Drugs of abuse hijack a mesolimbic pathway that processes homeostatic need.

Drugs of abuse are thought to promote addiction in part by "hijacking" brain reward systems, but the underlying mechanisms remain undefined. Using whole-brain FOS mapping and in vivo single-neuron calcium imaging, we found that drugs of abuse augment dopaminoceptive ensemble activity in the nucleus accumbens (NAc) and disorganize overlapping ensemble responses to natural rewards in a cell type-specific manner. Combining FOS-Seq, CRISPR-perturbation, and single-nucleus RNA sequencing, we identified Rheb as a molecular substrate that regulates cell type-specific signal transduction in NAc while enabling drugs to suppress natural reward consumption. Mapping NAc-projecting regions activated by drugs of abuse revealed input-specific effects on natural reward consumption. These findings characterize the dynamic, molecular and circuit basis of a common reward pathway, wherein drugs of abuse interfere with the fulfillment of innate needs.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33 Virus-infected Mammalian Cells.

BACKGROUND: The Mammalian Target of Rapamycin (mTOR) signaling pathway regulates protein phosphorylation and exerts control over major cellular processes. mTOR is activated by the small G-protein Ras Homolog Enriched in Brain (Rheb), which is encoded by the Rheb1 and Rheb-like-1 (RhebL1) genes. There is currently a paucity of information on the role of RhebL1, and specifically its involvement in viral infection. In the present study we investigated the role of RhebL1 during human influenza A/NWS/33 (NWS/33) (H1N1) virus infection of rhesus monkey-kidney (LLC-MK2) cells and human type II alveolar epithelial (A549) cells. METHODS: To assess the efficiency of NWS/33 virus replication, the expression of viral nucleoprotein was examined by indirect immunofluorescence (IIF) and the viral yield by fifty percent tissue culture infectious dose assay. An RNA-mediated RNA interference approach was used to investigate the role of RhebL1 during NWS/33 infection. RhebL1 expression was evaluated by IIF, Western blotting, and enzyme-linked immunosorbent assays. A two-tailed Student's t-test was applied to evaluate differences between groups. RESULTS: RhebL1 was differentially expressed in the cell models used in this study. Silencing of the RhebL1 gene led to increased NWS/33 virus infection in A549 cells, but not in LLC-MK2 cells. Moreover, the expression of hyperphosphorylated cytokeratin 8, a marker of NWS/33 virus infection efficiency, increased in A549 cells depleted of RhebL1 but remained almost unchanged in LLC-MK2 cells. CONCLUSIONS: These are the first results showing involvement of the endogenous RhebL1 protein during viral infection. Our data suggests that RhebL1 exerts a host cell-dependent modulatory role during influenza virus infection. RhebL1 appears to be a restrictive factor against NWS/33 virus replication in A549 cells, but not in LLC-MK2.

The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

Unraveling the Role of Ras Homolog Enriched in Brain (Rheb1 and Rheb2): Bridging Neuronal Dynamics and Cancer Pathogenesis through Mechanistic Target of Rapamycin Signaling.

Ras homolog enriched in brain (Rheb1 and Rheb2), small GTPases, play a crucial role in regulating neuronal activity and have gained attention for their implications in cancer development, particularly in breast cancer. This study delves into the intricate connection between the multifaceted functions of Rheb1 in neurons and cancer, with a specific focus on the mTOR pathway. It aims to elucidate Rheb1's involvement in pivotal cellular processes such as proliferation, apoptosis resistance, migration, invasion, metastasis, and inflammatory responses while acknowledging that Rheb2 has not been extensively studied. Despite the recognized associations, a comprehensive understanding of the intricate interplay between Rheb1 and Rheb2 and their roles in both nerve and cancer remains elusive. This review consolidates current knowledge regarding the impact of Rheb1 on cancer hallmarks and explores the potential of Rheb1 as a therapeutic target in cancer treatment. It emphasizes the necessity for a deeper comprehension of the molecular mechanisms underlying Rheb1-mediated oncogenic processes, underscoring the existing gaps in our understanding. Additionally, the review highlights the exploration of Rheb1 inhibitors as a promising avenue for cancer therapy. By shedding light on the complicated roles between Rheb1/Rheb2 and cancer, this study provides valuable insights to the scientific community. These insights are instrumental in guiding the identification of novel targets and advancing the development of effective therapeutic strategies for treating cancer.

CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

Design of negative-regulating proteins of Rheb/mTORC1 with much-reduced sizes of the tuberous sclerosis protein complex.

The mTORC1 signaling pathway regulates cell growth and metabolism in a variety of organisms from yeast to human, and inhibition of the mTORC1 pathway has the prospect to treat cancer or achieve longevity. The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700 kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2-GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG-MTM (short stabilization domain and GAP domain-membrane targeting motif) and SSG-TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much-reduced sizes (~1/15 and ~ 1/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down-regulating catabolism and autophagy.

Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis.

How neuronal signaling affects brain myelination remains poorly understood. We show dysregulated neuronal RHEB-mTORC1-DLK1 axis impairs brain myelination. Neuronal Rheb cKO impairs oligodendrocyte differentiation/myelination, with activated neuronal expression of the imprinted gene Dlk1. Neuronal Dlk1 cKO ameliorates myelination deficit in neuronal Rheb cKO mice, indicating that activated neuronal Dlk1 expression contributes to impaired myelination caused by Rheb cKO. The effect of Rheb cKO on Dlk1 expression is mediated by mTORC1; neuronal mTor cKO and Raptor cKO and pharmacological inhibition of mTORC1 recapitulate elevated neuronal Dlk1 expression. We demonstrate that both a secreted form of DLK1 and a membrane-bound DLK1 inhibit the differentiation of cultured oligodendrocyte precursor cells into oligodendrocytes expressing myelin proteins. Finally, neuronal expression of Dlk1 in transgenic mice reduces the formation of mature oligodendrocytes and myelination. This study identifies Dlk1 as an inhibitor of oligodendrocyte myelination and a mechanism linking altered neuronal signaling with oligodendrocyte dysfunction.

Arf5-mediated regulation of mTORC1 at the plasma membrane.

The mechanistic target of rapamycin (mTOR) kinase regulates a major signaling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localized at other locations. However, little is known about the recruitment and activation of mTORC1 at nonlysosomal sites. To identify regulators of mTORC1 recruitment to nonlysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signaling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2-enriched membrane ruffles together with the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor, and Arf5 was shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and refeeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.

GOLPH3 protein controls organ growth by interacting with TOR signaling proteins in Drosophila.

The oncoprotein GOLPH3 (Golgi phosphoprotein 3) is an evolutionarily conserved phosphatidylinositol 4-phosphate effector, mainly localized to the Golgi apparatus, where it supports organelle architecture and vesicular trafficking. Overexpression of human GOLPH3 correlates with poor prognosis in several cancer types and is associated with enhanced signaling downstream of mTOR (mechanistic target of rapamycin). However, the molecular link between GOLPH3 and mTOR remains elusive. Studies in Drosophila melanogaster have shown that Translationally controlled tumor protein (Tctp) and 14-3-3 proteins are required for organ growth by supporting the function of the small GTPase Ras homolog enriched in the brain (Rheb) during mTORC1 (mTOR complex 1) signaling. Here we demonstrate that Drosophila GOLPH3 (dGOLPH3) physically interacts with Tctp and 14-3-3ζ. RNAi-mediated knockdown of dGOLPH3 reduces wing and eye size and enhances the phenotypes of Tctp RNAi. This phenotype is partially rescued by overexpression of Tctp, 14-3-3ζ, or Rheb. We also show that the Golgi localization of Rheb in Drosophila cells depends on dGOLPH3. Consistent with dGOLPH3 involvement in Rheb-mediated mTORC1 activation, depletion of dGOLPH3 also reduces levels of phosphorylated ribosomal S6 kinase, a downstream target of mTORC1. Finally, the autophagy flux and the expression of autophagic transcription factors of the TFEB family, which anti correlates with mTOR signaling, are compromised upon reduction of dGOLPH3. Overall, our data provide the first in vivo demonstration that GOLPH3 regulates organ growth by directly associating with mTOR signaling proteins.

