RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Effect of MEHP on testosterone synthesis via Sirt1/Foxo1/Rab7 signaling pathway inhibition of lipophagy in TM3 cells.

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.

The vesicle trafficking gene, OsRab7, is critical for pollen development and male fertility in cytoplasmic male-sterility rice.

Rice cytoplasmic male sterility (CMS) provides an exceptional model for studying genetic interaction within plant nuclei given its inheritable trait of non-functional male gametophyte. Gaining a comprehensive understanding of the genes and pathways associated with the CMS mechanism is imperative for improving the vigor of hybrid rice agronomically, such as its productivity. Here, we observed a significant decrease in the expression of a gene named OsRab7 in the anther of the CMS line (SJA) compared to the maintainer line (SJB). OsRab7 is responsible for vesicle trafficking and loss function of OsRab7 significantly reduced pollen fertility and setting rate relative to the wild type. Meanwhile, over-expression of OsRab7 enhanced pollen fertility in the SJA line while a decrease in its expression in the SJB line led to the reduced pollen fertility. Premature tapetum and abnormal development of microspores were observed in the rab7 mutant. The expression of critical genes involved in tapetum development (OsMYB103, OsPTC1, OsEAT1 and OsAP25) and pollen development (OsMSP1, OsDTM1 and OsC4) decreased significantly in the anther of rab7 mutant. Reduced activities of the pDR5::GUS marker in the young panicle and anther of the rab7 mutant were also observed. Furthermore, the mRNA levels of genes involved in auxin biosynthesis (YUCCAs), auxin transport (PINs), auxin response factors (ARFs), and members of the IAA family (IAAs) were all downregulated in the rab7 mutant, indicating its impact on auxin signaling and distribution. In summary, these findings underscore the importance of OsRab7 in rice pollen development and its potential link to cytoplasmic male sterility.

Activation of lipophagy is required for RAB7 to regulate ferroptosis in sepsis-induced acute kidney injury.

Sepsis-induced acute kidney injury (S-AKI) is the most common type of acute kidney injury (AKI), accompanied by elevated morbidity and mortality rates. This study investigated the mechanism by which lipid droplets (LDs) degraded via autophagy (lipophagy)required for RAB7 regulated ferroptosis in the pathogenesis of S-AKI. Here, we constructed the S-AKI model in vitro and in vivo to elucidate the potential relationship of lipophagy and ferroptosis, and we first confirmed that the activation of lipophagy promoted renal tubular epithelial cell ferroptosis and renal damage in S-AKI. The results showed that lipopolysaccharide (LPS) induced a marked increase in lipid peroxidation and ferroptosis, which were rescued by ferrstain-1 (Fer-1), an inhibitor of ferroptosis. In addition, LPS induced the remarkable activation of RAB7-mediated lipophagy. Importantly, silencing RAB7 alleviated LPS-induced lipid peroxidation and ferroptosis. Thus, the present study demonstrated the potential significant role of ferroptosis and lipophagy in sepsis-induced AKI, and contributed to better understanding of the pathogenesis and treatment targets of AKI.

Acetyl-11-keto-beta-boswellic acid inhibits cell proliferation and growth of oral squamous cell carcinoma via RAB7B-mediated autophagy.

Natural products can overcome the limitations of conventional chemotherapy. Acetyl-11-keto-beta-boswellic acid (AKBA) as a natural product extracted from frankincense, exhibited chemotherapeutic activities in different cancers. However, whether AKBA exerts inhibiting effect of oral squamous cell carcinoma (OSCC) cells growth and the mechanism need to be explored. We attempted to investigate the therapeutic effects of AKBA against OSCC and explore the mechanism involved. Here we attempt to disclose the cell-killing effect of AKBA on OSCC cell lines and try to figure out the specifical pathway. The presence of increase autophagosome and the production of mitochondrial reactive oxygen species were confirmed after the application of AKBA on OSCC cells, and RAB7B inhibition enhanced autophagosome accumulation. Though the increase autophagosome was detected induced by AKBA, autophagic flux was inhibited as the failure fusion of autophagosome and lysosome. Cal27 xenografts were established to verify the role of anti-OSCC cells of AKBA in vivo. Based above findings, we speculate that natural product AKBA suppresses OSCC cells growth via RAB7B-mediated autophagy and may serve as a promising strategy for the therapy of OSCC.

