Regulator Of Endosomal Trafficking Research Articles

Pharmacologic enhancement of retromer rescues endosomal pathology induced by defects in the Alzheimer’s gene SORL1

The SORL1 gene (SORLA) is strongly associated with risk of developing Alzheimer's disease (AD). SORLA is a regulator of endosomal trafficking in neurons and interacts with retromer, a complex that is a "master conductor" of endosomal trafficking. Small molecules can increase retromer expression invitro, enhancing its function. We treated hiPSC-derived cortical neurons that are either fully deficient, haploinsufficient, or that harbor one copy of SORL1 variants linked to AD with TPT-260, a retromer-enhancing molecule. We show significant increases in retromer subunit VPS26B expression. We tested whether endosomal, amyloid, and TAU pathologies were corrected. We observed that the degree of rescue by TPT-260 treatment depended on the number of copies of functional SORL1 and which SORL1 variant was expressed. Using a disease-relevant preclinical model, our work illuminates how the SORL1-retromer pathway can be therapeutically harnessed.

Regulation of Endosomal Trafficking by Rab7 and Its Effectors in Neurons: Clues from Charcot-Marie-Tooth 2B Disease.

Intracellular endosomal trafficking controls the balance between protein degradation and synthesis, i.e., proteostasis, but also many of the cellular signaling pathways that emanate from activated growth factor receptors after endocytosis. Endosomal trafficking, sorting, and motility are coordinated by the activity of small GTPases, including Rab proteins, whose function as molecular switches direct activity at endosomal membranes through effector proteins. Rab7 is particularly important in the coordination of the degradative functions of the pathway. Rab7 effectors control endosomal maturation and the properties of late endosomal and lysosomal compartments, such as coordination of recycling, motility, and fusion with downstream compartments. The spatiotemporal regulation of endosomal receptor trafficking is particularly challenging in neurons because of their enormous size, their distinct intracellular domains with unique requirements (dendrites vs. axons), and their long lifespans as postmitotic, differentiated cells. In Charcot-Marie-Tooth 2B disease (CMT2B), familial missense mutations in Rab7 cause alterations in GTPase cycling and trafficking, leading to an ulcero-mutilating peripheral neuropathy. The prevailing hypothesis to account for CMT2B pathologies is that CMT2B-associated Rab7 alleles alter endocytic trafficking of the neurotrophin NGF and its receptor TrkA and, thereby, disrupt normal trophic signaling in the peripheral nervous system, but other Rab7-dependent pathways are also impacted. Here, using TrkA as a prototypical endocytic cargo, we review physiologic Rab7 effector interactions and control in neurons. Since neurons are among the largest cells in the body, we place particular emphasis on the temporal and spatial regulation of endosomal sorting and trafficking in neuronal processes. We further discuss the current findings in CMT2B mutant Rab7 models, the impact of mutations on effector interactions or balance, and how this dysregulation may confer disease.

Genetic Variants in Protein Tyrosine Phosphatase Non-Receptor Type 23 Are Responsible for Mesiodens Formation.

A mesiodens is a supernumerary tooth located in the midline of the premaxilla. To investigate the genetic cause of mesiodens, clinical and radiographic examination were performed on 23 family members of a two-generation Hmong family. Whole exome sequencing (WES) or Sanger sequencing were performed in 22 family members and two unrelated Thai patients with mesiodens. WES in the Hmong family revealed a missense mutation (c.1807G>A;p.Glu603Lys) in PTPN23 in seven affected members and six unaffected members. The mode of inheritance was autosomal dominance with incomplete penetrance (53.84%). Two additional mutations in PTPN23, c.2248C>G;p.Pro750Ala and c.3298C>T;p.Arg1100Cys were identified in two unrelated patients with mesiodens. PTPN23 is a regulator of endosomal trafficking functioning to move activated membrane receptors, such as EGFR, from the endosomal sorting complex towards the ESCRT-III complex for multivesicular body biogenesis, lysosomal degradation, and subsequent downregulation of receptor signaling. Immunohistochemical study and RNAscope on developing mouse embryos showed broad expression of PTPN23 in oral tissues, while immunofluorescence showed that EGFR was specifically concentrated in the midline epithelium. Importantly, PTPN23 mutant protein was shown to have reduced phosphatase activity. In conclusion, mesiodens were associated with genetic variants in PTPN23, suggesting that mesiodens may form due to defects in endosomal trafficking, leading to disrupted midline signaling.

