Mito-kaede photoactivation and chase experiment for mitophagy: optimizing flux measurement via fluid exchange system.

Modulating autophagy and mitophagy, vital cellular quality control systems, offer therapeutic potential for critical illnesses. However, limited drug screening options hinder progress. We present a novel assay using the photoswitchable fluorescent reporter, mito-Kaede, to quantify mitophagy flux. Mito-Kaede's superior UV-induced photoconversion and brightness post-conversion make it ideal for prolonged mitochondrial dynamics tracking. Its specificity in responding to mitophagy, confirmed by parkin-knockout cells, adds value. When coupled with a custom fluid exchange system, enabling efficient medium changes, precise mitophagy observations become feasible. This mitophagy assay, alongside our methodological insights, can decipher mitophagy's role in pathology and supports drug screening efforts.

Our method introduces a novel systematic approach for chronologically tracking the fluorescent decay of a photoactivatable fluorescent protein, mito-Kaede. This is combined with a fluid-exchange method to enable fixed-point observations before and after mitophagy stimulation.

Ubiquitination of ATAD3A by TRIM25 exacerbates cerebral ischemia-reperfusion injury via regulating PINK1/Parkin signaling pathway-mediated mitophagy.

BACKGROUND: Cerebral ischemia-reperfusion injury (CI/RI) is a complex process leading to neuronal damage and death, with mitophagy implicated in its pathogenesis. However, the significance of mitophagy in CI/RI remains debated. HYPOTHESIS: We hypothesized that TRIM25 reduces ATAD3A expression by ubiquitinating ATAD3A, promoting mitochondrial autophagy via the PINK1/Parkin pathway, and aggravating CI/RI. STUDY DESIGN: Rat middle cerebral artery occlusion (MCAO) followed by reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) in PC12 cells were used as animal and cell models, respectively. METHODS: To evaluate the success of the CI/R modeling, TTC and HE staining were employed. The determination of serum biochemical indexes was carried out using relative assay kits. The Western Blot analysis was employed to assess the expression of ATAD3A, TRIM25, as well as mitophagy-related proteins (PINK1, Parkin, P62, and LC3II/LC3I). The mRNA levels were detected using QRT-PCR. Mitochondrial membrane potential was assessed through JC-1 staining. Mitosox Red Assay Kit was utilized to measure mitochondrial reactive oxygen species levels in PC12 cells. Additionally, characterization of the mitophagy structure was performed using transmission electron microscopy (TEM). RESULTS: Our findings showed down-regulation of ATAD3A and up-regulation of TRIM25 in both in vivo and in vitro CI/RI models. Various experimental techniques such as Western Blot, JC-1 staining, Mitosox assay, Immunofluorescence assay, and TEM observation supported the occurrence of PINK1/Parkin signaling pathway-mediated mitophagy in both models. ATAD3A suppressed mitophagy, while TRIM25 promoted it during CI/RI injury. Additionally, the results indicated that TRIM25 interacted with and ubiquitinated ATAD3A via the proteasome pathway, affecting ATAD3A protein stability and expression. CONCLUSION: TRIM25 promoted Pink1/Parkin-dependent excessive mitophagy by destabilizing ATAD3A, exacerbating CI/RI. Targeting TRIM25 and ATAD3A may offer therapeutic strategies for mitigating CI/RI and associated neurological damage.

Parkin deficiency exacerbates particulate matter-induced injury by enhancing airway epithelial necroptosis.

Exposure to fine particulate matter (PM) disrupts the function of airway epithelial barriers causing cellular stress and damage. However, the precise mechanisms underlying PM-induced cellular injury and the associated molecular pathways remain incompletely understood. In this study, we used intratracheal instillation of PM in C57BL6 mice and PM treatment of the BEAS-2B cell line as in vivo and in vitro models, respectively, to simulate PM-induced cellular damage and inflammation. We collected lung tissues and bronchoalveolar lavage fluids to assess histopathological changes, necroptosis, and airway inflammation. Our findings reveal that PM exposure induces necroptosis in mouse airway epithelial cells. Importantly, concurrent administration of a receptor interacting protein kinases 3 (RIPK3) inhibitor or the deletion of the necroptosis effector mixed-lineage kinase domain-like protein (MLKL) effectively attenuated PM-induced airway inflammation. PM exposure dose-dependently induces the expression of Parkin, an E3 ligase we recently reported to play a pivotal role in necroptosis through regulating necrosome formation. Significantly, deletion of endogenous Parkin exacerbates inflammation by enhancing epithelial necroptosis. These results indicate that PM-induced Parkin expression plays a crucial role in suppressing epithelial necroptosis, thereby reducing airway inflammation. Overall, these findings offer valuable mechanistic insights into PM-induced airway injury and identify a potential target for clinical intervention.

Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans.

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

Protective effects of Parkin knockout on asthma-induced changes in juvenile mice: inflammation, airway resistance, and oxidative stress.

OBJECTIVE: This study aimed to explore the effects of Parkin (Prkn) knockout in a juvenile mouse model of asthma. METHODS: Prkn knockout (KO) and wild type (WT) mice were utilized to establish a juvenile mouse asthma model. The asthma model involved exposure to hyperoxia/ovalbumin (OVA), encompassing hyperoxia from postnatal day 1 (P1) to P7, sensitization on P21 and P28, and challenge from P36 to P42. Room air/phosphate-buffered saline (PBS) served as the control condition. Following airway resistance measurement, bronchoalveolar lavage fluid (BALF) was collected for cellular analysis, and lung tissues were subjected to histological examination and oxidative stress assessment. Serum levels of ovalbumin-specific immunoglobulin E (IgE), total IgE, interleukin-4 (IL-4), IL-5, and IL-13 were quantified using enzyme-linked immunosorbent assay (ELISA). RESULTS: WT mice exposed to hyperoxia/OVA showed decreased body weight and increased airway resistance compared to those exposed to control condition. Conversely, KO mice exhibited increased body weight under asthma conditions. KO mice with asthma had reduced total cell counts, along with lower levels of lymphocytes, eosinophils, and neutrophils, compared to WT asthma mice. Histological assessment showed attenuated inflammation and reduced collagen deposition in KO mice relative to WT mice, with lower serum levels of inflammatory markers and improved lung oxidative stress profiles. No significant differences were observed between KO and WT mice under room air/PBS conditions. CONCLUSIONS: Parkin knockout in juvenile mice mitigates asthma-related alterations in airway resistance, histopathological changes, inflammation status, and oxidative stress. These findings highlight a protective role of Parkin deficiency against asthma-associated pathologies.

SIRT1-mediated deacetylation of FOXO3 enhances mitophagy and drives hormone resistance in endometrial cancer.

BACKGROUND: The complex interplay between Sirtuin 1 (SIRT1) and FOXO3 in endometrial cancer (EC) remains understudied. This research aims to unravel the interactions of deacetylase SIRT1 and transcription factor FOXO3 in EC, focusing on their impact on mitophagy and hormone resistance. METHODS: High-throughput sequencing, cell experiments, and bioinformatics tools were employed to investigate the roles and interactions of SIRT1 and FOXO3 in EC. Co-immunoprecipitation (Co-IP) assay was used to assess the interaction between SIRT1 and FOXO3 in RL95-2 cells. Functional assays were used to assess cell viability, proliferation, migration, invasion, apoptosis, and the expression of related genes and proteins. A mouse model of EC was established to evaluate tumor growth and hormone resistance under different interventions. Immunohistochemistry and TUNEL assays were used to assess protein expression and apoptosis in tumor tissues. RESULTS: High-throughput transcriptome sequencing revealed a close association between SIRT1, FOXO3, and EC development. Co-IP showed a protein-protein interaction between SIRT1 and FOXO3. Overexpression of SIRT1 enhanced FOXO3 deacetylation and activity, promoting BNIP3 transcription and PINK1/Parkin-mediated mitophagy, which in turn promoted cell proliferation, migration, invasion, and inhibited apoptosis in vitro, as well as increased tumor growth and hormone resistance in vivo. These findings highlighted SIRT1 as an upstream regulator and potential therapeutic target in EC. CONCLUSION: This study reveals a novel molecular mechanism underlying the functional relevance of SIRT1 in regulating mitophagy and hormone resistance through the deacetylation of FOXO3 in EC, thereby providing valuable insights for new therapeutic strategies.

Parkin plays a crucial role in acute viral myocarditis by regulating mitophagy activity.

