Emerging Roles of Extracelluar Vesicles Derived from Bacteria, Mammalian or Plant Cells in the Pathogenesis and Clinical Application of Neurodegenerative Diseases.

A growing number of studies have indicated that extracellular vesicles (EVs), such as exosomes, are involved in the development of neurodegenerative diseases. Components of EVs with biological effects like proteins, nucleic acids, or other molecules can be delivered to recipient cells to mediate physio-/pathological processes. For instance, some aggregate-prone proteins, such as ß-amyloid and α-synuclein, had been found to propagate through exosomes. Therefore, either an increase of detrimental molecules or a decrease of beneficial molecules enwrapped in EVs may fully or partly indicate disease progression. Numerous studies have demonstrated that dysbiosis of the gut microbiota and neurodegeneration are tightly correlated, well-known as the "gut-brain axis". Accumulating evidence has revealed that the gut bacteria-derived EVs play a pivotal role in mediating microbe-host interactions and affect the function of the "gut-brain axis", which subsequently contributes to the pathogenesis of neurodegenerative diseases. In this review, we first briefly discuss the role of EVs from mammalian cells and microbes in mediating the progression of neurodegenerative diseases, and then propose a novel strategy that employs EVs of plants (plant cell-derived exosome-like nanoparticles) for treating neurodegeneration.

Exosome-shuttled miR-126 mediates ethanol-induced disruption of neural crest cell-placode cell interaction by targeting SDF1.

During embryonic development, 2 populations of multipotent stem cells, cranial neural crest cells (NCCs) and epibranchial placode cells (PCs), are anatomically adjacent to each other. The coordinated migration of NCCs and PCs plays a major role in the morphogenesis of craniofacial skeletons and cranial nerves. It is known that ethanol-induced dysfunction of NCCs and PCs is a key contributor to the defects of craniofacial skeletons and cranial nerves implicated in fetal alcohol spectrum disorder (FASD). However, how ethanol disrupts the coordinated interaction between NCCs and PCs was not elucidated. To fill in this gap, we established a well-designed cell coculture system to investigate the reciprocal interaction between human NCCs (hNCCs) and human PCs (hPCs), and also monitored the migration behavior of NCCs and PCs in zebrafish embryos. We found that ethanol exposure resulted in a disruption of coordinated hNCCs-hPCs interaction, as well as in zebrafish embryos. Treating hNCCs-hPCs with exosomes derived from ethanol-exposed hNCCs (ExoEtOH) mimicked ethanol-induced impairment of hNCCs-hPCs interaction. We also observed that SDF1, a chemoattractant, was downregulated in ethanol-treated hPCs and zebrafish embryos. Meanwhile, miR-126 level in ExoEtOH was significantly higher than that in control exosomes (ExoCon). We further validated that ExoEtOH-encapsulated miR-126 from hNCCs can be transferred to hPCs to suppress SDF1 expression in hPCs. Knockdown of SDF1 replicated ethanol-induced abnormalities either in vitro or in zebrafish embryos. On the contrary, overexpression of SDF1 or inhibiting miR-126 strongly rescued ethanol-induced impairment of hNCCs-hPCs interaction and developmental defects.

Ethanol exposure disrupted the formation of radial glial processes and impaired the generation and migration of outer radial glial cells in forebrain organoids derived from human embryonic stem cells.

Radial glial cells (RGCs) play a pivotal role in cerebral cortical development by functioning as a source of new neurons and by supporting the migration of newborn neurons. These functions are primarily dependent on the apical-basolateral structures of radial glial processes. This study aims to investigate the effects of ethanol exposure on the development of radial glial processes and the generation, migration, and transformation of outer radial glial cells (oRGCs). For this purpose, forebrain organoids were developed from human embryonic stem cells. These forebrain organoids contain abundant neural progenitor cells (SOX2+), express high levels of neural epithelial markers ß-catenin and PKCλ, and dorsal forebrain marker PAX6, and display well-organized cortical architectures containing abundant apical and basal RGCs, intermediate progenitors (IPCs), and neurons. Exposure of forebrain organoids to ethanol resulted in a significant increase in apoptosis in Nestin-positive radial glial cells. Ethanol exposure also remarkably decreased the levels of radial glial process-associated proteins, including Nestin, GFAP, and Vimentin, in radial glial cells and distinctly impaired the integrity and morphologies of radial glial processes. In addition, the ethanol-induced impairment of the radial glial processes is associated with decreased migration and proliferation of radial glial cells, reduction in the generation of HOPX+ oRGCs, and the accelerated transformation of oRGCs into astrocytes. These results demonstrate that ethanol exposure can disrupt cerebral cortex development by impairing the formation of radial glial processes and the generation, migration, and transformation of oRGCs.

