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Endoplasmic reticulum (ER)–endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER–endolysosome membrane contact sites mediate the ER–endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP–ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER–endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER–endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy. [ABSTRACT FROM AUTHOR]

Glutamate functions as the major excitatory neurotransmitter for primary sensory neurons and has a crucial role in sensitizing peripheral nociceptor terminals producing sensitization. Glutaminase (GLS) is the synthetic enzyme that converts glutamine to glutamate. GLS-immunoreactivity (-ir) and enzyme activity are elevated in dorsal root ganglion (DRG) neuronal cell bodies during chronic peripheral inflammation, but the mechanism for this GLS elevation is yet to be fully characterized. It has been well established that, after nerve growth factor (NGF) binds to its high-affinity receptor tropomyosin receptor kinase A (TrkA), a retrograde signaling endosome is formed. This endosome contains the late endosomal marker Rab7GTPase and is retrogradely transported via axons to the cell soma located in the DRG. This complex is responsible for regulating the transcription of several critical nociceptive genes. Here, we show that this retrograde NGF signaling mediates the expression of GLS in DRG neurons during the process of peripheral inflammation. We disrupted the normal NGF/TrkA signaling in adjuvant-induced arthritic (AIA) Sprague Dawley rats by the pharmacological inhibition of TrkA or blockade of Rab7GTPase, which significantly attenuated the expression of GLS in DRG cell bodies. The results indicate that NGF/TrkA signaling is crucial for the production of glutamate and has a vital role in the development of neurogenic inflammation. In addition, our pain behavioral data suggest that Rab7GTPase can be a potential target for attenuating peripheral inflammatory pain. [ABSTRACT FROM AUTHOR]

Mutations in PLEKHM1 cause osteopetrosis in humans and rats. The germline and osteoclast conditional deletions of Plekhm1 gene in mice lead to defective osteoclast bone resorption and increased trabecular bone mass without overt abnormalities in other organs. As an adaptor protein, pleckstrin homology and RUN domain containing M1 (PLEKHM1) interacts with the key lysosome regulator small GTPase RAB7 via its C-terminal RUBICON homologous (RH) domain. In this study, we have conducted a structural-functional study of the PLEKHM1 RH domain and RAB7 interaction in osteoclasts in vitro. The single mutations of the key residues in the Plekhm1 RH predicted from the crystal structure of the RUBICON RH domain and RAB7 interface failed to disrupt the Plekhm1-Rab7 binding, lysosome trafficking, and bone resorption. The compound alanine mutations at Y949-R954 and L1011-I1018 regions decreased Plekhm1 protein stability and Rab7-binding, respectively, thereby attenuated lysosome trafficking and bone resorption in osteoclasts. In contrast, the compound alanine mutations at R1060-Q1068 region were dispensable for Rab7-binding and Plekhm1 function in osteoclasts. These results indicate that the regions spanning Y949-R954 and L1011-I1018 of Plekhm1 RH domain are functionally important for Plekhm1 in osteoclasts and offer the therapeutic targets for blocking bone resorption in treatment of osteoporosis and other metabolic bone diseases. [ABSTRACT FROM AUTHOR]

Mutations or triplication of the alpha synuclein (ASYN) gene contribute to synucleinopathies including Parkinsons disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent evidence suggests that ASYN also plays an important role in amyloid-induced neurotoxicity, although the mechanism(s) remains unknown. One hypothesis is that accumulation of ASYN alters endolysosomal pathways to impact axonal trafficking and processing of the amyloid precursor protein (APP). To define an axonal function for ASYN, we used a transgenic mouse model of synucleinopathy that expresses a GFP-human ASYN (GFP-hASYN) transgene and an ASYN knockout (ASYN-/-) mouse model. Our results demonstrate that expression of GFP-hASYN in primary neurons derived from a transgenic mouse impaired axonal trafficking and processing of APP. In addition, axonal transport of BACE1, Rab5, Rab7, lysosomes and mitochondria were also reduced in these neurons. Interestingly, axonal transport of these organelles was also affected in ASYN-/- neurons, suggesting that ASYN plays an important role in maintaining normal axonal transport function. Therefore, selective impairment of trafficking and processing of APP by ASYN may act as a potential mechanism to induce pathological features of Alzheimers disease (AD) in PD patients.

Abstract Background The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T. reesei not only relies on the efficient transcription of cellulase genes but also their efficient secretion after being translated. However, little attention has been paid to the functional roles of the involved secretory pathway in the high-level production of cellulases in T. reesei. Rab GTPases are key regulators in coordinating various vesicle trafficking associated with the eukaryotic secretory pathway. Specifically, Rab7 is a representative GTPase regulating the transition of the early endosome to the late endosome followed by its fusion to the vacuole as well as homotypic vacuole fusion. Although crosstalk between the endosomal/vacuolar pathway and the secretion pathway has been reported, the functional role of Rab7 in cellulase production in T. reesei remains unknown. Results A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7 KD) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7 KD on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose. Conclusions TrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production.

