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.

RAB7L1 Participates in Secondary Brain Injury Induced by Experimental Intracerebral Hemorrhage in Rats.

RAB7, a member of RAS oncogene family-like 1 (RAB7L1), is a GTPase belonging to the Rab family and acts as an upstream regulator to regulate the kinase activity of leucine-rich repeat kinase 2 (LRRK2). Although LRRK2 has been shown to aggravate secondary brain injury (SBI) after intracerebral hemorrhage (ICH), it is unknown whether RAB7L1 is also involved in this process. The purpose of the present study was to investigate the role of RAB7L1 in ICH-induced SBI in vivo. Autologous blood was injected into adult male Sprague-Dawley rats to induce an ICH model in vivo. The results showed that the protein levels of RAB7L1 increased after ICH. Overexpression of RAB7L1 induced neuronal apoptosis and damage, as demonstrated by TUNEL-positive and FJB-positive cells, and exacerbated ICH-induced learning and cognitive dysfunctions; in contrast, downregulation of RAB7L1 via RNA interference yielded comparatively opposite changes in these parameters. In summary, this study demonstrates that RAB7L1 promotes SBI after ICH and may represent a potential target for ICH therapy.

Rab22a is a novel prognostic marker for cell progression in breast cancer.

Breast cancer (BC) is the most common female malignant tumor worldwide. The mechanism of tumorigenesis is still unclear. Ras­related proteins in brain (Rab)22a belongs to the Ras superfamily, which may act as an oncogene and participate in carcinogenesis. The present study aims to identify whether Rab22a could be a novel biomarker of prognosis and determine the effects of Rab22a on BC cell progression. A total 258 BC and 56 para­tumor or non­tumor formalin fixed paraffin embedded tissues were stained through immunohistochemistry. The association between Rab22a expression and clinicopathological features, as well as overall survival status were analyzed. The expression level of Rab22a in breast cell lines were detected using reverse transcription­quantitative PCR and western blotting. SK­BR­3 cells were infected with Rab22a short hairpin RNA lenti­virus and the ability of cell proliferation, migration and invasion were measured. Gene Set Enrichment Analysis (GSEA) was employed to analyze the pathways involved in the Rab22a mRNA high level group. Rab22a was found to be overexpressed in BC tissues and upregulated in BC cells. High expression of Rab22a was related to a poor prognosis of patients with BC. Knockdown of Rab22a decreased the proliferation, migration and invasion ability of BC cells. GSEA indicated that certain pathways, including mammalian target of rapamycin complex 1 and protein secretion were upregulated, while pathways, such as hypoxia and KRas were downregulated in the Rab22a high level group. Rab22a is of prognostic value for BC and necessary for BC cell proliferation.

Identification of GIMAP7 and Rabl3 as Putative Biomarkers for Oral Squamous Cell Carcinoma Through Comparative Proteomic Approach.

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers and has been listed as sixth most common human cancer. Due to late diagnosis and insufficient therapeutic response among patients, the survival rate remains very low accentuating the importance of early diagnostic markers. The study aimed to identify differentially expressed proteins in search for putative serum biomarkers and drug targets. Serum samples (n = 45) were depleted and resolved on two dimensional gel electrophoresis. Among differentially expressed proteins, two were identified using MALDI-TOF mass spectrometry. Gene expression levels of identified proteins were quantified in malignant and normal tissue using RT-qPCR. To validate serum Rabl3 expression, sandwich ELISA was performed. Proteomics analysis revealed two proteins which were found to be associated with oral cancer. The expression of GIMAP7 was found to be down regulated in serum of patients suffering from oral cancer while the expression of Rabl3 was found to be up-regulated. Gene expression analysis in malignant tissue and adjacent normal tissue revealed the same pattern. Quantitative ELISA was used to validate expression of Rabl3 in serum from oral cancer patients and healthy subjects which demonstrated significant up-regulation in cancer patients. Findings in current study demonstrate differential expression of novel putative biomarkers GIMAP7 and Rabl3 in oral cancer which suggests their potential role in oral cancer pathology and can be considered as predictive biomarkers.