Rationale: Parkin (an E3 ubiquitin protein ligase) is an important regulator of mitophagy. However, the role of Parkin in viral myocarditis (VMC) remains unclear. Methods: Coxsackievirus B3 (CVB3) infection was induced in mice to create VMC. Cardiac function and inflammatory response were evaluated by echocardiography, histological assessment, and molecular analyses. AAV9 (adeno-associated virus 9), transmission electron microscopy (TEM) and western blotting were used to investigate the mechanisms by which Parkin regulates mitophagy and cardiac inflammation. Results: Our data indicated that Parkin- and BNIP3 (BCL2 interacting protein 3 like)-mediated mitophagy was activated in VMC mice and neonatal rat cardiac myocytes (NRCMs) infected with CVB3, which blocked autophagic flux by inhibiting autophagosome-lysosome fusion. Parkin silencing aggravated mortality and accelerated the development of cardiac dysfunction in CVB3-treated mice. While silencing of Parkin did not significantly increase inflammatory response through activating NF-κB pathway and production of inflammatory cytokines post-VMC, the mitophagy activity were reduced, which stimulated the accumulation of damaged mitochondria. Moreover, Parkin silencing exacerbated VMC-induced apoptosis. We consistently found that Parkin knockdown disrupted mitophagy activity and inflammatory response in NRCMs. Conclusion: This study elucidated the important role of Parkin in maintaining cardiac function and inflammatory response by regulating mitophagy activity and the NF-κB pathway during acute VMC. Although the functional impact of mitophagy remains unclear, our findings suggest that Parkin silencing may accelerate VMC development.

TUBB4A Inhibits Glioma Development by Regulating ROS-PINK1/Parkin-Mitophagy Pathway.

Glioma is a refractory malignant tumor with a powerful capacity for invasiveness and a poor prognosis. This study aims to investigate the role and mechanism of tubulin beta class IVA (TUBB4A) in glioma progression. The differential expression of TUBB4A in humans was obtained from databases and analyzed. Glioma cells U251-MG and U87-MG were intervened by pcDNA3.1(+) and TUBB4A overexpression plasmid. MTT, CCK8, LDH, wound healing, transwell, and western blotting were used to explore whether TUBB4A participates in the development of glioma. Reactive oxygen species (ROS) were detected by the DCFH-DA probe. Mitochondrial membrane potential (MMP) was examined by JC-1. It was found that TUBB4A expression level correlated with tumor grade, IDH1 status, 1p/19q status, and poor survival in glioma patients. In addition, TUBB4A overexpression inhibited the proliferation, migration, and invasion of U251-MG and U87-MG, while increasing the degree of apoptosis. Notably, TUBB4A overexpression promotes ROS generation and MMP depolarization, and induces mitophagy through the PINK1/Parkin pathway. Interestingly, mitochondria-targeted ROS scavenger reversed the effect of TUBB4A overexpression on PINK1/Parkin expression and mitophagy, whereas mitophagy inhibitor did not affect ROS production. And the effect of TUBB4A overexpression on mitophagy and glioma progression was consistent with that of PINK1/Parkin agonist. In conclusion, TUBB4A is a molecular marker for predicting the prognosis of glioma patients and an effective target for inhibiting glioma progression by regulating ROS-PINK1/Parkin-mitophagy pathway.

[Retracted] Tanshinone IIA regulates colorectal cancer apoptosis via attenuation of Parkin­mediated mitophagy by suppressing AMPK/Skp2 pathways.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the immunofluorescence assay data shown in Fig. 4A on p. 1698 were strikingly similar to data that had already been submitted for publication in different form in another article written by different authors at different research institutes. In addition, there was an instance of apparent duplication of western blot data comparing between Fig. 5A and 5G, and the reader also had concerns regarding the presentation of the flow­cytometry cell­count histograms in Fig. 2A. Owing to the fact that the contentious data in the above article had already been submitted for publication elsewhere prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 18: 1692­1703, 2018; DOI: 10.3892/mmr.2018.9087].

Deficiency in the mitophagy mediator Parkin accelerates murine skin allograft rejection.