LRP2 contributes to planar cell polarity-dependent coordination of motile cilia function

Motile cilia are protruding organelles on specialized epithelia that beat in a synchronous fashion to propel extracellular fluids. Coordination and orientation of cilia beating on individual cells and across tissues is a complex process dependent on planar cell polarity (PCP) signaling. Asymmetric sorting of PCP pathway components, essential to establish planar polarity, involves trafficking along the endocytic path, but the underlying regulatory processes remain incompletely understood. Here, we identified the endocytic receptor LRP2 as regulator of PCP component trafficking in ependyma, a multi-ciliated cell type that is involved in facilitating flow of the cerebrospinal fluid in the brain ventricular system. Lack of receptor expression in gene-targeted mice results in a failure to sort PCP core proteins to the anterior or posterior cell side and, consequently, in the inability to coordinate cilia arrangement and to aligned beating (loss of rotational and translational polarity). LRP2 deficiency coincides with a failure to sort NHERF1, a cytoplasmic LRP2 adaptor to the anterior cell side. As NHERF1 is essential to translocate PCP core protein Vangl2 to the plasma membrane, these data suggest a molecular mechanism whereby LRP2 interacts with PCP components through NHERF1 to control their asymmetric sorting along the endocytic path. Taken together, our findings identified the endocytic receptor LRP2 as a novel regulator of endosomal trafficking of PCP proteins, ensuring their asymmetric partition and establishment of translational and rotational planar cell polarity in the ependyma.

A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. Inthis study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

Two siblings, distinct characteristics

In this issue of Structure, Healy etal. discovered the PDLIM family of proteins as novel interactors of the sorting nexin SNX17, determined the NMR structure of PDLIM7 with the bound SNX17 C terminus, and suggest that PDLIM proteins could represent novel regulators of endosomal trafficking together with SNX17.

Pharmacologic Stabilization of Retromer Rescues Endosomal Pathology Induced by Defects in the Alzheimer’s gene SORL1

AbstractBackgroundDefects in the Sortilin‐related receptor 1 gene, SORL1, are highly pathogenic Alzheimer’s disease (AD), on par with other causal AD genes. SORL1 is a key regulator of endosomal trafficking in neurons, and loss of SORL1 function disrupts trafficking pathways that are vital for neuronal health. SORL1 has been found to interact with retromer, a trafficking complex that is a ‘master conductor’ of endosomal outflow. Here we rely on retromer pharmacological chaperones that stabilize retromer in vitro, and thus enhance retromer function in cells to test the hypothesis that they will correct AD‐associated neuronal pathologies.MethodWe used an isogenic series of human induced pluripotent cell lines either deficient in SORL1 expression (SORL1 KO or SORL1+/−) or harboring a one copy of a missense variant linked to increased risk for AD (SORL1Var). We treated neurons derived from these hiPSCs with the established retromer chaperone, TPT‐260, and tested whether they corrected indicators of AD’s defining endosomal, amyloid, and tau pathologies.ResultWe observed that TPT‐260 treatment was able to correct all of these pathological indicators, however the degree of rescue of some of the phenotypes was dependent one whether the neurons harbor at least one functional copy of SORL1 or whether they are fully deficient.ConclusionMore than just validating that SORL1‐retromer acts a trafficking functional unit in neurons, our work sheds new light on how SORL1‐retromer is linked to AD’s cardinal pathologies and illuminates how this pathway can be therapeutically harnessed.

