Microtubule-associated Protein Research Articles

StableMARK-decorated microtubules in cells have expanded lattices

Microtubules are crucial in cells and are regulated by various mechanisms like posttranslational modifications, microtubule-associated proteins, and tubulin isoforms. Recently, the conformation of the microtubule lattice has also emerged as a potential regulatory factor, but it has remained unclear to what extent different lattices co-exist within the cell. Using cryo-electron tomography, we find that, while most microtubules have a compacted lattice (∼41 Å monomer spacing), approximately a quarter of the microtubules displayed more expanded lattice spacings. The addition of the microtubule-stabilizing agent Taxol increased the lattice spacing of all microtubules, consistent with results on reconstituted microtubules. Furthermore, correlative cryo-light and electron microscopy revealed that the stable subset of microtubules labeled by StableMARK, a marker for stable microtubules, predominantly displayed a more expanded lattice spacing (∼41.9 Å), further suggesting a close connection between lattice expansion and microtubule stability. The coexistence of different lattices and their correlation with stability implicate lattice spacing as an important factor in establishing specific microtubule subsets.

Functional Relationships between L1CAM, LC3, ATG12, and Aβ

Abnormal protein accumulations in the brain are linked to aging and the pathogenesis of dementia of various types, including Alzheimer’s disease. These accumulations can be reduced by cell indigenous mechanisms. Among these is autophagy, whereby proteins are transferred to lysosomes for degradation. Autophagic dysfunction hampers the elimination of pathogenic protein aggregations that contribute to cell death. We had observed that the adhesion molecule L1 interacts with microtubule-associated protein 1 light-chain 3 (LC3), which is needed for autophagy substrate selection. L1 increases cell survival in an LC3-dependent manner via its extracellular LC3 interacting region (LIR). L1 also interacts with Aβ and reduces the Aβ plaque load in an AD model mouse. Based on these results, we investigated whether L1 could contribute to autophagy of aggregated Aβ and its clearance. We here show that L1 interacts with autophagy-related protein 12 (ATG12) via its LIR domain, whereas interaction with ubiquitin-binding protein p62/SQSTM1 does not depend on LIR. Aβ, bound to L1, is carried to the autophagosome leading to Aβ elimination. Showing that the mitophagy-related L1-70 fragment is ubiquitinated, we expect that the p62/SQSTM1 pathway also contributes to Aβ elimination. We propose that enhancing L1 functions may contribute to therapy in humans.

Exploring the Benzazoles Derivatives as Pharmacophores for AChE, BACE1, and as Anti-Aβ Aggregation to Find Multitarget Compounds against Alzheimer’s Disease

Despite the great effort that has gone into developing new molecules as multitarget compounds to treat Alzheimer’s disease (AD), none of these have been approved to treat this disease. Therefore, it will be interesting to determine whether benzazoles such as benzimidazole, benzoxazole, and benzothiazole, employed as pharmacophores, could act as multitarget drugs. AD is a multifactorial disease in which several pharmacological targets have been identified—some are involved with amyloid beta (Aβ) production, such as beta secretase (BACE1) and beta amyloid aggregation, while others are involved with the cholinergic system as acetylcholinesterase (AChE) and butirylcholinesterase (BChE) and nicotinic and muscarinic receptors, as well as the hyperphosphorylation of microtubule-associated protein (tau). In this review, we describe the in silico and in vitro evaluation of benzazoles on three important targets in AD: AChE, BACE1, and Aβ. Benzothiazoles and benzimidazoles could be the best benzazoles to act as multitarget drugs for AD because they have been widely evaluated as AChE inhibitors, forming π–π interactions with W286, W86, Y72, and F338, as well as in the AChE gorge and catalytic site. In addition, the sulfur atom from benzothiazol interacts with S286 and the aromatic ring from W84, with these compounds having an IC50 value in the μM range. Also, benzimidazoles and benzothiazoles can inhibit Aβ aggregation. However, even though benzazoles have not been widely evaluated on BACE1, benzimidazoles evaluated in vitro showed an IC50 value in the nM range. Therefore, important chemical modifications could be considered to improve multitarget benzazoles’ activity, such as substitutions in the aromatic ring with electron withdrawal at position five, or a linker 3 or 4 carbons in length, which would allow for better interaction with targets.

