Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol.

The microtubule-associated protein tau has a critical role in Alzheimer's disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived 'tau biosensor cells'. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

Apolipoprotein E4 genotype compromises brain exosome production.

In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the ɛ4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-ß and tau pathology. In human post-mortem tissue and mouse models humanized for apolipoprotein E, we examined the impact of apolipoprotein E4 expression on brain exosomes, vesicles that are produced within and secreted from late-endocytic multivesicular bodies. Compared to humans or mice homozygous for the risk-neutral ɛ3 allele we show that the ɛ4 allele, whether homozygous or heterozygous with an ɛ3 allele, drives lower exosome levels in the brain extracellular space. In mice, we show that the apolipoprotein E4-driven change in brain exosome levels is age-dependent: while not present at age 6 months, it is detectable at 12 months of age. Expression levels of the exosome pathway regulators tumor susceptibility gene 101 (TSG101) and Ras-related protein Rab35 (RAB35) were found to be reduced in the brain at the protein and mRNA levels, arguing that apolipoprotein E4 genotype leads to a downregulation of exosome biosynthesis and release. Compromised exosome production is likely to have adverse effects, including diminishing a cell's ability to eliminate materials from the endosomal-lysosomal system. This reduction in brain exosome levels in 12-month-old apolipoprotein E4 mice occurs earlier than our previously reported brain endosomal pathway changes, arguing that an apolipoprotein E4-driven failure in exosome production plays a primary role in endosomal and lysosomal deficits that occur in apolipoprotein E4 mouse and human brains. Disruption of these interdependent endosomal-exosomal-lysosomal systems in apolipoprotein E4-expressing individuals may contribute to amyloidogenic amyloid-ß precursor protein processing, compromise trophic signalling and synaptic function, and interfere with a neuron's ability to degrade material, all of which are events that lead to neuronal vulnerability and higher risk of Alzheimer's disease development. Together, these data suggest that exosome pathway dysfunction is a previously unappreciated component of the brain pathologies that occur as a result of apolipoprotein E4 expression.

HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity.

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.

Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base.

Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A.

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

Intraflagellar transport 27 is essential for hedgehog signaling but dispensable for ciliogenesis during hair follicle morphogenesis.

Hair follicle morphogenesis requires precisely controlled reciprocal communications, including hedgehog (Hh) signaling. Activation of the Hh signaling pathway relies on the primary cilium. Disrupting ciliogenesis results in hair follicle morphogenesis defects due to attenuated Hh signaling; however, the loss of cilia makes it impossible to determine whether hair follicle phenotypes in these cilia mutants are caused by the loss of cilia, disruption of Hh signaling, or a combination of these events. In this study, we characterized the function of Ift27, which encodes a subunit of intraflagellar transport (IFT) complex B. Hair follicle morphogenesis of Ift27-null mice was severely impaired, reminiscent of phenotypes observed in cilia and Hh mutants. Furthermore, the Hh signaling pathway was attenuated in Ift27 mutants, which was in association with abnormal ciliary trafficking of SMO and GLI2, and impaired processing of Gli transcription factors; however, formation of the ciliary axoneme was unaffected. The ciliary localization of IFT25 (HSPB11), the binding partner of IFT27, was disrupted in Ift27 mutant cells, and Ift25-null mice displayed hair follicle phenotypes similar to those of Ift27 mutants. These data suggest that Ift27 and Ift25 operate in a genetically and functionally dependent manner during hair follicle morphogenesis. This study suggests that the molecular trafficking machineries underlying ciliogenesis and Hh signaling can be segregated, thereby providing important insights into new avenues of inhibiting Hh signaling, which might be adopted in the development of targeted therapies for Hh-dependent cancers, such as basal cell carcinoma.

A Role for Macro-ER-Phagy in ER Quality Control.

The endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20-50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole.

The overexpression of Rabl3 is associated with pathogenesis and clinicopathologic variables in hepatocellular carcinoma.

