Microtubule-associated Protein Research Articles

Characterization of multiple binding sites on microtubule associated protein 2c recognized by dimeric and monomeric 14-3-3ζ.

Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.

Roles of C/EBPβ/AEP in Neurodegenerative Diseases.

In recent years, an increasing number of studies have shown that increased activation of aspartic endopeptidases (AEPs) is a common symptom in neurodegenerative diseases (NDDs). AEP cleaves amyloid precursor protein (APP), tau (microtubule-associated protein tau), α- synuclein (α-syn), SET (a 39-KDa phosphoprotein widely expressed in various tissues and localizes predominantly in the nucleus), and TAR DNA-binding protein 43 (TDP-43), and promotes their aggregation, contributing to Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) pathogenesis. Abundant evidence supports the notion that CCAAT/enhancer-binding protein β (C/EBPβ)/AEP may play an important role in NDDs. Developing its small molecule inhibitors is a promising treatment of NDDs. However, current research suggests that the pathophysiological mechanism of the C/EBPβ/AEP pathway is very complex in NDDs. This review summarizes the structure of C/EBPβ and AEP, their major physiological functions, potential pathogenesis, their small molecule inhibitors, and how C/EBPβ/AEP offers a novel pathway for the treatment of NDDs.

Autophagy Attenuates Oxidative Stress-Induced Collagen Degradation in Vaginal Fibroblasts: Implications for Pelvic Organ Prolapse.

The relationship between autophagy and pelvic organ prolapse (POP) remains unknown. The aim of this novel experimental study, utilizing tissue samples derived from women undergoing gynecological surgery, is to investigate the role of autophagy in mitigating collagen degradation in human vaginal fibroblasts induced by oxidative stress, with particular emphasis on its implications in the pathogenesis of POP. Exploring the role of autophagy in protecting against collagen degradation and cellular senescence in human vaginal fibroblasts under oxidative stress may offer new insights into therapeutic strategies for conditions such as POP. This study consists of laboratory-based experimental research that utilizes tissue samples collected from female patients undergoing gynecological surgery to analyze the role of autophagy in collagen degradation induced by oxidative stress. By treating with different concentrations of hydrogen peroxide (H2O2) and using rapamycin (RAPA) and 3-methyladenine (3-MA) as autophagy activators and inhibitors respectively, the effects on human vaginal fibroblasts (HVFs) were evaluated. Cell viability was determined using the Cell Counting Kit-8 test. Cellular senescence was determined with senescence-associated-β-galactosidase labeling and western blotting to identify the expression of p21 and p53. Reactive oxygen species (ROS) were determined with 2,7-dichlorofluorescin diacetate. Additionally, western blotting was used to establish collagen I, collagen III, microtubule-associated protein 1A/1B-light chain 3 (LC3), Beclin-1, and p62 and reverse transcription-quantitative polymerase chain reaction was used to determine the mRNA levels of COL3A1, COL1A1,TIMP1,MMP9,LC3,andBeclin-1to investigate collagen metabolism and autophagic activity. The results showed that high-dose H2O2 significantly increased ROS levels, cell senescence, and collagen degradation in HVFs. The combined use of RAPA significantly reduced ROS levels, collagen degradation, and cell senescence, but this protective effect disappeared when 3-MA was added. Nevertheless, co-treatment of HVFs with RAPA, H2O2, and 3-MA abolished the positive impact of RAPA via boosting autophagy resistance. Autophagy inhibits collagen degeneration and cellular senescence caused by oxidative stress.