Protective effect of aspirin and gentisic acid, a plant-derived phenolic acid, on acrylamide-induced neurotoxicity by inhibiting apoptosis and autophagy.

Acrylamide (ACR) is a toxic agent for humans and animals. Gentisic acid, anaspirinmetabolite, has antioxidant activity. Therefore, the present study investigated the probable protective effects of aspirin and gentisic acid on ACR-induced neurotoxicity in PC12 cells and rats. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the effects of aspirin and gentisic acid (1.25, 2.5, 5 µM) on ACR (5 mM) toxicity. Male Wistar rats were randomly divided into 13 groups: (1) Control group, (2) ACR (50 mg/kg, 11 days, i.p.), (3-5) ACR + aspirin (25, 50, 75 mg/kg, 11 days, p.o.), (6-8) ACR + gentisic acid (25, 50, 75 mg/kg, 11 days, p.o.), (9) ACR + vitamin E (200 mg/kg, every other day, i.p.), (10, 11) Aspirin (75, 100 mg/kg, 11 days, p.o.), (12, 13) Gentisic acid (75, 100 mg/kg, 11 days, p.o.). Behavioral tests were assessed on the final day of the study. In the cerebral cortex, malondialdehyde (MDA), glutathione (GSH), cleaved-caspase-3, and microtubule-associated protein 1A/1B-light chain 3 (LC3) protein levels were evaluated. When compared with the ACR group, aspirin (2.5, 5 µM) and gentisic acid (2.5 µM) significantly enhanced cell viability. In comparison to the control group, ACR induced severe motor impairment, elevated MDA, cleaved-caspase-3, LC3 II/I ratio, and decreased GSH levels in the cerebral cortex of rats. ACR-induced changes were significantly reversed by aspirin and gentisic acid (25 mg/kg). Oxidative stress, apoptosis, and autophagy play important roles in the neurotoxicity of ACR. Aspirin and gentisic acid significantly reduced ACR-induced toxicity by inhibiting the mentioned mechanisms.

Plasma Proteomics of Type 2 Diabetes, Hypertension, and Co-Existing Diabetes/Hypertension in Thai Adults

The study explored proteomics to better understand the relationship between type 2 diabetes (T2DM) and hypertension (HT) in Thai adults, using shotgun proteomics and bioinformatics analysis. Plasma samples were taken from 61 subjects: 14 healthy subjects (mean age = 40.85 ± 7.12), 13 with T2DM (mean age = 57.38 ± 6.03), 16 with HT (mean age = 66.87 ± 10.09), and 18 with coexisting T2DM/HT (mean age = 58.22 ± 10.65). Proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein–protein interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 11.5. We identified six unique proteins in T2DM patients, including translationally controlled 1 (TPT1) and nibrin (NBN), which are associated with the DNA damage response. In HT patients, seven unique proteins were identified, among them long-chain fatty acid-CoA ligase (ASCL), which functions in the stimulation of triacylglycerol and cholesterol synthesis, and NADPH oxidase activator 1 (NOXA1), which is involved in high blood pressure via angiotensin II-induced reactive oxygen species (ROS)-generating systems. In coexisting T2DM/HT patients, six unique proteins were identified, of which two—microtubule-associated protein 1A (MAP1A)—might be involved in dementia via RhoB-p53 and diacylglycerol kinase beta (DGKB), associated with lipid metabolism. This study identified new candidate proteins that are possibly involved in the pathology of these diseases.

