Microtubule-associated Protein Research Articles

Sestrin2 Restricts Endothelial-to-Mesenchymal Transition Induced by Lipolysaccharide via Autophagy.

Endothelial-to-mesenchymal transition (EndoMT) is a significant biological phenomenon wherein endothelial cells undergo a loss of their endothelial traits and progressively acquire mesenchymal characteristics. Consequently, this transformation leads to both a compromised ability to maintain lumen permeability and alterations in vascular structure, which hampers the preservation of blood-brain barrier integrity. This study aimed to investigate inflammation-induced EndoMT and its etiology, with the goal of impeding the infiltration of peripheral inflammation into the central nervous system. Lipolysaccharide (LPS) was administered intraperitoneally to mice several times to establish a chronic inflammatory model. A cellular inflammatory model was established by LPS in human brain microvascular endothelial cells (HBMECs). The mRNA expressions of inflammatory cytokines interleukin-1β (IL-1β) and IL-6 were detected by real-time polymerase chain reaction (PCR). Immunofluorescence staining of platelet endothelial cell adhesion molecule-1 (CD31) and alpha smooth muscle actin (α-SMA) was conducted to assess the level of EndoMT. The expression levels of Occludin, zona occludens protein 1 (ZO-1), Sestrin2, microtubule-associated protein1 light chain 3 (LC3) and inducible nitric oxide synthase (iNOS) were detected by western blotting. LPS treatment induced the downregulation of ZO-1 and Occludin, which was accompanied by the elevated expressions of iNOS, α-SMA, Sestrin2 and LC3-II in the mouse cortex and HBMECs. Mechanistically, the knockdown of Sestrin2 in HBMECs exacerbated the EndoMT induced by LPS treatment, while the overexpression of Sestrin2 inhibited this process. Moreover, the induction of autophagy by rapamycin rescued the EndoMT induced by Sestrin2 knockdown. This study revealed that Sestrin2 inhibited endothelial inflammation and EndoMT via enhanced autophagy, which may provide a potential drug target for cerebrovascular inflammatory injury.

Dysregulated Apoptosis and Autophagy in Childhood Epilepsy: Correlation to Clinical and Pharmacological Patterns.

We aimed to assess the serum levels of caspase-3 as a marker of apoptosis and microtubule-associated protein 1A/1B-light chain 3 (MAP1-LC3) as an autophagy marker in epileptic children with various clinical and pharmacological types. This case-control study was carried out on 90 participants (50 pediatric patients with epilepsy and 40 healthy matched children), the patients were categorized into three groups: Group (A): 25 pharmacosensitive epilepsy, Group (B): 25 pharmacoresistant epilepsy, and Group (C): 40 (age, sex, and body mass index) matched healthy children selected as controls. Serum caspase-3 and MAP1-LC3 were measured in all study groups, using commercially available ELISA kits. Serum caspase-3 was significantly higher among epileptic children, especially in the pharmacoresistant group, cases managed with multiple antiepileptic drugs, and cases with abnormal EEG findings. Conversely, circulating MAP1-LC3 levels showed a significant reduction in epilepsy cases, particularly in pharmacoresistant cases, in cases treated with multiple antiepileptic drugs, and in cases with abnormal EEG data. A significant negative correlation between serum caspase-3 and MAP1-LC3 was found among epileptic children (r = -0.369, p = 0.0083). Serum caspase-3 was a more valid biomarker in helping diagnose childhood epilepsy, while serum MAP1-LC3 was more valid in predicting pharmacoresistant type. The study reveals that serum caspase-3 levels were significantly elevated, particularly in pharmacoresistant cases and those managed with multiple drugs. Conversely, MAP1-LC3 levels were significantly reduced in epilepsy cases, suggesting potential involvement of altered apoptosis and autophagy in childhood epilepsy.

A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation.

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein.

High-Speed Clearing and High-Resolution Staining for Analysis of Various Markers for Neurons and Vessels.

