Nanomaterials-mediated lysosomal regulation:a robust protein-clearance approach for the treatment of Alzheimer’s disease

Alzheimer’s disease is a debilitating,progressive neurodegenerative disorder characterized by the progressive accumulation of abnormal proteins,including amyloid plaques and intracellular tau tangles,primarily within the brain.Lysosomes,crucial intracellular organelles responsible for protein degradation,play a key role in maintaining cellular homeostasis.Some studies have suggested a link between the dysregulation of the lysosomal system and pathogenesis of neurodegenerative diseases,including Alzheimer’s disease.Restoring the normal physiological function of lysosomes hold the potential to reduce the pathological burden and improve the symptoms of Alzheimer’s disease.Currently,the efficacy of drugs in treating Alzheimer’s disease is limited,with major challenges in drug delivery efficiency and targeting.Recently,nanomaterials have gained widespread use in Alzheimer’s disease drug research owing to their favorable physical and chemical properties.This review aims to provide a comprehensive overview of recent advances in using nanomaterials(polymeric nanomaterials,nanoemulsions,and carbon-based nanomaterials)to enhance lysosomal function in treating Alzheimer’s disease.This review also explores new concepts and potential therapeutic strategies for Alzheimer’s disease through the integration of nanomaterials and modulation of lysosomal function.In conclusion,this review emphasizes the potential of nanomaterials in modulating lysosomal function to improve the pathological features of Alzheimer’s disease.The application of nanotechnology to the development of Alzheimer’s disease drugs brings new ideas and approaches for future treatment of this disease.

Enhanced autophagic clearance of amyloid-βvia histone deacetylase 6-mediated V-ATPase assembly and lysosomal acidification protects against Alzheimer's disease in vitro and in vivo

Recent studies have suggested that abnormal acidification of lysosomes induces autophagic accumulation of amyloid-βin neurons,which is a key step in senile plaque formation.Therefore,resto ring normal lysosomal function and rebalancing lysosomal acidification in neurons in the brain may be a new treatment strategy for Alzheimer's disease.Microtubule acetylation/deacetylation plays a central role in lysosomal acidification.Here,we show that inhibiting the classic microtubule deacetylase histone deacetylase 6 with an histone deacetylase 6 shRNA or thehistone deacetylase 6 inhibitor valproic acid promoted lysosomal reacidification by modulating V-ATPase assembly in Alzheimer's disease.Fu rthermore,we found that treatment with valproic acid markedly enhanced autophagy.promoted clearance of amyloid-βaggregates,and ameliorated cognitive deficits in a mouse model of Alzheimer's disease.Our findings demonstrate a previously unknown neuroprotective mechanism in Alzheimer's disease,in which histone deacetylase 6 inhibition by valproic acid increases V-ATPase assembly and lysosomal acidification.

Roles of neuronal lysosomes in the etiology of Parkinson’s disease

Therapeutic progress in neurodegenerative conditions such as Parkinson’s disease has been hampered by a lack of detailed knowledge of its molecular etiology.The advancements in genetics and genomics have provided fundamental insights into specific protein players and the cellular processes involved in the onset of disease.In this respect,the autophagy-lysosome system has emerged in recent years as a strong point of convergence for genetics,genomics,and pathologic indications,spanning both familial and idiopathic Parkinson’s disease.Most,if not all,genes linked to familial disease are involved,in a regulatory capacity,in lysosome function(e.g.,LRRK2,alpha-synuclein,VPS35,Parkin,and PINK1).Moreover,the majority of genomic loci associated with increased risk of idiopathic Parkinson’s cluster in lysosome biology and regulation(GBA as the prime example).Lastly,neuropathologic evidence showed alterations in lysosome markers in autoptic material that,coupled to the alpha-synuclein proteinopathy that defines the disease,strongly indicate an alteration in functionality.In this Brief Review article,I present a personal perspective on the molecular and cellular involvement of lysosome biology in Parkinson’s pathogenesis,aiming at a larger vision on the events underlying the onset of the disease.The attempts at targeting autophagy for therapeutic purposes in Parkinson’s have been mostly aimed at“indiscriminately”enhancing its activity to promote the degradation and elimination of aggregate protein accumulations,such as alpha-synuclein Lewy bodies.However,this approach is based on the assumption that protein pathology is the root cause of disease,while pre-pathology and pre-degeneration dysfunctions have been largely observed in clinical and pre-clinical settings.In addition,it has been reported that unspecific boosting of autophagy can be detrimental.Thus,it is important to understand the mechanisms of specific autophagy forms and,even more,the adjustment of specific lysosome functionalities.Indeed,lysosomes exert fine signaling capacities in addition to their catabolic roles and might participate in the regulation of neuronal and glial cell functions.Here,I discuss hypotheses on these possible mechanisms,their links with etiologic and risk factors for Parkinson’s disease,and how they could be targeted for disease-modifying purposes.