Up-regulation of microRNA-34a mediates ethanol-induced impairment of neural crest cell migration in vitro and in zebrafish embryos through modulating epithelial-mesenchymal transition by targeting Snail1.

Prenatal ethanol exposure can impair neural crest cell (NCC) development, including NCC survival, differentiation and migration, contributing to the craniofacial dysmorphology in Fetal Alcohol Spectrum Disorders (FASD). Epithelial-mesenchymal transition (EMT) plays an important role in regulating the migration of NCCs. The objective of this study is to determine whether ethanol exposure can suppress NCC migration through inhibiting EMT and whether microRNA-34a (miR-34a) is involved in the ethanol-induced impairment of EMT in NCCs. We found that exposure to 100 mM ethanol significantly inhibited the migration of NCCs. qRT-PCR and Western Blot analysis revealed that exposure to ethanol robustly reduced the mRNA and protein expression of Snail1, a critical transcriptional factor that has a pivotal role in the regulation of EMT. Ethanol exposure also significantly increased the mRNA expression of the Snail1 target gene E-cadherin1 and inhibited EMT in NCCs. We also found that exposure to ethanol significantly elevated the expression of miR-34a that targets Snail1 in NCCs. In addition, down-regulation of miR-34a prevented ethanol-induced repression of Snail1 and diminished ethanol-induced upregulation of Snail1 target gene E-cadherin1 in NCCs. Inhibition of miR-34a restored EMT and prevented ethanol-induced inhibition of NCC migration in vitro and in zebrafish embryos in vivo. These results demonstrate that ethanol-induced upregulation of miR-34a contributes to the impairment of NCC migration through suppressing EMT by targeting Snail1.

Sulforaphane Protects Against Ethanol-Induced Apoptosis in Human Neural Crest Cells Through Diminishing Ethanol-Induced Hypermethylation at the Promoters of the Genes Encoding the Inhibitor of Apoptosis Proteins.

The neural crest cell (NCC) is a multipotent progenitor cell population that is sensitive to ethanol and is implicated in the Fetal Alcohol Spectrum Disorders (FASD). Studies have shown that sulforaphane (SFN) can prevent ethanol-induced apoptosis in NCCs. This study aims to investigate whether ethanol exposure can induce apoptosis in human NCCs (hNCCs) through epigenetically suppressing the expression of anti-apoptotic genes and whether SFN can restore the expression of anti-apoptotic genes and prevent apoptosis in ethanol-exposed hNCCs. We found that ethanol exposure resulted in a significant increase in the expression of DNMT3a and the activity of DNMTs. SFN treatment diminished the ethanol-induced upregulation of DNMT3a and dramatically reduced the activity of DNMTs in ethanol-exposed hNCCs. We also found that ethanol exposure induced hypermethylation at the promoter regions of two inhibitor of apoptosis proteins (IAP), NAIP and XIAP, in hNCCs, which were prevented by co-treatment with SFN. SFN treatment also significantly diminished ethanol-induced downregulation of NAIP and XIAP in hNCCs. The knockdown of DNMT3a significantly enhanced the effects of SFN on preventing the ethanol-induced repression of NAIP and XIAP and apoptosis in hNCCs. These results demonstrate that SFN can prevent ethanol-induced apoptosis in hNCCs by preventing ethanol-induced hypermethylation at the promoter regions of the genes encoding the IAP proteins and diminishing ethanol-induced repression of NAIP and XIAP through modulating DNMT3a expression and DNMT activity.

MicroRNA-135a Protects Against Ethanol-Induced Apoptosis in Neural Crest Cells and Craniofacial Defects in Zebrafish by Modulating the Siah1/p38/p53 Pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in various biological processes, including apoptosis, by regulating gene expression. This study was designed to test the hypothesis that ethanol-induced downregulation of miR-135a contributes to ethanol-induced apoptosis in neural crest cells (NCCs) by upregulating Siah1 and activating the p38 mitogen-activated protein kinase (MAPK)/p53 pathway. We found that treatment with ethanol resulted in a significant decrease in miR-135a expression in both NCCs and zebrafish embryos. Ethanol-induced downregulation of miR-135a resulted in the upregulation of Siah1 and the activation of the p38 MAPK/p53 pathway and increased apoptosis in NCCs and zebrafish embryos. Ethanol exposure also resulted in growth retardation and developmental defects that are characteristic of fetal alcohol spectrum disorders (FASD) in zebrafish. Overexpression of miRNA-135a significantly reduced ethanol-induced upregulation of Siah1 and the activation of the p38 MAPK/p53 pathway and decreased ethanol-induced apoptosis in NCCs and zebrafish embryos. In addition, ethanol-induced growth retardation and craniofacial defects in zebrafish larvae were dramatically diminished by the microinjection of miRNA-135a mimics. These results demonstrated that ethanol-induced downregulation of miR-135a contributes to ethanol-induced apoptosis in NCCs by upregulating Siah1 and activating the p38 MAPK/p53 pathway and that the overexpression of miRNA-135a can protect against ethanol-induced apoptosis in NCCs and craniofacial defects in a zebrafish model of FASD.