Tissue clearing enables deep imaging in various tissues by increasing the transparency of tissues, but there were limitations of immunostaining of the large-volume tissues such as the whole brain. Here, we cleared and immune-stained whole mouse brain tissues using a novel clearing technique termed high-speed clearing and high-resolution staining (HCHS). We observed neural structures within the cleared brains using both a confocal microscope and a light-sheet fluorescence microscope (LSFM). The reconstructed 3D images were analyzed using a computational reconstruction algorithm. Various neural structures were well observed in three-dimensional (3D) images of the cleared brains from Gad-green fluorescent protein (GFP) mice and Thy 1-yellow fluorescent protein (YFP) mice. The intrinsic fluorescence signals of both transgenic mice were preserved after HCHS. In addition, large-scale 3D imaging of brains, immune-stained by the HCHS method using a mild detergent-based solution, allowed for the global topological analysis of several neuronal markers such as c-Fos, neuronal nuclear protein (NeuN), Microtubule-associated protein 2 (Map2), Tuj1, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) in various anatomical regions in the whole mouse brain tissues. Finally, through comparisons with various existing tissue clearing methodologies such as CUBIC, Visikol, and 3DISCO, it was confirmed that the HCHS methodology results in relatively less tissue deformation and higher fluorescence retention. In conclusion, the development of 3D imaging based on novel tissue-clearing techniques (HCHS) will enable detailed spatial analysis of neural and vascular networks present within the brain.

Cryo-EM unveils kinesin KIF1A’s processivity mechanism and the impact of its pathogenic variant P305L

Mutations in the microtubule-associated motor protein KIF1A lead to severe neurological conditions known as KIF1A-associated neurological disorders (KAND). Despite insights into its molecular mechanism, high-resolution structures of KIF1A-microtubule complexes remain undefined. Here, we present 2.7-3.5 Å resolution structures of dimeric microtubule-bound KIF1A, including the pathogenic P305L mutant, across various nucleotide states. Our structures reveal that KIF1A binds microtubules in one- and two-heads-bound configurations, with both heads exhibiting distinct conformations with tight inter-head connection. Notably, KIF1A’s class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and β-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12’s conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A’s superprocessive motility. Our findings advance the understanding of KIF1A’s molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa.

Porphyromonas gingivalis , a major oral pathobiont, evades canonical host pathogen clearance in human primary gingival epithelial cells (GECs) by initiating a non-canonical variant of autophagy consisting of Microtubule-associated protein 1A/1B-light chain 3 (LC3)-rich autophagosomes, which then act as replicative niches. Simultaneously, P. gingivalis inhibits apoptosis and oxidative-stress, including extracellular-ATP (eATP)-mediated reactive-oxygen-species (ROS) production via phosphorylating Heat Shock Protein 27 (HSp27) with the bacterial nucleoside-diphosphate-kinase (Ndk). Here, we have mechanistically identified that P. gingivalis -mediated induction of HSp27 is crucial for the recruitment of the LC3 isoform, LC3C, to drive the formation of live P. gingivalis -containing Beclin1-ATG14-rich autophagosomes that are redox sensitive and non-degrading. HSp27 depletions of both infected GECs and gingiva-mimicking organotypic-culture systems resulted in the collapse of P. gingivalis -mediated autophagosomes, and abolished P. gingivalis -induced LC3C-specific autophagic-flux in a HSp27-dependent manner. Concurrently, HSp27 depletion accompanied by eATP treatment abrogated protracted Beclin 1-ATG14 partnering and decreased live intracellular P. gingivalis levels. These events were only partially restored via treatments with the antioxidant N-acetyl cysteine (NAC), which rescued the cellular redox environment independent of HSp27. Moreover, the temporal phosphorylation of HSp27 by the bacterial Ndk results in HSp27 tightly partnering with LC3C, hindering LC3C canonical cleavage, extending Beclin 1-ATG14 association, and halting canonical maturation. These findings pinpoint how HSp27 pleiotropically serves as a major platform-molecule, redox regulator, and stepwise modulator of LC3C during P. gingivalis -mediated non-canonical autophagy. Thus, our findings can determine specific molecular strategies for interfering with the host-adapted P. gingivalis ' successful mucosal colonization and oral dysbiosis.