Abstract Background Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion. Methods Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively. Results We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity. Conclusions Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

Background: The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T. reesei not only relies on the efficient transcription of cellulase genes but also their efficient secretion after being translated. However, little attention has been paid to the functional roles of the involved secretory pathway in the high-level production of cellulases in T. reesei. Rab GTPases are key regulators in coordinating various vesicle trafficking associated with the eukaryotic secretory pathway. Specifically, Rab7 is a representative GTPase regulating the transition of the early endosome to the late endosome followed by its fusion to the vacuole as well as homotypic vacuole fusion. Although crosstalk between the endosomal/vacuolar pathway and the secretion pathway has been reported, the functional role of Rab7 in cellulase production in T. reesei remains unknown. Results: A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7KD) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7KD on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose. Conclusions: TrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production. [ABSTRACT FROM AUTHOR]

Background: Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion. Methods: Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively. Results: We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity. Conclusions: Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance. [ABSTRACT FROM AUTHOR]

Lipophagy is a selective autophagy that regulates lipid metabolism and reduces hepatic lipid deposition. However, the underlying mechanism has not been understood in fish. In this study, we used micronutrient zinc (Zn) as a regulator of autophagy and lipid metabolism and found that Ras-related protein 7 (rab7) was involved in Zn-induced lipophagy in hepatocytes of yellow catfish Pelteobagrus pelteobagrus. We then characterized the rab7 promoter and identified binding sites for a series of transcription factors, including Forkhead box O3 (FOXO3). Site mutation experiments showed that the −1358/−1369 bp FOXO3 binding site was responsible for Zn-induced transcriptional activation of rab7. Further studies showed that inhibition of rab7 significantly inhibited Zn-induced lipid degradation by lipophagy. Moreover, rab7 inhibitor also mitigated the Zn-induced increase of cpt1α and acadm expression. Our results suggested that Zn exerts its lipid-lowering effect partly through rab7-mediated lipophagy and FA β-oxidation in hepatocytes. Overall, our findings provide novel insights into the FOXO3/rab7 axis in lipophagy regulation and enhance the understanding of lipid metabolism by micronutrient Zn, which may help to reduce excessive lipid accumulation in fish. [ABSTRACT FROM AUTHOR]

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of the plasticizer di-2-ethylhexyl phthalic acid (DEHP), and studies have shown that MEHP causes serious reproductive effects. However, its exact mechanisms of action remain elusive. In this study, we aimed to investigate the reproductive effects of MEHP and preliminarily explore its underlying molecular mechanisms. We found that TM3 cells gradually secreted less testosterone and intracellular free cholesterol with increasing MEHP exposure. MEHP exposure inhibited lipophagy and the Sirt1/Foxo1/Rab7 signaling pathway in TM3 cells, causing aberrant accumulation of intracellular lipid droplets. Addition of the Sirt1 agonist SRT1720 and Rab7 agonist ML-098 alleviated the inhibition of lipophagy and increased free cholesterol and testosterone contents in TM3 cells. SRT1720 alleviated the inhibitory effect of MEHP on the Sirt1/Foxo1/Rab7 signaling pathway, whereas ML-098 only alleviated the inhibition of Rab7 protein expression by MEHP and had no effect on Sirt1 and Foxo1 protein expression. This suggests that MEHP inhibits lipophagy in TM3 cells by suppressing the Sirt1/Foxo1/Rab7 signaling pathway, ultimately leading to a further decrease in cellular testosterone secretion. This study improves our current understanding of the toxicity and molecular mechanisms of action of MEHP and provides new insights into the reproductive effects of phthalic acid esters.

Endoplasmic reticulum (ER)–endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER–endolysosome membrane contact sites mediate the ER–endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP–ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER–endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER–endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy.

In B cells, antigen processing and peptide-antigen (pAg) presentation is essential to ignite high-affinity antibody responses with the help of cognate T cells. B cells efficiently internalize and direct specific antigens for processing and loading onto MHCII. This critical step, which enables pAg presentation, occurs in MHCII compartments (MIICs) which possess the enzymatic machinery for pAg loading on MHCII. The intracellular transport systems that guide antigen and maintain this unique compartment remain enigmatic. Here, we probed the possible functional role of two known endosomal proteins, the Rab family small GTPases Rab7 and Rab9, that are both reported to colocalize with internalized antigen. As compared to Rab9, we found Rab7 to exhibit a higher overlap with antigen and MIIC components. Rab7 also showed a higher association with antigen degradation. The inhibition of Rab7 drastically decreased pAg presentation. Additionally, we detected the strong colocalization of perinuclearly clustered and presumably MIIC-associated antigen with autophagy protein LC3. When we pharmacologically inhibited autophagy, pAg presentation was inhibited. Together, our data promote Rab7 as an important regulator of antigen processing and, considering the previously reported functions of Rab7 in autophagy, this also raises the possibility of the involvement of autophagy-related machinery in this process. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The ras gene from rat brain (RAB) family of small GTPases is highly conserved among eukaryotes and regulates endomembrane trafficking pathways. RAB7, in particular, has been linked to various processes involved in regulating endocytic and autophagic pathways. Plants have several copies of RAB7 proteins that reflect the intricacy of their endomembrane transport systems. RAB7 activity regulates different pathways of endomembrane trafficking in plants: (1) endocytic traffic to the vacuole; (2) biosynthetic traffic to the vacuole; and (3) recycling from the late endosome to the secretory pathway. During certain developmental and stress related processes another pathway becomes activated (4) autophagic trafficking towards the vacuole that is also regulated by RAB7. RAB7s carry out these functions by interacting with various effector proteins. Current research reveals many unexplored RAB7 functions in connection with stress responses. Thus, this review describes a comprehensive summary of current knowledge of plant RAB7's functions, discusses unresolved challenges, and recommends prospective future research directions. [ABSTRACT FROM AUTHOR]

