scholarly journals AP-4 mediated ATG9A sorting underlies axonal and autophagosome biogenesis defects in a mouse model of AP-4 deficiency syndrome

2017 ◽  
Author(s):  
Davor Ivankovic ◽  
Guillermo López-Doménech ◽  
James Drew ◽  
Sharon A. Tooze ◽  
Josef T. Kittler
Keyword(s):  
Mouse Model ◽  
Transmembrane Protein ◽  
Adaptor Protein ◽  
Deficiency Syndrome ◽  
Cellular Level ◽  
Loss Of Function ◽  
Golgi Network ◽  
Autophagosome Maturation

AbstractAdaptor protein (AP) complexes have critical roles in transmembrane protein sorting. AP-4 remains poorly understood in the brain despite its loss of function leading to a hereditary spastic paraplegia termed AP-4 deficiency syndrome. Here we demonstrate that knockout (KO) of AP-4 in a mouse model leads to thinning of the corpus callosum and ventricular enlargement, anatomical defects previously described in patients. At the cellular level, we find that AP-4 KO leads to defects in axonal extension and branching, in addition to aberrant distal swellings. Interestingly, we show that ATG9A, a key protein in autophagosome maturation, is critically dependent on AP-4 for its sorting from the trans-golgi network. Failure of AP-4 mediated ATG9A sorting results in its dramatic retention in the trans-golgi network in vitro and in vivo leading to a specific reduction of the axonal pool of ATG9A. As a result, autophagosome biogenesis is aberrant in the axon of AP-4 deficient neurons. The specific alteration to axonal integrity and axonal autophagosome maturation in AP-4 knockout neurons may underpin the pathology of AP-4 deficiency.

scholarly journals The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

2005 ◽  
Vol 25 (23) ◽  
pp. 10533-10542 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Christian Schmedt ◽  
Masato Okada ◽  
Alexander Tarakhovsky
Keyword(s):  
T Cell ◽  
Adaptor Protein ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Thymic Development ◽  
Cell Functions ◽  
Carboxy Terminal ◽  
And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

scholarly journals Mesenchymal stem cell-derived exosomal microRNA-136-5p inhibits chondrocyte degeneration in traumatic osteoarthritis by targeting ELF3

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Xue Chen ◽  
Yuanyuan Shi ◽  
Pan Xue ◽  
Xinli Ma ◽  
Junfeng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Mouse Model ◽  
Cartilage Degeneration ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Post Traumatic ◽  
Chondrocyte Migration

Abstract Background Emerging evidence suggests that microRNAs (miRs) are associated with the progression of osteoarthritis (OA). In this study, the role of exosomal miR-136-5p derived from mesenchymal stem cells (MSCs) in OA progression is investigated and the potential therapeutic mechanism explored. Methods Bone marrow mesenchymal stem cells (BMMSCs) and their exosomes were isolated from patients and identified. The endocytosis of chondrocytes and the effects of exosome miR-136-5p on cartilage degradation were observed and examined by immunofluorescence and cartilage staining. Then, the targeting relationship between miR-136-5p and E74-like factor 3 (ELF3) was analyzed by dual-luciferase report assay. Based on gain- or loss-of-function experiments, the effects of exosomes and exosomal miR-136-5p on chondrocyte migration were examined by EdU and Transwell assay. Finally, a mouse model of post-traumatic OA was developed to evaluate effects of miR-136-5p on chondrocyte degeneration in vivo. Results In the clinical samples of traumatic OA cartilage tissues, we detected increased ELF3 expression, and reduced miR-136-5p expression was determined. The BMMSC-derived exosomes showed an enriched level of miR-136-5p, which could be internalized by chondrocytes. The migration of chondrocyte was promoted by miR-136-5p, while collagen II, aggrecan, and SOX9 expression was increased and MMP-13 expression was reduced. miR-136-5p was verified to target ELF3 and could downregulate its expression. Moreover, the expression of ELF3 was reduced in chondrocytes after internalization of exosomes. In the mouse model of post-traumatic OA, exosomal miR-136-5p was found to reduce the degeneration of cartilage extracellular matrix. Conclusion These data provide evidence that BMMSC-derived exosomal miR-136-5p could promote chondrocyte migration in vitro and inhibit cartilage degeneration in vivo, thereby inhibiting OA pathology, which highlighted the transfer of exosomal miR-136-5p as a promising therapeutic strategy for patients with OA.

scholarly journals The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

2018 ◽  
Vol 29 (8) ◽  
pp. 2110-2122 ◽  
Author(s):  
Gentzon Hall ◽  
Brandon M. Lane ◽  
Kamal Khan ◽  
Igor Pediaditakis ◽  
Jianqiu Xiao ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Adaptor Protein ◽  
Pi3k Pathway ◽  
Actin Binding ◽  
Loss Of Function ◽  
Signaling Axis ◽  
Phosphoinositide 3 Kinase

BackgroundWe previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.MethodsWe conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.ResultsExperiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.ConclusionsThe ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

scholarly journals Fingolimod phosphate inhibits astrocyte inflammatory activity in mucolipidosis IV