The Role of LC3-Associated Phagocytosis Inhibits the Inflammatory Response in Aspergillus fumigatus Keratitis.

The purpose of this study was to investigate the role and mechanism of microtubule-associated protein light chain-3 (LC3)-associated phagocytosis (LAP) in the immune response to Aspergillus fumigatus (A. fumigatus) keratitis. The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1β, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence. Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1β and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody. LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.

Mechanism of Yishen Chuchan decoction intervention of Parkinson's disease based on network pharmacology and experimental verification

The incidence of Parkinson's disease (PD) rises rapidly with the increase of age. With the advent of global aging, the number of patients with PD is rising along with the elderly population, especially in China. Previously, we found that Yishen chuchan decoction (YCD), prescribed based on clinical experience, has the potential of alleviating symptoms, delaying the progression, and controlling the development of PD. Nonetheless, the underlying mechanistic role is yet to be explored. AimThis research examined the possible therapeutic effects of YCD in alleviating PD via a systematic approach with network pharmacology and experimental validation, aiming at providing a new understanding of traditional Chinese medicine management regarding PD. MethodsThe chemical structure and properties of YCD were adopted from Traditional Chinese Medicine System Pharmacology Database (TCMSP), SwissADME, PubChem, and PubMed. The potential targets for YCD and PD were identified using Swiss Target Prediction, GeneCard, PubChem, and UniProt. The herbal-component-target network was created via the Cytoscape software. Moreover, by using the STRING database, the protein-protein interaction (PPI) network was screened. Gene function GO and KEGG pathway enrichment analyses were performed via the Metascape database. YCD-medicated Rat Serum from Sprague-Dawley (SD) Rats was prepared, and SH-SY5Y cells were preconditioned with rotenone to develop the PD model. To examine the impact of YCD on these cells and explore the mechanistic role of the p38 mitogen-activated protein kinase (MAPK) pathway, the cells were pretreated with either serum or a p38 MAPK pathway inhibitor. This study employed the Cell Counting Kit (CCK)-8 assay and Hoechst 33,342 staining to evaluate the viability and morphological changes induced by the YCD-medicated rat serum on rotenone-treated SH-SY5Y cells. Apoptosis was assessed by Flow cytometry. Immunofluorescence staining assessed the microtubule-associated protein 2 (MAP2) level. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the concentrations of inflammatory mediators interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Also, reactive oxygen species (ROS) and superoxide dismutase (SOD) levels were determined. Western Blotting measured the expression of total and phospho-p38 MAPK (p-p38). ResultsThis study identified 65 active components in YCD, which were found to target 801 specific genes. By screening, 63 potential core targets were identified from a pool of 172 overlapping targets between PD and YCD. These targets were examined by GO and KEGG analyses revealing their substantial correlation to MAPK, PI3K-Akt signaling pathways, positively controlling protein phosphorylation, and pathways of neurodegenerative diseases. SH-SY5Y cells were treated with 2 μM rotenone for 48 h, which reduced cell viability to 50 %, and reduced MAP2 expression, increased the rate of apoptosis, oxidative stress, inflammation, and p-p38 expressions. YCD-medicated rat serum significantly improved the viability, reduced the apoptosis rate, and increased the MAP2 expression. YCD-medicated serum increased SOD, reduced ROS and suppressed IL-6, IL-1β and TNF-α levels, thus inhibiting oxidative stress and inflammation in rotenone-treated SH-SY5Y cells. Moreover, YCD-medicated serum substantially lowered the p-p38 expression induced by rotenone. SB203580, a specific inhibitor of p38 MAPK, could also inhibit the p-p38 expression, apoptosis, and restore morphological damage of cells, also improve inflammation and oxidative stress. ConclusionYCD enhanced cell viability and reduced apoptosis rate, inflammation, and oxidative stress in vitro. These beneficial effects could potentially involve the suppression of p38 pathway and suppressed the phosphorylation of p38 MAPK.