During phagocytosis, endosomes both contribute with membrane to forming phagosomes and promote phagosome maturation. However, how these vesicles are delivered to the phagocytic cup and the phagosome has been unknown. Here, we show that Protrudin-mediated endoplasmic reticulum (ER)-endosome contact sites facilitate anterograde translocation of FYCO1 and VAMP7-positive late endosomes and lysosomes (LELys) to forming phagocytic cups in a retinal pigment epithelial-derived cell line (RPE1). Protrudin-dependent phagocytic cup formation required SYT7, which promotes fusion of LELys with the plasma membrane. RPE1 cells perform phagocytosis of dead cells (efferocytosis) that expose phosphatidylserine (PS) on their surface. Exogenous addition of apoptotic bodies increased the formation of phagocytic cups, which further increased when Protrudin was overexpressed. Overexpression of Protrudin also led to elevated uptake of silica beads coated with PS. Conversely, Protrudin depletion or abrogation of ER-endosome contact sites inhibited phagocytic cup formation resulting in reduced uptake of PS-coated beads. Thus, the Protrudin pathway delivers endosomes to facilitate formation of the phagocytic cup important for PS-dependent phagocytosis. [ABSTRACT FROM AUTHOR]

Lipophagy is a selective autophagy that regulates lipid metabolism and reduces hepatic lipid deposition. However, the underlying mechanism has not been understood in fish. In this study, we used micronutrient zinc (Zn) as a regulator of autophagy and lipid metabolism and found that Ras-related protein 7 (rab7) was involved in Zn-induced lipophagy in hepatocytes of yellow catfish Pelteobagrus pelteobagrus. We then characterized the rab7 promoter and identified binding sites for a series of transcription factors, including Forkhead box O3 (FOXO3). Site mutation experiments showed that the −1358/−1369 bp FOXO3 binding site was responsible for Zn-induced transcriptional activation of rab7. Further studies showed that inhibition of rab7 significantly inhibited Zn-induced lipid degradation by lipophagy. Moreover, rab7 inhibitor also mitigated the Zn-induced increase of cpt1α and acadm expression. Our results suggested that Zn exerts its lipid-lowering effect partly through rab7-mediated lipophagy and FA β-oxidation in hepatocytes. Overall, our findings provide novel insights into the FOXO3/rab7 axis in lipophagy regulation and enhance the understanding of lipid metabolism by micronutrient Zn, which may help to reduce excessive lipid accumulation in fish.

The ras gene from rat brain (RAB) family of small GTPases is highly conserved among eukaryotes and regulates endomembrane trafficking pathways. RAB7, in particular, has been linked to various processes involved in regulating endocytic and autophagic pathways. Plants have several copies of RAB7 proteins that reflect the intricacy of their endomembrane transport systems. RAB7 activity regulates different pathways of endomembrane trafficking in plants: (1) endocytic traffic to the vacuole; (2) biosynthetic traffic to the vacuole; and (3) recycling from the late endosome to the secretory pathway. During certain developmental and stress related processes another pathway becomes activated (4) autophagic trafficking towards the vacuole that is also regulated by RAB7. RAB7s carry out these functions by interacting with various effector proteins. Current research reveals many unexplored RAB7 functions in connection with stress responses. Thus, this review describes a comprehensive summary of current knowledge of plant RAB7’s functions, discusses unresolved challenges, and recommends prospective future research directions.

Autophagy is a lysosomal-dependent degradation process of eukaryotic cells responsible for breaking down unnecessary and damaged intracellular components. Autophagic activity gradually declines with age due to genetic control, and this change contributes to the accumulation of cellular damage at advanced ages, thereby causing cells to lose their functionality and viability. This could be particularly problematic in post-mitotic cells including neurons, the mass destruction of which leads to various neurodegenerative diseases. Here, we aim to uncover new regulatory points where autophagy could be specifically activated and test these potential drug targets in neurodegenerative disease models of Drosophila melanogaster. One possible way to activate autophagy is by enhancing autophagosome–lysosome fusion that creates the autolysosome in which the enzymatic degradation happens. The HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) protein complexes regulate the fusion process. The HOPS complex forms a bridge between the lysosome and autophagosome with the assistance of small GTPase proteins. Thus, small GTPases are essential for autolysosome maturation, and among these proteins, Rab2 (Ras-associated binding 2), Rab7, and Arl8 (Arf-like 8) are required to degrade the autophagic cargo. For our experiments, we used Drosophila melanogaster as a model organism. Nerve-specific small GTPases were silenced and overexpressed. We examined the effects of these genetic interventions on lifespan, climbing ability, and autophagy. Finally, we also studied the activation of small GTPases in a Parkinson's disease model. Our results revealed that GTP-locked, constitutively active Rab2 (Rab2-CA) and Arl8 (Arl8-CA) expression reduces the levels of the autophagic substrate p62/Ref(2)P in neurons, extends lifespan, and improves the climbing ability of animals during ageing. However, Rab7-CA expression dramatically shortens lifespan and inhibits autophagy. Rab2-CA expression also increases lifespan in a Parkinson's disease model fly strain overexpressing human mutant (A53T) α-synuclein protein. Data provided by this study suggests that Rab2 and Arl8 serve as potential targets for autophagy enhancement in the Drosophila nervous system. In the future, it might be interesting to assess the effect of Rab2 and Arl8 coactivation on autophagy, and it would also be worthwhile to validate these findings in a mammalian model and human cell lines. Molecules that specifically inhibit Rab2 or Arl8 serve as potent drug candidates to modulate the activity of the autophagic process in treating neurodegenerative pathologies. In the future, it would be reasonable to investigate which GAP enzyme can inhibit Rab2 or Arl8 specifically, but not affect Rab7, with similar medical purposes. [ABSTRACT FROM AUTHOR]