Overexpression of Rabl3 is associated with some malignancies. However, their relationship with hepatocellular carcinoma remains unclear. In this study, the expression of Rabl3 in hepatocellular carcinoma cell lines, and four pairs of matched hepatocellular carcinoma tissues and their adjacent normal hepatic tissues were detected by quantitative reverse transcription polymerase chain reaction and western blot. In addition, the protein expression of Rabl3 was examined in 162 cases of hepatocellular carcinoma by immunohistochemistry. Rabl3 in hepatocellular carcinoma cell lines was elevated at both messenger RNA and protein levels, and the Rabl3 protein was significantly upregulated by upto 3.3-fold in hepatocellular carcinoma compared with the paired normal hepatic tissues. Immunohistochemical analysis revealed that overexpressions of Rabl3 were 80.2% in hepatocellular carcinoma. Rabl3 is expressed at significantly higher rates in hepatocellular carcinoma compared with adjacent normal hepatic tissue (p < 0.01). Statistical analysis suggested the upregulation of Rabl3 was significantly associated with lymph node metastasis, tumor thrombus of the portal vein, and advanced clinical stage (p < 0.05). Furthermore, we found that overexpression of Rabl3 in hepatocellular carcinoma cells could significantly enhance cell proliferation and growth ability. Conversely, silencing Rabl3 by small hairpin RNA interference caused an inhibition of cell proliferation and growth. Our studies suggest that the Rabl3 is a valuable marker of hepatocellular carcinoma progression and that the overexpression of Rabl3 plays an important role in the development and pathogenesis of hepatocellular carcinoma.

Digital expression analysis of the genes associated with salinity resistance after overexpression of a stress-responsive small GTP-binding RabG protein in peanut.

The Rab protein family is the largest family of the small GTP-binding proteins. Among them, the RabG genes are known to be responsive to abiotic stresses, but the molecular mechanisms of the stress responses mediated by RabG genes in plants is poorly understood. To investigate the molecular mechanism of AhRabG gene in peanut, transgenic plants overexpressing the AhRabG gene (S6) with relatively higher salinity resistance than the non-transgenic plants (S7) were obtained. Digital gene expression (DGE) sequencing was performed with the leaves of S6 and S7 plants before and after salinity-stress treatment. The AhRabG gene in peanut was found to be involved in a few pathways such as "photosynthesis", "oxidative phosphorylation", "AMPK signaling pathway", "plant hormone signal transduction", etc. A total of 298 differentially expressed genes (DEGs) were found to be upregulated or downregulated at five sampling time points based on the comparison between S6 and S7 plants. Among them, 132 DEGs were responsive to salinity stress in S6 and/or S7 after salinity-stress treatment. These 132 DEGs included genes encoding various transcription factors and proteins involved in resistance to salinity stress such as MYB, AP2, RING-H2 zinc finger proteins, late embryogenesis abundant (LEA) proteins, dehydration-responsive protein RD22, peroxidases, CBL-interacting protein kinases, calcium-binding proteins, and others. The information from this study will be useful for further studies on elucidating the mechanism of salinity resistance conferred by RabG genein peanut.

Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

Rab23 is overexpressed in human astrocytoma and promotes cell migration and invasion through regulation of Rac1.

Rab23 overexpression has been implicated in several human cancers. However, its biological roles and molecular mechanism in astrocytoma have not been elucidated. The aim of this study is to explore clinical significance and biological roles of Rab23 in astrocytoma. We observed negative Rab23 staining in normal astrocytes and positive staining in 39 out of 86 (45 %) astrocytoma specimens using immunohistochemistry. The positive rate of Rab23 was higher in grades III and IV (56.5 %, 26/46) than grades I + II astrocytomas (32.5 %, 13/40, p < 0.05). Transfection of Rab23 plasmid was performed induced A172 cell proliferation, colony formation, invasion, and migration, while Rab23 depletion with siRNA reduced these abilities of U87 cells. In addition, we found that Rab23 transfection upregulated while its depletion reduced Rac1 activity. Treatment of transfected cells with a Rac1 inhibitor decreased Rac1 activity and invasion. In conclusion, Rab23 serves as an important oncoprotein in human astrocytoma by regulating cell invasion and migration through Rac1 activity.

Overexpression of Rab25 promotes hepatocellular carcinoma cell proliferation and invasion.

Rab25 was reported to be associated with several human cancers and malignant biological behavior of cancer cells. The goal of the present study was to determine its expression pattern and biological function in human hepatocellular carcinoma (HCC). We examined Rab25 protein in 92 cases of HCC tissues and 3 HCC cell lines. The results showed that Rab25 was upregulated in HCC tissues and cells compared with normal liver tissues and cell line. Rab25 overexpression correlated with advanced tumor stage and nodal metastasis. Rab25 small interfering RNA (siRNA) was employed in Bel7402 and SK-Hep-1 cell lines. Cell Counting Kit-8 (CCK-8) assay and colony formation assay showed that Rab25 depletion blocked cell growth rate and inhibited colony formation ability. Transwell assay showed that Rab25 depletion negatively regulated the invading ability of HCC cells. To explore the possible mechanisms, we checked several signaling pathways and found that Rab25 depletion downregulated AKT phosphorylation. In addition, luciferase reporter assay showed that Rab25 depletion inhibited the Wnt signaling pathway and its target genes such as cyclin D1, c-myc, and MMP7. In conclusion, Rab25 is overexpressed in human HCC and contributes to cancer cell proliferation and invasion possibly through regulation of the Wnt signaling pathway.