Therapeutic Effect of Donepezil on Neuroinflammation and Cognitive Impairment after Moderate Traumatic Brain Injury.

Despite the important clinical issue of cognitive impairment after moderate traumatic brain injury (TBI), there is currently no suitable treatment. Here, we used in vitro and in vivo models to investigate the effect of Donepezil-an acetylcholinesterase (AChE) inhibitor-on cognitive impairment in the acute period following injury, while focusing on neuroinflammation and autophagy- and mitophagy-related markers. The purpose of the in vitro study was to investigate potential neuroprotective effects in TBI-induced cells after donepezil treatment, and the in vivo study, the purpose was to investigate therapeutic effects on cognitive impairment in the acute period after injury by analyzing neuroinflammation and autophagy- and mitophagy-related markers. The in vitro TBI model involved injuring SH-SY5Y cells using a cell-injury controller and then investigating the effect of donepezil at a concentration of 80 μM. The in vivo TBI model was made using a stereotaxic impactor for male C57BL/6J mice. Immuno-histochemical markers and cognitive functions were compared after 7 days of donepezil treatment (1 mg/kg/day). Mice were divided into four groups: sham operation with saline treatment, sham operation with donepezil treatment, TBI with saline treatment, and TBI with donepezil treatment (18 mice in each group). Donepezil treatment was administered within 4 h post-TBI. In vitro, donepezil was found to lead to increased cell viability and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), along with decreased reactive oxygen species (ROS), lactate-dehydrogenase (LDH), 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA)-positive cells, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. The mRNA and protein expressions of neuroinflammation (Cyclooxygenase-2, COX-2; NOD-like receptor protein 3, NLRP3; Caspase-1; and Interleukin-1 beta, IL-1β), as well as autophagy- and mitophagy-related markers (death-associated protein kinase 1, DAPK1; PTEN-induced kinase 1, PINK1; BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like, BNIP3L; Beclin-1, BECN1; BCL2-associated X protein, BAX; microtubule-associated protein 1A/1B-light chain 3B (LC3B); Sequestosome-1; and p62) were all found to decrease after donepezil treatment. The in vivo study also showed that donepezil treatment resulted in decreased levels of cortical tissue losses and brain swelling in TBI compared to the TBI group without donepezil treatment. Donepezil treatment was also shown to decrease the mRNA and Western blotting expressions of all markers, and especially COX-2 and BNIP3L, which showed the most significant decreases. Moreover, TBI mice showed an decreased escape latency, increased alteration rate, and improved preference index, altogether pointing to better cognitive performance after donepezil treatment. Donepezil treatment may be beneficial in improving cognitive impairment in the early phase of moderate traumatic brain injury by ameliorating neuroinflammation, as well as autophagy and mitophagy.

Effect of Tianma Gouteng Decoction on PINK1/Parkin pathway in oxidative stress in vascular smooth muscle cell induced by AngⅡ

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.

Effect of Peitu Yifei Granules on autophagy mechanism in rats with idiopathic pulmonary fibrosis based on PI3K/Akt/mTOR signaling pathway