Cerliponase alfa decreases Aβ load and alters autophagy- related pathways in mouse hippocampal neurons exposed to fAβ1–42

Extracellular aggregation of amyloid-beta (Aβ) in the brain plays a central role in the onset and progression of Alzheimer's disease (AD). Moreover, intraneuronal accumulation of Aβ via oligomer internalization might play an important role in the progression of AD. Deficient autophagy, which is a lysosomal degradation process, occurs during the early stages of AD. Tripeptidyl peptidase-1 (TPP1) functions as a lysosomal enzyme, and TPP1 gene mutations are associated with type 2 late infantile neuronal ceroid lipofuscinosis (LINCL). Nevertheless, there is little information about the role of TPP1 in the pathogenesis of AD; therefore, the present study aimed to measure the decrease in intraneuronal Aβ accumulation by a recombinant analog of the TPP1 enzyme, cerliponase alfa (CER) (Brineura®), and to determine whether autophagy pathways play a role in this decrease. In this study, endogenous Aβ accumulation was induced by fAβ1–42 (a toxic fragment of full-length Aβ) exposure, and mouse hippocampal neuronal cells (HT-22) were treated with CER (human recombinant rhTPP1 1 mg mL−1). Soluble Aβ, TPP1, and the proteins involved in autophagy, including mammalian target of rapamycin (p-mTOR/mTOR), p62/sequestosome-1 (p62/SQSTM1), and microtubule-associated protein 1 A/1B-light chain 3 (LC3), were evaluated using western blotting. The sirtuin-1, beclin-1, and Atg5 genes were also studied using RT-PCR. Aβ and TPP1 localizations were observed via immunocytochemistry. CER reduced the Aβ load in HT-22 cells by inducing TPP1 expression and converting pro-TPP1 into the mature form. Furthermore, exposure to CER and fAβ1–42 induced the autophagy-regulatory/related pathways in HT-22 cells and exposure to CER alone increased sirtuin-1 activity. Based on the present findings, we suggest that augmentation of TPP1 with enzyme replacement therapy may be a potential therapeutic option for the treatment of AD.

Neuroprotective effect of Naochuxue prescription on intracerebral hemorrhage: inhibition of autophagy downregulating high mobility group box-1.

To determine the molecular mechanisms underlying the neuroprotective effects of Naochuxue prescription (,NCXP) in rats with intracerebral hemorrhage (ICH). Sprague-Dawley rats were injected with collagenase to generate ICH models, which were then randomly divided into six groups, including control, sham, model, and three intervention groups. The intervention groups received different doses of NCXP (0.13, 0.26, and 0.52 g/kg) daily for 10 d. High-performance liquid chromatography (HPLC) was used to analyze the chemical characteristics of NCXP. The neurobehavioral outcomes of the rats were evaluated using neurological deficit scores (Zea Longa 5) and the corner turn test. Pathomorphological changes in perihematomal tissues after ICH were observed using hematoxylin and eosin staining. Immunohistochemistry (IHC) was used to detect the inflammation expression of interleukin 6 (IL-6) and toll-like receptor 4 (TLR4). High mobility group box-1 (HMGB1), Beclin1, microtubule-associated protein 1 light chain 3 beta (LC3), and sequestosome 1 (p62) were detected using real-time quantitative polymerase chain reaction and Western blotting in perihematomal tissues. HPLC showed that the NCXP had good stability. Rats with ICH had severe neurological function deficits compared to the control group. IHC results showed that NCXP significantly downregulated the expression of the inflammatory proteins IL-6 and TLR4. ICH rats treated with NCXP showed less neurological injury than the model group, accompanied by a significantly decreased expression of HMGB1, Beclin1, and LC3 and an increased expression of p62. The neuroprotective effect of NCXP alleviated inflammation and autophagy possibly by downregulating HMGB1 expression. However, further research on the signaling pathways is required to verify this hypothesis.

Effect of phosphatase and tensin homolog-induced putative kinase 1/ E3 ubiquitin ligase Parkin mediated mitochondrial autophagy on chronic kidney disease myocardial injury and the intervention mechanism of Shenshuai recipe.

