Sodium alginate coating simultaneously increases the biosafety and immunotherapeutic activity of the cationic mRNA nanovaccine

The extraordinary advantages associated with mRNA vaccines, including their high efficiency, relatively low severity of side effects, and ease of manufacture, have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers. Nevertheless, most mRNA delivery carriers have many disadvantages, such as high toxicity, poor biocompatibility, and low efficiency in vivo, which have hindered the widespread use of mRNA vaccines. To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier, a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate (SA). Intriguingly, the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA, which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA. In addition, we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting. Finally, we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E. G7-OVA tumor-bearing mice, dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect. Therefore, we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.

Transcription factor EB attenuates neuronal damage following cerebral ischemia via regulating autophagy-lysosomal pathway / 国际脑血管病杂志

The disorder of autophagy lysosomal pathway (ALP) is an important pathogenesis of neuronal damage after cerebral ischemia, and the restoration of ALP may alleviate neuronal damage after cerebral ischemia. As the main transcription factor regulating ALP, transcription factor EB (TFEB) can directly regulate autophagosome generation, autophagosome-lysosome fusion, and autophagic flux by regulating the expression of autophagic genes and lysosomal genes. Therefore, regulating TFEB can alleviate ALP dysfunction and thereby reduce cerebral ischemic damage. This article reviews the structure, biological function of TFEB and its role in regulating ALP to alleviate neuronal damage after cerebral ischemia.

Efecto de la ausencia de suero fetal bovino sobre la morfología y organelas de células fibroblásticas / Effect of the absence of fetal bovine serum on the morphology and organelles of fibroblast cells

RESUMEN Los fibroblastos son células constituyentes de los tejidos conectivo. Los fibroblastos gingivales (FGs), células responsables de la síntesis de la matriz extracelular (MEC) en el tejido conectivo gingival, participan en la regulación de los procesos de cicatrización y de reparación de la encía. Debido a su potencial regenerativo, los FGs podrían ser células capaces de contribuir a mejorar los procesos de cicatrización, a nivel local y sistémico e, incluso, ser utilizadas como un modelo celular útil en la comprensión de los aspectos fisiopatológicos de la cavidad oral. El objetivo del presente trabajo fue describir el impacto de la concentración del suero fetal bovino (SFB), en la supervivencia, el crecimiento y la expresión de marcadores celulares en los FGs. Cultivos celulares de FGs fueron realizados durante 7 días, utilizando medio de cultivo DMEM (Dulbecco's Modified Eagle's médium), en ausencia y presencia de 10% de SFB. Análisis morfológicos e inmunohistoquímicos de la actina, mitocondrias, lisosomas y retículo endoplasmático (RE) fueron usados para evaluar el impacto de la concentración del SFB sobre los FGs. Los resultados indican que los FGs cultivados en presencia de 10% de SFB tuvieron una tasa de crecimiento más elevada en comparación con los FGs, cultivados en ausencia de SFB. El marcaje de los elementos celulares indica la ausencia de alteraciones en las organelas celulares de los FGs, cuando son cultivados en ausencia de SFB. En conclusión, los FGs son capaces de sobrevivir, proliferar y conservar sus características morfológicas, cuando son cultivados en presencia y ausencia de SFB.

ABSTRACT Fibroblasts are constituent cells of connective tissues. Gingival fibroblasts (GFs), cells responsible for the synthesis of the extracellular matrix (ECM) in the gingival connective tissue, participate in the regulation of healing and repair processes of the gingiva. Due to their regenerative potential, GFs could be cells capable of contributing to improve the healing processes at the local and systemic level and even be used as a useful cellular model in understanding of the physiopathological aspects of the oral cavity. The aim of the present work was to describe the impact of the concentration of fetal bovine serum (FBS) on the survival, growth and expression of cell markers in GFs. Cell cultures of GFs were performed for 7 days using DMEM culture medium (Dulbecco's Modified Eagle's medium) in the absence and presence of 10% FBS. Morphological and immunohistochemistry analyzes of actin, mitochondria, lysosomes and endoplasmic reticulum (ER) were used to evaluate the impact of FBS concentration on GFs. The results indicate that GFs cultured in the presence of 10% FBS had a higher growth rate compared to GFs cultured in the absence of FBS. The marking of the cellular elements indicates the absence of alterations in the cellular organelles of the GFs when they are cultured in the absence of FBS. In conclusion, GFs are capable to surviving, proliferating and conserving their morphological characteristics when they are cultured in the presence and absence of FBS.