To investigate the mechanism by which Peitu Yifei Granules inhibit idiopathic pulmonary fibrosis(IPF) in rats, fifty specific-pathogen-free(SPF) grade male Wistar rats were randomly divided into blank group and modeling group. IPF was induced in the modeling group rats by tracheal infusion of 5 mg·kg~(-1) bleomycin(BLM) and then randomly divided into model group, pirfenidone group, and high-dose, medium-dose, and low-dose groups treated with Peitu Yifei Granules. After 24 hours of modeling, the treatment groups received intragastric administration of either Peitu Yifei Granules or pirfenidone as a positive control drug; meanwhile, the model group received an equal volume of normal saline. After 21 days of treatment administration, lung tissue samples were collected for analysis. Pathological changes in lung tissues were assessed using hematoxylin-eosin(HE) staining and Masson's trichrome staining. The expression levels of protein kinase B(Akt), mammalian target of rapamycin(mTOR), their phosphorylated forms, and sequestosome 1(p62) were determined through Western blot(WB). Fluorescent quantitative real-time polymerase chain reaction(RT-qPCR) was used to measure messenger ribonucleic acid(mRNA) expression levels of Beclin-1, microtubule-associated proteins 1A/1B light chain 3B(LC3B), and p62. Immunohistochemistry was performed to assess protein expression levels of Beclin-1 and LC3B in lung tissue samples. RESULTS:: demonstrated that lung tissue structure appeared normal without significant collagen deposition in the blank group rats. In contrast, rats from the model group exhibited thickened alveolar septa along with evident inflammatory changes and collagen deposition. Compared to the model group rats, those treated with Peitu Yifei Granules or pirfenidone showed significantly improved lung tissue structure with reduced inflammation and collagen deposition observed histologically. Furthermore, compared with those of the blank group, the expressions of p62 and its mRNA, p-Akt and p-mTOR protein in lung tissues of the model group were significantly increased, while Beclin-1, LC3B and their mRNA levels were significantly decreased. Compared with those of the model group, the expressions of p62 and its mRNA, p-Akt and p-mTOR in lung tissues of the pirfenidone group and Peitu Yifei Granules high-dose and medium-dose groups were significantly decreased, while Beclin-1, LC3B and their mRNA expressions were significantly increased. The above results indicate that Peitu Yifei Granules can improve autophagy levels in lung tissues by inhibiting the phosphoinositide 3-kinase(PI3K)/Akt/mTOR signaling pathway and delay the development of IPF disease.

Identification of MAP65 family genes in tomato and the member SlMAP65-1 is involved in fruit morphogenesis

The plant growth process is accompanied by dynamic changes in the microtubules, and the rearrangement of microtubules is regulated by diverse microtubule-associated proteins (MAPs). Plant MAP65s have been exhaustively characterized in some species, but the information about MAP65 family members in tomato (Solanum lycopersicum) is limited. In this study, nine SlMAP65 family genes were identified in the tomato genome. Then a systematic analysis including physio-chemical properties, evolution, conserved motifs, domains, gene structure, and cis-regulatory elements of SlMAP65 family members was conducted. The member SlMAP65-1, which had the highest expression, was knocked out by CRISPR/Cas9. The tomato fruit of slmap65-1 loss of function lines showed an elongated morphology, and the data indicated that SlMAP65-1 was involved in fruit morphogenesis at early fruit development stage. These results provided insights for fruit morphogenesis-related research areas and future functional studies of SlMAP65 family members in tomato.

Anti-inflammatory mixture alleviates acute lung injury induced by sepsis in rats by modulating Beclin-1-mediated autophagy