LncRNA SNHG3/miRNA-151a-3p/RAB22A axis regulates invasion and migration of osteosarcoma.

To elucidate the potential function of lncRNA SNHG3 in the development of osteosarcoma. Quantitative real-time polymerase chain reaction was conducted for detection of SNHG3, miRNA-151a-3p and RAB22 A in osteosarcoma tissues and cells. Receiver operating characteristic curve was introduced to analyze the diagnostic potential of SNHG3 in osteosarcoma. Correlation between SNHG3 expression and the overall survival of osteosarcoma patients was evaluated using Kaplan-Meier method. Invasive and migratory potentials of osteosarcoma cells were examined by Transwell assay. Furthermore, dual-luciferase reporter gene assay, RNA-pull down and RIP assay were used to verify the binding of SNHG3/RAB22 A to miRNA-151a-3p. The function of SNHG3/miRNA-151a-3p/RAB22 A axis in osteosarcoma was finally confirmed by rescue experiments. SNHG3 and RAB22 A were highly expressed in osteosarcoma patients, while miRNA-151a-3p was lowly expressed. The overall survival of osteosarcoma patients with high expression of SNHG3 was shorter than those with low expression. SNHG3 overexpression markedly promoted invasive and migratory potentials of osteosarcoma cells. Through dual-luciferase reporter gene assay, both SNHG3 and RAB22 A could bind to miRNA-151a-3p. RAB22 A expression was positively regulated by SNHG3, but negatively regulated by miRNA-151a-3p. Finally, rescue experiments confirmed that RAB22 A overexpression could reverse the promotive effects of miRNA-151a-3p knockdown on invasive and migratory potentials of osteosarcoma cells. SNHG3 is highly expressed in osteosarcoma, and promotes the invasive and migratory potentials of osteosarcoma cells by absorbing miRNA-151a-3p to upregulate RAB22 A expression.

Nicastrin/miR-30a-3p/RAB31 Axis Regulates Keratinocyte Differentiation by Impairing EGFR Signaling in Familial Acne Inversa.

Nicastrin (NCSTN) mutations are associated with familial acne inversa (AI), and emerging evidence suggests that microRNAs (miRNAs) are involved in various skin diseases. However, whether NCSTN mutations affect miRNA levels and their subsequent signaling pathways in familial AI patients has not been studied. We aimed to elucidate the relationship between NCSTN mutations and familial AI pathogenesis by investigating differential miRNA expression and their related pathways. Combined with miRNA microarray data from familial AI patients, Ncstn keratinocyte-specific-knockout (NcstnΔKC) mice and bioinformatics predictions showed that NCSTN mutations led to decreased miR-30a-3p levels, which negatively regulated RAB31 expression. Moreover, enhanced RAB31 levels accelerated degradation of activated EGFR, leading to abnormal differentiation in keratinocytes. The impaired EGFR signaling and its effects on epidermal differentiation were also observed in familial AI patients and NcstnΔKC mice. Thus, our study showed that miR-30a-3p/RAB31/EGFR signaling pathway may play a key role in the pathogenesis of familial AI with NCSTN mutations.

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.

High expression of RAB38 promotes malignant progression of pancreatic cancer.

Ras­Related Protein Rab­38 (RAB38), which belongs to the RAB family, is involved in the biogenesis of lysosome­related organelles and defense against certain microbial infections. However, the clinical significance and potential function of RAB38 in pancreatic adenocarcinoma remain unclear. In the present study, an immunohistochemical assay was performed to analyze the expression of RAB38 in pancreatic adenocarcinoma tumor specimens from 82 patients, and the clinicopathological characteristics and survival rate of these patients were further examined. To validate the role of RAB38 in tumors, the effect of RAB38 on tumor cell proliferation, migration and invasion was assessed by establishing RAB38 knockdown cell lines. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to examine the expression levels of proteins associated with the cancer cell behavior. In addition, the inhibitory effect of RAB38 silencing on pancreatic cancer was examined in mice. The immunohistochemistry results revealed that RAB38 was upregulated and positively correlated with the grade of progression in pancreatic adenocarcinoma patients. Further investigation indicated that RAB38 downregulation significantly suppressed the proliferation, migration and invasive capacity of pancreatic cancer cells, as well as decreased the expression levels of Ki67, proliferating cell nuclear antigen, and matrix metalloproteinases 2 and 9. RAB38 silencing also inhibited the development of pancreatic cancer in vivo. Taken together, a high level of RAB38 was significantly associated with the malignant phenotypes of pancreatic cancer, suggesting that RAB38 may serve as a novel biomarker and a potential therapeutic target for pancreatic cancer.