Role of the Cytosolic Domain of the a3 Subunit of V-ATPase in the Interaction with Rab7 and Secretory Lysosome Trafficking in Osteoclasts.

We previously reported that the a3 subunit of proton-pumping vacuolar-type ATPase (V-ATPase) interacts with Rab7 and its guanine nucleotide exchange factor, Mon1a-Ccz1, and recruits them to secretory lysosomes in osteoclasts, which is essential for anterograde trafficking of secretory lysosomes. The a3 subunit interacts with Mon1a-Ccz1 through its cytosolic N-terminal domain. Here, we examined the roles of this domain in the interaction with Rab7 and trafficking of secretory lysosomes. Immunoprecipitation experiments showed that a3 interacted with Rab7 through its cytosolic domain, similar to the interaction with Mon1a-Ccz1. We connected this domain with a lysosome localization signal and expressed it in a3-knockout (a3KO) osteoclasts. Although the signal connected to the cytosolic domain was mainly detected in lysosomes, impaired lysosome trafficking in a3KO osteoclasts was not rescued. These results indicate that the cytosolic domain of a3 can interact with trafficking regulators, but is insufficient to induce secretory lysosome trafficking. The C-terminal domain of a3 and other subunits of V-ATPase are likely required to form a fully functional complex for secretory lysosome trafficking.

The Small GTPase Rab7 Regulates Antigen Processing in B Cells in a Possible Interplay with Autophagy Machinery.

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.

Super-resolution microscopy: Insights into mitochondria-lysosome crosstalk in health and disease.

Live super-resolution microscopy has allowed for new insights into recently identified mitochondria-lysosome contact sites, which mediate crosstalk between mitochondria and lysosomes, including co-regulation of Rab7 GTP hydrolysis and Drp1 GTP hydrolysis. Here, we highlight recent findings and future perspectives on this dynamic pathway and its roles in health and disease.

Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines.

The interaction manner and biological function of Rab7 and its effector, Rab-interacting lysosomal protein (RILP), remain unclear in invertebrates. We provide a protocol for detecting the effects of Rab7 and RILP terminals on lysosome and autophagy in Spodoptera frugiperda Sf9 cells with overexpression and RNA interference. We describe steps for overexpressing plasmids, generating long double-stranded RNA, and transfecting them into Sf9 cells. We then detail procedures for cell immunofluorescence imaging with harmine treatment and fluorescence analysis. For complete details on the use and execution of this protocol, please refer to Cui et al. (2023).1.

Misregulation of mitochondria-lysosome contact dynamics in Charcot-Marie-Tooth Type 2B disease Rab7 mutant sensory peripheral neurons.

Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.

Disruption of Golgi markers by two RILP-directed shRNAs in neurons: A new role for RILP or a neuron-specific off-target phenotype?

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. To test if the dynein adapter RAB-interacting lysosomal protein (RILP) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents previously validated in non-neuronal cells. Striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of staining. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. It would be interesting to identify the actual target for this neuronal Golgi phenotype. Cell type-specific off-target phenotypes therefore likely occur in neurons, necessitating revalidation of reagents that were previously validated in other cell types.

[Silencing SIRT1 reduces 5-fluorouracil resistance of cholangiocarcinoma cells by inhibiting the FOXO1/Rab7 autophagy pathway].