The Rheb GTPase promotes pheromone blindness via a TORC1-independent pathway in the phytopathogenic fungus Ustilago maydis.

The target of the rapamycin (TOR) signaling pathway plays a negative role in controlling virulence in phytopathogenic fungi. However, the actual targets involved in virulence are currently unknown. Using the corn smut fungus Ustilago maydis, we tried to address the effects of the ectopic activation of TOR on virulence. We obtained gain-of-function mutations in the Rheb GTPase, one of the conserved TOR kinase regulators. We have found that unscheduled activation of Rheb resulted in the alteration of the proper localization of the pheromone receptor, Pra1, and thereby pheromone insensitivity. Since pheromone signaling triggers virulence in Ustilaginales, we believe that the Rheb-induced pheromone blindness was responsible for the associated lack of virulence. Strikingly, although these effects required the concourse of the Rsp5 ubiquitin ligase and the Art3 α-arrestin, the TOR kinase was not involved. Several eukaryotic organisms have shown that Rheb transmits environmental information through TOR-dependent and -independent pathways. Therefore, our results expand the range of signaling manners at which environmental conditions could impinge on the virulence of phytopathogenic fungi.

Purification and biochemical characterization of the Rag GTPase heterodimer.

The mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient levels in the cell and based on the availability, regulates cellular growth and proliferation. Its activity is tightly modulated by two GTPase units, the Rag GTPases and the Rheb GTPase. The Rag GTPases are the central hub of amino acid sensing as they summarize the amino acid signals from upstream regulators and control the subcellular localization of mTORC1. Unique from canonical signaling GTPases, the Rag GTPases are obligatory heterodimers, and the two subunits coordinate their nucleotide loading states to regulate their functional states. Robust biochemical analysis is indispensable to understanding the molecular mechanism governing the GTPase cycle. This chapter discusses protocols for purifying and biochemically characterizing the Rag GTPase heterodimer. We described two purification protocols to recombinantly produce the Rag GTPase heterodimer in large quantities. We then described assays to quantitatively measure the nucleotide binding and hydrolysis by the Rag GTPases. These assays allow for a thorough investigation of this unique heterodimeric GTPase, and they could be applicable to investigations of other noncanonical GTPases.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

miR-199a-3p/5p regulate tumorgenesis via targeting Rheb in non-small cell lung cancer.

Lung cancer is one of the deadliest cancers, in which non-small cell lung cancer (NSCLC) accounting for 85% and has a low survival rate of 5 years. Dysregulation of microRNAs (miRNAs) can participate in tumor regulation and many major diseases. In this study, we found that miR-199a-3p/5p were down-expressed in NSCLC tissue samples, cell lines, and the patient sample database. MiR-199a-3p/5p overexpression could significantly suppress cell proliferation, migration ability and promote apoptosis. Through software prediction, ras homolog enriched in brain (Rheb) was identified as a common target of miR-199a-3p and miR-199a-5p, which participated in regulating mTOR signaling pathway. The same effect of inhibiting NSCLC appeared after down-regulating the expression of Rheb. Furthermore, our findings revealed that miR-199a can significantly inhibit tumor growth and metastasis in vivo, which fully demonstrates that miR-199a plays a tumor suppressive role in NSCLC. In addition, miR-199a-3p/5p has been shown to enhance the sensitivity of gefitinib to EGFR-T790M in NSCLC. Collectively, these results prove that miR-199a-3p/5p can act as cancer suppressor genes to inhibit the mTOR signaling pathway by targeting Rheb, which in turn inhibits the regulatory process of NSCLC. Thus, to investigate the anti-cancer effect of pre-miR-199a/Rheb/mTOR axis in NSCLC, miR-199a-3p and miR-199a-5p have the potential to become an early diagnostic marker or therapeutic target for NSCLC.