The black tiger shrimp, Penaeus monodon, is the most economically important penaeid shrimp in Bangladesh for both culture and capture fishery. The culture of these penaeid shrimps is always risky due to different diseases and environmental stressors. This study aimed to assess the relative expression of the Penaeus monodon Rab7 gene in the post larvae (PL) of the P. monodon cultured under different salinity conditions. We challenged the PL of the P. monodon in aquariums with two different salinity and dissolved oxygen conditions during a 7-day test. The relative expression of Rab7 was upregulated during the experimental period of 36 hours. Average differences in the relative expression of Rab7 were 0.3-folds and 0.5-folds, and 0.4-folds and 0.6-folds after 18 and 36 hours of rearing in 2 and 54 ppt salinity treated shrimp PL, respectively, when ß-actin was used as an internal control. On the other hand, when elongation factor 1 alpha (EF1a) was used for normalization, the average relative expression of Rab7's fold differences were 0.2-folds and 0.5-folds, and 0.3-folds and 0.6-folds after 18 and 36 hours of rearing in 2 and 54 ppt salinity treated shrimp PL, respectively. Therefore, our results suggested that the relative expression of the Rab7 was positively correlated with an increased level of salinity and challenge duration. The significant increase in the expression level of Rab7 in the PL of P. monodon exposed to high salinity stress (54 ppt) conditions indicated its functional role in osmotic stress in shrimps. However, further studies are necessary to elucidate the mechanisms of how Rab7 is upregulated in shrimp cells after applying different levels of salinity using a larger sample group. [ABSTRACT FROM AUTHOR]

Dendrites differ from axons in multiple ways, including the presence of minus-end out microtubules intermixed with the more conventional plus-end out microtubules. The mixed microtubule polarity makes regulation of directional transport in dendrites a challenge. Dynein can in principle be a retrograde and anterograde motor in dendrites. We show in our recent paper that dynein supports bi-directional transport of late endosomes in dendrites. We also show that overexpression of the RAB7 effector RILP which recruits dynein to late endosomes imparts retrograde bias onto late endosomes. Inhibition of dynein leads to a decrease in bi-directional motility of late endosomes, an expected result. Unexpectedly, inhibition of dynein also impairs endosome maturation as evidenced by increased association of GTP-RAB7 with late endosomes. Ultimately, dynein inhibition causes degradation defects of short-lived dendritic receptors and stunted dendrite morphologies. Much more work is required to fully understand how endosomal pathways are regulated in time and space in dendrites. Given the prevalence of neurological disorders where endosome-lysosome functions are impaired, this is a topic of great translational relevance. Abbreviations: RILP: Rab-interacting lysosomal protein; EE: early endosome; LE: late endosome; lys: lysosome; NSG2: neural-specific gene 2; RILP-CT: RILP C-terminal truncation.

The composition of the plasma membrane (PM) must be tightly controlled despite constant, rapid endocytosis, which requires active, selective recycling of endocytosed membrane components. For many proteins, the mechanisms, pathways, and determinants of this PM recycling remain unknown. We report that association with ordered, lipid-driven membrane microdomains (known as rafts) is sufficient for PM localization of a subset of transmembrane proteins and that abrogation of raft association disrupts their trafficking and leads to degradation in lysosomes. Using orthogonal, genetically encoded probes with tunable raft partitioning, we screened for the trafficking machinery required for efficient recycling of engineered microdomain-associated cargo from endosomes to the PM. Using this screen, we identified the Rab3 family as an important mediator of PM localization of microdomain-associated proteins. Disruption of Rab3 reduced PM localization of raft probes and led to their accumulation in Rab7-positive endosomes, suggesting inefficient recycling. Abrogation of Rab3 function also mislocalized the endogenous raft-associated protein Linker for Activation of T cells (LAT), leading to its intracellular accumulation and reduced T cell activation. These findings reveal a key role for lipid-driven microdomains in endocytic traffic and suggest Rab3 as a mediator of microdomain recycling and PM composition. [ABSTRACT FROM AUTHOR]

Obesity is one of the most prevalent chronic metabolic diseases, and induction of apoptosis in preadipocytes and adipocytes is a potential strategy to treat obesity. Celastrol represents one of the most robust anti-obesity phytochemicals so far, yet its direct binding target remains elusive. Here, we determined that celastrol could induce apoptosis in preadipocytes via mitochondrial mediated pathway. Further study clarified that celastrol inhibited the fusion of autophagosome and lysosome to prohibit autophagy, leading to cell apoptosis. By conducting virtual screening and genetic manipulation, we verified that overexpression of VAMP7 and RAB7 could block the effects of celastrol on inhibiting autophagy and inducing apoptosis. The Surface Plasmon Resonance study confirmed the direct binding of celastrol with VAMP7 and RAB7. The functional study illustrated the inhibition of RAB7 GTPase activity after celastrol treatment. Moreover, celastrol induced comparable apoptosis in murine epididymal adipose tissue, human preadipocytes and adipocytes, but not in human hepatocytes. An inhibitory effect on differentiation of human primary visceral preadipocytes was also observed. In conclusion, celastrol exhibited inhibitory effect of autophagy via direct binding with VAMP7 and RAB7, leading to an increase in preadipocytes apoptosis. These results advance our understanding in the potential application of celastrol in treating obesity. [ABSTRACT FROM AUTHOR]

ABSTRACT Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)–related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC (P

Abstract Objective SARS CoV-2, the etiologic agent of coronavirus disease-2019 (COVID-19) is well-known to use ACE2 to begin internalization. Some viruses enter the host cell through the endocytosis process and involve some endocytosis proteins, such as the Rab family. However, the relationship between SARS CoV-2 infection with endocytic mRNA RAB5, RAB7, and RAB11B is unknown. This study aims to compare the expression of RAB5, RAB7, and RAB11B between positive and negative COVID-19 patient groups. Results Both viral and human epithelial RNA Isolation and RT-PCR were performed from 249 samples. The genes expression was analysed using appropriate statistical tests. We found the Median (inter-quartile range/IQR) of RAB5, RAB7, and RAB11B expression among the COVID-19 patient group was 2.99 (1.88), 0.17 (0.47), 0.47 (1.49), and 1.60 (2.88), 1.05 (2.49), 1.10 (3.96) among control group respectively. We proceeded with Mann Whitney U Test and found that RAB5 expression was significantly increased (P

Abstract Parkinson’s disease is mainly caused by aggregation of α-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain-barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinson’s disease and related synucleinopathies.