Expression, purification and guanine nucleotide binding characterization of Arabidopsis RabE1d13-185 GTPase.

Arabidopsis RabE1d subclass plays important plant-specific functions in plant growth and development, response to ethylene and defence to plant pathogen, besides their basic cellular role in membrane trafficking. In this study, we present the expression, purification, and characterization of the recombinant core domain of AtRabE1d13-185. AtRabE1d13-185 was successfully expressed in Escherichia coli and purified via two-step nickel affinity chromatography followed by gel filtration, and identified single band in SDS-PAGE. The resultant protein was functionally active, as determined by interaction with guanine nucleotide by a fluorescence-based assay. The intrinsic tryptophan of AtRabE1d13-185 showed fluorescence resonance energy transfer (FRET) effect upon forming complex with fluorescent methylanthraniloyl (mant)-GDP, but quenched when binding with non-labelled guanine nucleotide. The association rate of mantGDP with AtRabE1d13-185 was determined to be 3.48 × 10(7) s(-1) M(-1). The dissociation rates of GDP and mantGDP from the complex with AtRabE1d13-185 were similar. The koff values were determined to be 4.02 × 10(-4) s(-1) based on the FRET effect for the AtRabE1d13-185:GDP and 5.41 × 10(-4) s(-1) for mantGDP excited directly.

Phenotypic characterisation of RAB6A knockout mouse embryonic fibroblasts.

BACKGROUND INFORMATION: Rab6 is one of the most conserved Rab GTPaes throughout evolution and the most abundant Rab protein associated with the Golgi complex. The two ubiquitous Rab isoforms, Rab6A and Rab6A', that are generated by alternative splicing of the RAB6A gene, regulate several transport steps at the Golgi level, including retrograde transport between endosomes and Golgi, anterograde transport between Golgi and the plasma membrane, and intra-Golgi and Golgi to endoplasmic reticulum transport. RESULTS: We have generated mice with a conditional null allele of RAB6A. Mice homozygous for the RAB6A null allele died at an early stage of embryonic development. Mouse embryonic fibroblasts (MEFs) were isolated from RAB6A(loxP/loxP) Rosa26-CreERT2 and incubated with 4-hydroxy tamoxifen, resulting in the efficient depletion of Rab6A and Rab6A'. We show that Rab6 depletion affects cell growth, alters Golgi morphology and decreases the Golgi-associated levels of some known Rab6 effectors such as Bicaudal-D and myosin II. We also show that Rab6 depletion protects MEFs against ricin toxin and delays VSV-G secretion. CONCLUSIONS: Our study shows that RAB6 is an essential gene required for normal embryonic development. We confirm in MEF cells most of the functions previously attributed to the two ubiquitous Rab6 isoforms.

miR-184 and miR-150 promote renal glomerular mesangial cell aging by targeting Rab1a and Rab31.

The molecular mechanism of kidney aging is not well understood, but the abnormal expression of miRNAs with aging is considered to be an important contributor. miR-184 and miR-150 were screened using a miRNA microarray and qRT-PCR and found to be significantly upregulated in 24-month-old rats. Rat renal primary glomerular mesangial cells (GMCs) were isolated from 3-month and 24-month-old rats for the in vitro analysis of the roles of miR-184 and miR-150 in kidney aging. Bioinformatics analyses suggested that Rab1a and Rab31, which are associated with cell autophagy, were targeted by both miR-184 and miR-150. miR-184 and miR-150 were increased significantly in aging GMCs versus young cells, while Rab1a and Rab31 were significantly lower in aging cells. Furthermore, dual luciferase reporter assays revealed that miR-184 and miR-150 bound to the 3'-UTR of Rab1a and Rab31 mRNAs. Transfection of miR-184 and miR-150 mimics into young GMCs suppressed the expression of Rab1a and Rab31. Transfected cells showed lower autophagy activities and higher levels of cellular oxidative products, leading to the aging of young GMCs. However, miR-184 and miR-150 inhibitors promoted autophagy and reduced oxidative damage by upregulating Rab1a and Rab31 in old GMCs. In conclusion, miR-184 and miR-150 inhibited autophagy, promoting GMC aging.

