Targeting lon protease to inhibit persister cell formation in Salmonella Typhimurium: a drug repositioning approach.

Objective: Persister cells are a specific subset of bacteria capable of surviving exposure to lethal doses of antibiotics, leading to antibiotic therapy failures and infection relapses. This research explores the utilization of drug repositioning to target the Lon protease in Salmonella Typhimurium. Method: In this study, FDA-approved drugs sourced from the Drug Bank database were screened to identify existing pharmaceuticals with the potential to combat the Lon protease. The formation of persister cells in the presence of antibiotics, as well as the combination of antibiotics with potential Lon protease inhibitors, was examined. Furthermore, the expression of type II toxin-antitoxin system genes was analyzed to enhance our comprehension of the inhibitors' effects. Result: Molecular docking analysis revealed that Diosmin and Nafcillin exhibited strong binding affinity to the Lon protease. Molecular dynamics simulation trajectories analysis demonstrated that the interaction of these ligands with the enzyme did not induce instability; rather, the enzyme's structure remained stable. Combinations of ceftazidime and ciprofloxacin with either Nafcillin or Diosmin led to significant reductions in bacterial cell counts. Furthermore, the effectiveness of these combinations, when compared to antibiotics alone, highlighted the substantial impact of Nafcillin and Diosmin in reducing type II TA system gene expression. Conclusion: These findings suggest promising prospects for developing novel therapeutic approaches targeting persister cells to mitigate treatment failures in Salmonella infections.

Intracellular Survival and Pathogenicity Modulation of Salmonella Lon, CpxR, and RfaL Mutants Used as Live Bacterial Vectors under Abiotic Stress, Unveiling the Link between Stress Response and Virulence in Epithelial Cells.

In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL. These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology.

Proteolytic control of FixT by the Lon protease impacts FixLJ signaling in Caulobacter crescentus.

Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter. IMPORTANCE: The Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.

Suppression of the Escherichia coli rnpA49 conditionally lethal phenotype by different compensatory mutations.

RNase P is an essential enzyme found across all domains of life that is responsible for the 5'-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli In E. coli, the temperature-sensitive (ts) rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme's activity in vivo. Here, we report for the first time naturally occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the ts defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.

Polyphosphate and tyrosine phosphorylation in the N-terminal domain of the human mitochondrial Lon protease disrupts its functions.

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.

A system for inducible mitochondria-specific protein degradation in vivo.

Targeted protein degradation systems developed for eukaryotes employ cytoplasmic machineries to perform proteolysis. This has prevented mitochondria-specific analysis of proteins that localize to multiple locations, for example, the mitochondria and the nucleus. Here, we present an inducible mitochondria-specific protein degradation system in Saccharomyces cerevisiae based on the Mesoplasma florum Lon (mf-Lon) protease and its corresponding ssrA tag (called PDT). We show that mitochondrially targeted mf-Lon protease efficiently and selectively degrades a PDT-tagged reporter protein localized to the mitochondrial matrix. The degradation can be induced by depleting adenine from the medium, and tuned by altering the promoter strength of the MF-LON gene. We furthermore demonstrate that mf-Lon specifically degrades endogenous, PDT-tagged mitochondrial proteins. Finally, we show that mf-Lon-dependent PDT degradation can also be achieved in human mitochondria. In summary, this system provides an efficient tool to selectively analyze the mitochondrial function of dually localized proteins.

LONP1 ameliorates liver injury and improves gluconeogenesis dysfunction in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe liver disease with complex pathogenesis. Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis. Accumulating evidence suggests that glucose metabolic disturbance, especially gluconeogenesis dysfunction, plays a critical role in the disease progression of ACLF. Lon protease-1 (LONP1) is a novel mediator of energy and glucose metabolism. However, whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown. METHODS: In this study, we collected liver tissues from ACLF patients, established an ACLF mouse model with carbon tetrachloride (CCl 4 ), lipopolysaccharide (LPS), and D-galactose (D-gal), and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model. LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation. Liver histopathology, biochemical index, mitochondrial morphology, cell viability and apoptosis, and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF. RESULTS: We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues. Furthermore, LONP1 overexpression remarkably attenuated liver injury, which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ACLF mice. Moreover, mitochondrial morphology was improved upon overexpression of LONP1. Meanwhile, the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression. Similarly, the hepatoprotective effect was also observed in the hepatocyte injury model, as evidenced by improved cell viability, reduced cell apoptosis, and improved gluconeogenesis level and activity, while LONP1 knockdown worsened liver injury and gluconeogenesis disorders. CONCLUSION: We demonstrated that gluconeogenesis dysfunction exists in ACLF, and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction, which would provide a promising therapeutic target for patients with ACLF.