Alterations in mitochondrial function and associated quality control programs, including mitochondrial-specific autophagy, termed mitophagy, are gaining increasing recognition in the context of disease. However, the role of mitophagy in organ transplant rejection remains poorly understood. Using mice deficient in Parkin, a ubiquitin ligase that tags damaged or dysfunctional mitochondria for autophagic clearance, we assessed the impact of Parkin-dependent mitophagy on skin-graft rejection. We observed accelerated graft loss in Parkin-deficient mice across multiple skin graft models. Immune cell distributions posttransplant were largely unperturbed compared to wild-type; however, the CD8+ T cells of Parkin-deficient mice expressed more T-bet, IFNγ, and Ki67, indicating greater priming toward effector function. This was accompanied by increased circulating levels of IL-12p70 in Parkin-deficient mice. Using a mixed leukocyte reaction, we demonstrated that naïve Parkin-deficient CD4+ and CD8+ T cells exhibit enhanced activation marker expression and proliferative responses to alloantigen, which were attenuated with administration of a pharmacological mitophagy inducer (p62-mediated mitophagy inducer), known to increase mitophagy in the absence of a functional PINK1-Parkin pathway. These findings indicate a role for Parkin-dependent mitophagy in curtailing skin-graft rejection.

Ginsenoside Rg1 ameliorates cerebral ischemia-reperfusion injury by regulating Pink1/ Parkin-mediated mitochondrial autophagy and inhibiting microglia NLRP3 activation.

OBJECTIVE: This study aimed to further elucidate the mechanism of ginsenoside Rg1 in the treatment of cerebral ischemia-reperfusion. METHODS: In this study, we observed the apoptosis of RM cells (microglia) after oxygen-glucose deprivation/reoxygenation (OGD/R) modeling before and after Rg1 administration, changes in mitochondrial membrane potential, changes in the content of Reactive oxygen species (ROS) and inflammatory vesicles NLR Family Pyrin Domain Containing 3 (NLRP3), and the expression levels of autophagy-related proteins, inflammatory factors, and apoptosis proteins. We further examined the pathomorphological changes in brain tissue, neuronal damage, changes in mitochondrial morphology and mitochondrial structure, and the autophagy-related proteins, inflammatory factors, and apoptosis proteins expression levels in CI/RI rats before and after administration of Rg1 in vivo experiments. RESULTS: In vitro experiments showed that Rg1 induced mitochondrial autophagy, decreased mitochondrial membrane potential, and reduced ROS content thereby inhibiting NLRP3 activation, decreasing secretion of inflammatory factors and RM cell apoptosis by regulating the PTEN induced putative kinase 1(Pink1) /Parkin signaling pathway. In vivo experiments showed that Rg1 induced mitochondrial autophagy, inhibited NLRP3 activation, improved inflammatory response, and reduced apoptosis by regulating the Pink1/Parkin signaling pathway, and Rg1 significantly reduced the area of cerebral infarcts, improved the pathological state of brain tissue, and attenuated the neuronal damage, thus improving cerebral ischemia/reperfusion injury in rats. CONCLUSION: Our results suggest that ginsenoside Rg1 can ameliorate cerebral ischemia-reperfusion injury by modulating Pink1/ Parkin-mediated mitochondrial autophagy in microglia and inhibiting microglial NLRP3 activation.

Protocatechuic aldehyde attenuates chondrocyte senescence via the regulation of PTEN-induced kinase 1/Parkin-mediated mitochondrial autophagy.

This study aimed to investigate whether the beneficial effects of PCA on chondrocyte senescence are mediated through the regulation of mitophagy. Chondrocyte senescence plays a significant role in the development and progression of knee osteoarthritis (OA). The compound protocatechuic aldehyde (PCA), which is abundant in the roots of Salvia miltiorrhiza, has been reported to have antioxidant properties and the ability to protect against cellular senescence. To achieve this goal, a destabilization of the medial meniscus (DMM)-induced mouse OA model and a lipopolysaccharide (LPS)-induced chondrocyte senescence model were used, in combination with PINK1 gene knockdown or overexpression. After treatment with PCA, cellular senescence was assessed using Senescence-Associated ß-Galactosidase (SA-ß-Gal) staining, DNA damage was evaluated using Hosphorylation of the Ser-139 (γH2AX) staining, reactive oxygen species (ROS) levels were measured using Dichlorodihydrofluorescein diacetate (DCFH-DA) staining, mitochondrial membrane potential was determined using a 5,5',6,6'-TETRACHLORO-1,1',3,3'-*. TETRAETHYBENZIMIDA (JC-1) kit, and mitochondrial autophagy was examined using Mitophagy staining. Western blot analysis was also performed to detect changes in senescence-related proteins, PINK1/Parkin pathway proteins, and mitophagy-related proteins. Our results demonstrated that PCA effectively reduced chondrocyte senescence, increased the mitochondrial membrane potential, facilitated mitochondrial autophagy, and upregulated the PINK1/Parkin pathway. Furthermore, silencing PINK1 weakened the protective effects of PCA, whereas PINK1 overexpression enhanced the effects of PCA on LPS-induced chondrocytes. PCA attenuates chondrocyte senescence by regulating PINK1/Parkin-mediated mitochondrial autophagy, ultimately reducing cartilage degeneration.