To study whether Shenshuai recipe (, SSR) can play a protective role on chronic kidney disease myocardial injury model through phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/E3 ubiquitin ligase Parkin (Parkin) mitochondrial autophagy pathway. Forty-eight nephrectomized rats were randomly divided into six groups: sham-operated group, model group, Benazepril group, low, medium and high-dose groups of SSR. The rats were given the cor-responding intervention for six weeks, then were sacrificed. Serum was examined by enzyme linked immunosorbent assay (ELISA). Cardiac ultrasound was used to detect cardiac function in 5/6 nephrectomized rats. Myocardial tissue was examined by light and electron microscopy; PINK1, Parkin, microtubule-associated protein1 light chain 3 II (LC3B), sequestosome 1 (P62), BECN1 (Beclin-1) and dynamin-related protein 1 (Drp-1) were measured by real time polymerase chain reaction (RT-PCR), Western blot (WB) and immunohistochemistry (IHC). The expression levels of blood urea nitrogen (BUN) and creatinine (SCr) in the model group were significantly higher than those in the sham-operated group, indicating that modeling was successful. SSR can protect myocardium by reducing the relative expression of creatine kinase myocardial isoenzyme and hypersensitivity cardiac troponin I (P<0.05). SSR can improve cardiac function in rats after ultrasound testing. SSR can improve the pathological manifestations of myocardial tissue after Masson staining. SSR can increase the number of autophagosomes and autophagiclysosomes in 5/6 nephrectomized rats (P<0.05). Determined by RT-PCR, WB and IHC, SSR can increase the relative expression of PINK1, Parkin, and LC3B (P<0.05), and decrease the relative expression of P62, Beclin-1 and Drp-1 (P<0.05). The PINK1/Parkin mitochondrial autophagy pathway in myocardial tissues in 5/6 nephrectomy CKD myocardial injury rats was inhibited. SSR can activate PINK1/Parkin mitochondrial autophagy to enhance mitochondrial autophagy, and play a protective role in myocardial tissues.

The GhEB1C gene mediates resistance of cotton to Verticillium wilt.

The GhEB1C gene of the EB1 protein family functions as microtubule end-binding protein and may be involved in the regulation of microtubule-related pathways to enhance resistance to Verticillium wilt. The expression of GhEB1C is induced by SA, also contributing to Verticillium wilt resistance. Cotton, as a crucial cash and oil crop, faces a significant threat from Verticillium wilt, a soil-borne disease induced by Verticillium dahliae, severely impacting cotton growth and development. Investigating genes associated with resistance to Verticillium wilt is paramount. We identified and performed a phylogenetic analysis on members of the EB1 family associated with Verticillium wilt in this work. GhEB1C was discovered by transcriptome screening and was studied for its function in cotton defense against V. dahliae. The RT-qPCR analysis revealed significant expression of the GhEB1C gene in cotton leaves. Subsequent localization analysis using transient expression demonstrated cytoplasmic localization of GhEB1C. VIGS experiments indicated that silencing of the GhEB1C gene significantly increased susceptibility of cotton to V. dahliae. Comparative RNA-seq analysis showed that GhEB1C silenced plants exhibited altered microtubule-associated protein pathways and flavonogen-associated pathways, suggesting a role for GhEB1C in defense mechanisms. Overexpression of tobacco resulted in enhanced resistance to V. dahliae as compared to wild-type plants. Furthermore, our investigation into the relationship between the GhEB1C gene and plant disease resistance hormones salicylic axid (SA) and jasmonic acid (JA) revealed the involvement of GhEB1C in the regulation of the SA pathway. In conclusion, our findings demonstrate that GhEB1C plays a crucial role in conferring immunity to cotton against Verticillium wilt, providing valuable insights for further research on plant adaptability to pathogen invasion.