Mutations or triplication of the alpha synuclein (ASYN) gene contribute to synucleinopathies including Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent evidence suggests that ASYN also plays an important role in amyloid-induced neurotoxicity, although the mechanism(s) remains unknown. One hypothesis is that accumulation of ASYN alters endolysosomal pathways to impact axonal trafficking and processing of the amyloid precursor protein (APP). To define an axonal function for ASYN, we used a transgenic mouse model of synucleinopathy that expresses a GFP-human ASYN (GFP-hASYN) transgene and an ASYN knockout (ASYN−/−) mouse model. Our results demonstrate that expression of GFP-hASYN in primary neurons derived from a transgenic mouse impaired axonal trafficking and processing of APP. In addition, axonal transport of BACE1, Rab5, Rab7, lysosomes and mitochondria were also reduced in these neurons. Interestingly, axonal transport of these organelles was also affected in ASYN−/− neurons, suggesting that ASYN plays an important role in maintaining normal axonal transport function. Therefore, selective impairment of trafficking and processing of APP by ASYN may act as a potential mechanism to induce pathological features of Alzheimer's disease (AD) in PD patients.

IntroductionMacrophages are the preferential target of Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of ruminant paratuberculosis. Uptake of pathogens by intestinal macrophages results in their trafficking through endosomal compartments, ultimately leading to fusion with an acidic lysosome to destroy the pathogen. MAP possesses virulence factors which disrupt these endosomal pathways. Additionally, levels of serum vitamin D3 have proven relevant to host immunity. Dynamics of endosomal trafficking and vitamin D3 metabolism have been largely unexplored in bovine paratuberculosis.MethodsThis study aimed to characterize expression of early and late endosomal markers Rab5 and Rab7, respectively, within CD68+ macrophages in frozen mid-ileum sections harvested from cows at different stages of natural paratuberculosis infection. Additionally, factors of vitamin D3 signaling and metabolism were characterized through expression of vitamin D3 activating enzyme 1α-hydroxylase (CYP27B1), vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and vitamin D3 receptor (VDR) within CD68+ ileal macrophages.Results and discussionCows with clinical paratuberculosis had significantly greater macrophage and MAP burden overall, as well as intracellular MAP. Total expression of Rab5 within macrophages was reduced in clinical cows; however, Rab5 and MAP colocalization was significantly greater in this group. Intracellular Rab7 colocalization with MAP was not detected in subclinical or Johne's Disease negative (JD-) control cows but was present in clinical cows. Additionally, macrophage CYP27B1 expression was significantly reduced in clinical cows. Taken together, the results from this study show disparate patterns of expression for key mediators in intracellular MAP trafficking and vitamin D metabolism for cows at different stages of paratuberculosis.

Introduction: Leucine-rich repetitive kinase-2 (LRRK2) is a Parkinson's disease-related gene that also participates in many inflammatory diseases. However, the functional role of LRRK2 in cardiovascular disease is not clear. Objective: In this study, we aimed to elucidate the role of LRRK2 in cardiac remodelling under pressure overload. Methods: Aortic banding surgery was performed to induce cardiac remodelling in a LRRK2 knockout mouse model. A cardiomyocyte remodelling model was established by phenylephrine (PE) stimulation in neonatal rat cardiomyocytes. Results: LRRK2 was upregulated in remodelled mouse hearts and cardiomyocytes. Cardiac hypertrophy, fibrosis and dysfunction were ameliorated in LRRK2 knockout mice. LRRK2 silencing protected against the PE-induced cardiomyocyte hypertrophic response, while LRRK2 over-expression worsened the PE-induced hypertrophic response in cardiomyocytes. Decreased autophagy was observed in remodelled cardiomyocytes, whereas LRRK2 silencing increased autophagy levels and LRRK2 overexpression reduced autophagy levels. The autophagy inhibitors 3-MA, bafilomycin and chloroquine reversed the protective effects of LRRK2 deficiency. The autophagy activator rapamycin reversed the deleterious effects of LRRK2 overexpression. We found that LRRK2 inhibited Bcl-2 phosphorylation, thus decreasing the phosphorylation of Beclin1. The protective effects of LRRK2 knockout were partly counteracted by Beclin1(+/−) in vivo and Beclin1 silencing in vitro. We also observed an interaction between LRRK2 and Rab7, an autolysosome degradation-associated protein, which caused Rab7 downregulation. Rab7 knockdown almost completely reversed LRRK2 silencing-induced protection of cardiomyocytes Conclusion: LRRK2 deficiency protected against cardiac remodelling under pressure overload by increasing Bcl-2/Beclin1 and Rab7-regulated autophagy levels in the heart.

Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants. [Display omitted] • VPS41 converts from condensates to VAPVs upon autophagy induction • Autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway • The condensates-to-VAPVs conversion pathway is essential for autophagy degradation • The condensates-to-VAPVs conversion pathway is crucial for normal autophagy progression In plants, the process through which autophagosomes are transported to the vacuole for degradation remains largely unknown. Jiang et al. report a condensates-to-VAPVs conversion pathway, which plays an essential role in the vacuolar transport of autophagosomes and the normal progression of autophagy. [ABSTRACT FROM AUTHOR]

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.