Gentiana decoction inhibits liver fibrosis and the activation of hepatic stellate cells via upregulating the expression of Parkin.

Liver fibrosis is a wound-healing process. It can be induced by various chronic liver diseases. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs), a key event. However, no effective treatment strategies to cure or alleviate liver fibrosis-induced pathologic changes have yet been developed. Traditional Chinese medicine (TCM) exhibits a good anti-fibrosis action, with few side effects. Gentiana decoction, a TCM also called Longdan Xiegan Tang (LXT), is used for purging the liver in clinical settings. However, the role of LXT in preventing liver fibrosis and the underlying regulatory mechanism have not yet been investigated. This study demonstrates that LXT treatment can protect the liver from the injuries resulting from CCl4-induced liver fibrosis in mice and suppress the activation of HSCs. The mice in the LXT group exhibit litter collagen I and HSC activation marker α-smooth muscle actin (α-SMA) expression. Transcriptome sequencing of the mouse liver tissue reveals that the level of Parkin, a mitophagy marker, decreased in CCl4-induced liver fibrosis. Further study shows that the injection of Parkin-overexpression adeno-associated virus (Parkin-AAV) via the tail vein can reduce CCl4-induced liver fibrogenesis in mice. We conducted a mechanistic study also, which suggests that LXT treatment suppresses the activation of HSCs by upregulating the expression of Parkin. Hence, it can be suggested that LXT inhibits liver fibrosis by activating the Parkin signaling pathway.

Dysfunction of Mitochondrial Dynamics Induces Endocytosis Defect and Cell Damage in Drosophila Nephrocytes.

Mitochondria are crucial for cellular ATP production. They are highly dynamic organelles, whose morphology and function are controlled through mitochondrial fusion and fission. The specific roles of mitochondria in podocytes, the highly specialized cells of the kidney glomerulus, remain less understood. Given the significant structural, functional, and molecular similarities between mammalian podocytes and Drosophila nephrocytes, we employed fly nephrocytes to explore the roles of mitochondria in cellular function. Our study revealed that alterations in the Pink1-Park (mammalian PINK1-PRKN) pathway can disrupt mitochondrial dynamics in Drosophila nephrocytes. This disruption led to either fragmented or enlarged mitochondria, both of which impaired mitochondrial function. The mitochondrial dysfunction subsequently triggered defective intracellular endocytosis, protein aggregation, and cellular damage. These findings underscore the critical roles of mitochondria in nephrocyte functionality.

[Effect of Tianma Gouteng Decoction on PINK1/Parkin pathway in oxidative stress in vascular smooth muscle cell induced by Angâ ¡].