2018 ◽  
Vol 27 (15) ◽  
pp. 2725-2738 ◽  
Author(s):  
Laura D Weinstock ◽  
Amanda M Furness ◽  
Shawn S Herron ◽  
Sierra S Smith ◽  
Sitara B Sankar ◽  
...  
Keyword(s):  
Mouse Model ◽  
Protein Signaling ◽  
Loss Of Function ◽  
Neurodevelopmental Disease ◽  
Mapk Pathways ◽  
Inflammatory Activation ◽  
Mucolipidosis Iv ◽  
Pro Inflammatory Cytokines

Abstract Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1−/− astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1−/− mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1−/− astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1−/− astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

scholarly journals Canonical Interaction of Cyclin G–associated Kinase with Adaptor Protein 1 Regulates Lysosomal Enzyme Sorting

2007 ◽  
Vol 18 (8) ◽  
pp. 2991-3001 ◽  
Author(s):  
Satoshi Kametaka ◽  
Kengo Moriyama ◽  
Patricia V. Burgos ◽  
Evan Eisenberg ◽  
Lois E. Greene ◽  
...  
Keyword(s):  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Adaptor Protein ◽  
Physiological Relevance ◽  
Lysosomal Sorting ◽  
Canonical Motif ◽  
Cyclin G ◽  
Golgi Network

The adaptor protein 1 (AP1) complex is a heterotetramer that participates in cargo sorting into clathrin-coated vesicles at the trans-Golgi network (TGN) and endosomes. The γ subunit of AP1 possesses a C-terminal “ear” domain that recruits a cohort of accessory proteins through recognition of a shared canonical motif, ΨG[PDE][ΨLM] (where Ψ is an aromatic residue). The physiological relevance of these ear-motif interactions, however, remains to be demonstrated. Here we report that the cyclin G–associated kinase (GAK) has two sequences fitting this motif, FGPL and FGEF, which mediate binding to the AP1-γ-ear domain in vitro. Mutation of both γ-ear–binding sequences or depletion of AP1-γ by RNA interference (RNAi) decreases the association of GAK with the TGN in vivo. Depletion of GAK by RNAi impairs the sorting of the acid hydrolase, cathepsin D, to lysosomes. Importantly, expression of RNAi-resistant GAK restores the lysosomal sorting of cathepsin D in cells depleted of endogenous GAK, whereas expression of a similar construct bearing mutations in both γ-ear–binding sequences fails to correct the sorting defect. Thus, interactions between the ΨG[PDE][ΨLM]-motif sequences in GAK and the AP1-γ-ear domain are critical for the recruitment of GAK to the TGN and the function of GAK in lysosomal enzyme sorting.

scholarly journals Mitogen-Inducible Gene 6 Inhibits Angiogenesis by Binding to SHC1 and Suppressing Its Phosphorylation

2021 ◽  
Vol 9 ◽  
Author(s):  
Lixian Liu ◽  
Liying Xing ◽  
Rongyuan Chen ◽  
Jianing Zhang ◽  
Yuye Huang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Adaptor Protein ◽  
Model Systems ◽  
Vascular Endothelial ◽  
Loss Of Function ◽  
Angiogenic Effect ◽  
Inducible Gene

The mitogen-inducible gene 6 (MIG6) is an adaptor protein widely expressed in vascular endothelial cells. However, it remains unknown thus far whether it plays a role in angiogenesis. Here, using comprehensive in vitro and in vivo model systems, we unveil a potent anti-angiogenic effect of MIG6 in retinal development and neovascularization and the underlying molecular and cellular mechanisms. Loss of function assays using genetic deletion of Mig6 or siRNA knockdown increased angiogenesis in vivo and in vitro, while MIG6 overexpression suppressed pathological angiogenesis. Moreover, we identified the cellular target of MIG6 by revealing its direct inhibitory effect on vascular endothelial cells (ECs). Mechanistically, we found that the anti-angiogenic effect of MIG6 is fulfilled by binding to SHC1 and inhibiting its phosphorylation. Indeed, SHC1 knockdown markedly diminished the effect of MIG6 on ECs. Thus, our findings show that MIG6 is a potent endogenous inhibitor of angiogenesis that may have therapeutic value in anti-angiogenic therapy.

scholarly journals Spinosin Attenuates Osteoclastogenesis Through Suppressing NF-κB by the Activation of Nrf2/HO-1 Signaling in Osteoporosis

2021 ◽  
Author(s):  
Bo Liu ◽  
Yuna Zhang
Keyword(s):  
Bone Resorption ◽  
Mouse Model ◽  
Transmembrane Protein ◽  
Tartrate Resistant Acid Phosphatase ◽  
Potential Candidate ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Trabecular Thickness