MicroRNA-34a mediates ethanol-induced impairment of neural differentiation of neural crest cells by targeting autophagy-related gene 9a.

Neural crest cells (NCCs) are multipotent progenitor cells that are sensitive to ethanol and are implicated in Fetal Alcohol Spectrum Disorders (FASD). The objective of this study is to test whether ethanol exposure can inhibit the neural differentiation of NCCs by inhibiting autophagy and whether miR-34a is involved in ethanol-induced inhibition of autophagy in NCCs. We found that ethanol exposure resulted in the inhibition of neural differentiation of NCCs. Exposure to ethanol also significantly decreased autophagy in NCCs, as indicated by a decreased LC3II/I ratio and an elevated expression of p62 protein. Knockdown of p62 restored the expression of the neurogenesis genes, NF and Mash1, in ethanol-exposed NCCs, suggesting that ethanol exposure can inhibit the neural differentiation of NCCs by inhibiting autophagy. We also found that ethanol exposure resulted in a significant increase in miR-34a expression in NCCs. Inhibition of miR-34a restored the expression of Atg9a, a direct target of miR-34a and significantly decreased ethanol-induced inhibition of autophagy in NCCs. Down-regulation of miR-34a also prevented ethanol-induced inhibition of neural differentiation of NCCs. These results demonstrate that ethanol-induced inhibition of neural differentiation of NCCs is mediated by the miR-34a through targeting Atg9a.

Graphene Oxide Modulates B Cell Surface Phenotype and Impairs Immunoglobulin Secretion in Plasma Cell.

Since discovery, graphene oxide (GO) has been used in all aspects of human life and revealed promising applications in biomedicine. Nevertheless, the potential risks of GO were always being revealed. Although GO was found to induce immune cell death and innate immune response, little is known regarding its toxicity to the specific adaptive immune system that is crucial for protecting against exotic invasion. The B-cell mediated adaptive immune system, which composed of highly specialized cells (B and plasma cell) and specific immune response (antibody response) is the focus in our present study. Using diverse standard immunological techniques, we found that GO modulated B cell surface phenotype, both costimulatory molecules (CD80, CD86 and especially CD40) and antigen presenting molecules (both classical and nonclassical) under the condition without causing cell death. Meanwhile, the terminal differentiated immunoglobulin (Ig) secreting plasma cell was affected by GO, which displayed a less secretion of Ig and more severe ER stress caused by the retention of the secreted form of Ig in cell compartment. The combined data reveal that GO has a particular adverse effect to B cell and the humoral immunity, directly demonstrating the potential risk of GO to the specific adaptive immunity.

Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation.

AIM: Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro. METHODS: HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells. RESULTS: Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 µg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells. CONCLUSION: The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.

The acidic transformed nano-VO2 causes macrophage cell death by the induction of lysosomal membrane permeabilization and Ca2+ efflux.

Because of its outstanding thermochromic characteristics and metal-insulator transition (MIT) property, nano-vanadium dioxide (abbreviated as nano-VO2 or nVO2) has been applied widely in electrical/optical devices and design of intelligent window. However, the biological effect of nVO2 is not well understood, especially when affected by environmental factors or living organisms. For VO2 is an amphoteric oxide, we simulated pH's influence to nVO2's physicochemical properties by exposure nVO2 in water of different pH values. We found that nVO2 transformed to a new product after exposure in acidic water for two weeks, as revealed by physicochemical characterization such as SEM, TEM, XRD, and DLS. This transformation product formed in acidic water was referred as (acidic) transformed nVO2). Both pristine/untransformed and transformed nVO2 displayed no obvious toxicity to common epithelial cells; however, the acidic transformed nVO2 rapidly induced macrophage cell death. Further investigation demonstrated that transformed nVO2 caused macrophage apoptosis by the induction of Ca2+ efflux and the following mitochondrial membrane permeabilization (MMP) process. And a more detailed time course study indicated that transformed nVO2 caused lysosomal membrane permeabilization (LMP) at the earlier stage, indicating LMP could be chosen as an earlier and sensitive end point for nanotoxicological study. We conclude that although nVO2 displays no acute toxicity, its acidic transformation product induces macrophage apoptosis by the induction of LMP and Ca2+ efflux. This report suggests that the interplay with environmental factors or living organisms can results in physicochemical transformation of nanomaterials and the ensuing distinctive biological effects.