This study explored the effect of Tianma Gouteng Decoction on oxidative stress induced by angiotensin Ⅱ(AngⅡ) in vascular smooth muscle cell(VSMC) and its molecular mechanism. Primary rat VSMC were cultured using tissue block method, and VSMC were identified by α-actin immunofluorescence staining. AngⅡ at a concentration of 1×10~(-6) mol·L~(-1) was used as the stimulating factor, and Sprague Dawley(SD) rats were orally administered with Tianma Gouteng Decoction to prepare drug serum. Rat VSMC were divided into normal group, model group, Chinese medicine group, and inhibitor(3-methyladenine, 3-MA) group. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation activity. Bromodeoxyuridine(BrdU) flow cytometry was used to detect cell cycle. Transwell assay was used to detect cell migration ability. Enzyme-linked immunosorbent assay(ELISA) was used to detect the activity of superoxide dismutase(SOD), catalase(CAT), and malondialdehyde(MDA) in VSMC. The intracellular reactive oxygen species(ROS) fluorescence intensity was detected using DCFH-DA fluorescent probe. Western blot was used to detect the expression of PTEN-induced putative kinase 1(PINK1), Parkin, p62, and microtubule-associated protein 1A/1B-light chain 3(LC3-Ⅱ) proteins in VSMC. The results showed that Tianma Gouteng Decoction-containing serum at a concentration of 8% could significantly inhibit VSMC growth after 48 hours of intervention. Compared with the normal group, the model group showed significantly increased cell proliferation activity and migration, significantly decreased levels of SOD and CAT, significantly increased levels of MDA, significantly enhanced ROS fluorescence intensity, significantly decreased expression of PINK1, Parkin, and LC3-Ⅱ proteins, and significantly increased expression of p62 protein. Compared with the model group, the Chinese medicine group showed significantly reduced cell proliferation activity and migration, significantly increased levels of SOD and CAT, significantly decreased levels of MDA, significantly weakened ROS fluorescence intensity, significantly increased expression of PINK1, Parkin, and LC3-Ⅱ proteins, and significantly decreased expression of p62 protein. Compared with the Chinese medicine group, the addition of the mitochondrial autophagy inhibitor 3-MA could block the intervention of Tianma Gouteng Decoction-containing serum on VSMC proliferation, migration, mitochondrial autophagy, and oxidative stress levels, with statistically significant differences. In summary, Tianma Gouteng Decoction has good antioxidant activity and can inhibit cell proliferation and migration. Its mechanism of action may be related to the activation of the mitochondrial autophagy PINK1/Parkin signaling pathway.

Knocking down EGR1 inhibits nucleus pulposus cell senescence and mitochondrial damage through activation of PINK1-Parkin dependent mitophagy, thereby delaying intervertebral disc degeneration.

Mitophagy plays a crucial role in maintaining the homeostasis of intervertebral disc (IVD). Early Growth Response 1 (EGR1), a conservative transcription factor, is commonly upregulated under oxidative stress conditions and participates in regulating cellular senescence, apoptosis, and inflammatory responses. However, the specific role of EGR1 in nucleus pulposus (NP) cell senescence and mitophagy remains unclear. In this study, through bioinformatics analysis and validation using human tissue specimens, we found that EGR1 is significantly upregulated in IVD degeneration (IDD). Further experimental results demonstrate that knockdown of EGR1 inhibits TBHP-induced NP cell senescence and mitochondrial dysfunction while promoting the activation of mitophagy. The protective effect of EGR1 knockdown on NP cell senescence and mitochondrion disappears upon inhibition of mitophagy with mdivi1. Mechanistic studies reveal that EGR1 suppresses NP cell senescence and mitochondrial dysfunction by modulating the PINK1-Parkin dependent mitophagy pathway. Additionally, EGR1 knockdown delays acupuncture-induced IDD in rats. In conclusion, our study demonstrates that under TBHP-induced oxidative stress, EGR1 knockdown mitigates NP cell senescence and mitochondrial dysfunction through the PINK1-Parkin dependent mitophagy pathway, thereby alleviating IDD.

Taurine rescues intervertebral disc degeneration by activating mitophagy through the PINK1/Parkin pathway.

Intervertebral disc degeneration (IDD) is a common cause of low back pain and disability. Recent studies have highlighted the critical role of mitochondrial dysfunction in the progression of IDD. In this study, we investigated the therapeutic potential of taurine in delaying IDD through the activation of mitophagy via the PINK1/Parkin pathway. Our in vitro and in vivo experiments demonstrate that taurine treatment significantly enhances mitophagy, reduces oxidative stress, delays cell senescence, and promotes the removal of damaged mitochondria in nucleus pulposus cells (NPC). Additionally, taurine-mediated activation of the PINK1/Parkin pathway leads to improved mitochondrial homeostasis and slows the progression of disc degeneration. These findings provide new insights into the protective effects of taurine and highlight its potential as a therapeutic agent for IDD.