The role of PI3K signaling pathway in Alzheimer’s disease

Alzheimer’s disease (AD) is a debilitating progressively neurodegenerative disease. The best-characterized hallmark of AD, which is marked by behavioral alterations and cognitive deficits, is the aggregation of deposition of amyloid-beta (Aβ) and hyper-phosphorylated microtubule-associated protein Tau. Despite decades of experimental progress, the control rate of AD remains poor, and more precise deciphering is needed for potential therapeutic targets and signaling pathways involved. In recent years, phosphoinositide 3-kinase (PI3K) and Akt have been recognized for their role in the neuroprotective effect of various agents, and glycogen synthase kinase 3 (GSK3), a downstream enzyme, is also crucial in the tau phosphorylation and Aβ deposition. An overview of the function of PI3K/Akt pathway in the pathophysiology of AD is provided in this review, along with a discussion of recent developments in the pharmaceuticals and herbal remedies that target the PI3K/Akt signaling pathway. In conclusion, despite the challenges and hurdles, cumulative findings of novel targets and agents in the PI3K/Akt signaling axis are expected to hold promise for advancing AD prevention and treatment.

Enhancement of cognitive function in mice with Alzheimer's disease through hyperbaric oxygen-induced activation of cellular autophagy.

In this study, we examined the effectiveness of hyperbaric oxygen (HBO) therapy in ameliorating cognitive deficits in mice with Alzheimer's disease (AD), while also assessing its impact on the autophagic pathway within the context of AD. 20 double-transgenic mice expressing the amyloid precursor protein and presenilin 1 (APP/PS1) were purposefully selected and randomly assigned to groups A and B. Concurrently, 20 C57BL/6 mice were chosen and randomly categorized into groups C and D, each consisting of 10 mice. Mice in groups B and D received HBO treatment. The Morris water maze assay was used to assess changes in mouse behavior. Immunohistochemistry techniques were used to quantify the expression levels of amyloid-beta 42 (Aβ42) and microtubule-associated protein 1A/1B-light chain 3 (LC3) in hippocampal tissues, while western blot analysis was used to investigate the levels of LC3-II, p62, phosphoinositide 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) proteins within hippocampal tissues. Mice allocated to group B exhibited reduced escape latency and prolonged dwell time in the target quadrant compared to other groups. Histological examination revealed conspicuous plaque-like deposits of Aβ42 in the hippocampal tissues of mice in groups A and B. Group B displayed diminished Aβ42-positive reactants and augmented microtubule-associated protein 1A/1B-LC3-positive reactants compared to group A. LC3-positive reactants were also detected in the hippocampal tissues of mice in groups C and D, surpassing the levels observed in groups A and B. Furthermore, group B demonstrated significantly lower expression of mTOR protein and markedly higher expression of LC3-II protein in mouse hippocampal tissues when compared to group A (P < 0.05). Conversely, there were no significant disparities noted in PI3K and p62 protein expression between groups B and A. Notably, no discernible discrepancies were observed in the expression levels of mTOR, PI3K, LC3-II, and p62 proteins between groups C and D within mouse hippocampal tissues. HBO treatment demonstrates efficacy in enhancing cognitive function in mice with AD and holds promise as a potential therapeutic intervention for AD by facilitating the activation of the mTOR pathway-mediated autophagy.

Further validation of the association between MAPT haplotype-tagging polymorphisms and Alzheimer's disease: neuropsychological tests, cerebrospinal fluid biomarkers, and APOE genotype.