Abstract ObjectiveOsteoporosis is a prevalent metabolic skeletal disorder featured by microarchitecture bone injury and excessive osteoclastic activity.Here, we aimed to explore the effect of Spinosin on osteoclastogenesis of osteoporosis.DesignThe receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis model was established in bone marrow macrophages (BMMs) in vitro. The ovariectomy (OVX)-induced bone loss mouse model was constructed in vivo, followed by micro-CT analysis, Histomorphometric analysis, Hematoxylin and Eosin (H&E) and TRAP staining.ResultsOur data showed that the treatment of Spinosin significantly inhibited the TRAP positive osteoclast and bone resorption induced by RANKL in the BMMs. Spinosin significantly reduced the expression of osteoclast-specific factors, including osteoclast stimulatory transmembrane protein (OC-STAMP), dendritic cell-specific transmembrane protein (DC-STAMP), cathepsin K (CTSK), TRAP, c-Fos and nuclear factor of activated T cells cytoplasm 1 (NFATc1)， in the RANKL-treated BMMs. Mechanically, Spinosin was able to inactivate NF-κB by stimulating Nrf2/HO-1 signaling in BMMs. The trabecular space (Tb.Sp), trabecular number (Tb.N), trabecular thickness (Tb.Th), and bone volume to total volume (BV/TV) were inhibited by OVX treatment, and Spinosin could reverse the effect in the bone resorption mouse model. The OVX-induced serum levels of tumor necrosis factor-α (TNF-α) and tartrate-resistant acid phosphatase 5 B (TRAcp5B) were blocked by Spinosin in the mice. Moreover, Spinosin was able to alleviate OVX-induced loss of femur bone and osteoclasts in vivo.ConclusionsIn conclusion, Spinosin attenuates osteoclastogenesis of osteoporosis through inhibiting NF-κB by activating Nrf2/HO-1 expression. Spinosin may serve as the potential candidate for the treatment of osteoporosis.

scholarly journals Gene editing preserves visual function in a mouse model of retinal degeneration

2019 ◽  
Author(s):  
Paola Vagni ◽  
Laura E. Perlini ◽  
Naïg A. L. Chenais ◽  
Tommaso Marchetti ◽  
Martina Parrini ◽  
...  
Keyword(s):  
Mouse Model ◽  
Retinitis Pigmentosa ◽  
Gene Editing ◽  
Point Mutations ◽  
Loss Of Function ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Function Point

AbstractInherited retinal dystrophies are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favourable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. We used a tailored CRISPR/Cas9-based gene editing system to prevent retinal photoreceptor death in the Rd10 mouse model of retinitis pigmentosa. We tested the gene editing toolin vitroand then usedin vivosubretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations.

Abstract 13575: Lack of Both Mitochondrial Proteins Sirt3 and Ucp2 in Mice Recapitulates Many Critical Features of Human Pulmonary Arterial Hypertension (PAH), Including Inflammatory Plexogenic Lesions, Supporting the Metabolic Theory of PAH

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Yongneng Zhang ◽  
Sotirios D Zervopoulos ◽  
Aristeidis E Boukouris ◽  
Bruno Saleme ◽  
Yongsheng Liu ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Mouse Model ◽  
Uncoupling Protein ◽  
Mitochondrial Proteins ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Metabolic Theory ◽  
Apoptosis Resistance

Loss-of function SNPs for Sirt3 (a mitochondrial deacetylase) and Ucp2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with insulin resistance and obesity in humans; and patients with PAH have insulin resistance without being obese. In a cohort of PAH patients (n=60) we found these SNPs often both in the same patient in a homozygous or heterozygous manner and their presence correlated positively with the degree of PAH upon referral, the presence of type II diabetes and outcomes (death, transplantation). We generated and studied double KO mice for Sirt3 and Ucp2 using closed-chest right heart catheterization and echocardiography and found increasing severity of pulmonary hypertension (PHT) in Sirt3 +/- -Ucp2 +/ - , Sirt3 -/- -Ucp2 +/ - , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- , associated with decreasing cardiac output and increasing right ventricular hypertrophy, dilatation and dysfunction (TAPSE), compared to wild-type (WT) mice. The LVEDP in all mice was normal. There was increasing severity of vascular remodeling with increasing levels of CD4 + cell infiltration, while Sirt3 -/- -Ucp2 +/- , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- mice also developed frequent plexogenic lesions. In vivo and in vitro pulmonary artery smooth muscle cells (PASMC) expressed higher levels of Ki67, compared to WT mice. In vitro, the Sirt3 -/- -Ucp2 +/- , Sirt3 +/- -Ucp2 -/- and Sirt3 -/- -Ucp2 -/- PASMC exhibited more apoptosis-resistance, expressed higher nuclear levels of proliferative transcription factors (HIF1, NFATc2), exhibited decreased respiration and higher levels of glycolysis, similarly to previous reports of animal and human PAH PASMC. The Sirt3 -/- -Ucp2 -/- , but not the WT mice, also developed in vivo evidence of insulin resistance. Our work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PHT (without environmental or chemical triggers) in a gene dose-response dependent manner that mimics human PAH. No other mouse model of PHT has shown frequent and predictable plexogenic lesions. Our study is relevant to the PAH patients that carry loss-of-function SNPs of mitochondrial proteins and offers a new mouse model of PAH, with more features of human PAH than previous mice models.

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