Deciphering the neuroprotective mechanisms of RACK1 in cerebral ischemia-reperfusion injury: Pioneering insights into mitochondrial autophagy and the PINK1/Parkin axis.

INTRODUCTION: Cerebral ischemia-reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy. METHODS: We utilized high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) to identify core genes associated with CIRI. In vitro experiments used human neuroblastoma SK-N-SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. In vivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis. RESULTS: Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. In vitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. In vivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them. CONCLUSION: This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments.

Protection of Parkin over-expression on lung in rats with exertional heat stroke by activating mitophagy.

OBJECTIVE: To investigate the role of Parkin overexpression-induecd mitophagy in alleviating acute lung injury of exertional heat stroke(EHS) rats. METHODS: Eighty SD rats were divided into four groups: Control group (CON group), Control Parkin overexpression group (CON + Parkin group), exertional heat stroke group (EHS group), and exertional heat stroke Parkin overexpression group (EHS + Parkin group). Adeno-associated virus carrying the Parkin gene was intravenously injected into the rats to overexpress Parkin in the lung tissue. An exertional heat stroke rat model was established, and survival curves were plotted. Lung Micro-CT was performed, and lung coefficient and pulmonary microvascular permeability were measured. Enzyme-linked immunosorbent assays(ELISA) were used to determine the levels of interleukin-6(IL-6), interleukin-1ß(IL-1ß), Tumor necrosis factor-α(TNF-α), and reactive oxygen species(ROS). The morphology of mitochondria in type II epithelial cells of lung tissue was observed using transmission electron microscopy. The apoptosis of lung tissue, the level of mitophagy, and the co-localization of Pink1 and Parkin were determined using immunofluorescence. The expression of Pink1, Parkin, MFN2, PTEN-L, PTEN, p62, and microtubule associated protein 1 light chain 3 (LC3) in rat lung tissue was measured by western blot. RESULTS: Compared with the CON group, there were more severe lung injury and more higher levels of IL-6, IL-1ß, TNF-α in EHS rats. Both of the LC3-II/LC3-I ratio and the co-localization of LC3 and Tom20 in the lung tissue of EHS rats decreased. Compared with the EHS group, the survival rate of rats in the EHS + Parkin overexpression group was significantly increased, lung coefficient and pulmonary microvascular permeability were reduced, and pathological changes such as exudation and consolidation were significantly alleviated. The levels of IL-6, IL-1ß, TNF-α, and ROS were significantly decreased; the degree of mitochondrial swelling in type II alveolar epithelial cells was reduced, and no vacuolization was observed. Lung tissue apoptosis was reduced, and the colocalization fluorescence of Pink1 and Parkin, as well as LC3 and Tom20, were increased. The expression of Parkin and LC3-II/LC3-I ratio in lung tissue were both increased, while the expression of P62, Pink1, MFN2, and PTEN-L was decreased. CONCLUSION: Pink1/Parkin-mediated mitophagy dysfunction is one of the mechanisms underlying acute lung injury in rats with EHS, and activation of Parkin overexpression induced-mitophagy can alleviate acute lung injury caused by EHS.

Low-dose hexavalent chromium induces mitophagy in rat liver via the AMPK-related PINK1/Parkin signaling pathway.

Hexavalent chromium (Cr(VI)) is a hazardous metallic compound commonly used in industrial processes. The liver, responsible for metabolism and detoxification, is the main target organ of Cr(VI). Toxicity experiments were performed to investigate the impacts of low-dose exposure to Cr(VI) on rat livers. It was revealed that exposure of 0.05 mg/kg potassium dichromate (K2Cr2O7) and 0.25 mg/kg K2Cr2O7 notably increased malondialdehyde (MDA) levels and the expressions of P-AMPK, P-ULK, PINK1, P-Parkin, and LC3II/LC3I, and significantly reduced SOD activity and P-mTOR and P62 expression levels in liver. Electron microscopy showed that CR(VI) exposure significantly increased mitophagy and the destruction of mitochondrial structure. This study simulates the respiratory exposure mode of CR(VI) workers through intratracheal instillation of CR(VI) in rats. It confirms that autophagy in hepatocytes is induced by low concentrations of CR(VI) and suggest that the liver damage caused by CR(VI) may be associated with the AMPK-related PINK/Parkin signaling pathway.