Genetic studies have shown that variants in the microtubule-associated protein tau (MAPT) gene, which encodes tau protein, can increase the risk for Alzheimer's disease (AD). Additionally, two haplotypes of the MAPT gene (H1 and H2) are associated with various neurodegenerative disorders, including AD. This study aimed to test the association of MAPT haplotypes (H1 and H2) and MAPT haplotype-tagging polymorphisms (rs1467967, rs242557, rs3785883, rs2471738, del-In9, rs7521) with AD. The study included 964 individuals: 113 with AD, 53 with mild cognitive impairment (MCI), 54 with other dementias, and 744 healthy controls. The results showed that individuals carrying the A allele in the MAPT rs1467967 polymorphism, the GG genotype in the MAPT rs7521 polymorphism, and the G allele in the MAPT rs242557 polymorphism had worse performance on various neuropsychological tests. Carriers of the C allele in MAPT rs2471738 polymorphism and CC homozygotes also showed worse performance on neuropsychological tests and pathological levels of several cerebrospinal fluid (CSF) biomarkers. However, T allele carriers in the MAPT rs2471738 polymorphism were more represented among patients with dementia and apolipoprotein E (APOE) ɛ4 carriers. Carriers of the H2 MAPT haplotype had worse performance on various neuropsychological tests, consistent with our previous study, which associated the H2 MAPT haplotype with pathological levels of CSF AD biomarkers. Regarding the MAPT rs3785883 polymorphism, further research is needed since both the AA and GG genotypes were associated with pathological levels of CSF and plasma AD biomarkers. In conclusion, further genetic studies are needed to elucidate the role of MAPT haplotypes and MAPT haplotype-tagging polymorphisms in the development of AD.

Forebrain Eml1 depletion reveals early centrosomal dysfunction causing subcortical heterotopia.

Subcortical heterotopia is a cortical malformation associated with epilepsy, intellectual disability, and an excessive number of cortical neurons in the white matter. Echinoderm microtubule-associated protein like 1 (EML1) mutations lead to subcortical heterotopia, associated with abnormal radial glia positioning in the cortical wall, prior to malformation onset. This perturbed distribution of proliferative cells is likely to be a critical event for heterotopia formation; however, the underlying mechanisms remain unexplained. This study aimed to decipher the early cellular alterations leading to abnormal radial glia. In a forebrain conditional Eml1 mutant model and human patient cells, primary cilia and centrosomes are altered. Microtubule dynamics and cell cycle kinetics are also abnormal in mouse mutant radial glia. By rescuing microtubule formation in Eml1 mutant embryonic brains, abnormal radial glia delamination and heterotopia volume were significantly reduced. Thus, our new model of subcortical heterotopia reveals the causal link between Eml1's function in microtubule regulation and cell position, both critical for correct cortical development.

Synergistic Effect between the APOE ε4 Allele with Genetic Variants of GSK3B and MAPT: Differential Profile between Refractory Epilepsy and Alzheimer Disease.

Temporal Lobe Epilepsy (TLE) is a chronic neurological disorder characterized by recurrent focal seizures originating in the temporal lobe. Despite the variety of antiseizure drugs currently available to treat TLE, about 30% of cases continue to have seizures. The etiology of TLE is complex and multifactorial. Increasing evidence indicates that Alzheimer's disease (AD) and drug-resistant TLE present common pathological features that may induce hyperexcitability, especially aberrant hyperphosphorylation of tau protein. Genetic polymorphic variants located in genes of the microtubule-associated protein tau (MAPT) and glycogen synthase kinase-3β (GSK3B) have been associated with the risk of developing AD. The APOE ε4 allele is a major genetic risk factor for AD. Likewise, a gene-dose-dependent effect of ε4 seems to influence TLE. The present study aimed to investigate whether the APOE ɛ4 allele and genetic variants located in the MAPT and GSK3B genes are associated with the risk of developing AD and drug-resistant TLE in a cohort of the Mexican population. A significant association with the APOE ε4 allele was observed in patients with AD and TLE. Additional genetic interactions were identified between this allele and variants of the MAPT and GSK3B genes.

TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b−/− mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.

Investigation of Persistent Photoconductivity of Gallium Nitride Semiconductor and Differentiation of Primary Neural Stem Cells.