Deletion of Rheb1 in Osteocytes Leads to Osteopenia Characterized by Reduced Bone Formation and Enhanced Bone Resorption.

Ras homologue enriched in brain 1 (Rheb1), an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), is known to modulate various cellular processes. However, its impact on bone metabolism in vivo remains unknown. The study aimed at understanding the role of Rheb1 on bone homeostasis. We measured the serum parameters and performed histomorphometry, quantitative real-time polymerase chain reaction, and Western blotting, along with the generation of mouse gene knockout (KO) model, and conducted a microcomputed tomography analysis and tartrate-resistant acid phosphatase staining, to delineate the impacts of Rheb1 on bone homeostasis. In the Rheb1 KO mice, the results showed that Rheb1 KO caused significant damage to the bone microarchitecture, indicating that mTORC1 activity was essential for the regulation of bone homeostasis. Specifically, suppressed mineralization activity in primary osteoblasts and a decreased osteoblast number were observed in the Rheb1 KO mice, demonstrating that loss of Rheb1 led to impaired osteoblastic differentiation. Furthermore, the higher apoptotic ratio in Rheb1-null osteocytes could promote Tnfsf11 expression and lead to an increase in osteoclasts, indicating increased bone resorption activity in the KO mice. The findings confirmed that Rheb1 deletion in osteoblasts/osteocytes led to osteopenia due to impaired bone formation and enhanced bone resorption.

Translation initiation factor eIF3a regulates glucose metabolism and cell proliferation via promoting small GTPase Rheb synthesis and AMPK activation.

Eukaryotic translation initiation factor 3 subunit A (eIF3a), the largest subunit of the eIF3 complex, has been shown to be overexpressed in malignant cancer cells, potentially making it a proto-oncogene. eIF3a overexpression can drive cancer cell proliferation but contributes to better prognosis. While its contribution to prognosis was previously shown to be due to its function in suppressing synthesis of DNA damage repair proteins, it remains unclear how eIF3a regulates cancer cell proliferation. In this study, we show using genetic approaches that eIF3a controls cell proliferation by regulating glucose metabolism via the phosphorylation and activation of AMP-activated protein kinase alpha (AMPKα) at Thr172 in its kinase activation loop. We demonstrate that eIF3a regulates AMPK activation mainly by controlling synthesis of the small GTPase Rheb, largely independent of the well-known AMPK upstream liver kinase B1 and Ca2+/calmodulin-dependent protein kinase kinase 2, and also independent of mammalian target of rapamycin signaling and glucose levels. Our findings suggest that glucose metabolism in and proliferation of cancer cells may be translationally regulated via a novel eIF3a-Rheb-AMPK signaling axis.

Rheb regulates nuclear mTORC1 activity independent of farnesylation.

The small GTPase Ras homolog enriched in brain (Rheb) plays a critical role in activating the mechanistic target of rapamycin complex 1 (mTORC1), a signaling hub that regulates various cellular functions. We recently observed nuclear mTORC1 activity, raising an intriguing question as to how Rheb, which is known to be farnesylated and localized to intracellular membranes, regulates nuclear mTORC1. In this study, we found that active Rheb is present in the nucleus and required for nuclear mTORC1 activity. We showed that inhibition of farnesyltransferase reduced cytosolic, but not nuclear, mTORC1 activity. Furthermore, a farnesylation-deficient Rheb mutant, with preferential nuclear localization and specific lysosome tethering, enables nuclear and cytosolic mTORC1 activities, respectively. These data suggest that non-farnesylated Rheb is capable of interacting with and activating mTORC1, providing mechanistic insights into the molecular functioning of Rheb as well as regulation of the recently observed, active pool of nuclear mTORC1.

miR-155-5p in Extracellular Vesicles Derived from Choroid Plexus Epithelial Cells Promotes Autophagy and Inflammation to Aggravate Ischemic Brain Injury in Mice.