Macrophages are important host defense cells in ruminant paratuberculosis (Johne's Disease; JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). Classical macrophage functions of pathogen trafficking, degradation, and antigen presentation are interrupted in mycobacterial infection. Immunologic stimulation by 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D3 on macrophage phenotype and endosomal trafficking of MAP in monocyte-derived macrophages (MDMs) cultured from JD-, JD+ subclinical, and JD+ clinically infected cattle. MDMs were pre-treated 100 ng/ml 25(OH) D3 or 4 ng/ml 1,25(OH)2D3 and incubated 24 hrs with MAP at 10:1 multiplicity of infection (MOI). In vitro MAP infection upregulated pro-inflammatory (M1) CD80 and downregulated resolution/repair (M2) CD163. Vitamin D3 generally decreased CD80 and increased CD163 expression. Furthermore, early endosomal marker Rab5 was upregulated 140x across all stages of paratuberculosis infection following in vitro MAP infection; however, Rab5 was reduced in MAP-activated MDMs from JD+ subclinical and JD+ clinical cows compared to healthy controls. Rab7 expression decreased in control and clinical cows following MDM infection with MAP. Both forms of vitamin D3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH)2D3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro. Vitamin D3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D3 analog. Results from this study show exogenous vitamin D3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture. [ABSTRACT FROM AUTHOR]

Abstract Background Neuroinflammation and oxidative stress plays an important role in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). This study is the first to show that activation of autophagy protein nuclear receptor binding factor 2 (NRBF2) could reduce endoplasmic reticulum stress (ERS)-associated inflammation and oxidative stress after SAH. Methods Male C57BL/6J mice were subjected to endovascular perforation to establish a model of SAH. NRBF2 overexpression adeno-associated virus (AAV), NRBF2 small interfering RNAs (siRNA), lysosomal inhibitor-chloroquine (CQ), and late endosome GTPase Rab7 receptor antagonist-CID1067700 (CID) were used to investigate the role of NRBF2 in EBI after SAH. Neurological tests, brain water content, western blotting and immunofluorescence staining were evaluated. Results Our study found that the level of NRBF2 was increased after SAH and peaked at 24 h after SAH. In addition, we found that the overexpression of NRBF2 significantly improved neurobehavioral scores and reduced ERS, oxidative stress, and neuroinflammation in SAH, whereas the inhibition of NRBF2 exacerbated these phenotypes. In terms of mechanism, NRBF2 overexpression significantly promoted autophagosome maturation, with the downregulation of CHOP, Romo-1, TXNIP, NLRP3, TNF-α, and IL-1β expression through interaction with Rab7. The protective effect of NRBF2 on ERS-associated neuroinflammation and oxidative stress after SAH was eliminated by treatment with CQ. Meanwhile, it was also reversed by intraperitoneal injection of CID. Moreover, the MIT domain of NRBF2 was identified as a critical binding site that interacts with Rab7 and thereby promotes autophagosome maturation. Conclusion Our data provide evidence that the autophagy protein NRBF2 has a protective effect on endoplasmic reticulum stress-associated neuroinflammation and oxidative stress by promoting autophagosome maturation through interactions with Rab7 after SAH.

Macrophages are important host defense cells in ruminant paratuberculosis (Johne’s Disease; JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). Classical macrophage functions of pathogen trafficking, degradation, and antigen presentation are interrupted in mycobacterial infection. Immunologic stimulation by 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D3 on macrophage phenotype and endosomal trafficking of MAP in monocyte-derived macrophages (MDMs) cultured from JD-, JD+ subclinical, and JD+ clinically infected cattle. MDMs were pre-treated 100 ng/ml 25(OH)D3 or 4 ng/ml 1,25(OH)2D3 and incubated 24 hrs with MAP at 10:1 multiplicity of infection (MOI). In vitro MAP infection upregulated pro-inflammatory (M1) CD80 and downregulated resolution/repair (M2) CD163. Vitamin D3 generally decreased CD80 and increased CD163 expression. Furthermore, early endosomal marker Rab5 was upregulated 140× across all stages of paratuberculosis infection following in vitro MAP infection; however, Rab5 was reduced in MAP-activated MDMs from JD+ subclinical and JD+ clinical cows compared to healthy controls. Rab7 expression decreased in control and clinical cows following MDM infection with MAP. Both forms of vitamin D3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH)2D3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro. Vitamin D3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D3 analog. Results from this study show exogenous vitamin D3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture.

Gastrulation denotes a very important developmental process, which includes significant structural tissue rearrangements and patterning events that shape the emerging vertebrate organism. At the end of gastrulation, the three body axes are spatially defined while the left-right axis still lacks any molecular or morphological polarity. In most vertebrates, this is established during neurulation by a symmetry breaking LR organizer. However, this mesoderm-derived structure depends on proper induction and specification of the mesoderm, which in turn requires involvement of several signaling pathways. Endocytosis and the endosomal machinery offer manifold platforms for intracellular pathway regulation, especially late endosomes claim increasing attention. The late endosomal regulator Rab7 has been linked to mesoderm specification during gastrulation. Distinct axial defects due to compromised dorsal mesoderm development in rab7-deficient Xenopus embryos suggested a requirement of Rab7 for FGF-dependent mesoderm patterning and LR asymmetry. Here we specifically addressed such a role of Rab7, demonstrating a functional requirement for LR organizer development and symmetry breakage. Using different FGF/MAPK pathway components we show that Rab7 participates in dorsal mesoderm patterning. We suggest a hierarchical classification of Rab7 upstream of MAPK-dependent mesoderm specification, most probably at the level of the small GTPase Ras. Thus, this study affords an insight on how the Rab7-regulated endosomal machinery could participate in signal transduction to enable correct mesoderm specification and left-right asymmetry.