OBJECTIVE: To investigate the mechanism by which SIRT1 silencing reduces 5-fluorouracil (5-FU) resistance of cholangiocarcinoma cells and the role of FOXO1/Rab7 autophagy pathway in mediating this effect. METHODS: Human cholangiocarcinoma HCCC-9810 cells were treated with 50, 100, 150, and 200 µg/mL 5-FU to construct a 5-FU-resistant cell model, whose expressions of SIRT1, FOXO1 and Rab7 were detected with immunofluorescence assay, Western blotting and RTqPCR, and the expression levels of autophagy related proteins (Beclin1, LC3, and p62) were detected with Western blotting. The 5-FU resistant cells were transfected with a SIRT1 siRNA, and the changes in 5-Fu resistance and migration ability of the cells were evaluated using CCK-8 assay and wound healing assay; The changes in FOXO1 and Rab7 mRNA levels and protein expressions of SIRT1, FOXO1, Rab7, Beclin1, LC3 and P62 were detected with RT-qPCR and Western blotting. RESULTS: Treatments with 5-FU at 50, 100, 150, and 200 µg/mL all inhibited the proliferation of HCCC-9810 cells. Immunofluorescence assay revealed significantly enhanced SIRT1 expression in 5-FU-resistant HCC-9810 cells, and Western blotting also showed significantly up-regulated protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin 1 (P < 0.001) and an increased LC3II/LC3I ratio in the cells (P < 0.001). The mRNA levels of SIRT1, Rab7 and FOXO1 were also up-regulated in 5-Fu-resistant cells (P < 0.05). SIRT1 silencing significantly attenuated 5-FU resistance and migration ability of HCCC-9810 cells, and obviously decreased the protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin1 and the LC3II/LC3I ratio as well (P < 0.001). FOXO1 and Rab7 mRNA levels were significantly decreased in 5-FU-resistant HCC-9810 cells after SIRT1 silencing (P < 0.05). CONCLUSION: Silencing SIRT1 attenuates 5-FU resistance in HCC-9810 cells by inhibiting the activation of the FOXO1/Rab7 autophagy pathway.

Rab7a-mTORC1 signaling-mediated cholesterol trafficking from the lysosome to mitochondria ameliorates hepatic lipotoxicity induced by aflatoxin B1 exposure.

Aflatoxin B1 (AFB1) is a common contaminant in many foodstuffs and is considered a public health concern worldwide due to its hepatotoxicity caused by lipid metabolism disorders. However, the molecular mechanism underlying AFB1-induced lipotoxicity-dependent liver injury via regulating cholesterol metabolism remains unclear. We established a cholesterol trafficking disorder-mediated hepatic lipotoxicity model with AFB1 mixture exposure in vitro (HepaRG and HepG2 cells, 1.6 µM for 36 h) and in vivo (C57BL/6 mice, 3 mg kg-1, i.g., every other day for 6 weeks). In vitro, the interaction between lysosomal Niemann-Pick type C1 (NPC1) protein and mitochondrial translocator protein (TSPO) regulated lipotoxicity induced by AFB1 mixture exposure, including lysosomal membrane permeabilization and mitochondria-dependent necroptosis. Moreover, the downregulation of lysosomal Ras-associated protein 7a (Rab7a) enhanced the mammalian target of rapamycin complex 1 (mTORC1)-mediated disorders of cholesterol trafficking from the lysosome to mitochondria. Furthermore, cholesterol trafficking disorder-mediated hepatic lipotoxicity induced by the low-dose level of AFB1 exposure was relieved by genetic or pharmaceutic activation of Rab7a to inhibit mTORC1 in vitro and ex vivo. In vivo, mTORC1 inhibitor (Torin1, 4 mg kg-1, i.p., every other day for 3 weeks) alleviated the cholesterol trafficking disorder-mediated hepatic lipotoxicity via upregulating the molecular machinery of lysosomes and mitochondria contact mediated by NPC1 and TSPO interaction in the low dose of AFB1 exposure. Altogether, our data suggested a novel mechanism that lysosomal Rab7a-mTORC1 signaling determined the cholesterol trafficking regulated by NPC1-TSPO from the lysosome to mitochondria, which promoted hepatic lipotoxicity via lysosomal quality control and mitochondria-dependent necroptosis signaling pathways in chemical mixture exposure.

Micro/nano-modified titanium surfaces accelerate osseointegration via Rab7-dependent mitophagy.