A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.

Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show here, using cryo-electron microscopy (cryo-EM), that the activation process of the Lon AAA+ protease may involve a pentameric assembly and a substrate-dependent incorporation of the sixth protomer to form the substrate-pore-loop contacts seen in the translocating state. Based on the structural results, we design truncated monomeric mutants that inhibit Lon activity by binding to the native pentamer and demonstrated that expressing these monomeric mutants in Escherichia coli cells containing functional Lon elicits specific phenotypes associated with lon deficiency, including the inhibition of persister cell formation. These findings uncover a substrate-dependent assembly process for the activation of a AAA+ protein and demonstrate a targeted approach to selectively inhibit its function within cells.

The heat shock protein LarA activates the Lon protease in response to proteotoxic stress.

The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that high levels of LarA cause growth inhibition in a Lon-dependent manner and that Lon-mediated degradation of LarA itself ensures low LarA levels in the absence of stress. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand in response to protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.

Regulation of the general stress response sigma factor σT by Lon-mediated proteolysis.

IMPORTANCE: Regulated protein degradation is a critical process in all cell types, which contributes to the precise regulation of protein amounts in response to internal and external cues. In bacteria, protein degradation is carried out by ATP-dependent proteases. Although past work revealed detailed insights into the operation principles of these proteases, there is limited knowledge about the substrate proteins that are degraded by distinct proteases and the regulatory role of proteolysis in cellular processes. This study reveals a direct role of the conserved protease Lon in regulating σT, a transcriptional regulator of the general stress response in α-proteobacteria. Our work is significant as it underscores the importance of regulated proteolysis in modulating the levels of key regulatory proteins under changing conditions.

Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease.

Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.

Mitochondrial Lon protease promotes CD4+ T cell activation by activating the cGAS-STING-TBK1 axis in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4+ T cells play an essential role. We extracted CD4+ T cells from SLE-prone Fcgr2b-/- mice to elaborate the mechanism of mitochondrial Lon protease in CD4+ T cell activation in SLE. Transcriptome sequencing was performed in SLE-prone Fcgr2b-/- mice, and the stimulator of interferon gene (STING) related to SLE was obtained. It was demonstrated that STING expression was elevated in CD4+ T cells in SLE-prone Fcgr2b-/- mice. The downstream genes and pathways of STING were predicted by GO and KEGG approaches. The data indicated that STING regulated IFN signaling to promote CD4+ T cell activation in SLE-prone Fcgr2b-/- mice. Next, the interaction of cGAS, STING, TBK1, and IFN-I was verified by Co-IP assay. Moreover, the roles of cGAS, STING, and TBK1 in activating CD4+ T cells from SLE-prone Fcgr2b-/- mice were evaluated using gain- or loss-of-function experiments. Mechanistically, cGAS upregulated the IFN-I signaling pathway by directly interacting with STING and TBK1, contributing to CD4+ T cell activation. Besides, cytosolic mtDNA could activate CD4+ T cell activation in SLE-prone Fcgr2b-/- mice by upregulating the cGAS-STING-TBK1 axis. The function of mitochondrial Lon protease in oxidative damage and mtDNA release in CD4+ T cells of SLE-prone Fcgr2b-/- mice were explored. Mitochondrial Lon protease enhanced mtDNA release into the cytoplasm under oxidative stress. Collectively, our work indicates that mitochondrial Lon protease enhances CD4+ T cell activation by inducing mtDNA leakage and offers new candidate targets for developing diagnostic and therapeutic strategies.

Antibiotic-induced degradation of antitoxin enhances the transcription of acetyltransferase-type toxin-antitoxin operon.