Cultivo de células epiteliales dentales: impacto del suero fetal bovino / Culture of dental epithelial cells: impact of fetal bovine serum

RESUMEN Objetivo. Describir la influencia del Suero Fetal Bovino (SFB) en la supervivencia, crecimiento y expresión de organelas celulares en las células epiteliales dentales de rata. Materiales y métodos. Cultivos de células epiteliales dentales de rata fueron llevados a cabo a 37°C en una atmosfera húmeda, en ausencia y a una concentración de 10% de SFB. Una evaluación morfológica fue realizada durante la proliferación y confluencia de las células en cultivo. Dobles marcajes por inmunofluorencia fueron efectuados haciendo uso de anticuerpos anti-actina, anti-TOMM20 y anti-LAMP1. Resultados. Se evidenciaron células epiteliales dentales circulares u ovoides con núcleos voluminosos durante la proliferación y confluencias de manera similar en las células cultivas en presencia y ausencia de SFB. La carencia de SFB impactó negativamente la proliferación de las células epiteliales. No fueron observadas alteraciones en la localización de los inmunomarcajes anti-actina, anti-TOMM20 y anti-LAMP1 en las dos condiciones de cultivos experimentales. Conclusiones. La supresión del SFB en el cultivo de células epiteliales dentales de rata disminuyó la supervivencia, proliferación y sugiere no tener un impacto sobre las organelas evaluadas.

ABSTRACT Objective. Describe the influence of Fetal bovine serum (FBS) on the survival, growth and expression of cellular organelles in rat dental epithelial cells. Material and methods. Cell cultures of rat dental epithelial cells were carried out at 37°C in a humid atmosphere, in the absence and at a concentration of 10% FBS. Morphological evaluation was performed during the proliferation and confluence of cell in culture. Double immunofluorescence labels were made using anti-Actin, anti-TOMM20A, and anti-LAMP1 antibodies. Results. Circular or ovoid dental epithelial cells with bulky nuclei were evidenced during proliferation and confluences in a similar manner in culturing cells in the presence and absence of FBS. The lack of FBS negatively impacts the proliferation of epithelial cells. No alterations were observed in the localization of the anti-actin, anti-TOMM20 and anti-LAMP1 immunomarkers in both conditions of experimental cultures. Conclusion. FBS suppression in rat dental epithelial cells decreased survival, proliferation and suggests not having an impact on the organelles evaluated.

Expresión inmunohistoquímica de la proteína de membrana asociada a lisosomas en ameloblastos de ratones

Introducción: La formación del esmalte dental comprenden cambios morfofuncionales de los ameloblastos. Estas modificaciones involucran la participación activa de organelas celulares, entre ellas los lisosomas. Objetivo: Identificar la distribución de la proteína de membrana asociada a lisosomas en los ameloblastos. Materiales y métodos: Incisivos en crecimiento continuo de ratones con edades de 7, 14 días y células epiteliales dentales de rata fueron utilizadas para identificar la proteína de membrana asociada a lisosomas tipo 1 (Lamp1) mediante la técnica de inmunohistoquímica. Resultados: Un marcaje citoplasmático de Lamp1 fue identificado en las células ameloblásticas durante todas las etapas del proceso de diferenciación de los ameloblastos, independientemente de sus edades. Lamp1 también fue detectado en las células epiteliales dentales. Conclusiones: Se evidencia la expresión de Lamp1 en los ameloblastos en incisivos de crecimiento continuo y células epiteliales dentales de rata. Próximos estudios deberían abordar el rol funcional de Lamp1 en la formación del esmalte.