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.

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface.

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human placenta, with novel localization at the maternal-fetal interface.

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.

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.

Mevastatin blockade of autolysosome maturation stimulates LBH589-induced cell death in triple-negative breast cancer cells.

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, and combining a HDACi with other agents is an attractive therapeutic strategy in solid tumors. We report here that mevastatin increases HDACi LBH589-induced cell death in triple-negative breast cancer (TNBC) cells. Combination treatment inhibited autophagic flux by preventing Vps34/Beclin 1 complex formation and downregulating prenylated Rab7, an active form of the small GTPase necessary for autophagosome-lysosome fusion. This means that co-treatment with mevastatin and LBH589 activated LKB1/AMPK signaling and subsequently inhibited mTOR. Co-treatment also led to cell cycle arrest in G2/M phase and induced corresponding expression changes of proteins regulating the cell cycle. Co-treatment also increased apoptosis both in vitro and in vivo, and reduced tumor volumes in xenografted mice. Our results indicate that disruption of autophagosome-lysosome fusion likely underlies mevastatin-LBH589 synergistic anticancer effects. This study confirms the synergistic efficacy of, and demonstrates a potential therapeutic role for mevastatin plus LBH589 in targeting aggressive TNBC, and presents a novel therapeutic strategy for further clinical study. Further screening for novel autophagy modulators could be an efficient approach to enhance HDACi-induced cell death in solid tumors.

miR-148a increases the sensitivity to cisplatin by targeting Rab14 in renal cancer cells.

MicroRNA (miR) can exert various biological functions by targeting oncogenes or tumor suppressor genes in numerous human malignancies. Recent evidence has shown that miR-148a increases the drug sensitivity of various cancer cells. Herein, we show that ectopic expression of miR-148a induces apoptosis, reduces clonogenicity, and increases the sensitivity to TRAIL and cisplatin in renal cancer cells. The luciferase reporter assay showed that miR-148a negatively regulated ras-related protein 14 (Rab14) expression by binding to the miR-148a binding site in the 3' untranslated region (3'UTR) of Rab14. Rab14-specific siRNA-induced downregulation of Rab14 increases the sensitivity to cisplatin, while forced expression of Rab14 lacking 3'-UTR abrogated the pro-apoptotic function of miR-148a in renal cancer cells. These findings suggest that miR-148a acts as a tumor suppressor and holds great potential for renal cancer therapy by directly targeting Rab14.

Strain-Specific Altered Regulatory Response of Rab7a and Tau in Creutzfeldt-Jakob Disease and Alzheimer's Disease.

There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the disease, we checked the regulatory interactive response of Rab7a, Rab9, and known biomarkers PrPC and tau forms in frontal cortex at pre-symptomatic stage of the disease in tg340 mice expressing about fourfold of human PrP-M129 with PrP-null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, we analyzed 5XFAD mice, exhibiting five mutations in the APP and presenilin genes related to familial Alzheimer's disease (FAD), to validate specific regulatory response of Rab7a, Rab9, tau, and phosphorylated form of tau by immunostaining 5XFAD mice in comparison with the wild-type age-matched mice brain. The cortical region of 5XFAD mice brain showed accumulated form of Rab7a in puncta that co-label for p-Tau, indicating colocalization by using confocal laser-scanning microscopy and was confirmed by using reverse co-immunoprecipitation. Furthermore, synthetic RNA (siRNA) against the Rab7a gene decreased expression of Rab7a protein, in cortical primary neuronal cultures of PrPC wild type. This depleted expression of Rab7a led to the increased accumulation of PrPC in Rab9-positive endosomal compartments and consequently an increased co-localization between PrPC/Rab9; however, total tau level decreased. Interestingly, siRNA against tau gene in cortical primary neuronal cultures of PrPC wild-type mice showed enhanced Rab7a and Rab9 expression and increase formation of dendritic spines. The work described highlighted the selective involvement of late endosomal compartment marker Rab7a in CJD, slow and rapid progressive forms of AD pathogenesis.