Jumonji domain-containing protein 6 promotes gastric cancer progression: Modulating immune evasion through autophagy and oxidative stress pathways

Objective: Immune response is crucial in the development of gastric cancer (GC), and Jumonji domain-containing protein 6 (JMJD6) plays an important role in mediating GC cell behavior. This study aims to elucidate the mechanisms through which JMJD6 affects autophagy and immune evasion in GC cells. Material and Methods: Immunocytochemistry was employed to assess JMJD6 and programmed death-ligand 1 (PD-L1) levels in gastric cancer cell line (MKN-45) and gastric epithelial cell line cells. MKN-45 cells with JMJD6 knockdown and overexpression were generated. The effect of JMJD6 on MKN-45 cells was evaluated using cell counting kit-8 assay, cellular fluorescence staining, and Transwell assays. Western blot analysis and immunofluorescence techniques were employed to investigate the regulation of autophagy by JMJD6. Reactive oxygen species (ROS) levels were evaluated by applying ROS fluorescence staining. Meanwhile, the protein and gene expression levels of molecules related to antioxidant stress responses were assessed through immunofluorescence assays and quantitative real-time polymerase chain reactions, respectively. Results: The expression levels of JMJD6 and PD-L1 were elevated in GC cells (P < 0.001). JMJD6 overexpression enhanced MKN-45 cell migration, invasion, and colony formation in vitro (P < 0.001). In MKN-45 cells, the epithelial-mesenchymal transition was promoted by JMJD6 upregulation but was notably inhibited by JMJD6 knockdown (P < 0.001). JMJD6 overexpression increased the expression levels of Sequestosome 1, Microtubule-associated protein 1A/1B-light chain 3 (LC3)II/LC3I, and PD-L1 in MKN-45 cells, and autophagy activation further elevated PD-L1 levels (P < 0.001). In addition, JMJD6 overexpression reduced ROS production and increased the expression of molecules related to antioxidant stress response, with the reverse effects observed on JMJD6 knockdown (P < 0.001). Conclusion: JMJD6 notably facilitates GC progression and immune evasion by modulating autophagy and oxidative stress pathways.

Effect of invivo reprogramming of astrocytes combined with exercise training on neurorepair in rats with spinal cord injury.

The inability of damaged neurons to regenerate and of axons to establish new functional connections leads to permanent functional deficits after spinal cord injury (SCI). Although astrocyte reprogramming holds promise for neurorepair in various disease models, it is not sufficient on its own to achieve significant functional recovery. A rat SCI model was established using a spinal cord impactor. Seven days postsurgery, adeno-associated virus were injected to overexpress the transcription factors NeuroD1 and Neurogenin-2 (Ngn2) in the spinal cord. The rats were then trained to walk on a weight-supported treadmill for 4 weeks, starting 14 days after modeling. The effects of these interventions on motor and sensory functions, as well as spinal cord tissue repair, were subsequently evaluated. The combination of NeuroD1 and Ngn2 overexpression with weight-supported exercise training significantly improved gait compared to either intervention alone. The group receiving the combined intervention exhibited enhanced sensitivity in sensory assessments. Immunofluorescence analysis revealed increased colocalization of astrocytes and microtubule-associated protein 2-positive neurons in the injury area. These effects were more pronounced than those observed with spinal cord tissue repair alone. Additionally, the combined intervention significantly reduced glial scarring and the size of the injury area. Exercise intervention enhances the reprogramming effects of astrocytes and restores motor function, yielding better results than either intervention alone.

P0210 ORMDL proteins regulate intestinal epithelial cell homeostasis by modulating ER architecture, autophagy, and DNA damage response