Introduction: Tooth enamel formation includes morphological changes in ameloblasts. These modifications involve the active participation of cellular organelles, including lysosomes. Objective: To identify the lysosomal membrane protein distribution in ameloblasts. Materials and method: The lysosomal membrane protein type 1 (Lamp 1) was identified in continuous growth incisor from mice (7, 14 days old) and epithelial rat's cells using the immunohistochemistry technic. Results: A cytoplasmic Lamp 1 marker was identified in ameloblast cells during all the stages of the process, no matter the age. Lamp 1 was detected, too, in the epithelial dental cells. Conclusion: It is evident the expression of Lamp 1 in ameloblasts of continuous growth incisor and epithelial dental rat's cells. Next studies must be done about the functional role of Lamp 1 in tooth enamel formation.

The role of lysosomes in manganese-induced toxicity in SK-N-SH cells / 中华劳动卫生职业病杂志

Objective@#To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells.@*Methods@#SK-N-SH cells were treated with MnCl2 at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl2 at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit.@*Results@#Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl2 treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl2 treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl2 treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl2, while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn’t observed significant difference of the activity of CTSD between different MnCl2 treatment groups.@*Conclusion@#MnCl2 could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.

Release and uptake mechanisms of vesicular Ca stores

Cells utilize calcium ions (Ca) to signal almost all aspects of cellular life, ranging from cell proliferation to cell death, in a spatially and temporally regulated manner. A key aspect of this regulation is the compartmentalization of Ca in various cytoplasmic organelles that act as intracellular Ca stores. Whereas Ca release from the large-volume Ca stores, such as the endoplasmic reticulum (ER) and Golgi apparatus, are preferred for signal transduction, Ca release from the small-volume individual vesicular stores that are dispersed throughout the cell, such as lysosomes, may be more useful in local regulation, such as membrane fusion and individualized vesicular movements. Conceivably, these two types of Ca stores may be established, maintained or refilled via distinct mechanisms. ER stores are refilled through sustained Ca influx at ER-plasma membrane (PM) membrane contact sites (MCSs). In this review, we discuss the release and refilling mechanisms of intracellular small vesicular Ca stores, with a special focus on lysosomes. Recent imaging studies of Ca release and organelle MCSs suggest that Ca exchange may occur between two types of stores, such that the small stores acquire Ca from the large stores via ER-vesicle MCSs. Hence vesicular stores like lysosomes may be viewed as secondary Ca stores in the cell.

Lysosomal enzymes and mannose 6-phosphate receptors in the lacrimal drainage system: Evidence and its potential implications

Purpose: To investigate the presence and patterns of lysosomal enzymes and mannose 6-phosophate receptor (MPRs) in human lacrimal drainage system. Methods: The study was performed on healthy lacrimal sacs and nasolacrimal ducts obtained from exenteration samples immediately after surgery and frozen at ?80°C for subsequent analysis. Soluble proteins' extract was used for enzyme assays, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE), native PAGE, activity staining, and western blot analysis. Membrane proteins were separately assessed for detection of mannose 6-phosphate receptors, MPR 46. Sepharose gels, 4-methylumbelliferyl substrates, and antibodies against common lysosomal enzymes and MPRs were used. Enzyme assays were carried out in triplicate to ascertain the results. Results: Differential lysosomal enzyme activities were documented, and among them acid phosphatase and ?-hexosaminidase were found to be high. Western blot analysis using enzyme antibodies and subsequent activity staining confirmed strong signals for moderately expressed enzymes such as fucosidase, glucuronidase, and mannosidase. Membrane extracts demonstrated the presence of MPR 46, which indicates the possible roles of cation-dependent MPRs in lysosomal targeting in human lacrimal drainage system. Conclusion: This study provides a proof of principle for the presence of differential lysosomal activity and mannose 6-phosphate ligand transport receptors in human lacrimal drainage system and hypothesizes the potential implications of their dysfunctions.