The endoplasmic reticulum (ER)-associated degradation system regulates aggregation and degradation of mutant neuroserpin.

Familial encephalopathy with neuroserpin inclusion bodies is a neurodegenerative disorder characterized by the accumulation of neuroserpin polymers in the endoplasmic reticulum (ER) of cortical and subcortical neurons in the CNS because of neuroserpin point mutations. ER-associated degradation (ERAD) is involved in mutant neuroserpin degradation. In this study, we demonstrate that two ER-associated E3 ligases, Hrd1 and gp78, are involved in the ubiquitination and degradation of mutant neuroserpin. Overexpression of Hrd1 and gp78 decreases the mutant neuroserpin protein level, whereas Hrd1 and gp78 knockdown increases mutant neuroserpin stability. Moreover, ERAD impairment by mutant valosin-containing protein increases the mutant neuroserpin protein level and aggregate formation. Thus, these findings identify mutant neuroserpin as an ERAD target and show that Hrd1 and gp78 mediate mutant neuroserpin turnover through the ERAD pathway.

Degradation of TDP-43 and its pathogenic form by autophagy and the ubiquitin-proteasome system.

TAR DNA-binding protein-43 (TDP-43) is a nuclear protein functioning in the regulation of transcription and mRNA splicing. TDP-43 is accumulated in ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) diseased brains. However, the pathways involved in the clearance of TDP-43 and its pathogenic form (TDP-25), a truncated form of TDP-43, are still not elucidated. In this study, we demonstrated that the protein levels of TDP-43 and TDP-25 were increased in cells treated with a proteasome inhibitor, MG132, or an autophagy inhibitor, 3-MA, whereas, they were decreased in cells treated with an enhancer of autophagy, trehalose. Furthermore, more protein level changes of TDP-25 than TDP-43 were observed in cells treated with above inhibitors or enhancer. Thus, our data suggest that TDP-43 and TDP-25 are degraded by both proteasome and autophagy with TDP-25 being more regulated.

Gp78, an ER associated E3, promotes SOD1 and ataxin-3 degradation.

Superoxide dismutase-1 (SOD1) and ataxin-3 are two neurodegenerative disease proteins in association with familial amyotrophic lateral sclerosis and Machado-Joseph disease/spinocerebellar ataxia type 3. Both normal and mutant types of SOD1 and ataxin-3 are degraded by the proteasome. It was recently reported that these two proteins are associated with the endoplasmic reticulum (ER). Mammalian gp78 is an E3 ubiquitin ligase involved in ER-associated degradation (ERAD). Here, we show that gp78 interacts with both SOD1 and ataxin-3. Overexpression of gp78 promotes the ubiquitination and degradation of these two proteins, whereas knockdown of gp78 stabilizes them. Moreover, gp78 represses aggregate formation of mutant SOD1 and protect cells against mutant SOD1-induced cell death. Furthermore, gp78 is increased in cells transfected with these two mutant proteins as well as in ALS mice. Thus, our results suggest that gp78 functions in the regulation of SOD1 and ataxin-3 to target them for ERAD.

[Missed diagnostic cholesteattomaous otitismedia].

OBJECTIVE: To explore the missed diagnostic cholesteattomaous otitismedia lacked of clinical symptoms in order to minimize the mistake in clinical diagnose. METHODS: A retrospective study of 31 cases with the missed diagnostic cholesteattomaous otitismedia, confirmed by surgery and pathology, was conducted. RESULTS: Twenty-one cases (67.7%) had not obvious otorrhea. There were only slight finding such as attic retraction, apophysis, small granulation tissue, ear wax covered or tiny, even non, perforation in the pars flaccida or the superior and posterior pars tensa of the ear-drum. In general, the hearing loss is slight or medium conductive deafness. The type B curve was showed in tympanogram. The diagnosis rate of X-ray was only 41.9%, and 80.7% for the computed tomography. The middle ear structures aggressed by cholesteames was epitympanum, ossicular chain, tympanic antrum, mastoid process and mesotympanum. The complications occurred in 11 cases (35.5%) were the expose of facial nerve or and the meninges, or, and the labyrinth fistula. CONCLUSION: The cases with the cholesteatoma lack obvious clinic symptoms would result in the missed diagnosis because of slow and long term during cholesteatoma development. Sometimes misdiagnosed or missed diagnosis happened to in such cases of cholesteatoma with little clinical manifestation, and it is so dangerous for the patients in such case. Therefore, otolaryngologist must pay more attention to the missed diagnostic cholesteattomaous otitismedia.