Role of lysosomal trafficking regulator in autophagic lysosome reformation in neurons:a disease perspective

Lysosomes are discrete organelles that act as recycling centers for extracellular and intracellular materials,playing a pivotal role in maintaining cellular homeostasis.Their acidic environment,maintained by numerous hydrolytic enzymes,facilitates substrate degradation.Dysfunction in lysosomal processes can lead to abnormal substrate degradation,significantly impacting cellular homeostasis.High energy-demanding cells,such as post-mitotic neurons,are especially vulnerable to these changes,often resulting in neurological diseases.Autophagy,a conserved catabolic process,requires extensive lysosomal utilization.It plays a key role in removing unnecessary intracellular components,ensuring cellular homeostasis,and promoting cell survival during stress conditions such as starvation,infection,or cellular damage.

SIRT1 facilitates amyloid beta peptide degradation by upregulating lysosome number in primary astrocytes

Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer＇s disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.

Association of Lysosome Associated Protein Transmembrane 4 Beta Gene Polymorphism with the Risk of Pancreatic Cancer

Objective： Lysosome associated protein transmembrane 4 beta （LAPTM4B） was originally identified as a gene in human hepatocellular carcinoma （HCC）. It was successfully cloned by fluorescence differential display, rapid amplification of cDNA ends （RACE） and reverse transcription polymerase chain reaction （RT-PCR）. Previous study showed that the novel gene played an important role in the occurrence, development, migration and prognosis of tumors. Pancreatic cancer is an aggressive malignancy with the majority of patients dying within one year after diagnosis. This study tries to find out the relationship between lysosome associated protein transmembrane 4 beta gene polymorphism and the susceptibility of pancreatic cancer. Methods： A case-control study was conducted in China, including 58 pancreatic cancer cases and 156 healthy controls. Human genomic DNA was used as the template, polymerase chain reaction （PCR） was used to detect the distribution of LAPTM4B genotype. Analyses Odds ratio （OR） and corresponding 95% confidence interval （95%CI） with logistic regression were performed. Results： Two alleles of LAPTM4B generated three kinds of genotypes in population, ＊1/1, ＊1/2, and ＊2/2. The genotype frequency of ＊1/1, ＊1/2 and ＊2/2 in the pancreatic cancer group were 41.4%, 44.8% and 13.8% respectively, which were not significantly different from those of healthy group （47.4%, 42.9%, 9.6%） （P=0.773, P=0.291）. Also the ＊2 allele frequency of LAPTM4B among pancreatic cancer had no significantly difference with the controls （P=0.354）. When compared to the ＊1 allele, the people with ＊2 allele had no increased risk of pancreatic cancer. Conclusion： The gene polymorphism of LAPTM4B may not influence the susceptibility of pancreatic cancer.

S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3

The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosisinducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I （PCD I, apoptosis） and PCD II （autophagy）-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atgl2-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class Ⅲ inhibitor, 3-methyladenine （3-MA）, and by the vacuole H＋-ATPase inhibitor, bafilomycin-A1 （Baf-A1）. S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, ATM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species （ROS） generation, and partially pro- tected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated ceils. In addition, either ATM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.

KCNJ15 deficiency promotes drug resistance via affecting the function of lysosomes

The altered lysosomal function can induce drug redistribution which leads to drug resistance and poor prognosis for cancer patients.V-ATPase,an ATP-driven proton pump positioned at lysosomal surfaces,is responsible for maintaining the stability of lysosome.Herein,we reported that the potassium voltage-gated channel subfamily J member 15(KCNJ15)protein,which may bind to V-ATPase,can regulate the function of lysosome.The deficiency of KCNJ15 protein in breast cancer cells led to drug aggregation as well as reduction of drug efficacy.The application of the V-ATPase inhibitor could inhibit the binding between KCNJ15 and V-ATPase,contributing to the amelioration of drug resistance.Clinical data analysis revealed that KCNJ15 deficiency was associated with higher histological grading,advanced stages,more metastases of lymph nodes,and shorter disease free survival of patients with breast cancer.KCNJ15 expression level is positively correlated with a high response rate after receiving neoadjuvant chemotherapy.Moreover,we revealed that the small molecule drug CMA/BAF can reverse drug resistance by disrupting the interaction between KCNJ15 and lysosomes.In conclusion,KCNJ15 could be identified as an underlying indicator for drug resistance and survival of breast cancer,which might guide the choice of therapeutic strategies.