Quercetin alleviates chronic alcohol exposure induced liver autophagy dysfunction / 中国药理学通报

Aim To investigate the potential protective effect of quercetin on chronic ethanol exposure induced autophagy dysfunction .Methods The male C57 BL/6 J mice were divided randomly into four groups: Nor-mal control group ( Con);Ethanol group ( Eth);Etha-nol plus Quercetin group ( Eth+Qu); Quercetin con-trol group ( Qu ) .The mice were pair-fed with either regular or ethanol-containing Lieber De Carli liquids diets for 15 weeks and the related parameters were as-sayed .Result Compared to Con group , chronic etha-nol feeding led to serious aminotransferases leakage , hepatic lipid accumulation , and liver oxidative dam-age.Additionally, long-term ethanol exposure caused lysosome damage , cathepsin B leakage to cytoplasm and decreased the expression of LAMP 2A which was the key protein for the autophagosome-lysosome fusion . As expect , quercetin supplementation evidently allevia-ted lysosome damage and decreased the abnormal LC 3-II and p62 accumulation , accelerated the autophago-some-lysosome fusion and increased the expression of LAMP2A.Conclusion Quercetin may alleviate the liver injury induced by chronic alcohol exposure by a-meliorating the lysosomal damage and up-regulating LAMP2 A expression .

Mechanism of microphthalmia family of transcription factors in tumorigenesis / 国际肿瘤学杂志

Microphthalmia family of transcription factors (MiT/TFE) is very important for the regulation of cancer cell proliferation and energy metabolism.The MiT/TFE promotes the genesis and development of tumors by up-regulating the expression of lysosomal genes as well as acting on the oxidative metabolism and the oxidative stress response.MiT/TFE can also regulate lysosomal signaling including the mTORC1 and Wnt/3-catenin pathways.The relationship between MiT/TFE and folliculin (FLCN) is associated with the tumorigenesis.

A Novel Homozygous LIPA Mutation in a Korean Child with Lysosomal Acid Lipase Deficiency / 대한소아소화기영양학회지

Patients with lysosomal acid lipase (LAL) deficiency and glycogen storage disease (GSD) demonstrated hepatomegaly and dyslipidemia. In our case, a 6-year-old boy presented with hepatosplenomegaly. At 3 years of age, GSD had been diagnosed by liver biopsy at another hospital. He showed elevated serum liver enzymes and dyslipidemia. Liver biopsy revealed diffuse microvesicular fatty changes in hepatocytes, septal fibrosis and foamy macrophages. Ultrastructural examination demonstrated numerous lysosomes that contained lipid material and intracytoplasmic cholesterol clefts. A dried blood spot test revealed markedly decreased activity of LAL. LIPA gene sequencing identified the presence of a novel homozygous mutation (p.Thr177Ile). The patient's elevated liver enzymes and dyslipidemia improved with enzyme replacement therapy. This is the first report of a Korean child with LAL deficiency, and our findings suggest that this condition should be considered in the differential diagnosis of children with hepatosplenomegaly and dyslipidemia.

A Novel Homozygous LIPA Mutation in a Korean Child with Lysosomal Acid Lipase Deficiency / 대한소아소화기영양학회지

Patients with lysosomal acid lipase (LAL) deficiency and glycogen storage disease (GSD) demonstrated hepatomegaly and dyslipidemia. In our case, a 6-year-old boy presented with hepatosplenomegaly. At 3 years of age, GSD had been diagnosed by liver biopsy at another hospital. He showed elevated serum liver enzymes and dyslipidemia. Liver biopsy revealed diffuse microvesicular fatty changes in hepatocytes, septal fibrosis and foamy macrophages. Ultrastructural examination demonstrated numerous lysosomes that contained lipid material and intracytoplasmic cholesterol clefts. A dried blood spot test revealed markedly decreased activity of LAL. LIPA gene sequencing identified the presence of a novel homozygous mutation (p.Thr177Ile). The patient's elevated liver enzymes and dyslipidemia improved with enzyme replacement therapy. This is the first report of a Korean child with LAL deficiency, and our findings suggest that this condition should be considered in the differential diagnosis of children with hepatosplenomegaly and dyslipidemia.