KDM4B-mediated epigenetic silencing of miRNA-615-5p augments RAB24 to facilitate malignancy of hepatoma cells.

Emerging evidence indicates that dysregulation of microRNAs (miRNAs) contributes to hepatocellular carcinoma (HCC) tumorigenesis and development. Here, we found that miR-615-5p was obviously downregulated in HCC. Furthermore, the deficiency of demethylase KDM4B stimulated the CpG methylation of miR-615-5p promoter and then decreased the miR-615-5p expression. The Ras-related protein RAB24 was found to be downregulated by miR-615-5p. The low level of miR-615-5p increased the expression of RAB24 and facilitated HCC growth and metastasis in vitro and in vivo. Moreover, miR-615-5p suppresses HCC cell growth by influencing cell cycle progression and apoptosis. Downregulation of miR-615-5p and upregulation of RAB24 promotes the epithelial-mesenchymal transition (EMT), adhesion and vasculogenic mimicry (VM) of HCC cells, all of which contribute to cell motility and metastasis. Thus, miR-615-5p, who is downregulated by KDM4B-mediated hypermethylation in its promoter, functions as a tumor suppressor by inhibiting RAB24 expression in HCC. In conclusion, our findings characterize miR-615-5p as an important epigenetically silenced miRNA involved in the Rab-Ras pathway in hepatocellular carcinoma and expand our understanding of the molecular mechanism underlying hepatocarcinogenesis and metastasis.

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.

Characterization of Heparan Sulfate Proteoglycan-positive Recycling Endosomes Isolated from Glioma Cells.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs)-dependent endocytic events have been involved in glioma progression. Thus, comprehensive understanding of the intracellular trafficking complexes formed in presence of HSPGs would be important for development of glioma treatments. MATERIALS AND METHODS: Subcellular fractionation was used to separate vesicles containing HSPGs from the rat C6 glioma cell line. Isolated HSPG-positive vesicles were further characterized with liquid chromatography-mass spectrometry. RESULTS: The HSPG-positive vesicular fractions, distinct from plasma membrane-derived material, were enriched in endocytic marker, Rab11. Proteomic analysis identified more than two hundred proteins to be associated with vesicular membrane, among them, over eighty were related to endosomal uptake, recycling or vesicular transport. CONCLUSION: Part of HSPGs in glioma cells is internalized through clathrin-dependent endocytosis and undergo recycling. The development of compounds regulating HSPG-mediated trafficking will likely enable design of effective glioma treatment.

RAB23, regulated by miR-92b, promotes the progression of esophageal squamous cell carcinoma.

RAB23, a member of Ras-related small GTPase family, has been reported to be up-regulated in several cancer types. However, its biological functions and the underlying molecular mechanisms for its oncogenic roles in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, we have shown that the expression of RAB23 was elevated in ESCC tissues and ESCC cells. Overexpression of RAB23 promoted the growth and migration of the ESCC cells, while knocking down the expression RAB23 inhibited the growth, migration and metastasis of the ESCC cells. The molecular mechanism study showed that RAB23 activated beta-catenin/TCF signaling and regulated the expression of several target genes. In the further study, it was found that the expression of RAB23 was regulated by the miR-92b. Forced expression of MiR-92b decreased the mRNA and protein level of RAB23, and RAB23 rescued the biological functions of miR-92b. Taken together, this study revealed the oncogenic roles and the regulation of RAB23 in ESCC, suggesting RAB23 might be a therapeutic target.