BACKGROUND: Bacterial toxin-antitoxin (TA) modules respond to various stressful conditions. The Gcn5-related N-acetyltransferase-type toxin (GNAT) protein encoded by the GNAT-RHH TA locus is involved in the antibiotic tolerance of Klebsiella pneumoniae. OBJECTIVES: To investigate the transcriptional mechanism of the GNAT-RHH operon kacAT under antibiotic stress. METHODS: The transcriptional level of the kacAT operon of K. pneumoniae was measured by quantitative real-time (qRT) PCR assay. The degradation of antitoxin KacA was examined by western blot and fluorescent protein. The ratio of [KacA]:[KacT] was calculated by the fluorescence intensity of KacA-eGFP and mCherry-KacT. Mathematical modelling predicted protein and transcript synthesis dynamics. RESULTS: A meropenem-induced increase in transcript levels of kacA and kacT resulted from the relief from transcriptional autoregulation of the kacAT operon. Meropenem induces the degradation of KacA through Lon protease, resulting in a reduction in the ratio of [KacA]:[KacT]. The decreased ratio causes the dissociation of the KacAT complex from its promoter region, which eliminates the repression of kacAT transcription. In addition, our dynamic model of kacAT expression regulation quantitatively reproduced the experimentally observed reduction of the [KacA]:[KacT] ratio and a large increase in kacAT transcript levels under the condition of strong promoter autorepression by the KacAT complex. CONCLUSIONS: Meropenem promotes the degradation of antitoxin by enhancing the expression of Lon protease. Degradation of antitoxin reduces the ratio of intracellular [antitoxin]:[toxin], leading to detachment of the TA complex from its promoter, and releasing repression of TA operon transcription. These results may provide an important insight into the transcriptional mechanism of GNAT-RHH TA modules under antibiotic stress.

Sirt3 restricts tumor initiation via promoting LONP1 deacetylation and K63 ubiquitination.

BACKGROUND: Sirtuin 3 (Sirt3) is a controversial regulator of carcinogenesis. It residents in the mitochondria and gradually decays during aging. In this study, we tried to investigate the role of Sirt3 in carcinogenesis and to explore its involvement in metabolic alteration. METHODS: We generated conditional intestinal epithelium Sirt3-knockout mice by crossing ApcMin/+; Villin-Cre with Sirt3fl/fl (AVS) mice. The deacetylation site of Lon protease-1 (LONP1) was identified with Mass spectrometry. The metabolic flux phenotype was determined by Seahorse bioanalyzer. RESULTS: We found that intestinal epithelial cell-specific ablation of Sirt3 promotes primary tumor growth via stabilizing mitochondrial LONP1. Notably, we newly identified that Sirt3 deacetylates human oncogene LONP1 at N terminal residue lysine 145 (K145). The LONP1 hyperacetylation-mutant K145Q enhances oxidative phosphorylation to accelerate tumor growth, whereas the deacetylation-mutant K145R produces calorie-restriction like phenotype to restrain tumorigenesis. Sirt3 deacetylates LONP1 at K145 and subsequently facilitates the ESCRT0 complex sorting and K63-ubiquitination that resulted in the degradation of LONP1. Our results sustain the notion that Sirt3 is a tumor-suppressor to maintain the appropriate ubiquitination and degradation of oncogene LONP1. CONCLUSION: Sirt3 represents a targetable metabolic checkpoint of oncogenesis, which produces energy restriction effects via maintaining LONP1 K145 deacetylation and subsequent K63 ubiquitination.

LONP1 targets HMGCS2 to protect mitochondrial function and attenuate chronic kidney disease.

Mitochondria comprise the central metabolic hub of cells and their imbalance plays a pathogenic role in chronic kidney disease (CKD). Here, we studied Lon protease 1 (LONP1), a major mitochondrial protease, as its role in CKD pathogenesis is unclear. LONP1 expression was decreased in human patients and mice with CKD, and tubular-specific Lonp1 overexpression mitigated renal injury and mitochondrial dysfunction in two different models of CKD, but these outcomes were aggravated by Lonp1 deletion. These results were confirmed in renal tubular epithelial cells in vitro. Mechanistically, LONP1 downregulation caused mitochondrial accumulation of the LONP1 substrate, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), which disrupted mitochondrial function and further accelerated CKD progression. Finally, computer-aided virtual screening was performed, which identified a novel LONP1 activator. Pharmacologically, the LONP1 activator attenuated renal fibrosis and mitochondrial dysfunction. Collectively, these results imply that LONP1 is a promising therapeutic target for treating CKD.

Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

Unique Structural Fold of LonBA Protease from Bacillus subtilis, a Member of a Newly Identified Subfamily of Lon Proteases.

ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.

Construction of Mitochondrial Protection and Monitoring Model of Lon Protease Based on Machine Learning under Myocardial Ischemia Environment.

The localization of a protein's submitochondrial structure is important for therapeutic design of associated disorders caused by mitochondrial abnormalities because many human diseases are directly tied to mitochondria. When Lon protease expression changes, glycolysis replaces respiratory metabolism in the cell, which is a common occurrence in cancer cells. The fact that protein formation is a dynamic research object makes it impossible to reproduce the unique living environment of proteins in an experimental setting, which surely makes it more challenging to determine protein function through experiments. This research suggests a model of Lon protease-based mitochondrial protection under myocardial ischemia based on ML (machine learning). To ensure the balance of all submitochondrial proteins, the data set is processed using a random oversampling method, each overlapping fixed-length subsequence that is created from the protein sequence functions as a channel in the convolution layer. The results demonstrate that applying the oversampling strategy increases the ROC value by 17.6%-21.3%. Our prediction method is successful as evidenced by the fact that ML prediction outperforms the predictions of other conventional classifiers.

Transcranial Direct-Current Stimulation Regulates MCT1-PPA-PTEN-LONP1 Signaling to Confer Neuroprotection After Rat Cerebral Ischemia-Reperfusion Injury.

Propionic acid (PPA) is a critical metabolite involved in microbial fermentation, which functions to reduce fat production, inhibit inflammation, and reduce serum cholesterol levels. The role of PPA in the context of cerebral ischemia-reperfusion (I/R) injury has yet to be clarified. Increasing evidence indicate that transcranial direct-current stimulation (tDCS) is a safe approach that confers neuroprotection in cerebral ischemia injury. Here, we show that the levels of PPA were reduced in the ischemic brain following a rat cerebral I/R injury and in the cultured rat cortical neurons after oxygen-glucose deprivation (OGD), an in vitro model of ischemic injury. We found that the decreased levels of transporter protein monocarboxylate transporter-1 (MCT1) were responsible for the OGD-induced reduction of PPA. Supplementing PPA reduced ischemia-induced neuronal death after I/R. Moreover, our results revealed that the neuroprotective effect of PPA is mediated through downregulation of phosphatase PTEN and subsequent upregulation of Lon protease 1 (LONP1). We demonstrated that direct-current stimulation (DCS) increased MCT1 expression and PPA level in OGD-insulted neurons, while tDCS decreased the brain infarct volume in the MCAO rats via increasing the levels of MCT1 expression and PPA. This study supports a potential application of tDCS in ischemic stroke.

LONP1 downregulation with ageing contributes to osteoarthritis via mitochondrial dysfunction.

Osteoarthritis (OA) is an age-related disorder and an important cause of disability that is characterized by a senescence-associated secretory phenotype and matrix degradation leading to a gradual loss of articular cartilage integrity. Mitochondria, as widespread organelles, are involved in regulation of complex biological processes such as energy synthesis and cell metabolism, which also have bidirectional communication with the nucleus to help maintain cellular homeostasis and regulate adaptation to a broad range of stressors. In light of the evidence that OA is strongly associated with mitochondrial dysfunction. In addition, mitochondria are considered to be the culprits of cell senescence, and mitochondrial function changes during ageing are considered to have a controlling role in cell fate. Mitochondrial dysfunction is also observed in age-related OA, however, the internal mechanism by which mitochondrial function changes with ageing to lead to the development of OA has not been elucidated. In this study, we found that the expression of Lon protease 1 (LONP1), a mitochondrial protease, was decreased in human OA cartilage and in ageing rat chondrocytes. Furthermore, LONP1 knockdown accelerated the progression and severity of osteoarthritis, which was associated with aspects of mitochondrial dysfunction including oxidative stress, metabolic changes and mitophagy, leading to downstream MAPK pathway activation. Antioxidant therapy with resveratrol suppressed oxidative stress and MAPK pathway activation induced by LONP1 knockdown to mitigate OA progression. Therefore, our findings demonstrate that LONP1 is a central regulator of mitochondrial function in chondrocytes and reveal that downregulation of LONP1 with ageing contributes to osteoarthritis.