Research progress on the roles of lysosome in the evolution of cancer / 中国医师杂志

Lysosomes are acidic catabolic organelles,containing over 50 acid hydrolases and many lysosomal specific membrane proteins.Lysosomes are responsible for the disposal and recycling of worn out and damaged cellular macromolecules and organelles as well as the digestion of extracellular and foreign materials delivered to them by endocytosis,autophagy and phagocytosis.Increased expression and function of various lysosomal hydrolases are common in human tumors,and they often correlate with a higher risk of rccurrence and poor prognosis.Inhibition of lysosomal exocytosis can inhibit tumor invasion and metastasis.It does not affect the activity of acid hydrolytic enzymes,but causes the instability of lysosomal membrane and increases the sensitivity of tumor cells to drugs.

Effects of change in the activity of vacuolar adenosine triphosphatase of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism / 中华烧伤杂志

Objective@#To explore the effects of change of activity of vacuolar adenosine triphosphatase (V-ATPase) of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism.@*Methods@#The myocardial lysosomes were extracted from the hearts of 12 SD rats with ultra-high speed gradient density centrifugation, then Western blotting and transmission electron microscope observation were conducted for identification. One hundred and twenty rats were divided into pure burn group, ATP group, normal control group, and bafilomycin group according to the random number table, with 30 rats in each group. Rats in pure burn group and ATP group were inflicted with 40% TBSA full-thickness scald on the back. Immediately after injury, rats in pure burn group were intraperitoneally injected with lactated Ringer′s solution in 4 mL·%TBSA-1·kg-1, and rats in ATP group were intraperitoneally injected with ATP in 0.4 mg/kg at 12 h before burn, immediately after burn, and 12 h after burn. Rats in normal control group did not receive any treatment, and rats in bafilomycin group were intraperitoneally injected with bafilomycin A1 in 0.3 mg/kg at the same time points as those of ATP group. At 24 h after burn, 30 rats from each group were collected for determining activity of V-ATPase of myocardial lysosome with coupled-enzyme assay and the expression of myocardium autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and P62 by Western blotting. Left ventricular arterial blood was collected to detect the content of 5 items of myocardial enzyme spectrum and cardiac troponin T (cTnT). Data were processed with one-way analysis of variance and t test.@*Results@#(1) After identification, both the expression level of lysosome-related membrane protein 1 and purity of lysosome in the sample were high, and the structure of lysosome was intact. (2) At 24 h after burn, the activity values of V-ATPase of myocardial lysosome in rats of pure burn group, ATP group, normal control group, and bafilomycin group were (2.03±0.67), (3.01±0.58), (4.29±0.26), and (1.83±0.52) μmol·mg-1·h-1, respectively. The activity value of V-ATPase of myocardial lysosome in rats of pure burn group was significantly lower than the values in ATP group and normal control group (with t values respectively 3.14 and 8.87, P values below 0.01). The activity values of V-ATPase of rats in normal control group were significantly higher than those in bafilomycin group (t=11.87, P<0.01). At 24 h after burn, the expressions of myocardial LC3 and P62 in pure burn group were significantly higher than those in ATP group and normal control group (with t values from 3.73 to 5.88, P values below 0.01). The expressions of myocardial LC3 and P62 in normal control group were significantly lower than those in bafilomycin group (with t values respectively 2.64 and 3.07, P<0.05 or P<0.01). At 24 h after burn, the content of 5 items of myocardial enzyme spectrum and cTnT in pure burn group was significantly higher than that in ATP group and normal control group (with t values from 3.24 to 16.72, P values below 0.01). The content of 5 items of myocardial enzyme spectrum and cTnT in normal control group was significantly lower than that in bafilomycin group (with t values from 2.39 to 10. 70, P values below 0.01).@*Conclusions@#The activity of V-ATPase of myocardial lysosome decreased in rats after severe burn, which can result in myocardial damage by inhibiting myocardial autophagy flux.