Enhancement of lysosome biogenesis as a potential therapeutic approach for neurodegenerative diseases

Millions of people are suffering from Alzheimer’s disease globally,but there is still no effective treatment for this neurodegenerative disease.Thus,novel therapeutic approaches for Alzheimer’s disease are needed,which requires further evaluation of the regulato ry mechanisms of protein aggregate degradation.Lysosomes are crucial degradative organelles that maintain cellular homeostasis.Transcription factor EB-mediated lysosome biogenesis enhances autolysosomedependent degradation,which subsequently alleviates neurodege nerative diseases,including Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease.In this review,we start by describing the key features of lysosomes,including their roles in nutrient sensing and degradation,and their functional impairments in different neurodegenerative diseases.We also explain the mechanisms—especially the post-translational modifications—which impact transcription factor EB and regulate lysosome biogenesis.Next,we discuss strategies for promoting the degradation of toxic protein aggregates.We describe Proteolysis-Ta rgeting Chimera and related technologies for the targeted degradation of specific proteins.We also introduce a group of LYsosome-Enhancing Compounds,which promote transcription factor EB-mediated lysosome biogenesis and improve learning,memory,and cognitive function in APP-PSEN1 mice.In summary,this review highlights the key aspects of lysosome biology,the mechanisms of transcription factor EB activation and lysosome biogenesis,and the promising strategies which are emerging to alleviate the pathogenesis of neurodegenerative diseases.

Enhanced lysosome escape mediated by 1,2-dicarboxylic-cyclohexene anhydride-modified poly-L-lysine dendrimer as a gene delivery system

Antisense oligodeoxynucleotide(ASODN)can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing,in turn,plays antitumor therapeutic roles.In the study,a novel HIF-1αASODN-loaded nanocomposite was formulated to efficiently deliver gene to the target RNA.The physicochemical properties of nanocomposite were characterized using TEM,FTIR,DLS and zeta potentials.The mean diameter of resulting GEL-DGL-FA-ASODN-DCA nanocomposite was about 170–192 nm,and according to the agarose gel retardation assay,the loading amount of ASODN accounted for 166.7 mg/g.The results of cellular uptake showed that the nanocomposite could specifically target to HepG2 and Hela cells.The cytotoxicity assay demonstrated that the toxicity of vectors was greatly reduced by using DCA to reversibly block the cationic DGL.The subcellular distribution images clearly displayed the lysosomal escape ability of the DCA-modified nanocomposite.In vitro exploration of molecular mechanism indicated that the nanocomposite could inhibit m RNA expression and HIF-1αprotein translation at different levels.In vivo optical images and quantitative assay testified that the formulation accumulated preferentially in the tumor tissue.In vivo antitumor efficacy research confirmed that this nanocomposite had significant antitumor activity and the tumor inhibitory rate was 77.99%.These results manifested that the GEL-DGL-FA-ASODNDCA nanocomposite was promising in gene therapeutics for antitumor by interacting directly with target RNA.

Combination of kaempferol and chloroquine induces glioma cell death via expansion and subsequent rupture of lysosomes

OBJECTIVE Chloroquine is considered as a potential chemotherapy and radiotherapy sensitizer,but the anticancer effect of chloroquine alone is limited.Since we found that the flavonoid kaempferol effectively sensitizes glioma cells to chloroquine-mediated cell death,we investigated the underlying mechanisms of glioma cell death induced by the combination of kaempferol and chloroquine.METHODS To examine the effect of kaempferol and/or chloroquine on various glioma cells,cell viability assay using calcein-AM and EthD-1was performed.The changes in the lysosomal structures following treatment with kaempferol and/or chloroquine were observed by electron microscopy and fluorescence microscopy using acridine orange or Lyso-tracker Red.The changes in cathepsin D proteins were analyzed by Western blotting,immunocytochemistry,and fluorescence microscopy using BODIPY FL-pepstatin.RESULTS Treatment with subtoxic doses of chloroquine,when combined with kaempferol,effectively induced cell death in various glioma cells,but not in normal astrocytes.While kaempferol treatment increased the numbers of lysosome,chloroquine treatment increased lysosomal masses.Combined treatment with kaempferol and chloroquine induced the expansion and subsequent rupture of lysosomes,leading to the spillage of the lysosomal contents into the cytosol.We found that while kaemfperol treatment increased the active mature forms of cathepsin D,chloroquine treatment completely blocked the processing of cathepsin D.The processing of cathepsin D was also blocked by the combined treatment,but the activity of cathepsin D,which was released from the lysosomes,was restored.The cell death induced by kaempferol and chloroquine in U251 MG cells was accompanied by mitochondrial dysfunction,ER stress,and DNA damage.CONCLUSION Disruption of lysosomal membrane integrity and a resultant release of lysosomal proteases may critically contribute to the irreparable damage of various organelles and glioma cell death by chloroquine plus kaempferol.