Interactome analysis reveals endocytosis and membrane recycling of EpCAM during differentiation of embryonic stem cells and carcinoma cells

SummaryTransmembrane epithelial cell adhesion molecule (EpCAM) is expressed in epithelia, carcinoma, teratoma, and embryonic stem cells (ESCs). EpCAM displays spatiotemporal patterning during embryogenesis, tissue morphogenesis, cell differentiation, and epithelial-to-mesenchymal transition (EMT) in carcinomas. Potential interactors of EpCAM were identified in murine F9 teratoma cells using a stable isotope labeling with amino acids in cell culture-based proteomic approach (n = 77, enrichment factor >3, p value ≤ 0.05). Kyoto Encyclopedia of Genes and Genomes and gene ontology terms revealed interactions with regulators of endosomal trafficking and membrane recycling, which were further validated for Rab5, Rab7, and Rab11. Endocytosis and membrane recycling of EpCAM were confirmed in mF9 cells, E14TG2α ESC, and Kyse30 carcinoma cells. Reduction of EpCAM during mesodermal differentiation and TGFβ-induced EMT correlated with enhanced endocytosis and block or reduction of recycling in ESCs and esophageal carcinoma cells. Hence, endocytosis and membrane recycling are means of regulation of EpCAM protein levels during differentiation of ESC and EMT induction in carcinoma cells.

Interactome Analysis Reveals Endocytosis and Membrane Recycling of Murine EpCAM During Differentiation of Embryonic Stem Cells

Transmembrane glycoprotein EpCAM (Epithelial cell adhesion molecule) is expressed in epithelia, carcinoma, teratoma, and embryonic stem cells (ESC). EpCAM is functional in tissue morphogenesis and integrity, proliferation, differentiation and pluripotency. Interactors of murine EpCAM (mEpCAM) were identified in mF9 teratoma cells using SILAC-MS (n = 77, enrichment factor > 3, p-value ≤ 0.05). KEGG- and GO-term analyses revealed prominent interactions with regulators of endosomal trafficking and membrane recycling including Ras-superfamily small G-proteins Rab5, Rab7 and Rab11. Accordingly, endocytosis (8.25%;10.67%) and membrane recycling (59.93%; 24.81%) were quantified in mF9 cells and ESC, respectively. Importantly, EpCAM endocytosis was instrumental in mesodermal differentiation of ESC, which is associated with loss of EpCAM expression at the plasma membrane. EpCAM endocytosis was increased to 19.38% in average and membrane recycling was completely blocked during mesodermal differentiation. Hence, endocytosis and membrane recycling are means of regulation of the availability of EpCAM during differentiation.

Toward Understanding the Molecular Role of SNX27/Retromer in Human Health and Disease.

Aberrations in membrane trafficking pathways have profound effects in cellular dynamics of cellular sorting processes and can drive severe physiological outcomes. Sorting nexin 27 (SNX27) is a metazoan-specific sorting nexin protein from the PX-FERM domain family and is required for endosomal recycling of many important transmembrane receptors. Multiple studies have shown SNX27-mediated recycling requires association with retromer, one of the best-known regulators of endosomal trafficking. SNX27/retromer downregulation is strongly linked to Down’s Syndrome (DS) via glutamate receptor dysfunction and to Alzheimer’s Disease (AD) through increased intracellular production of amyloid peptides from amyloid precursor protein (APP) breakdown. SNX27 is further linked to addiction via its role in potassium channel trafficking, and its over-expression is linked to tumorigenesis, cancer progression, and metastasis. Thus, the correct sorting of multiple receptors by SNX27/retromer is vital for normal cellular function to prevent human diseases. The role of SNX27 in regulating cargo recycling from endosomes to the cell surface is firmly established, but how SNX27 assembles with retromer to generate tubulovesicular carriers remains elusive. Whether SNX27/retromer may be a putative therapeutic target to prevent neurodegenerative disease is now an emerging area of study. This review will provide an update on our molecular understanding of endosomal trafficking events mediated by the SNX27/retromer complex on endosomes.