The relationship between cathepsin L and apoptosis cell in rats after cerebral ischemia reperfusion / 中国医师杂志

Objective To investigate the relationship between cathepsin L and apoptosis cell in rats after cerebral ischemia reperfusion.Methods Sixty healthy male Sprague-Dawley Rats (10-12 weeks old,260-300 g) were chosen.Based on the random number table method,the rats were randomly divided into sham-operated control group (Sham group,n =10),ischemia-reperfusion group (model group,n =25),and Z-FY-DMK intervention group (CLI group,n =25).Rats were randomly divided into 6 h,12 h,24 h,and 48 h four subgroups in model group and CLI group,respectively.Modified transient middle cerebral artery occlusion was made as Longa described,the intervention groups were injected intracerebroventricularly Z-FY-DMK (20 μg / 1μ1 ×5 μl) preoperative 30 min prior to surgery,Sham group and schemia reperfusion injury (IRI) group were injected intracerebroventricularly dimethyl sulfoxide (DMSO) 5 μ1 (10ml/L) at the same time.Cell apoptosis was detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) straining.Western blotting was used to detect the expression of cathepsin L and caspase-3.Results In the cortical area of ischemic brain,apoptosis cells of sham operation group were rare,while apoptosis of nerve cells of model group with 6 hours reperfusion were visible,and were gradually increased in the order of 12 hours,24 hours and 48 hours.At the same time point,the apoptosis cells of CL intervention group (6 h,12 h,24 h,48 h) were obviously less than model group (P ＜0.05).Western blotting found little visible cathepsin L protein expression in ischemic cerebral cortex preoptic in the sham group.For model group,the cathepsin L expression initially increased in sub groups with 6 hours reperfusion,reached to a peak in sub groups with 12 hours and 24 hours,and remained a high level in sub groups with 48 hours reperfusion.Compared to model group,the cathepsin L expressions of CL intervention group were obviously decreased at all time points (P ＜ O.05).Conclusions Cathepsin L may be involved in neuronal apoptosis by means of caspases 3 pathway.

Effect of cyclosporine A on autophagy-lysosomal pathway in tubular epithelial cells / 中华肾脏病杂志

Objective To investigate the effect of cyclosporine A (CsA) on autophagylysosomal pathway in tubular epithelial cells.Methods Human renal tubular epithelial cell line (HK-2 cell) was treated with different concentrations (3,5 and 10 μmol/L) of CsA for 24 h.Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV-PI staining followed by flow cytometry analysis,respectively.Autophagy-related protein LC3-Ⅱ and p62 were detected by immunofluorescence assay.Autophagic flux was analyzed in HK-2 cells transfected with a tandem mRFP-GFP fluorescent-tagged LC3 (ffLC3) plasmid by laser confocal microscope.The lysosomal degradation was evaluated by DQ-ovalbumin staining followed by flow cytometry analysis.Results The viability of HK-2 cells was significantly decreased with CsA stimulation when compared with control group (P ＜ 0.01),but the number of apoptotic cells was markedly increased by CsA treatment (P ＜ 0.05).Compared with the control group,different doses of CsA dramatically increased the expressions of LC3-Ⅱ (P ＜ 0.01) and p62 (P ＜ 0.05) in HK-2 cells.Moreover,HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes.In HK-2 cells,exposured to CsA caused a decrease in lysosomal degradation by DQ-ovalbumin staining when compared with control group (P ＜ 0.01).Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.

Virulence genes of Parkinson's disease and autophagy / 中华老年医学杂志

Parkinson's disease(PD)is a common neurodegenerative disease mainly affecting old persons.The pathogenesis of PD is still unclear, although it has been studied for many years.Recently, more and more evidences show that the dysfunction of autophagy plays a pivotal role in the pathogenesis of PD.Substantial progress in the genetic research of PD has showed that several pathogenic genes were identified correlated with autophagy, in particular, playing an important role in sporadic cases of PD.Here, we will discuss pathogenic genes-correlated dysfunctional autophagy-lysosomes system, so as to specify the pathogenesis of PD and provide a clue for its treatment.

Deacetylation of TFEB promotes fibrillar Aβ degradation by upregulating lysosomal biogenesis in microglia

Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.