To investigate the protective effects of an anti-inflammatory mixture on acute lung injury (ALI) induced by sepsis in rats, as well as its possible mechanisms. A total of 40 Sprague-Dawley (SD) rats were randomly divided into the sham group, septic ALI model group (model group), 3-methyladenine (3-MA) control group, and anti-inflammatory mixture pretreatment group, with 10 rats in each group. Cecal ligation and perforation (CLP) was performed to reproduce a septic ALI model. The rats in the sham group only underwent opening and closing the abdomen without perforation and ligation. Both groups were given saline gavage and intraperitoneal injection for 3 consecutive days before surgery. The 3-MA control group was given intraperitoneal injection of saline and autophagy inhibitor 3-MA 15 mg/kg for 3 consecutive days before modeling. The anti-inflammatory mixture pretreatment group was given 8.8 mL/kg of anti-inflammatory mixture by gavage [the composition of anti-inflammatory mixture: rhubarb 15 g (after the next), coptis chinensis 15 g, baical skullcap root 12 g, magnoliae cortex 12 g, dahurian patrinia herb 30 g] and saline intraperitoneal injection for 3 consecutive days before modeling. The rats in each group were anesthetized 24 hours after surgery and died due to abdominal aortic blood collection. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum inflammatory cytokines interleukins (IL-1β and IL-6). Lung tissue was taken and then the bronchoalveolar lavage fluid (BALF) was collected, and the levels of IL-1β and IL-6 were detected by ELISA. Lung wet/dry weight (W/D) ratio was measured. After hematoxylin-eosin (HE) staining, the histopathological changes of the lungs were observed under light microscopy. Western blotting was used to detect the expression of autophagy markers microtubule-associated protein 1 light chain 3- II/I (LC3- II/I) and Beclin-1 protein in lung tissue. Autophagosomes in lung tissue were observed with transmission electron microscopy. Compared with the sham group, the rats in the model group exhibited severe destruction of lung tissue structure, with significant infiltration of inflammatory cells, the lung W/D ratio and the levels of IL-1β and IL-6 in serum and BALF were significantly increased, the expressions of LC3- II/I and Beclin-1 protein were down-regulated, the autophagosomes were more. The rats in the 3-MA control group exhibited more severe lung tissue injury as compared with the model group, the lung W/D ratio and the levels of inflammatory cytokines in serum and BALF were further increased, the expressions of LC3- II/I and Beclin-1 protein still showed a decrease tendency as compared with the sham group, and the autophagosomes were less than that in the model group. Compared with the model group, the anti-inflammatory mixture pretreatment group showed milder lung tissue injury with a minimal amount of inflammatory cell infiltration, the lung W/D ratio was significantly reduced (7.07±1.02 vs. 11.33±1.85, P < 0.05), the levels of IL-1β and IL-6 in both serum and BALF were significantly decreased [IL-1β (ng/L): 26.04±3.86 vs. 40.83±5.46 in serum, 17.75±2.02 vs. 26.86±4.32 in BALF; IL-6 (ng/L): 91.28±10.15 vs. 129.44±13.05 in serum, 76.06±7.51 vs. 120.91±7.47 in BALF, all P < 0.05], and the ratio of LC3- II/I and Beclin-1 protein expression were significantly increased [LC3- II/I ratio: 1.23±0.02 vs. 0.60±0.02, Beclin-1 protein (Beclin-1/GAPDH): 2.37±0.33 vs. 0.62±0.05, both P < 0.05]. Furthermore, an increase in the number of autophagosomes was observed. The anti-inflammatory mixture improves lung injury in rats with sepsis induced by CLP and reduce inflammation levels, potentially through upregulation of Beclin-1-mediated autophagy.

Naringin inhibits cisplatin resistance of ovarian cancer cells by inhibiting autophagy mediated by the TGF-β2/smad2 pathway.

Resistance to cisplatin (DDP) in patients with ovarian cancer (OC) poses a great challenge to improving the quality of life of patients. Past reports have revealed that naringin can induce apoptosis of OC cells and delay the occurrence of drug resistance in OC cells. However, the molecular role by which naringin inhibits DDP resistance in OC has not been definitively proven by researchers. The objective of this study is to investigate the effect of naringin on DDP resistance in OC cells and the specific mechanism of naringin mediating autophagy. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were selected to evaluate the role of naringin or DDP on the proliferation and apoptosis of human OC cells (SKOV3/A2780). The protein levels of Sequestosome 1 (SQSTM1/p62, hereinafter referred to as p62), microtubule-associated protein 1 light chain 3 (LC3), transforming growth factor-β2 (TGF-β2) and SMAD family member 2 (smad2) were detected with Western blotting assay. Immunofluorescence assay was also used to evaluate the level of LC3 in different groups of cells. Besides, functional analyses were performed in vivo. Naringin was shown to promote DDP sensitivity and apoptosis of human OC DDP-resistant cell line (SKOV3/A2780-DDP cells). Significantly increased p62 expression and reduced LC3 expression were found in naringin-treated cells. The autophagy agonist, rapamycin, reversed the effect of naringin on the resistance of SKOV3-DDP cells to DDP. Naringin inhibited levels of TGF-β2/smad2 pathway-related proteins, and regulated autophagy in SKOV3-DDP cells. In vivo experiments demonstrated that injection of naringin inhibited DDP resistance and autophagy in mice xenograft model. In summary, naringin inhibits DDP resistance in OC cells by inhibiting autophagy mediated by the TGF-β2/smad2 pathway.