Objective: SARS CoV-2, the etiologic agent of coronavirus disease-2019 (COVID-19) is well-known to use ACE2 to begin internalization. Some viruses enter the host cell through the endocytosis process and involve some endocytosis proteins, such as the Rab family. However, the relationship between SARS CoV-2 infection with endocytic mRNA RAB5, RAB7, and RAB11B is unknown. This study aims to compare the expression of RAB5, RAB7, and RAB11B between positive and negative COVID-19 patient groups. Results: Both viral and human epithelial RNA Isolation and RT-PCR were performed from 249 samples. The genes expression was analysed using appropriate statistical tests. We found the Median (inter-quartile range/IQR) of RAB5, RAB7, and RAB11B expression among the COVID-19 patient group was 2.99 (1.88), 0.17 (0.47), 0.47 (1.49), and 1.60 (2.88), 1.05 (2.49), 1.10 (3.96) among control group respectively. We proceeded with Mann Whitney U Test and found that RAB5 expression was significantly increased (P < 0.001), and RAB7 and RAB11B expression was significantly decreased (P < 0.001 and P = 0.036) in the COVID-19 patient group compared to the control group. This first report showed significant differences in RAB5, RAB7, and RAB11B exist between COVID-19 positive and negative patients. [ABSTRACT FROM AUTHOR]

Rab7 is a GTPase that controls late endosome and lysosome trafficking. Recent studies have demonstrated that Rab7 is ubiquitinated, a post-translational modification mediated by an enzymatic cascade. To date, only one ubiquitin E3 ligase and one deubiquitinase have been identified in regulating Rab7 ubiquitination. Here, we report that RNF167, a transmembrane endolysosomal ubiquitin ligase, can ubiquitinate Rab7. Using immunoprecipitation and in vitro ubiquitination assays, we demonstrate that Rab7 is a direct substrate of RNF167. Subcellular fractionation indicates that RNF167 activity maintains Rab7′s membrane localization. Epifluorescence microscopy in HeLa cells shows that Rab7-positive vesicles are larger under conditions enabling Rab7 ubiquitination by RNF167. Characterization of its ubiquitination reveals that Rab7 must be in its GTP-bound active form for membrane anchoring and, thus, accessible for RNF167-mediated ubiquitin attachment. Cellular distribution analyses of lysosome marker Lamp1 show that vesicle positioning is independent of Rab7 and RNF167 expression and that Rab7 endosomal localization is not affected by RNF167 knockdown. However, both Rab7 and RNF167 depletion affect each other's lysosomal localization. Finally, this study demonstrates that the RNF167-mediated ubiquitination of Rab7 GTPase is impaired by variants of Charcot–Marie–Tooth Type 2B disease. This study identified RNF167 as a new ubiquitin ligase for Rab7 while expanding our knowledge of the mechanisms underlying the ubiquitination of Rab7. [ABSTRACT FROM AUTHOR]

Cancer cells undergo drastic metabolic adaptions to cover increased bioenergetic needs, contributing to resistance to therapies. This includes a higher demand for cholesterol, which often coincides with elevated cholesterol uptake from low-density lipoproteins (LDL) and overexpression of the LDL receptor in many cancers. This implies the need for cancer cells to accommodate an increased delivery of LDL along the endocytic pathway to late endosomes/lysosomes (LE/Lys), providing a rapid and effective distribution of LDL-derived cholesterol from LE/Lys to other organelles for cholesterol to foster cancer growth and spread. LDL-cholesterol exported from LE/Lys is facilitated by Niemann–Pick Type C1/2 (NPC1/2) proteins, members of the steroidogenic acute regulatory-related lipid transfer domain (StARD) and oxysterol-binding protein (OSBP) families. In addition, lysosomal membrane proteins, small Rab GTPases as well as scaffolding proteins, including annexin A6 (AnxA6), contribute to regulating cholesterol egress from LE/Lys. Here, we summarize current knowledge that links upregulated activity and expression of cholesterol transporters and related proteins in LE/Lys with cancer growth, progression and treatment outcomes. Several mechanisms on how cellular distribution of LDL-derived cholesterol from LE/Lys influences cancer cell behavior are reviewed, some of those providing opportunities for treatment strategies to reduce cancer progression and anticancer drug resistance. [ABSTRACT FROM AUTHOR]

E‐cadherin has a fundamental role in epithelial tissues by providing cell–cell adhesion. Polarised E‐cadherin exocytosis to the lateral plasma membrane is central for cell polarity and epithelial homeostasis. Loss of E‐cadherin secretion compromises tissue integrity and is a prerequisite for metastasis. Despite this pivotal role of E‐cadherin secretion, the transport mechanism is still unknown. Here we identify Myosin V as the motor for E‐cadherin secretion. Our data reveal that Myosin V and F‐actin are required for the formation of a continuous apicolateral E‐cadherin belt, the zonula adherens. We show by live imaging how Myosin V transports E‐cadherin vesicles to the plasma membrane, and distinguish two distinct transport tracks: an apical actin network leading to the zonula adherens and parallel actin bundles leading to the basal‐most region of the lateral membrane. E‐cadherin secretion starts in endosomes, where Rab11 and Sec15 recruit Myosin V for transport to the zonula adherens. We also shed light on the endosomal sorting of E‐cadherin by showing how Rab7 and Snx16 cooperate in moving E‐cadherin into the Rab11 compartment. Thus, our data help to understand how polarised E‐cadherin secretion maintains epithelial architecture and prevents metastasis. [ABSTRACT FROM AUTHOR]

In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 mm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux. [ABSTRACT FROM AUTHOR]

Parkinson's disease is mainly caused by aggregation of α-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain-barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinson's disease and related synucleinopathies. [ABSTRACT FROM AUTHOR]

Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.