Recent advances in genetic studies of stuttering

Speech and language are uniquely human-specific traits, which contributed to humans becoming the predominant species on earth. Disruptions in the human speech and language function may result in diverse disorders. These include stuttering, aphasia, articulation disorder, spasmodic dysphonia, verbal dyspraxia, dyslexia and specific language impairment. Among these disorders, stuttering is the most common speech disorder characterized by disruptions in the normal flow of speech. Twin, adoption, and family studies have suggested that genetic factors are involved in susceptibility to stuttering. For several decades, multiple genetic studies including linkage analysis were performed to connect causative gene to stuttering, and several genetic studies have revealed the association of specific gene mutation with stuttering. One notable genetic discovery came from the genetic studies in the consanguineous Pakistani families. These studies suggested that mutations in the lysosomal enzyme-targeting pathway genes (GNPTAB, GNPTG and NAPGA) are associated with non-syndromic persistent stuttering. Although these studies have revealed some clues in understanding the genetic causes of stuttering, only a small fraction of patients are affected by these genes. In this study, we summarize recent advances and future challenges in an effort to understand genetic causes underlying stuttering.

Estudo da degradação da proteína Tau hiperfosforilada por vias independentes do proteassoma, em modelo experimental de neurodegeneração / Study of hyperphosphorylated Tau protein degradation by proteasome-independent pathways, in an experimental model of neurodegeneration

O desenvolvimento das doenças neurodegenerativas, como a doença de Alzheimer, está associado à presença de agregados proteicos contendo Tau hiperfosforilada (p-Tau). Esta disfunção da Tau leva a prejuízos na homeostase celular. Um mecanismo chave para diminuir e/ou prevenir os danos promovidos pelos agregados contendo Tau seria o estímulo de sua degradação. Neste sentido, a proposta do presente estudo foi analisar a degradação da proteína Tau após aumento da expressão exógena da cochaperona Bag-2, a qual influencia o sistema proteassomal de degradação; bem como avaliar a ativação dos sistemas de degradação, a fim de correlacionar estes sistemas em cultura de células primárias e organotípica do hipocampo de ratos. Os resultados mostraram que a rotenona foi capaz de aumentar os níveis de p-Tau e que a superexpressão de Bag-2, foi eficiente em prevenir e degradar a p-Tau. O mecanismo envolvido neste processo envolve a coordenação dos sistemas proteassomal e lisossomal, já que a Rab7 e a Rab24 (envolvidas na via lisossomal) mostraram-se diminuídas na fase que antecede a agregação proteica, enquanto houve aumento da Rab24 na presença dos agregados proteicos. Com relação ao peptídeo beta amiloide, foi demonstrado tendência de aumento de p-Tau acompanhado de diminuição da atividade proteassomal e lisossomal. O tratamento com PADK (ativador lisossomal) foi capaz de reverter este efeito nestas diferentes condições. A análise da interrelação entre os sistemas mostrou que uma inibição do proteassoma favorece a via lisossomal e que o inverso não se repete. Os resultados sugerem que a modulação das vias de degradação pode ser interessante para o estudo, prevenção e tratamento das doenças neurodegenerativas associadas à agregação de proteínas...

Neurodegenerative diseases, such as Alzheimer's, are associated to protein inclusions containing hyperphosphorylated Tau (p-Tau). It is well established that Tau dysfunction impairs cell homeostasis. A key mechanism to prevent and/or reduce the damage promoted by aggregates of Tau might be its degradation. In view of this, the aims of the present study are to evaluate p- Tau clearance following exogenous expression of Bag-2, which stimulates proteasome; as well as to analyze the activation of both lysosome and proteasome pathways in order to understand the crosstalk between these two systems in primary and organotypic cultures of rat hippocampus. Results showed that rotenone was able of increasing p-Tau that was prevented and degraded by Bag-2 overexpression. Mechanisms involved in this process involve the coordination of cell degradation systems, depending upon aggregation status, since Rab7 and Rab24 (involved in lysosomal pathway) were decreased before protein aggregation, while Rab24 increased in the presence of protein inclusions. Amyloid-beta peptide also increased p-Tau accompanied by decreased proteasome and lysosome activity. PADK (lysosomal activator) treatment reverted the inhibition promoted by amyloidbeta peptide. Inhibition of proteasome leads to activation of lysosome, but lysosome inhibition does not affect proteasome. Overall, results suggest that targeting degradation pathways might be useful to understand, prevent and treat neurodegenerative diseases associated with protein deposits...