Mammalian VPS45 orchestrates trafficking through the endosomal system

Vacuolar protein sorting 45 homolog (VPS45), a member of the Sec1/Munc18 (SM) family, has been implicated in the regulation of endosomal trafficking. VPS45 deficiency in human patients results in congenital neutropenia, bone marrow fibrosis, and extramedullary renal hematopoiesis. Detailed mechanisms of the VPS45 function are unknown. Here, we show an essential role of mammalian VPS45 in maintaining the intracellular organization of endolysosomal vesicles and promoting recycling of cell-surface receptors. Loss of VPS45 causes defective Rab5-to-Rab7 conversion resulting in trapping of cargos in early endosomes and impaired delivery to lysosomes. In this context, we demonstrate aberrant trafficking of the granulocyte colony-stimulating factor receptor in the absence of VPS45. Furthermore, we find that lack of VPS45 in mice is not compatible with embryonic development. Thus, we identify mammalian VPS45 as a critical regulator of trafficking through the endosomal system and early embryogenesis of mice.

The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2

The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.

Tks5 SH3 domains exhibit differential effects on invadopodia development.

The Src substrate Tks5 helps scaffold matrix-remodeling invadopodia in invasive cancer cells. Focus was directed here on how the five SH3 domains of Tks5 impact that activity. Mutations designed to inhibit protein-protein interactions were created in the individual SH3 domains of Tks5, and the constructs were introduced into the LNCaP prostate carcinoma cell line, a model system with intrinsically low Tks5 expression and which our lab had previously showed the dependence of Src-dependent Tks5 phosphorylation on invadopodia development. In LNCaP cells, acute increases in wild-type Tks5 led to increased gelatin matrix degradation. A similar result was observed when Tks5 was mutated in its 4th or 5th SH3 domains. This was in contrast to the 1st, 2nd, and 3rd SH3 domain mutations of Tks5 where each had a remarkable accentuating effect on gelatin degradation. Conversely, in the invadopodia-competent Src-3T3 model system, mutations in any one of the first three SH3 domains had a dominant negative effect that largely eliminated the presence of invadopodia, inhibited gelatin degradation activity, and redistributed both Src, cortactin, and Tks5 to what are likely endosomal compartments. A hypothesis involving Tks5 conformational states and the regulation of endosomal trafficking is presented as an explanation for these seemingly disparate results.

Class III PI3K Vps34 Controls Thyroid Hormone Production by Regulating Thyroglobulin Iodination, Lysosomal Proteolysis, and Tissue Homeostasis.

Background: The production of thyroid hormones [triiodothyronine (T3), thyroxine (T4)] depends on the organization of the thyroid in follicles, which are lined by a monolayer of thyrocytes with strict apicobasal polarity. This polarization supports vectorial transport of thyroglobulin (Tg) for storage into, and recapture from, the colloid. It also allows selective addressing of channels, transporters, ion pumps, and enzymes to their appropriate basolateral [Na+/I- symporter (NIS), SLC26A7, and Na+/K+-ATPase] or apical membrane domain (anoctamin, SLC26A4, DUOX2, DUOXA2, and thyroperoxidase). How these actors of T3/T4 synthesis reach their final destination remains poorly understood. The PI 3-kinase isoform Vps34/PIK3C3 is now recognized as a main component in the general control of vesicular trafficking and of cell homeostasis through the regulation of endosomal trafficking and autophagy. We recently reported that conditional Vps34 inactivation in proximal tubular cells in the kidney prevents normal addressing of apical membrane proteins and causes abortive macroautophagy. Methods:Vps34 was inactivated using a Pax8-driven Cre recombinase system. The impact of Vps34 inactivation in thyrocytes was analyzed by histological, immunolocalization, and messenger RNA expression profiling. Thyroid hormone synthesis was assayed by 125I injection and plasma analysis. Results:Vps34 conditional knockout (Vps34cKO) mice were born at the expected Mendelian ratio and showed normal growth until postnatal day 14 (P14), then stopped growing and died at ∼1 month of age. We therefore analyzed thyroid Vps34cKO at P14. We found that loss of Vps34 in thyrocytes causes (i) disorganization of thyroid parenchyma, with abnormal thyrocyte and follicular shape and reduced PAS+ colloidal spaces; (ii) severe noncompensated hypothyroidism with extremely low T4 levels (0.75 ± 0.62 μg/dL) and huge thyrotropin plasma levels (19,300 ± 10,500 mU/L); (iii) impaired 125I organification at comparable uptake and frequent occurrence of follicles with luminal Tg but nondetectable T4-bearing Tg; (iv) intense signal in thyrocytes for the lysosomal membrane marker, LAMP-1, as well as Tg and the autophagy marker, p62, indicating defective lysosomal proteolysis; and (v) presence of macrophages in the colloidal space. Conclusions: We conclude that Vps34 is crucial for thyroid hormonogenesis, at least by controlling epithelial organization, Tg iodination as well as proteolytic T3/T4 excision in lysosomes.