Abstract Background ORM1-like protein 3 (ORMDL3) is a risk gene for asthma and inflammatory bowel disease (IBD), identified in genome-wide association studies (GWAS).1,2 It encodes an endoplasmic reticulum (ER)-resident transmembrane protein implicated in calcium homeostasis, sphingolipid biosynthesis, and regulation of the unfolded protein response (UPR) upon ER stress.3,4 However, the role of ORMDL3 and its homologous protein family members ORMDL1 and ORMDL2, which exhibit partly redundant functions, remains unclear in the context of intestinal inflammation. Methods Constitutive and conditional intestinal epithelial cell-specific knockout (KO) mice for Ormdl1, Ormdl2, and Ormdl3 were generated. Histological analysis of small intestinal (SI) Paneth cells (lysozyme immunohistochemistry), goblet cells (periodic acid-Schiff staining) and ER morphology in Paneth cells (transmission electron microscopy [TEM]) was conducted. Interactions between ORMDL proteins and autophagy-related proteins were examined by co-immunoprecipitation and immunoblot assays in irradiated mouse embryonic fibroblasts (iMEFs). Organoids derived from Ormdl1/2/3ΔIEC mice were used for bulk RNA sequencing. Results The Ormdl1/2/3 triple KO was embryonically lethal, while Ormdl3-KO and Ormdl1/3 double KO mice exhibited reduced body size, weight, and embryonic viability, along with fewer Paneth and goblet cells. To eliminate any compensatory effects of the multiple ORMDL proteins, the Ormdl1/2/3ΔIEC mice were analyzed. These mice showed no macroscopic phenotype but displayed microscopic inflammation, reduced Paneth and goblet cells, and increased SI tuft cells. Both constitutive and conditional Ormdl knockout mice showed massive dilation of the ER. While autophagy is a known compensatory mechanism resolving ER stress, we demonstrated that ORMDL3 co-localized and co-precipitated with the autophagy-related protein LC3 (microtubule-associated protein 1A/1B-light chain 3). Consistent with this, loss of ORMDL proteins compromised autophagic flux. Bulk RNA sequencing of SI organoids from Ormdl1/2/3ΔIEC mice revealed reduced expression of genes involved in lipid biosynthesis, tuft cell differentiation via BMP-signalling, and innate immunity, including antimicrobial peptides, likely due to loss of Paneth cells. In contrast, DNA damage response genes were upregulated, as reflected by an increase in γ-H2AX-positive epithelial cells. Conclusion The data suggest a critical regulatory role for ORMDL proteins in the ER-autophagy axis within epithelial cells, shaping mucosal homeostasis and expression of antimicrobial peptides, thereby conferring protection against IBD. Further studies are required to elucidate the molecular basis of the function of ORMDL proteins in intestinal inflammation.

The Kinesin Kar3 is Required for Endoplasmic Reticulum-Associated Degradation.

Degradation of aberrant, excess, and regulatory proteins at the endoplasmic reticulum (ER) is a conserved feature of eukaryotic cells, disruption of which contributes to disease. While remarkable progress has been made in recent years, mechanisms and genetic requirements for ER-Associated Degradation (ERAD) remain incompletely understood. We recently conducted a screen for genes required for turnover of a model ER translocon-associated substrate of the Hrd1 ubiquitin ligase in Saccharomyces cerevisiae. This screen revealed loss of Kar3 impedes degradation of Deg1*-Sec62, which persistently and aberrantly engages the translocon. Kar3 is a microtubule-associated kinesin 14 family member that impacts multiple aspects of microtubule dynamics during cell division and karyogamy. We investigated involvement of Kar3 and its cofactors in ERAD. Loss of Kar3 hindered ERAD mediated by three ubiquitin ligases but did not impair turnover of a soluble nuclear protein. Further, KAR3 deletion caused hypersensitivity to conditions associated with proteotoxic stress. Kar3's cytoplasmic cofactor Vik1 was also required for efficient degradation of Deg1*-Sec62. Our results reveal a profound and underappreciated role for microtubule-associated proteins in ERAD.

Luteolin ameliorates rat model of metabolic syndrome-induced cardiac injury by apoptosis suppression and autophagy promotion via NR4A2/p53 regulation