To achieve rapid and successful osseointegration of titanium (Ti) implants, the underlying mechanisms of surface modification-mediated bone metabolism need to be clarified. Given that the microenvironment surrounding Ti implants may be altered after implant insertion, mitophagy as a key control system for cellular homeostasis is most likely to regulate osseointegration. Recent findings suggest that PTEN-induced putative kinase 1 (Pink1)/Parkin-mediated mitophagy plays a key role in bone metabolism. Since the micro/nano-modified surfaces of Ti implants have been widely appreciated for osseointegration acceleration, we used two common micro/nano-modified techniques and demonstrated elevations of both the osteo-differentiation potential and Pink1/Parkin pathway of osteoblasts. Moreover, the Pink1/Parkin pathway exhibited an upward trend during osteoblast differentiation. However, when osteoblasts were treated with CCCP, a Pink1/Parkin inducer, the osteo-differentiation potential decreased. Our further study showed that the small GTPase Rab7, which was inhibited by CCCP, was essential for the Pink1/Parkin pathway. Upon Pink1 or Rab7 knockdown, the pro-osteogenic effect of micro/nano-modified Ti surfaces was significantly weakened. The present results demonstrated that Rab7 activation was essential for active mitophagy and osteogenesis. In addition, Rab7 was confirmed to mediate the process of autophagosome formation. Our findings provide novel insights into new targets for osseointegration promotion, regardless of Ti surface characteristics.

RAB7A GTPase Is Involved in Mitophagosome Formation and Autophagosome-Lysosome Fusion in N2a Cells Treated with the Prion Protein Fragment 106-126.

Failed communication between mitochondria and lysosomes causes dysfunctional mitochondria, which may induce mitochondria-related neurodegenerative diseases. Here, we show that RAB7A, a small GTPase of the Rab family, mediates the crosstalk between these two important organelles to maintain homeostasis in N2a cells treated with PrP106-126. Specifically, we demonstrate that mitophagy deficiency in N2a cells caused by PrP106-126 is associated with dysregulated RAB7A localization in mitochondria. Cells lacking RAB7A display decreased mitochondrial colocalization with lysosomes and significantly increased mitochondrial protein expression, resulting in inhibited mitophagy. In contrast, overexpression of GTP-bound RAB7A directly induces lysosome colocalization with mitochondria. Further study revealed that GTP-bound RAB7A protects mitochondrial homeostasis by supporting autophagosome biogenesis. Moreover, we suggest that depletion of RAB7A leads to gross morphological changes in lysosomes, which prevents autophagosome-lysosome fusion and interferes with the breakdown of autophagic cargo within lysosomes. Overexpression of GTP-bound RAB7A can also alleviate PrP106-126-induced morphological damage and dysfunction of mitochondria, reducing neuronal apoptosis. Collectively, our data demonstrate that RAB7A successfully drives mitochondria to the autophagosomal lumen for degradation, suggesting that the communication of proteotoxic stress from mitochondria to lysosomes requires RAB7A, as a signaling molecule, to establish a link between the disturbed mitochondrial network and its remodeling. These findings indicate that small molecules regulating mitophagy have the potential to modulate cellular homeostasis and the clinical course of neurodegenerative diseases. Proposed model of mitophagy regulated by RAB7A. (1) Accumulating PrP106-126 induced mitophagy. (2) RAB7A is recruited to mitochondria. (3) ATG5-12 and ATG9A (5) vesicles are recruited to the autophagosome formation sites in a RAB7A-dependent manner. The ATG5-12 complex recruits and anchors LC3-I to form active LC3-II (4), accelerating mitophagosomal formation. The ATG9A vesicles are thought to be a source of membranes for autophagosome assembly. The recruitment of proteins and lipids induces membrane expansion and subsequent closure to form the mitophagosome. (6) Maintenance of the normal low lysosomal PH depends on active (GTP-bound) RAB7A. (7) RAB7A recruits effector molecules responsible for tight membrane interactions, and directly or indirectly, the subsequent autophagosome merges with the lysosome, and the cargo is completely degraded.