A gallium nitride (GaN) semiconductor is one of the most promising materials integrated into biomedical devices to play the roles of connecting, monitoring, and manipulating the activity of biological components, due to its excellent photoelectric properties, chemical stability, and biocompatibility. In this work, it was found that the photogenerated free charge carriers of the GaN substrate, as an exogenous stimulus, served to promote neural stem cells (NSCs) to differentiate into neurons. This was observed through the systematic investigation of the effect of the persistent photoconductivity (PPC) of GaN on the differentiation of primary NSCs from the embryonic rat cerebral cortex. NSCs were directly cultured on the GaN surface with and without ultraviolet (UV) irradiation, with a control sample consisting of tissue culture polystyrene (TCPS) in the presence of fetal bovine serum (FBS) medium. Through optical microscopy, the morphology showed a greater number of neurons with the branching structures of axons and dendrites on GaN with UV irradiation. The immunocytochemical results demonstrated that GaN with UV irradiation could promote the NSCs to differentiate into neurons. Western blot analysis showed that GaN with UV irradiation significantly upregulated the expression of two neuron-related markers, βIII-tubulin (Tuj-1) and microtubule-associated protein 2 (MAP-2), suggesting that neurite formation and the proliferation of NSCs during differentiation were enhanced by GaN with UV irradiation. Finally, the results of the Kelvin probe force microscope (KPFM) experiments showed that the NSCs cultured on GaN with UV irradiation displayed about 50 mV higher potential than those cultured on GaN without irradiation. The increase in cell membrane potential may have been due to the larger number of photogenerated free charges on the GaN surface with UV irradiation. These results could benefit topical research and the application of GaN as a biomedical material integrated into neural interface systems or other bioelectronic devices.

HN1 expression contributes to mitotic fidelity through Aurora A-PLK1-Eg5 axis.

Hematological and neurological expressed 1 (HN1) is homolog of Jupiter protein from Drosophila melanogaster where it functions as a microtubule-associated protein. However, in mammalian cells, HN1 is associated partially with y-tubulin in centrosomes, Stathmin for stabilizing microtubules, and Cdh1 for regulating Cyclin B1 for cell cycle regulation. Moreover, HN1 overexpression leads to early mitotic exit as well. Other molecular functions and interactions of HN1 are not clear yet. Here, based on our previous analysis where HN1 was shown to cluster supernumerary centrosomes and maintain mitotic spindle assembly, we further investigated the role of HN1 in centrosome maintenance and mitotic fidelity in PC-3 prostate and MDA-MB231 mammary cancer cell lines. The maturation-associated roles of HN1 during cell division by examining the AuroraA-PLK1 axis involving a plus end kinesin, Eg5 as well as pericentriolar matrix protein (PCM1) as components of centrosomes were established. We found that HN1 co-localized to centrioles with Eg5 and Aurora A to suppress aberrant spindle formation to ensure the fidelity of centriole/centrosome duplication when overexpressed. Consistently, depleting the HN1 expression using siRNA or shRNA resulted in an increased number of dysregulated mitotic spindle structures, where Aurora A as well as PLK1 co-localizations with Eg5 and PCM1 were disrupted. Further, the PLK1 and Aurora A kinase's phosphorylations also decreased, confirming the hypothesis that the cells struggle in mitotic progression, display nuclear and cytokinetic abnormalities with supernumerary but immature mononucleated centrosomes. In summary, we described the role of HN1 in centrosome nucleation/maturation in PLK1-Eg5 axis and concomitant mitotic spindle formation in human cells.

Etomidate protects retinal ganglion cells from hydrogen peroxide-induced injury via Nrf2/HO-1 pathway.