The Flavoring Agent Ethyl Vanillin Induces Cellular Stress Responses in HK-2 Cells.

Flavored e-cigarettes are a popular alternative to cigarette smoking; unfortunately, the extrapulmonary effects are not well-characterized. Human proximal tubule cells were cultured for 24 or 48 h with 0-1000 µM ethyl vanillin (ETH VAN) and cytotoxicity evaluated. Mitochondrial health was significantly diminished following 48 h of exposure, accompanied by significantly decreased spare capacity, coupling efficiency, and ATP synthase expression. ETH VAN at 24 h inhibited glycolysis. The endoplasmic reticulum (ER) stress marker C/EBP homologous protein (CHOP) was increased at 100 μM relative to 500-1000 μM. The downstream proapoptotic marker cleaved caspase-3 subsequently showed a decreasing trend in expression after 48 h of exposure. The autophagy biomarkers microtubule-associated proteins 1A/1B light chain 3 (LC3B-I and LC3B-II) were measured by Western blot. LC3B-II levels and the LC3B-II/LC3B-I ratio increased at 24 h, which suggested activation of autophagy. In contrast, by 48 h, the autophagy biomarker LC3B-II decreased, resulting in no change in the LC3B-II/LC3B-I ratio. Mitophagy biomarker PTEN-induced putative kinase 1 (PINK1) expression decreased after 48 h of exposure. The downstream marker Parkin was not significantly changed after 24 or 48 h. These findings indicate that the flavoring ETH VAN can induce energy pathway dysfunction and cellular stress responses in a renal model.

Integrated Spatial Multi-Omics Study of Postmortem Brains of Alzheimer's Disease.

Pathological hallmark of Alzheimer's disease (AD) is characterized by the accumulation and aggregation of amyloid β (Aβ) peptides into extracellular plaques of the brain. Clarification of the process of how soluble Aβ starts to assemble into amyloid fibrils is an essential step in elucidating the pathogenesis of AD. In our previous study, Aβ proteoforms including full-length Aβ40 and Aβ42/43 with N- and C-terminal truncated forms were visualized in postmortem brains from AD patients with matrix-assisted laser desorption/ionization-based mass spectrometry imaging (MALDI-MSI). In this study, Aβ proteoforms were consistently visualized by an updated protocol, and uncharacterized peptides such as Aβ1-29 and Aβ10-40 in AD brains were also visualized. To decipher neurotoxic effects of Aβ in patients' brains, here we integrate liquid chromatography tandem mass spectrometry (LC-MS/MS) based shotgun proteomics with laser microdissection (LMD) excised tissue samples as well as direct tissue imaging with MALDI-MSI. With this approach, we have highlighted dynamic alterations of microtubule associating proteins (MAPs) including MAP1A, MAP1B and MAP2 as well as AD dominant proteins including APP, UCHL1, SNCA, and APOE. Of note, as lipid dysregulation has been implicated with AD pathology, we have challenged to integrate proteomics and lipid imaging for AD and control brain tissue. Spatial multi-omics is also valid to uncover molecular pathology of white matter as well as grey matter and leptomeningeal area, for example, by visualizing heme in patients' postmortem brains.