The LMTK1-TBC1D9B-Rab11A Cascade Regulates Dendritic Spine Formation via Endosome Trafficking.

Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons and in vivo LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENT Dendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.

Structure and Dynamics of Mono- vs. Doubly Lipidated Rab5 in Membranes.

The Rab5 small GTPase is a regulator of endosomal trafficking and vesicle fusion. It possesses two adjacent cysteine residues for post-translational geranylgeranylation at its C-terminus for the protein to associate with the early endosome membrane. We compare the effect of mono-lipidification of only one cysteine residue with the doubly modified, fully functional Rab protein in both guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states and in different membranes (one, three, and six-component membranes). Molecular simulations show that the mono-geranylgeranylated protein is less strongly associated with the membranes and diffuses faster than the doubly lipidated protein. The geranylgeranyl anchor membrane insertion depth is smaller and the protein–membrane distance distribution is broad and uncharacteristic for the membrane composition. The mono-geranylgeranylated protein reveals an unspecific association with the membrane and an orientation at the membrane that does not allow a nucleotide-specific recruitment of further effector proteins. This work shows that double-lipidification is critical for Rab5 to perform its physiological function and mono-geranylgeranylation renders it membrane-associated but non-functional.

P62/SQSTM1 - steering the cell through health and disease.

SQSTM1 (also known as p62) is a multifunctional stress-inducible scaffold protein involved in diverse cellular processes. Its functions are tightly regulated through an extensive pattern of post-translational modifications, and include the isolation of cargos degraded by autophagy, induction of the antioxidant response by the Keap1-Nrf2 system, as well as the regulation of endosomal trafficking, apoptosis and inflammation. Accordingly, malfunction of SQSTM1 is associated with a wide range of diseases, including bone and muscle disorders, neurodegenerative and metabolic diseases, and multiple forms of cancer. In this Review, we summarize current knowledge regarding regulation, post-translational modifications and functions of SQSTM1, as well as how they are dysregulated in various pathogenic contexts.

CLIC4/Arf6 Pathway.

Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood. To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension. Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition. Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

Modulation of cis- and trans- Golgi and the Rab9A-GTPase during infection by Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum

Like most intracellular pathogens, the apicomplexan parasites Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum scavenge metabolites from their host cells. Recruitment of the Golgi complex to the vicinity of the parasitophorous vacuole (PV) is likely to aid in this process. In this work, we comparatively assessed B. besnoiti, T. gondii and N. caninum infected human retinal pigmented epithelial (hTERT-RPE-1) cells at 24 h post-infection and used antibodies to confirm Golgi ribbon compaction in B. besnoiti, and Golgi ribbon dispersion in T. gondii, while no alteration in Golgi morphology was seen in N. caninum infected cells. In either case, the Golgi stacks of infected cells contained both cis- (GM130) and trans- (TGN46) Golgi proteins. The localization of Rab9A, an important regulator of endosomal trafficking, was also studied. GFP-tagged Rab9A was recruited to the vicinity of the PV of all three parasites. Toxoplasma-infected cells exhibited increased expression of Rab9A in comparison to non-infected cells. However, Rab9A expression levels remained unaltered upon infection with N. caninum and B. besnoiti tachyzoites. In contrast to Rab9A, a GFP-tagged dominant negative mutant form of Rab9A (Rab9A DN), was not recruited to the PV, and the expression of Rab9A DN did not affect host cell invasion nor replication by all three parasites. Thus, B. besnoiti, T. gondii and N. caninum show similarities but also differences in how they affect constituents of the endosomal/secretory pathways.

Endosomal Phosphatidylinositol 3-Kinase Is Essential for Canonical GPCR Signaling.

G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the β2-adrenoceptor (β2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by β2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.