true physiological role of PREP is not yet understood. In this paper, we report the phenotyping of a mouse line where the PREP gene has been knocked out. This work indicates that the lack of PREP in mice causes reduced anxiety but also hyperactivity. The cortical volumes of PREP knockout mice were smaller than those of wild type littermates. Additionally, we found increased expression of diazepam binding inhibitor protein in the cortex and of the somatostatin receptor-2 in the hippocampus of PREP knockout mice. Furthermore, immunohistochemistry and tail suspension test revealed lack of response of PREP knockout mice to lipopolysaccharide insult. Further analysis revealed significantly increased levels of polysialylated-neural cell adhesion molecule in PREP deficient mice. These findings might be explained as possible alteration in brain plasticity caused by PREP deficiency, which in turn affect behaviour and brain development., (Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.)

All-trans retinoic acid (ATRA) is a potent inducer of osteogenic differentiation in mouse adipose-derived stromal cells (mASCs), although the underlying mechanisms responsible for its mode of action have yet to be completely elucidated. High temperature requirement protease A1 (HtrA1) is a newly recognized modulator of human multipotent stromal cell (MSC) osteogenesis and as such, may play a role in regulating ATRA-dependent osteogenic differentiation of mASCs. In this study, we assessed the influence of small interfering RNA (siRNA)-induced repression of HtrA1 production on mASC osteogenesis and examined its effects on ATRA-mediated mammalian target of rapamycin (mTOR) signaling. Inhibition of HtrA1 production in osteogenic mASCs resulted in a significant reduction of alkaline phosphatase activity and mineralized matrix formation. Western blot analyses revealed the rapid activation of Akt (Ser473) and p70S6K (Thr389) in ATRA-treated mASCs, and that levels of phosphorylated p70S6K were noticeably reduced in HtrA1-deficient mASCs. Further studies using mTOR inhibitor rapamycin and siRNA specific for the p70S6K gene Rps6kb1 confirmed ATRA-mediated mASC osteogenesis as being dependent on p70S6K activation. Finally, transfection of cells with a constitutively active rapamycin-resistant p70S6K mutant could restore the mineralizing capacity of HtrA1-deficient mASCs. These findings therefore lend further support for HtrA1 as a positive mediator of MSC osteogenesis and provide new insights into the molecular mode of action of ATRA in regulating mASC lineage commitment.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that reproduces cardinal signs of clinical, histopathological, and immunological features found in Multiple Sclerosis (MS). Mast cells are suggested to be involved in the main inflammatory phases occurring during EAE development, possibly by secreting several autacoids and proteases. Among the latter, the chymase mouse mast cell protease 4 (mMCP-4) can contribute to the inflammatory response by producing endothelin-1 (ET-1). The aim of this study was to determine the impact of mMCP-4 on acute inflammatory stages in EAE. C57BL/6 wild type (WT) or mMCP-4 knockout (KO) mice were immunized with MOG35-55 plus complete Freund's adjuvant followed by pertussis toxin. Immunized WT mice presented an initial acute phase characterized by progressive increases in clinical score, which were significantly reduced in mMCP-4 KO mice. In addition, higher levels of spinal myelin were found in mMCP-4 KO as compared with WT mice. Finally, whereas EAE triggered significant increases in brain levels of mMCP-4 mRNA and immunoreactive ET-1 in WT mice, the latter peptide was reduced to basal levels in mMCP-4 KO congeners. Together, the present study supports a role for mMCP-4 in the early inflammatory phases of the disease in a mouse model of MS.