The Vac14-interaction network is linked to regulators of the endolysosomal and autophagic pathway.

The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositol-modifying enzymes are tightly interconnected.

High Expression of Rab-like 3 (Rabl3) is Associated with Poor Survival of Patients with Non-Small Cell Lung Cancer via Repression of MAPK8/9/10-Mediated Autophagy.

BACKGROUND Rab-like 3 (Rabl3) is a member of the Rab subfamily of small GTPases which are involved in controlling proliferation and vesicular trafficking. Recent studies suggest that Rab proteins might play a critical role in regulating cancer cell survival, but the underlying mechanisms remain largely unknown. MATERIAL AND METHODS We performed a bioinformatics analysis to examine the correlation between the expression level of Rabl3 and survival of non-small cell lung cancer (NSCLC) patients in three independent cohorts containing 484 patients. The function of Rabl3 was examined in NSCLC cell line A549 in vitro. Following Rabl3 knockdown, cells were stained with propidium iodine (PI) and Annexin V, followed by flow cytometry analysis (FACS) for cell death and autophagy induction. The activity of the MAPK signaling pathway was assessed by Western blotting of different MAPK phosphorylations, and modulated with different chemical inhibitors. RESULTS High expression of Rabl3 was significantly correlated with poor survival in all three independent NSCLC cohorts. In line with this result, Rabl3 was frequently overexpressed in lung cancer cell lines as compared with normal lung fibroblast cell lines. Knockdown of Rabl3 in lung cancer cells significantly enhanced cell death accompanied with autophagy induction, as evidenced by an increased level of autophagy marker LC3-II. Interestingly, Rabl3 knockdown was associated with enhanced activation of MAPK8/9/10 but not MAPK11/12/13/14. Treatment of MAPK8/9/10-specific inhibitor SP600125, but not MAPK11/12/13/14-specific inhibitor SB203580, largely abolished Rabl3 knockdown-induced LC3-I/LC3-II conversion and autophagic cell death. CONCLUSIONS Together, these results suggest that high expression of Rabl3 might inhibit cell death in NSCLCs via repression of MAPK8/9/10-mediated autophagy.

The nutrient stress-induced small GTPase Rab5 contributes to the activation of vesicle trafficking and vacuolar activity.

Rab family small GTPases regulate membrane trafficking by spatiotemporal recruitment of various effectors. However, it remains largely unclear how the expression and functions of Rab proteins are regulated in response to extracellular or intracellular stimuli. Here we show that Ypt53, one isoform of Rab5 in Saccharomyces cerevisiae, is up-regulated significantly under nutrient stress. Under non-stress conditions, Vps21, a constitutively expressed Rab5 isoform, is crucial to Golgi-vacuole trafficking and to vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, the up-regulated Ypt53 and the constitutive Vps21 function redundantly to maintain these activities, which, in turn, prevent the accumulation of reactive oxygen species and maintain mitochondrial respiration. Together, our results clarify the relative roles of these constitutive and nutrient stress-inducible Rab5 proteins that ensure adaptable vesicle trafficking and vacuolar hydrolase activity, thereby allowing cells to adapt to environmental changes.

A non-BRICHOS SFTPC mutant (SP-CI73T) linked to interstitial lung disease promotes a late block in macroautophagy disrupting cellular proteostasis and mitophagy.

Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged primary translation product of SP-C isoforms were generated to test the hypothesis that deposition of hSP-C(I73T) within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, wild-type hSP-C (hSP-C(WT)) colocalized with exogenously expressed human ATP binding cassette class A3 (hABCA3), an indicator of normal trafficking to lysosomal-related organelles. In contrast, hSP-C(I73T) was dissociated from hABCA3 but colocalized to the plasma membrane as well as the endosomal network. Cells expressing hSP-C(I73T) exhibited increases in size and number of cytosolic green fluorescent protein/microtubule-associated protein 1 light-chain 3 (LC3) vesicles, some of which colabeled with red fluorescent protein from the gene dsRed/hSP-C(I73T). By transmission electron microscopy, hSP-C(I73T) cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris, which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-C(I73T) cells exhibited increased expression of Atg8/LC3, SQSTM1/p62, and Rab7, consistent with a distal block in autophagic vacuole maturation, confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-C(I73T) cells showed an impaired degradative capacity for an aggregation-prone huntingtin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and parkin expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-C(I73T) induces an acquired block in macroautophagy-dependent proteostasis and mitophagy, which could contribute to the increased vulnerability of the lung epithelia to second-hit injury as seen in ILD.