BackgroundReduced cardiac autophagy, inflammation, and apoptosis contribute to cardiovascular complications caused by metabolic syndrome (MetS). It is documented that the nuclear receptor 4A2 (NR4A2) could modulate autophagy and apoptosis in cardiac complications. The aim of this investigation was to assess the therapeutic potential of luteolin, with documented beneficial properties, against MetS-associated cardiac injury.MethodsForty male albino Wistar rats were divided into 5 groups randomly as controls, MetS, and MetS animals treated with luteolin (25, 50, 100 mg/kg ip). The animal’s weight, blood pressure, lipid profile, tolerance to glucose and insulin, and cardiac histopathology were evaluated. Moreover, troponin T, creatine kinase-myocardial band (CK-MB), inflammatory profile (IL-6, IL-1β, TNF-α), transforming growth factor-β1 (TGF-β1), oxidative stress, and matrix metalloproteinase-9 (MMP-9) were analyzed to determine the cardiac state. Cardiac NR4A2 and p53, as well as apoptotic (B-cell leukemia/lymphoma 2 [BCL-2], Caspase [CASP]-3, and CASP-9) and autophagic mediators (Sequestosome-1/p62, Microtubule-associated protein 1 A/1B-light chain 3 [LC3], and Beclin-1) were measured by RT-qPCR and ELISA.ResultsLuteolin remarkably restored MetS-induced biochemical derangements and related cardiac injury via the suppression of apoptosis, inflammation, and stress but promotion of autophagy (p-value < 0.001).ConclusionCurrent findings revealed the promising therapeutical properties of luteolin against MetS-associated cardiovascular risks.

Novel synaptic markers predict early tau pathology and cognitive deficit in an asymptomatic population at risk of Alzheimer's disease.

Cognitive dysfunction in Alzheimer's disease (AD) correlates closely with pathology in the neuronal microtubule-associated protein tau. Tau pathology may spread via neural synapses. In a population of cognitively unimpaired elderly at elevated risk of AD, we investigated four cerebrospinal (CSF) markers of synaptic dysfunction and degeneration. Three of these (SYT1, SNAP25, and ADAM23) are derived from pre-synaptic structures, while ADAM22 reflects post-synaptic changes. All four markers correlated strongly with tau protein measures. In statistical models, SYT1 accounted for more than half the total variance in both total- and P(181)-tau levels. Observed correlations with CSF levels of Alzheimer amyloid-β (Aβ42) were somewhat weaker. In longitudinal data, baseline levels of ADAM22 and ADAM23 robustly predicted increase over time in both total- and P-tau. CSF SYT1 levels also correlated with PET image uptake of tau and (at a trend level) Aβ in areas of interest for early AD pathology. CSF SYT1 and SNAP25 levels correlated inversely with a global psychometric score and several of its domain subscales. In quantitative trait loci analyses, all four synaptic markers were associated with at least one AD genetic risk locus. Upon "staging" participants by their evidence of amyloid and tau pathology (A/T/N framework), the CSF synaptic markers were unexpectedly reduced in participants with CSF evidence of amyloid but not tau pathology. They were clearly elevated, however, in the CSF of persons with indications of both tau and amyloid pathology. These observations provide evidence for clear pre-synaptic degeneration in cognitively unimpaired persons with biomarker evidence of early AD pathology.

The Role of Autophagy in Copper-Induced Apoptosis and Developmental Neurotoxicity in SH-SY5Y Cells.

Copper (Cu) is a global environmental pollutant that poses a serious threat to humans and ecosystems. Copper induces developmental neurotoxicity, but the underlying molecular mechanisms are unknown. Neurons are nonrenewable, and they are unable to mitigate the excessive accumulation of pathological proteins and organelles in cells, which can be ameliorated by autophagic degradation. In this study, we established an in vitro model of Cu2+-exposed (0, 15, 30, 60 and 120 μM) SH-SY5Y cells to explore the role of autophagy in copper-induced developmental neurotoxicity. The results showed that copper resulted in the reduction and shortening of neural synapses in differentiated cultured SH-SY5Y cells, a downregulated Wnt signaling pathway, and nuclear translocation of β-catenin. Exposure to Cu2+ increased autophagosome accumulation and autophagic flux blockage in terms of increased sequestosome 1 (p62/SQSTM1) and microtubule-associated protein 1 light chain 3B (LC3B) II/LC3BI expressions and inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. Furthermore, copper induced apoptosis, characterized by increased expressions of Bcl2 X protein (Bax), caspase 3, and Poly (ADP-ribose) polymerase (PARP) and decreased expression of B-cell lymphoma 2 (Bcl2). Compared with the 120 μM Cu2+ exposure group alone, autophagy activator rapamycin pretreatment increased expression of Wnt and β-catenin nuclear translocation, decreased expression of LC3BII/LC3BI and p62, as well as upregulated expression of Bcl2 and downregulated expressions of caspase 3 and PARP. In contrast, after autophagy inhibitor chloroquine pretreatment, expressions of Wnt and β-catenin nuclear translocation were decreased, expression levels of LC3BII/LC3BI and p62 were upregulated, expression of Bcl2 was decreased, while expression levels of caspase 3, Bax, and PARP were increased. In conclusion, the study demonstrated that autophagosome accumulation and autophagic flux blockage were associated with copper-induced developmental neurotoxicity via the Wnt signaling pathway, which might deepen the understanding of the developmental neurotoxicity mechanism of environmental copper exposure.

Brain-derived tau oligomer polymorphs: distinct aggregations, stability profiles, and biological activities

Aggregation of microtubule-associated tau protein is a distinct hallmark of several neurodegenerative disorders such as Alzheimer’s disease (AD), dementia with Lewy bodies (DLB), and progressive supranuclear palsy (PSP). Tau oligomers are suggested to be the primary neurotoxic species that initiate aggregation and propagate prion-like structures. Furthermore, different diseases are shown to have distinct structural characteristics of aggregated tau, denoted as polymorphs. Here, we investigate the structural and functional differences of amplified brain-derived tau oligomers (aBDTOs) from AD, DLB, and PSP. Our results indicate that the aBDTOs possess different structural and morphological features that impact neuronal function, gene regulation, and ultimately disease progression. The distinct tau oligomeric polymorphs may thus contribute to the development of clinical phenotypes and shape the progression of diseases. Our results can provide insight into developing personalized therapy to target a specific neurotoxic tau polymorph.

Regulatory roles of eugenol in paraquat-altered SNCA/LZTS3/MAPT in the cerebellum of Wistar rats

BackgroundThe Microtubules-associated protein tau (MAPT), alpha-synuclein (SNCA), and leucine zipper tumor suppressor 3 (LZTS3) genes are implicated in neurodegeneration and tumor suppression, respectively. This study investigated the regulatory roles of eugenol on paraquat-altered genes.ResultsForty male Wistar rats divided into five groups of eight rats were used. The control group received normal saline; the Paraquat (PQ)-untreated group received only Paraquat. The low dose of eugenol was 200 mg/kg, the medium dose of eugenol was 400 mg/kg, and the high dose of eugenol was 600 mg/kg. All groups except the control group received 10 mg/kg of PQ orally for 14 days at one-day intervals, allowing PQ in the rats for 28 days. Eugenol treatment started on the 29th and lasted 14 days. Motor impairments were determined using wire string and beam-walk; biomarkers were estimated using cerebellar homogenates, while frozen cerebellum was used to study LZTS3, MAPT, and SNCA gene expression. LZTS3 was significantly suppressed in the PQ-untreated group and highly expressed in the eugenol-treated group. The MAPT and SNCA genes were overexpressed in the PQ-untreated group compared to the control group. Eugenol significantly decreased the expression of these genes compared to that in the PQ-untreated group. Antioxidants were reduced considerably, and oxidative stress markers were increased significantly, which could have caused increased protein fibrillation and reduced limb functionality. Histology revealed that eugenol mitigated the alterations caused by Paraquat.ConclusionsPQ can enhance tumor expression in addition to causing neurotoxicity, which decreases limb functionality, while eugenol, an antioxidant, can mitigate the effects of PQ.

The ER protein CANX (calnexin)-mediated autophagy protects against alzheimer disease

ABSTRACT Although the relationship between macroautophagy/autophagy and Alzheimer disease (AD) is widely studied, the underlying mechanisms are poorly understood, especially the regulatory role of the initiation signaling of autophagy on AD. Here, we find that the ER transmembrane protein CANX (calnexin) is a novel interaction partner of the autophagy-inducing kinase ULK1 and is required for ULK1 recruitment to the ER under basal or starved conditions. Loss of CANX results in the inactivity of ULK1 kinase and inhibits autophagy flux. In the brains of people with AD and APP-PSEN1 mice, the interaction of CANX and ULK1 declines. In mice, the lack of CANX in hippocampal neurons causes the accumulation of autophagy receptors and neuron damage, which affects the cognitive function of C57BL/6 mice. Conversely, overexpression of CANX in hippocampal neurons enhances autophagy flux and partially contributes to improving cognitive function of APP-PSEN1 mice, but not the CANX variant lacking the interaction domain with ULK1. These findings reveal a novel role of CANX in autophagy activity and cognitive function by cooperating with ULK1. Abbreviation: AD: Alzheimer disease; APEX: ascorbate peroxidase; APP: amyloid beta precursor protein; APP-PSEN1 mice: amyloid beta precursor protein-presenilin 1 transgenic mice; ATG: autophagy related; Aβ: amyloid-β; BiFC: bimolecular fluorescence complementation; CANX: calnexin; EBSS: Earle’s balanced salt solution; EM: electron microscopy; IP: immunopurification; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MWM: Morris water maze; PLA: proximity ligation assay; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1/p62, sequestosome 1.

CRMP/UNC-33 maintains neuronal microtubule arrays by promoting individual microtubule rescue.

Microtubules (MTs) are intrinsically dynamic polymers. In neurons, staggered individual microtubules form stable, polarized acentrosomal MT arrays spanning the axon and dendrite to support long-distance intracellular transport. How the stability and polarity of these arrays are maintained when individual MTs remain highly dynamic is still an open question. Here, we visualize MT arrays invivo in C.elegans neurons with single MT resolution. We find that the CRMP family homolog UNC-33 is essential for the stability and polarity of MT arrays in neurites. In unc-33 mutants, MTs exhibit dramatically reduced rescue after catastrophe, develop gaps in coverage, and lose their polarity, leading to trafficking defects. UNC-33 is stably anchored on the cortical cytoskeleton and forms patch-like structures along the dendritic shaft. These discrete and stable UNC-33 patches concentrate free tubulins and correlate with MT rescue sites. Invitro, purified UNC-33 preferentially associates with MT tips and increases MT rescue frequency. Together, we propose that UNC-33 functions as a microtubule-associated protein (MAP) to promote individual MT rescue locally. Through this activity, UNC-33 prevents the loss of individual MTs, thereby maintaining the coverage and polarity of MT arrays throughout the lifetime of neurons.

Non-mutated human tau stimulates Alzheimer's disease-relevant neurodegeneration in a microglia-dependent manner.

The accumulation of abnormal, non-mutated tau protein is a key pathological hallmark of Alzheimer's disease (AD). Despite its strong association with disease progression, the mechanisms by which tau drives neurodegeneration in the brain remain poorly understood. Here, we selectively expressed non-mutated or mutated human microtubule-associated protein tau ( hMAPT ) in neurons across the brain and observed neurodegeneration in the hippocampus, especially associated with non-mutated human tau. Single-nuclei RNA sequencing confirmed a selective loss of hippocampal excitatory neurons by the wild-type tau and revealed the upregulation of neurodegeneration-related pathways in the affected populations. The accumulation of phosphorylated tau was accompanied by cellular stress in neurons and reactive gliosis in multiple brain regions. Notably, the lifelong absence of microglia significantly and differentially influenced the extent of neurodegeneration in the hippocampus and thalamus. Therefore, our study established an AD-relevant tauopathy mouse model, elucidated both neuron-intrinsic and neuron-extrinsic responses, and highlighted critical and complex roles of microglia in modulating tau-driven neurodegeneration.

High glucose induces renal tubular epithelial cell senescence by inhibiting autophagic flux.

Autophagy, a cellular degradation process involving the formation and clearance of autophagosomes, is mediated by autophagic proteins, such as microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (p62), and modulated by 3-methyladenine (3-MA) as well as chloroquine (CQ). Senescence, characterised by permanent cell cycle arrest, is marked by proteins such as cyclin-dependent kinase inhibitor 1 (p21) and tumour protein 53 (p53). This study aims to investigate the relationship between cell senescence and renal function in diabetic kidney disease (DKD) and the effect of autophagy on high-glucose-induced cell senescence. We categorised 46 patients with DKD diagnosed by renal biopsy into classes I, IIa, IIb, III and IV and used four normal kidney specimens from patients with renal trauma as controls. We evaluated pathological changes, LC3 and p21. We used streptozotocin-induced DKD models in rats and 35mM glucose-cultured human proximal tubular epithelial cells (HK-2) with or without 3-MA and CQ. We assessed p53, p21, LC3 and p62. We observed autophagosomes and detected senescence-associated galactosidase (SA-β-gal) activity. In patients with DKD, p21 and LC3 expression levels increased over time and correlated positively with blood creatinine and proteinuria. In DKD rats and HK-2 cells, p21, p53, LC3 and p62 expression levels were higher than in the controls, as were SA-β-gal-positive cells, renal tubular autophagosomes and co-expression of p21 and LC3. The 3-MA reduced p16, p21 and p53 expression compared with the high glucose group, whereas CQ had the opposite effect. These results suggest that renal tubular cell senescence is associated with the progression of DKD. Additionally, autophagic flux may play a role in mediating high-glucose-induced senescence in renal tubular cells.

Bushen-Huoxue-Mingmu-Formula attenuates pressurization-induced retinal ganglion cell damage by reducing mitochondrial autophagy through the inhibition of the Pink1/Parkin pathway.

Bushen-Huoxue-Mingmu-Formula (MMF) has achieved definite clinical efficacy. However, its mechanism is still unclear. Investigating the molecular mechanism of MMF to protect retinal ganglion cells (RGCs). This study developed a pressurization-induced model of damaged RGCs, which were then treated with a serum supplemented with MMF. The effects of MMF on proliferation, apoptosis, adenosine 5'-triphosphate content, and mitochondrial structure of RGCs were investigated, and the underlying molecular mechanism was explored by RNA interference experiment. In the pressurization-induced RGC injury model, apoptosis rate increased, cell proliferation decreased, adenosine 5'-triphosphate content reduced, mitochondrial structure was disrupted, BCL2-associated X, cleaved caspase-3, and microtubule-associated proteins light chain 3 II/I protein expression enhanced, B cell lymphoma-2 and p62 protein expression decreased, and the Pink1/Parkin pathway was activated. The stress-induced damage to RGCs was, however, reversible following MMF-mediated inhibition of the Pink1/Parkin pathway. Pink1 short-hairpin RNA downregulated Pink1 expression in RGCs, which led to outcomes that aligned with those observed with MMF intervention. MMF altered the expression of apoptosis- and autophagy-related proteins and possibly inhibited the Pink1/Parkin signaling pathway, which led to reduced pressurization-induced mitochondrial autophagy in RGCs. This preventive effect of MMF on RGCs can be potentially useful to preserve the viability of RGCs.

Changes to the Autophagy-Related Muscle Proteome Following Short-Term Treatment with Ectoine in the Duchenne Muscular Dystrophy Mouse Model mdx.

The most severe form of muscular dystrophy (MD), known as Duchenne MD (DMD), remains an incurable disease, hence the ongoing efforts to develop supportive therapies. The dysregulation of autophagy, a degradative yet protective mechanism activated when tissues are under severe and prolonged stress, is critically involved in DMD. Treatments that harness autophagic capacities therefore represent a promising therapeutic approach. Osmolytes are protective organic molecules that regulate osmotic pressure and cellular homeostasis and may support tissue-repairing autophagy. We therefore explored the effects of the osmolyte ectoine in the standard mouse model of DMD, the mdx, focusing on the autophagy-related proteome. Mice were treated with ectoine in their drinking water (150 mg/kg) or through daily intraperitoneal injection (177 mg/kg) until they were 5.5 weeks old. Hind limb muscles were dissected, and samples were prepared for Western blotting for protein quantification and for immunofluorescence for an evaluation of tissue distribution. We report changes in the protein levels of autophagy-related 5 (ATG5), Ser366-phosphorylated sequestosome 1 (SQSTM1), heat shock protein 70 (HSP70), activated microtubule-associated protein 1A/1B-light chain 3 (LC3 II) and mammalian target of rapamycin (mTOR). Most importantly, ectoine significantly improved the balance between LC3 II and SQSTM1 levels in mdx gastrocnemius muscle, and LC3 II immunostaining was most pronounced in muscle fibers of the tibialis anterior from treated mdx. These findings lend support for the further investigation of ectoine as a potential therapeutic intervention for DMD.

GENI as an AMPK Activator Binds α and γ Subunits and Improves the Memory Dysfunction of Alzheimer's Disease Mouse Models via Autophagy and Neuroprotection.

Geniposidic 4-isoamyl ester (GENI) with anti-aging effects is a new iridoid glycoside derivative from Gardenia jasminoides Ellis found in our previous study. In this study, to indicate whether this compound has anti-Alzheimer's disease (AD) effect, the galactose-induced AD mice and naturally aging mice with AD were used to do drug efficacy evaluation. Furthermore, the Western blot, small interfering RNA (siRNA), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CESTA), liquid chromatography-tandem mass spectrometry (LC/MS-MS), adenosine 5'-monophosphate-activated protein kinase (AMPK) mutants and surface plasmon resonance (SPR) analysis were utilized to clarify the mechanism of action and identify target protein of this molecule. GENI exerts anti-AD efficacy in galactose-induced AD mice and naturally aging mice with AD through neuroprotection and modification of autophagy and neuron inflammation. Moreover, AMPK as the target protein of GENI to produce an anti-AD effect is identified and the ASP148, ASP157, and ASP166 of the AMPK α subunit and lysine (LYS)148, aspartic acid (ASP)156, LYS309, and ASP316 in the AMPK γ subunit as binding sites are confirmed. Meanwhile, the AMPK/unc-51-like autophagy-activating kinase 1 (ULK1)/microtubule-associated protein 1 light chain 3 beta (LC3B) and AMPK/mammalian target of rapamycin (mTOR) signaling pathways involved in anti-AD effects of GENI. The findings provide a new perspective on treating neurodegenerative diseases by activating AMPK for the energy metabolism disorder.

Efficient Biochemical Method for Characterizing and Classifying Related Amyloidogenic Peptides.

Amyloidosis is a group of proteinopathies characterized by the systemic or organ-specific deposition of proteins in the form of amyloid fibers. Nearly 40 proteins play a role in these pathologies, and the structures of the associated fibers are beginning to be determined by Cryo-EM. However, the molecular events underlying the process, such as fiber nucleation and elongation, are poorly understood, which impairs developing efficient therapies. In most cases, only a few dozen amino acids of the pathological protein are found in the final structure of the fibers, while amyloid peptides comprising five to 10 amino acids are involved in the fiber nucleation process. The identification and biochemical characterization of these peptides are therefore of major scientific and clinical importance. We demonstrated that in silico approaches are limited due to the peptides' small size and long-distance intra- and intermolecular interactions that occur during nucleation. To address this problem, we developed a novel biochemical method for characterizing and classifying batches of related peptides. Initial work to optimize our approach is based on the reference peptide PHF6 (β1) from Microtubule-Associated Protein Tau (MAPT) as compared to 22 related peptides. Depending on their biochemical properties and using the Garnier-Delamarche plot we propose, we classified these peptides into three groups: aggregative, amyloid, and soluble (neither aggregative nor amyloid). We emphasize that our biochemical classification method is applicable to any family of peptides and could be scaled up for high-throughput analyses.