To determine whether etomidate (ET) has a protective effect on retinal ganglion cells (RGCs) injured with hydrogen peroxide (H2O2) and to explore the potential mechanism underlying the antioxidative stress effect of ET. Cultured RGCs were identified by double immunofluorescent labeling of microtubule-associated protein 2 and Thy1.1. An injury model of H2O2-induced RGCs oxidative stress was established in vitro. Cells were pretreated with different concentrations of ET (1, 5, and 10 µmol/L) for 4h, followed by further exposure to H2O2 at 1000 µmol/L. Cell counting kit 8 and Annexin V/propidium iodide assays were applied to detect the viabilities and apoptosis rates of the RGCs at 12, 24, and 48h after H2O2 stimulation. The levels of nitric oxide, malondialdehyde, and glutathione in culture media were measured at these time points. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to observe the effects of ET on the messenger RNA and protein expression of inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase 1 and the level of conjugated acrolein in RGCs at 12, 24, and 48h after H2O2 stimulation and in the retina at 12h after optic nerve transection (ONT). The applications of 5 and 10 µmol/L of ET significantly increased the viability of RGCs. Results from qRT-PCR indicated a decrease in the expression of iNOS and an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs at 12, 24 and 48h after H2O2 stimulation, as well as in ET-treated retinas at 12h after ONT. Western blot analysis revealed a decrease in the expression of iNOS and levels of conjugated acrolein, along with an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs in vitro and ET-treated retinas in vivo. ET is a neuroprotective agent in primary cultured RGCs injured by H2O2. The effect of ET is dose-dependent with the greatest effect being at 10 µmol/L. ET plays an antioxidant role by inhibiting iNOS, up-regulating Nrf2/HO-1, decreasing the production of acrolein, and increasing the scavenge of acrolein.

Untangling the complexity and impact of tau protein ubiquitination.

The microtubule-associated protein tau is an intrinsically disordered protein highly expressed in neuronal axons. In healthy neurons, tau regulates microtubule dynamics and neurite outgrowth. However, pathological conditions can trigger aberrant tau aggregation into insoluble filaments, a hallmark of neurodegenerative disorders known as tauopathies. Tau undergoes diverse posttranslational modifications (PTMs), suggesting complex regulation and potentially varied functions. Among PTMs, the role and mechanisms of ubiquitination in physiology and disease have remained enigmatic. The past three decades have witnessed the emergence of key studies on tau protein ubiquitination. In this concept, we discuss how these investigations have begun to shed light on the ubiquitination patterns of physiological and pathological tau, the responsible enzymatic machinery, and the influence of ubiquitination on tau aggregation. We also provide an overview of the semi-synthetic methods that have enabledin vitroinvestigations of conformational transitions of tau induced by ubiquitin modification. Finally, we discuss future perspectives in the field necessary to elucidate the molecular mechanisms of tau ubiquitination and clearance.

Resistin alleviates lipopolysaccharide-induced inflammation in bovine alveolar macrophages by activating the AMPK/mTOR signaling pathway and autophagy

ObjectiveResistin (RETN) is an adipocyte-specific hormone that participates in metabolism and modulates cellular inflammation. Our study aimed to assess the effects of RETN treatment on autophagy and the underlying molecular and biological mechanisms in bovine alveolar macrophages (BAMs). MethodsThe optimal concentration of RETN + lipopolysaccharide (LPS) on macrophages was screened and then used to co-culture with alveolar macrophages. Autophagosomes in BAMs were examined using a transmission electron microscope (TEM). Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of microtubule-associated protein light chain 3 (LC3) and p62. Western blot (WB) was used to detect the protein expressions of LC3 and p62. The distribution of LC3 and p62 proteins in the cells was observed by immunofluorescence (IF). The concentrations of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expression of adenosine-monophosphate-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), and p-mTOR was detected using WB. ResultsThe treatment of BAMs with RETN or LPS increased the number of autophagosomes and the ratio of LC3II/LC3I and decreased the expression level of p62 protein. RETN treatment significantly triggered autophagy compared to LPS treatment. Moreover, the ratios of p-AMPK/AMPK and p-mTOR/mTOR were upregulated and downregulated, respectively, after RETN treatment, suggesting that AMPK/mTOR signaling pathway activation is required for RETN-mediated autophagy in BAMs. Additionally, the ratio of LC3-II/LC3-I was lower, and the concentrations of IL-1β, IL-6, and TNF-α significantly decreased in the LPS and RETN co-treatment groups compared to the single LPS treatment group. However, both autophagy- and LPS-induced inflammation were partially alleviated by RETN treatment. ConclusionRETN can promote autophagy in BAMs by activating the AMPK/mTOR signaling pathway, it may help prevent LPS-induced inflammation.