Charcot-Marie-Tooth 2B peripheral sensory neuropathy (CMT2B) is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M) in Rab7 GTPase. Strong evidence suggests that the Rab7 mutation(s) enhances the cellular levels of activated Rab7 proteins, thus resulting in increased lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor (NGF) in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A "gain of toxicity" model has, thus, been proposed based on these observations. However, studies of fly photo-sensory neurons indicate that the Rab7 mutation(s) causes a "loss of function", resulting in haploinsufficiency. In the review, we summarize experimental evidence for both hypotheses. We argue that better models (rodent animals and human neurons) of CMT2B are needed to precisely define the disease mechanisms.

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34-/- mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of the plasticizer di-2-ethylhexyl phthalic acid (DEHP), and studies have shown that MEHP causes serious reproductive effects. However, its exact mechanisms of action remain elusive. In this study, we aimed to investigate the reproductive effects of MEHP and preliminarily explore its underlying molecular mechanisms. We found that TM3 cells gradually secreted less testosterone and intracellular free cholesterol with increasing MEHP exposure. MEHP exposure inhibited lipophagy and the Sirt1/Foxo1/Rab7 signaling pathway in TM3 cells, causing aberrant accumulation of intracellular lipid droplets. Addition of the Sirt1 agonist SRT1720 and Rab7 agonist ML-098 alleviated the inhibition of lipophagy and increased free cholesterol and testosterone contents in TM3 cells. SRT1720 alleviated the inhibitory effect of MEHP on the Sirt1/Foxo1/Rab7 signaling pathway, whereas ML-098 only alleviated the inhibition of Rab7 protein expression by MEHP and had no effect on Sirt1 and Foxo1 protein expression. This suggests that MEHP inhibits lipophagy in TM3 cells by suppressing the Sirt1/Foxo1/Rab7 signaling pathway, ultimately leading to a further decrease in cellular testosterone secretion. This study improves our current understanding of the toxicity and molecular mechanisms of action of MEHP and provides new insights into the reproductive effects of phthalic acid esters. [Display omitted] • Lipophagy played a key role in MEHP-induced testosterone biosynthesis disruption in TM3 cell. • MEHP inhibits lipophagy in TM3 cells by suppressing the Sirt1/Foxo1/Rab7 signaling pathway. • Pretreatment with Sirt1 agonist SRT1720 and Rab7 agonist ML-098 attenuates MEHP-induced testosterone synthesis reduction. [ABSTRACT FROM AUTHOR]

Abstract Background Mesenchymal stem cells (MSCs) are pluripotent stromal cells that release extracellular vesicles (EVs). EVs contain various growth factors and antioxidants that can positively affect the surrounding cells. Nanoscale MSC-derived EVs, such as exosomes, have been developed as bio-stable nano-type materials. However, some issues, such as low yield and difficulty in quantification, limit their use. We hypothesized that enhancing exosome production using nanoparticles would stimulate the release of intracellular molecules. Results The aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified, positively charged nanoparticles and exosome generation from MSCs. We determined that Rab7, a late endosome and auto-phagosome marker, was increased upon exosome expression and was associated with autophagosome formation. Conclusions It was concluded that the nanoparticles we developed were transported to the lysosome by clathrin-mediated endocytosis. additionally, entered nanoparticles stimulated that autophagy related factors to release exosome from the MSC. MSC-derived exosomes using nanoparticles may increase exosome yield and enable the discovery of nanoparticle-induced genetic factors.

Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

Historically, the genetic analysis of mammalian cells entailed the isolation of randomly arising mutant cell lines with altered properties, followed by laborious genetic mapping experiments to identify the mutant gene responsible for the phenotype. In recent years, somatic cell genetics has been revolutionized by functional genomics screens, in which expression of every protein‐coding gene is systematically perturbed, and the phenotype of the perturbed cells is determined. We outline here a novel functional genomics screening strategy that differs fundamentally from commonly used approaches. In this strategy, we express libraries of artificial transmembrane proteins named traptamers and select rare cells with the desired phenotype because, by chance, a traptamer specifically perturbs the expression or activity of a target protein. Active traptamers are then recovered from the selected cells and can be used as tools to dissect the biological process under study. We also briefly describe how we have used this new strategy to provide insights into the complex process by which human papillomaviruses enter cells. [ABSTRACT FROM AUTHOR]

The Toll-Dorsal signaling pathway controls dorsal-ventral (DV) patterning in early Drosophila embryos, which defines specific cell fates along the DV axis and controls morphogenetic behavior of cells during gastrulation and beyond. The extent by which DV patterning information regulates subcellular organization in pre-gastrulation embryos remains unclear. We find that during Drosophila cleavage, the late endosome marker Rab7 is increasingly recruited to the yolk granules and promotes the formation of dynamic membrane tubules. The biogenesis of yolk granule tubules is positively regulated by active Rab7 and its effector complex HOPS, but negatively regulated by the Rab7 effector retromer. The occurrence of tubules is strongly biased towards the ventral side of the embryo, which we show is controlled by the Toll-Dorsal signaling pathway. Our work provides the first evidence for the formation and regulation of yolk granule tubulation in oviparous embryos and elucidates an unexpected role of Toll-Dorsal signaling in regulating this process. • Rab7 is recruited to yolk granules and promotes granule tubulation during cellularization. • Yolk granule tubulation is specifically upregulated at the ventral region. • Toll-Dorsal signaling triggers upregulation of yolk granule tubulation. • Yolk granule tubulation requires microtubules and the Rab7 effector complex HOPS. • The Rab7 effector complex retromer represses yolk granule tubulation. [ABSTRACT FROM AUTHOR]