Follicular fluid HD-sevs-mir-128–3p is a key molecule in regulating bovine granulosa cells autophagy

Follicular fluid (FF) is rich in extracellular vesicles (EVs). EVs carries a variety of miRNA involved in regulating follicular development, the function of cells in follicles, primordial follicular formation, follicular recruitment and selection, follicular atresia, oocyte communication, granulosa cells (GCs) function and luteinization and other biological processes of follicular development. Previous studies in our laboratory have shown that bovine follicular fluid (bFF) high density-small extracellular vesicles (HD-sEVs)-miRNA was enriched in autophagy-related pathways. However, the mechanism of bFF EVs carrying miRNA regulating GCs autophagy is not clear. Thus, this study carried out a series of studies on the previous HD-sEVs sequencing data and miR-128–3p contained in bFF HD-sEVs. A total of 38 differentially expressed genes were detected by RNA-Seq after overexpression of miR-128–3p in bovine GCs (bGCs). Through cell transfection, Western blot (WB) and Immunofluorescence (IF), it was proved that overexpression of miR-128–3p could promote the expression of LC3 (microtubule-associated protein I light chain 3), inhibit p62, promote the number of autophagosome, promote the formation of autophagy lysosome and autophagy flow, and activate bGCs autophagy. MiR-128–3p inhibitor significantly inhibited the expression of LC3 and monodansylcadaverine (MDC) in bGCs, and promoted the expression of autophagy substrate p62, indicating that HD-sEVs-miR-128–3p could activate bGCs autophagy. In addition, through double luciferase assay, bioinformatics analysis, WB and RT-qPCR, it was concluded that bFF HD-sEVs-miR-128–3p could target TFEB (transcription factor EB) and FoxO4 (Forkhead box O4) and activate GCs autophagy.

Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

Oligodendrocyte Dysfunction in Tauopathy: A Less Explored Area in Tau-Mediated Neurodegeneration.

Aggregation of the microtubule-associated protein tau (MAPT) is the hallmark pathology in a spectrum of neurodegenerative disorders collectively called tauopathies. Physiologically, tau is an inherent neuronal protein that plays an important role in the assembly of microtubules and axonal transport. However, disease-associated mutations of this protein reduce its binding to the microtubule components and promote self-aggregation, leading to formation of tangles in neurons. Tau is also expressed in oligodendrocytes, where it has significant developmental roles in oligodendrocyte maturation and myelin synthesis. Oligodendrocyte-specific tau pathology, in the form of fibrils and coiled coils, is evident in major tauopathies including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD). Multiple animal models of tauopathy expressing mutant forms of MAPT recapitulate oligodendroglial tau inclusions with potential to cause degeneration/malfunction of oligodendrocytes and affecting the neuronal myelin sheath. Till now, mechanistic studies heavily concentrated on elucidating neuronal tau pathology. Therefore, more investigations are warranted to comprehensively address tau-induced pathologies in oligodendrocytes. The present review provides the current knowledge available in the literature about the intricate relations between tau and oligodendrocytes in health and diseases.

Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice.

JOURNAL/nrgr/04.03/01300535-202508000-00025/figure1/v/2024-09-30T120553Z/r/image-tiff TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon. TAU is missorted and aggregated in an array of diseases known as tauopathies. Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications, changes of which affect microtubule stability and dynamics, microtubule interaction with other proteins and cellular structures, and mediate recruitment of microtubule-severing enzymes. As impairment of microtubule dynamics causes neuronal dysfunction, we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics. We therefore aimed to study the effects of a disease-causing mutation of TAU (P301L) on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics, to assess whether P301L-TAU causes stability-changing modifications to microtubules. To investigate TAU localization, phosphorylation, and effects on tubulin post-translational modifications, we expressed wild-type or P301L-TAU in human MAPT -KO induced pluripotent stem cell-derived neurons (iNeurons) and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU (pR5 mice). Human neurons expressing the longest TAU isoform (2N4R) with the P301L mutation showed increased TAU phosphorylation at the AT8, but not the p-Ser-262 epitope, and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons. P301L-TAU showed pronounced somatodendritic presence, but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU. P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation, but reduced acetylation, of microtubules compared with non-transgenic littermates. In sum, P301L-TAU results in changes in microtubule PTMs, suggestive of impairment of microtubule stability. This is accompanied by missorting and aggregation of TAU in mice but not in iNeurons. Microtubule PTMs/impairment may be of key importance in tauopathies.