Ischemic stroke is a common disease of the central nervous system, and ischemic brain injury (IBI) is its main manifestation. Recently, extracellular vesicles (EVs) have been strongly related to the diagnosis and treatment of IBI. However, the underlying mechanism of their effects remains enigmatic. In the present study, we aimed to study how miR-155-5p plays a role in choroid plexus epithelial (CPE) cell-derived EVs in IBI pathology. We found that miR-155-5p expression was enriched in CPE cell-derived EVs, which were subsequently internalized by neurons, enabling the delivery of miR-155-5p into neurons. An inducible oxygen and glucose deprivation and reoxygenation (OGD/R) cell model was developed to mimic ischemic neuronal injury in vitro. miR-155-5p overexpression led to reduced neuron viability, promoted apoptosis, elevated autophagic proteins' expression, and activated NLR family pyrin domain-containing 3- (NLRP3-) related inflammasomes, thereby aggravating OGD-induced neuronal injury. A dual-luciferase reporter assay exhibited that miR-155-5p could inhibit the Ras homolog enriched in brain (Rheb) expression, a mechanism critical for miR-155-5p-mediated neuronal injury. Furthermore, a mouse IBI model was developed using the transient middle cerebral artery occlusion (tMCAO) method. Animal experiments verified that miR-155p delivery via CPE cell-derived EVs aggravated IBI by suppressing Rheb expression. In conclusion, miR-155-5p in CPE-derived EVs can aggravate IBI pathology by suppressing Rheb expression and promoting NLRP3-mediated inflammasomes, suggesting its role as a potential therapeutic target in IBI.

Quercetin mediates TSC2-RHEB-mTOR pathway to regulate chondrocytes autophagy in knee osteoarthritis.

OBJECTIVE: This study aimed to explore the specific molecular mechanism of the therapeutic effect of quercetin in knee osteoarthritis (KOA). METHODS: The KOA rat model was constructed by excising the medial meniscus and transecting the anterior meniscus. Joint injuries in rats were determined by Hematoxylin-Eosin (H&E) and Safranin O staining. The severity of KOA was then assessed according to the Osteoarthritis Research Society International (OARSI). The expressions of TSC2 and LC2B in joint tissue were measured by immunohistochemistry. Besides, chondrocytes treated with 10 ng/ml IL-1ß were used to construct a chondrocyte arthritis model, while those treated with 4 or 8 µM quercetin were served as treatment groups. MTT, flow cytometry and toluidine blue staining were used to detect cell viability, apoptosis and mucopolysaccharide synthesis, respectively. qRT-PCR or Western blot was performed to determine the expressions of MMP-13, collagen II, Aggrecan, TSC2, RHEB, mTOR, p-mTOR, ULK1, p-ULK1, LC3B-I, LC3B-II and P62 in chondrocytes. RESULTS: Quercetin alleviated the joint injury and suppressed the increase in MMP-13 expression and the decreases in collagen II and Aggrecan expressions in KOA rats. In addition, quercetin suppressed RHEB, p-mTOR, p-ULK1 and P62 expressions but promoted TSC2 and LC3BII expressions in KOA rats. Furthermore, quercetin could relieve the decrease of cell viability and the increase of apoptosis that induced by IL-1ß, and promote the synthesis of IL-1ß-inhibited mucopolysaccharide in chondrocytes. Nevertheless, siTSC2 partially offset the therapeutic effects of quercetin in chondrocytes. CONCLUSION: Quercetin alleviated KOA by mediating the TSC2-RHBE-mTOR signaling pathway.

Development of a versatile HPLC-based method to evaluate the activation status of small GTPases.

Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood.