Microtubule-associated Protein Research Articles

HIPK3 maintains sensitivity to platinum drugs and prevents disease progression in gastric cancer

In the realm of cancer therapeutics and resistance, kinases play a crucial role, particularly in gastric cancer (GC). Our study focused on platinum-based chemotherapy resistance in GC, revealing a significant reduction in homeodomain-interacting protein kinase 3 (HIPK3) expression in platinum-resistant tumors through meticulous analysis of transcriptome datasets. In vitro and in vivo experiments demonstrated that HIPK3 knockdown enhanced tumor proliferation and metastasis, while upregulation had the opposite effect. We identified the myocyte enhancer factor 2C (MEF2C) as a transcriptional regulator of HIPK3 and uncovered HIPK3's role in downregulating the morphogenesis regulator microtubule-associated protein (MAP7) through ubiquitination. Phosphoproteome profiling revealed HIPK3's inhibitory effects on mTOR and Wnt pathways crucial in cell proliferation and movement. A combined treatment strategy involving oxaliplatin, rapamycin, and IWR1-1-endo effectively overcame platinum resistance induced by reduced HIPK3 expression. Monitoring HIPK3 levels could serve as a GC malignancy and platinum resistance indicator, with our proposed treatment strategy offering novel avenues for reversing resistance in gastric cancer.

Icariin ameliorates osteoporosis by activating autophagy in ovariectomized rats.

Osteoporosis (OP) is a major problem that increases the mortality and disability rate worldwide. With an increase in the aging population, OP has become a major public threat to human health. Searching for effective and suitable targets for drug treatment in OP has become an urgent need. Osteoporosis is a metabolic bone disease characterized by reduced bone mass and density as well as micro-architectural deterioration. Icariin is a flavonoid extracted from plants of the genus Epimedium and has been shown to exert potential anti-OP activity. The present study was designed to observe the effect of icariin on OP and to clarify the underlying mechanisms in ovariectomized (OVX) rats. Hematoxylin and eosin (H&E) staining, von Kossa staining and micro-computed tomography (micro-CT) confirmed significant bone loss in the OVX group. Protein expression level was detected with western blot analysis. Icariin reversed a trend of increased bone turnover by reducing serum alkaline phosphatase (ALP), procollagen type I N-terminal propeptide (PINP), tartrate-resistant acid phosphatase isoform 5b (TRACP-5b), and C-telopeptide of type I collagen (CTX-I). Furthermore, icariin decreased sequestosome 1 (p62) and increased microtubule-associated protein 1 light chain 3II/microtubule-associated protein 1 light chain 3I (LC3II/LC3I), autophagy-related protein 7 (Atg7), and Beclin 1 in the femur of OVX rats, improving the indicators of impaired autophagy in OP. Icariin reversed the significant upregulation of the serine/threonine protein kinase (Akt), mammalian target of rapamycin (mTOR) and unc-51-like autophagy activating kinase 1 (ULK1) at Ser757, and the downregulation of p-AMP-activated protein kinase (p-AMPK) and ULK1 phosphorylated at Ser555 in the OVX rats, suggesting that the mechanism of icariin action in OP treatment involves the activation and suppression of the AMPK/ULK1 and AKT/mTOR/ULK1 autophagy pathways, respectively.

Glioblastoma and its treatment are associated with extensive accelerated brain aging.

Progressive neurocognitive dysfunction is the leading cause of a reduced quality of life in patients with primary brain tumors. Understanding how the human brain responds to cancer and its treatment is essential to improve the associated cognitive sequelae. In this study, we performed integrated transcriptomic and tissue analysis on postmortem normal-appearing non-tumor brain tissue from glioblastoma (GBM) patients that had received cancer treatments, region-matched brain tissue from unaffected control individuals and Alzheimer's disease (AD) patients. We show that normal-appearing non-tumor brain regions of patients with GBM display hallmarks of accelerated aging, in particular mitochondrial dysfunction, inflammation, and proteostasis deregulation. The extent and spatial pattern of this response decreased with distance from the tumor. Gene set enrichment analyses and a direct comparative analysis with an independent cohort of brain tissue samples from AD patients revealed a significant overlap in differentially expressed genes and a similar biological aging trajectory. Additionally, these responses were validated at the protein level showing the presence of increased lysosomal lipofuscin, phosphorylated microtubule-associated protein Tau, and oxidative DNA damage in normal-appearing brain areas of GBM patients. Overall, our data show that the brain of GBM patients undergoes accelerated aging and shared AD-like features, providing the basis for novel or repurposed therapeutic targets for managing brain tumor-related side effects.

A circRNA ceRNA network involved in cognitive dysfunction after chronic cerebral hypoperfusion.

Chronic Cerebral Hypoperfusion (CCH) is associated with cognitive dysfunction, the underlying mechanisms of which remain elusive, hindering the development of effective therapeutic approaches. In this study, we employed an established CCH animal model to delve into neuropathological alterations like oxidative stress, inflammation, neurotransmitter synthesis deficits, and other morphological alterations. Our findings revealed that while the number of neurons remained unchanged, there was a significant reduction in neuronal fibers post-CCH, as evidenced by microtubule-associated protein 2 (MAP2) staining. Moreover, myelin basic protein (MBP) staining showed exacerbated demyelination of neuronal fibers. Furthermore, we observed increased neuroinflammation, proliferation, and activation of astrocytes and microglia, as well as synaptic loss and microglial-mediated synapse engulfment post-CCH. Utilizing RNA sequencing, differential expression analysis displayed alterations in both mRNAs and circRNAs. Following gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, both showed significant enrichment in immunological and inflammation-related terms and pathways. Importantly, the differentially expressed circular RNAs (DE circRNAs) exhibited a notable coexpression pattern with DE mRNAs. The ternary circRNA-miRNA-mRNA competing endogenous RNAs (ceRNA) network was constructed, and subsequent analysis reiterated the significance of neuroimmunological and neuroinflammatory dysfunction in CCH-induced neuropathological changes and cognitive dysfunction. This study underscores the potential role of circRNAs in these processes, suggesting them as promising therapeutic targets to mitigate the detrimental effects of CCH.

Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC& Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

The effects and mechanisms of modified Xiaoyaosan on chronic unpredictable mild stress (CUMS)-induced depressive mice based on network pharmacology

Ethnopharmacological relevanceClinical research and basic scientific experiments have shown that modified Xiaoyaosan (MXYS) has antidepressant effects, whose system mechanism however has not been thoroughly characterized. Aim of the studyThis research was aimed at evaluating the treatment effects of MXYS on chronic unpredictable mild stress (CUMS)-induced depressive mice and exploring underlying mechanisms. Materials and methodsWhether MXYS has effects on depression was investigated via the depressive behaviors of mice, electron microscopy, real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, immunofluorescence (IF) staining and the stereotaxic injection of adeno-associated viruses (AAVs). In addition, network pharmacology was applied to predict relevant molecular targets and possible mechanisms and perform further in vivo validation. ResultsMXYS is effective in ameliorating the depression-like symptoms of CUMS mice. It can stimulate autophagosome formation, activate the expression of microtubule-associated protein 1 light chain 3 (LC3B), autophagy-related gene 5 (Atg5), Atg7 and neuron-specific nuclear protein (NeuN), and decrease the protein expression sequestosome 1 (SQSTM1/p62). The autophagy-upregulating effect of MXYS was weakened by silencing. The network pharmacology analysis revealed that mitogen-activated protein kinase 1 (MAPK1), MAPK3, serine/threonine-protein kinase (AKT1), proto-oncogene tyrosine-protein kinase (SRC), PI 3 kinase p85 alpha (PIK3R1), catenin (cadherin-associated protein) beta 1 (CTNNB1) and human thrombin activator 1 (HRAS) may be of importance to treat depression by MXYS. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that metabolic and autophagy pathways, pathways in cancer and MAPK, phosphoinositide 3-kinase (PI3K)-Akt and rhoptry-associated protein 1 (Rap1) signaling pathways are involved in the antidepressant effects of MXYS. As suggested by Western blot, the anti-depression mechanism of MXYS is possibly associated with the extracellular signal-regulated protein kinase (ERK)/P38 MAPK signaling pathway. ConclusionThe findings indicate the possible antidepressant effects of MXYS on CUMS mice via triggering autophagy to alleviate neuronal apoptosis and prompting autophagy, which may involve the ERK/P38 MAPK signaling pathway.

Dictamnine Ameliorates DNFB-Induced Atopic Dermatitis Like Skin Lesions in Mice by Inhibiting M1 Macrophage Polarization and Promoting Autophagy.

Autophagy and M1 macrophage polarization play important roles in the regulation of inflammation in atopic dermatitis (AD). Dictamnine is one of the main ingredients in Cortex Dictamni, a widely used traditional Chinese medicine for the treatment of dermatitis. In the present study, we investigated the anti-inflammatory effects of dictamnine on AD like skin lesions and M1 macrophage polarization. A 2,4-dinitrofluorobenzene (DNFB) triggered AD like skin lesions models in mice was established to identify the ameliorative effects of dictamnine on AD in vivo. In addition, an M1 macrophage polarization model was co-stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) using phorbol myristate acetate (PMA) differentiated THP-1 cells, to investigate the effect of dictamnine on promoting autophagy and inhibiting inflammatory factor release. Dictamnine suppressed DNFB-induced skin inflammation by inhibiting M1 macrophage polarization, up-regulating the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3) expression, and promoting macrophage autophagy at inflammatory sites. Dictamnine also could reduce the release of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8), and down-regulate the mRNA expression of these genes in LPS-IFN-γ triggered M1 polarized macrophages. Dictamnine ameliorates AD like skin lesions by inhibiting M1 macrophage polarization and promoting autophagy. Hence, dictamnine is expected to be a potential therapeutic candidate for AD.

Dihydroartemisinin alleviates ammonia toxicity and autophagy in zebrafish (Danio rerio) gill

Ammonia is highly toxic to aquatic organisms and has become a consistent pollutant in intensive fish farming conditions. Dihydroartemisinin (DhA) has potential value in the treatment of ammonia poisoning in aquaculture. However, the application investigation of DhA is insufficient in the field of aquaculture. In the present study, 540 zebrafish with an average body weight of (1.00 ± 0.01) g were randomly assigned into 9 groups. The zebrafish were fed a basal diet supplemented with or without 0.25% or 0.5% DhA for 1 week. The zebrafish were then exposed to water supplemented with or without 40 or 80 mg/L ammonium acetate (NH4AC) for 4 days. The results showed that dietary supplementation with 0.5% DhA attenuated mortality and ammonia accumulation in zebrafish exposed to 80 mg/L NH4AC. In addition, the alleviative effects of 0.5% DhA were associated with enhanced mRNA levels for ammonia transporter (Rhesus-associated glycoprotein, Rh family C glycoprotein 1, Na+/K+-ATPase, carbonic anhydrase), anti-inflammatory cytokines (transforming growth factor β), as well as the down-regulation of mRNA expression of inflammatory factors (interleukin 1β), hypoxia-inducible factors (hypoxia-inducible factor 1 αb, hypoxia-inducible factor 2α), and protein abundances of autophagy (autophagy-related gene 5, BCL-2-interacting myosin-like coiled-coil protein-1, microtubule-associated protein 1 light chain 3 Ⅱ, cathepsin B, cathepsin L). Taken together, DhA has great potential in aquaculture as a defence against ammonia toxicity.

ADVANCEMENT IN THE TREATMENT OF ALZHEIMER'S DISEASE, 2023

Background: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by extracellular amyloid β deposition and intercellular neurofibrillary tangles. AD is a complex disease with a strong genetic component, and it is classified into three phases by the Food and Drug Administration (US-FDA): pre-pathology, plaque formation, and neural destruction. A number of therapeutic agents have been developed to reduce AD pathology and improve cognition. Objectives: The objective of this paper was to review recent advancements in AD treatment and cure. Methodology: A literature search was conducted using the keywords "therapeutic developments for AD" and "natural products as a drug for AD." Publications related to AD were retrieved, and the NCBI Clinical trial database was also searched to check drug status. Results: Monoclonal antibodies against amyloid β plaques, tau protein, and microtubule-associated protein have shown positive results in animal models, but they have failed in clinical trials. This is likely due to the fact that these antibodies are designed to target specific proteins that are not always present in the human brain. Conclusion: Based on the findings of this review, it is suggested that medicines based on natural extracts and combination therapy would be least toxic and more effective than single therapy. This is because combination therapy targets multiple pathways involved in AD pathogenesis. There is also a dire need to initiate AD research in Pakistan and facilitate the Pakistani community in early diagnosis and cure.

Lactate-induced autophagy activation: unraveling the therapeutic impact of high-intensity interval training on insulin resistance in type 2 diabetic rats

Impaired autophagy is a hallmark of diabetes. The current study proposed to investigate if high intensity interval training (HIIT) induced lactate accumulation could stimulate autophagy in type 2 diabetic male rats. 28 male Wistar rats were randomly assigned into four groups: Healthy Control (CO), Diabetes Control (T2D), Exercise (EX), and Diabetes + Exercise (T2D + EX). Diabetes was induced by feeding high-fat diet and administrating single dose of streptozotocin (35 mg/kg). After becoming diabetic, the animals in the exercise groups (EX and T2D + EX) performed an eight-week HIIT (4–10 interval, 80–100% Vmax, 5 days per week). Serum levels of lactate, glucose and insulin as well as the levels of lactate, pyruvate, lactate transporter monocarboxylate transporter 1 (MCT1), phosphorylated mitogen-activated protein kinases (p-MAP 1 and 2), phosphorylated extracellular signal-regulated protein kinases 1 and 2 (p-ERK 1 and 2), mammalian target of rapamycin (p-mTOR), ribosomal protein S6 kinase beta-1 (p-70S6k), p90 ribosomal S6 kinases (p-90RSK), autophagy related 7 (ATG7), Beclin-1, microtubule-associated protein 1A/1B, and 2A/2B -light chain 3 levels (LC3-I), (LC3- II), (LC3I/LC3II) in soleus muscle were measured. Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and serum glucose was lower in T2D + EX compared to T2D group (P < 0.0001). While serum and soleus muscle levels of lactate was not different between T2D and T2D + Ex, the levels of Pyruvate (P < 0.01), MCT1, p-ERK1/2, p-mTOR, p70S6k, P-90RSK, ATG7, LC3-II, and LC3-II/LC3I ratios were higher in T2D + EX compared to T2D group (P < 0.0001). We concluded that eight weeks of high-intensity interval training could activated ERK/P90SRK while inhibiting mTOR/P70S6K signaling pathway in lactate dependent manner. It means increased autophagy which resulted in improve insulin resistance (IR) and reduce blood glucose.

Serum level of proteins involved in apoptosis and autophagy in patients with type 2 diabetes and non-alcoholic fatty liver disease

Aim of the study was to estimate the blood serum level of microtubule-associated protein 1 light chain 3 alpha (MAP1LC3alpha), beclin-1 (BECN-1), Bcl-2 (BCL-2), caspase-3 (CASP3) and autophagy related 5 (ATG-5) protein as potential markers of liver fibrosis in type 2 diabetes (T2D) patients with non-alcoholic fatty liver disease (NAFLD).Material and methods. A single center cross-sectional observational study was performed. Seventy four patients with T2D and NAFLD were included, 23 men and 51 women, aged 18 to 74 years. Transient elastography was used for the assessment of liver fibrosis. The severity of fibrosis was estimated using the Metavir sсore. In addition, indirect liver fibrosis indices APRI and FIB-4 were calculated. Serum concentration of MAP1LC3alpha, BECN-1, BCL-2, CASP and ATG-5 was measured by ELISA. We applied ROC analysis to assess the value of studied indicators in diagnosis of liver fibrosis.Results. Liver fibrosis stage F1 was verified in 16 subjects, stage F2 in 12, stage F3 in 7, and stage F4 in 19 individuals. Patients with advanced liver fibrosis (stage 3-4) when compared to those without fibrosis (stage 0–2) had higher content of MAP1LC3alpha (p = 0.01) and BECN-1 (p = 0.01). There were no significant differences in the level of BCL-2, CASP3 and ATG-5 between patients with different severity of fibrosis. In the ROC analysis, MAP1LC3alpha and BECN-1 showed significance for the diagnosis of severe liver fibrosis, which, however, did not exceed that of the APRI index.Conclusions. In patients with T2D and NAFLD, serum levels of MAP1LC3alpha and BECN-1 are associated with severe liver fibrosis.

Dual isolation of primary neurons and oligodendrocytes from guinea pig frontal cortex.

Primary cell culture is a technique that is widely used in neuroscience research to investigate mechanisms that underlie pathologies at a cellular level. Typically, mouse or rat tissue is used for this process; however, altricial rodent species have markedly different neurodevelopmental trajectories comparatively to humans. The use of guinea pig brain tissue presents a novel aspect to this routinely used cell culture method whilst also allowing for dual isolation of two major cell types from a physiologically relevant animal model for studying perinatal neurodevelopment. Primary neuronal and oligodendrocyte cell cultures were derived from fetal guinea pig's frontal cortex brain tissue collected at a gestational age of 62 days (GA62), which is a key time in the neuronal and oligodendrocyte development. The major advantage of this protocol is the ability to acquire both neuronal and oligodendrocyte cellular cultures from the frontal cortex of one fetal brain. Briefly, neuronal cells were grown in 12-well plates initially in a 24-h serum-rich medium to enhance neuronal survival before switching to a serum-free media formulation. Oligodendrocytes were first grown in cell culture flasks using a serum-rich medium that enabled the growth of oligodendrocyte progenitor cells (OPCs) on an astrocyte bed. Following confluency, the shake method of differential adhesion and separation was utilized via horizontally shaking the OPCs off the astrocyte bed overnight. Therefore, OPCs were plated in 12-well plates and were initially expanded in media supplemented with growth hormones, before switching to maturation media to progress the lineage to a mature phenotype. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on both cell culture types to analyze key population markers, and the results were further validated using immunocytochemistry. Primary neurons displayed the mRNA expression of multiple neuronal markers, including those specific to GABAergic populations. These cells also positively stained for microtubule-associated protein 2 (MAP2; a dendritic marker specific to neurons) and NeuN (a marker of neuronal cell bodies). Primary oligodendrocytes expressed all investigated markers of the oligodendrocyte lineage, with a majority of the cells displaying an immature oligodendrocyte phenotype. This finding was further confirmed with positive oligodendrocyte transcription factor (OLIG2) staining, which serves as a marker for the overall oligodendrocyte population. This study demonstrates a novel method for isolating both neurons and oligodendrocytes from the guinea pig brain tissue. These isolated cells display key markers and gene expression that will allow for functional experiments to occur and may be particularly useful in studying neurodevelopmental conditions with perinatal origins.

Tau Protein Alterations Induced by Hypobaric Hypoxia Exposure.

Tauopathies are a group of neurodegenerative diseases whose central feature is dysfunction of the microtubule-associated protein tau (MAPT). Although the exact etiology of tauopathies is still unknown, it has been hypothesized that their onset may occur up to twenty years before the clear emergence of symptoms, which has led to questions about whether the prognosis of these diseases can be improved by, for instance, targeting the factors that influence tauopathy development. One such factor is hypoxia, which is strongly linked to Alzheimer's disease because of its association with obstructive sleep apnea and has been reported to affect molecular pathways related to the dysfunction and aggregation of tau proteins and other biomarkers of neurological damage. In particular, hypobaric hypoxia exposure increases the activation of several kinases related to the hyperphosphorylation of tau in neuronal cells, such as ERK, GSK3β, and CDK5. In addition, hypoxia also increases the levels of inflammatory molecules (IL-β1, IL-6, and TNF-α), which are also associated with neurodegeneration. This review discusses the many remaining questions regarding the influence of hypoxia on tauopathies and the contribution of high-altitude exposure to the development of these diseases.

Modulation of Tau Pathology in Alzheimer's Disease by Dietary Bioactive Compounds.

Tau is a microtubule-associated protein essential for microtubule assembly and stability in neurons. The abnormal intracellular accumulation of tau aggregates is a major characteristic of brains from patients with Alzheimer's disease (AD) and other tauopathies. In AD, the presence of neurofibrillary tangles (NFTs), which is composed of hyperphosphorylated tau protein, is positively correlated with the severity of the cognitive decline. Evidence suggests that the accumulation and aggregation of tau cause synaptic dysfunction and neuronal degeneration. Thus, the prevention of abnormal tau phosphorylation and elimination of tau aggregates have been proposed as therapeutic strategies for AD. However, currently tau-targeting therapies for AD and other tauopathies are limited. A number of dietary bioactive compounds have been found to modulate the posttranslational modifications of tau, including phosphorylation, small ubiquitin-like modifier (SUMO) mediated modification (SUMOylation) and acetylation, as well as inhibit tau aggregation and/or promote tau degradation. The advantages of using these dietary components over synthetic substances in AD prevention and intervention are their safety and accessibility. This review summarizes the mechanisms leading to tau pathology in AD and highlights the effects of bioactive compounds on the hyperphosphorylation, aggregation and clearance of tau protein. The potential of using these bioactive compounds for AD prevention and intervention is also discussed.

P75NTR regulates autophagy through the YAP-mTOR pathway to increase the proliferation of interfollicular epidermal cells and promote wound healing in diabetic mice

Wound healing is delayed in diabetic patients. Increased autophagy and dysfunction of interfollicular epidermal (IFE) cells are closely associated with delayed healing of diabetic wounds. Autophagy plays an important role in all stages of wound healing, but its role in diabetic wound healing and the underlying molecular mechanisms are not clear. Here, we found that diabetic mice had delayed wound healing and increased autophagy in wounds compared with normal mice and that chloroquine, an inhibitor of autophagy, decreased the level of autophagy, improved the function of IFE cells, and accelerated wound healing in diabetic mice. Treatment of IFE cells with advanced glycosylation end products (AGEs) resulted in increased microtubule-associated protein chain (LC3) expression and decreased prostacyclin-62 (P62) expression, indicating increased autophagy in AGE-treated IFE cells. Moreover, P75NTR reduced autophagy in IFE cells in the presence of AGEs and significantly increased the proliferation of IFE cells. In addition, P75NTR participated in regulating autophagy in IFE cells and in wounds in diabetic mice through the YAP-mTOR signalling pathway, which increased the functional activity of the cells and the healing rate of wounds in diabetic mice. Thus, our study suggests that P75NTR protects IFE cells against AGEs by affecting autophagy and accelerating wound healing in diabetic mice, providing a basis for understanding the role of autophagy in diabetic wound healing.

Cell death induced by nicotine in human neuroblastoma SH-SY5Y cells is mainly attributed to cytoplasmic vacuolation originating from the trans-Golgi network

Humans are usually exposed to nicotine through the use of tobacco products. Although it is generally believed that nicotine is relatively harmless in tobacco consumption, it is, in fact, a toxic substance that warrants careful consideration of its potential toxicity. However, the current understanding of the neurotoxicity of nicotine is still very limited. In this study, we aim to reveal the toxic risk of nicotine to key target neuronal cells and its potential toxic mechanisms. The results showed that nicotine induced cell death, ROS increase, mitochondrial membrane potential decrease, and DNA damage in SH-SY5Y human neuroblastoma cells at millimolar concentrations, but did not cause toxic effects at the physiological concentration. These toxic effects were accompanied by cytoplasmic vacuolation. The inhibition of cytoplasmic vacuolation by bafilomycin A1 greatly reduced nicotine-induced cell death, indicating that cytoplasmic vacuolation is the key driving factor of cell death. These cytoplasmic vacuoles originated from the trans-Golgi network (TGN) and expressed microtubule-associated protein 1 light chain 3-II (LC3-II) and lysosomal associated membrane protein 1(LAMP1). The presence of LC3-II and LAMP1 within these vacuoles serves as evidence of compromised TGN structure and function. These findings provide valuable new insights into the potential neurotoxic risk and mechanisms of nicotine.

Characterization of Posttranslationally Modified PHF-1 Tau Peptides Using Gaussian Accelerated Molecular Dynamics Simulation.

The microtubule-associated protein, Tau, is an intrinsically disordered protein that plays a crucial role in neurodegenerative diseases like Alzheimer's disease. The posttranslational modifications across the Tau protein domains are involved in regulating Tau protein's function and disease onset. Of the various posttranslational modifications at Ser, Thr, and Tyr sites, O-GlcNAcylation and phosphorylation are the most critical ones, playing a vital role in Tau aggregation and tauopathies. To understand the function, it is essential to characterize the structural changes associated withTaumodification. Previous experimental studies have focused on high-resolution nuclear magnetic resonance techniques to structurally characterize the effect of phosphorylation, O-GlcNAcylation, and combination of both PTMs on Tau conformation in small peptides centered on the PHF-1 epitope from amino acid 392 to 411. The structural characterization using atomistic molecular dynamics simulation of such disordered peptides requires long simulation time, proper sampling method, and utilization of appropriate force fields for accurate determination of conformational ensembles, resembling the experimental data. This chapter details the protocol for the structural characterization of modified Tau peptides using the CHARMM36m force field and enhanced sampling methods like Gaussian accelerated molecular dynamics (GaMD) simulation. We have focused on a detailed explanation of the GaMD method and analyses of molecular dynamics trajectories to explain the relationship between two modifications, phospho- and glyco-, at C-terminus of Tau protein and its stable conformation over the longer simulation timeframes. The analyses involve energetics reweighting, clustering of simulation trajectories, and characterization of secondary structure using circular dichroism data from the simulation. The reader can utilize this protocol to investigate the structures of complex proteins, especially the disordered ones.

α-Linolenic Acid Vesicles-Mediated Tau Internalization in Microglia.

In Alzheimer's disease, the synaptic loss is prominent due to the accumulation of Amyloid βeta (Aβ) protein in synapses, which affect neurotransmission, and thus ultimately causes neuronal loss. Tau, a microtubule-associated protein, is a vital protein of intracellular neurofibrillary tangles (NFTs) in AD. Along with the accumulation of aberrant proteins, glial cells, mainly astrocytes and microglia, play a major role in impairing neuronal network. Microglia have the ability to phagocytose Tau and rerelease in exosomes, which causes further spreading of Tau. Reduction in exosome synthesis can reduce spreading of Tau. Modulating microglia to clear the extracellular Tau seeds by its imported degradation would resolve the disease condition in Alzheimer's disease. In this study, we have shown the ability of α-linolenic acid (ALA) to inhibit the Tau aggregation and modulate their internalization property in microglial cells.

Protective effects of carvedilol against ovarian ischemia-reperfusion injury in rats

Ovarian torsion is a gynecological emergency that can lead to ovarian ischemia-reperfusion injury (IRI). This study aimed to investigate the potential protective effects of carvedilol against ovarian IRI in rats. A total of 24 female rats were divided into four groups: control, ischemia (I), ischemia-reperfusion (IR), and ischemia-reperfusion with carvedilol treatment (IR-C). Ovarian ischemia was induced by clamping the ovarian pedicle for 4 hours, followed by reperfusion for 4 hours. The IR-C group received carvedilol treatment 30 minutes before reperfusion. After the experiment, the ovaries were removed and evaluated for hemorrhage, edema, and immunopositivity of Caspase 3, apoptosis-inducing factor (AIF), Microtubule-associated protein 1 light chain 3b (LC3B), and Tumor necrosis factor-alpha (TNFa). In the staining performed with Caspase 3 and AIF, moderate immunopositivity was observed in the I and IR groups (p&lt;0.001), while the IR-C group showed mild immunopositivity. In the stainings performed with LC3B and TNFa, the I and IR groups exhibited severe immunopositivity (p&lt;0.001), whereas in the IR-C group, TNFa showed moderate immunopositivity (p&lt;0.001), and LC3B showed mild immunopositivity (p&lt;0.001). The results suggest that carvedilol may have protective effects against ovarian IRI in rats.

Identification of new microtubule small-molecule inhibitors and microtubule-associated genes against triple negative breast cancer.

Breast cancer represents the leading cancer type and leading cause of cancer-related death among women in the world. Triple-negative breast cancer (TNBC) is a subset of breast cancer with the poorest prognosis and still lacking of effective therapeutic options. We recently screened a natural product library and identified 3 new hit compounds with selective and prominent anti-TNBC activities on different subtype of TNBC cell lines. Interestingly, all of these 3 hit compounds belong to "cytoskeletal drugs" that target tubulin and microtubule function. Our data also showed that these hit compounds showed consistently effective on TNBC cells which are resistant to those currently used antimicrotubule agents such as Paclitaxel. RNA-Sequencing analyses revealed the anti-TNBC mechanisms of these hit compounds and identified a subset of new cellular factors commonly affected by hit compounds in different subtypes of TNBC cells. Among them, we demonstrated AHCYL1 and SPG21 as new microtubule-associated proteins, which were required for TNBC cell survival with clinical implication through tissue array analysis. Our studies provide new insights into the mechanisms of TNBC pathogenesis and offer promising therapeutic directions for this aggressive breast cancer.