Anti-sense RNA Inhibits the Expression of Synaptotagmin Ⅱ in RBL-2H3 and Enhances the Exocytosis of Lysosomes in RBL-2H3

Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.

Detection of distribution of copper inside and outside of lysosomes in cultured hepatolenticular degeneration fibroblasts by electron probe X-ray microanalysis

OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were clutured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu^(2+) inside lysosomes of HLD cells (14.6±2.1 mmol/kg) and of heterozygote cells (11.6±0.6 mmol/kg) was higher than that of normal cells (4.5±1.2 mmol/kg) (P<0.01). The content of Cu^(2+) outside lysosomes of HLD cells (17.5±4.2 mmol/kg) and of heterozygote cells (12.0±0.9 mmol/kg) was higher than that of normal cells (4.7±1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.

Concluding Step in Cell Restitution Cycle: ER Transport Vesicles with Sphingolipids in the Outer Leaflet of the Membrane Restore Lysosomes

Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer leaflet (OL) of endoplasmic reticulum (ER), is engaged in the synthesis of ER transport vesicles that recondition cell organelles, and the inner leaflet (IL) SPT in the restitution of the cell membrane. The OL SPT impacts assembly of sphingomyelinase (SMase)—susceptible ER vesicles but not the SMase-resistant and sphingolipid (SPhL) core—carrying vesicles that refurbish the cell membrane. The investigation of the SPT-initiated differences in the placement of SPhL in vesicular membranes by utilizing ER depleted of OL SPT, allows us to conclude that the restitution of endosomal and lysosomal membranes is achieved with the involvement of OL SPT, whereas the IL SPT is involved in formation of the lipid core for glycosphingolipids (GSL) and sphingomyelin (SM) of the apical and basolateral cell membrane. These findings along with our previously published report (Slomiany and Slomiany, Advances in Biological Chemistry, 2013, 3, 275-287), provide a clear distinction between the processes that renovate cell membrane and its organelles from that of the endocytotic cell debridement, and show that vesicles are navigated to the specific organelles and the cell membrane by the biomembrane constituents programmed in ER.

Mechanisms of lysosomal proteases participating in cerebral ischemia-induced neuronal death

There are three different types of cell death, including apoptosis （Type I）, autophagic cell death （Type II）, and necrosis （Type III）. Ischemic neuronal death influences stroke development and progression. Lysosomes are important organelles having an acidic milieu to maintain cellular metabolism by degrading unneeded extra- and intracellular substances. Lysosomal enzymes, including cathepsins and some lipid hydrolases, when secreted following rupture of the lysosomal membrane, can be very harmful to their environment, which results in pathological destruction of cellular structures. Since lysosomes contain catalytic enzymes for degrading proteins, carbohydrates and lipids, it seems natural that they should participate in cellular death and dismantling. In this review, we discuss the recent developments in ischemic neuronal death, and present the possible molecular mechanisms that the lysosomal enzymes participate in the three different types of cell death in ischemic brain damage. Moreover, the research related to the selective cathepsin inhibitors may provide a novel therapeutic target for treating stroke and promoting recovery.

线粒体tRNA-Lys(T7719G)基因变异影响绵羊颗粒细胞凋亡生理机制研究

本团队前期研究发现小尾寒羊线粒体tRNA-Lys基因(T7719G)变异与产羔数显著相关,并降低了颗粒细胞凋亡率,为此,开展了该变异影响颗粒细胞凋亡生理机制的研究。本研究选取G、T基因型绵羊各3只,屠宰后取卵巢组织培养颗粒细胞,颗粒细胞生理指标检测试验分为两组,每组3个重复,分别检测线粒体基因组拷贝数,线粒体呼吸酶复合物Ⅰ、复合物Ⅱ、复合物Ⅲ、复合物Ⅳ、复合物Ⅴ活性,线粒体耗氧量和胞外产酸能力、ROS产量、ATP水平和膜电位,以及tRNA-Lys总量和氨酰化水平,13个mtDNA编码多肽蛋白表达水平。结果表明,G基因型颗粒细胞线粒体拷贝数与T基因型差异极显著(P<0.01),为T基因型的1.78倍。G基因型细胞线粒体复合物Ⅲ活性是T型细胞的1.25倍(P<0.05),表明G基因型细胞氧化呼吸功能增强。G基因型颗粒细胞线粒体耗氧量、ATP偶联耗氧量比T基因型细胞分别提高了6.84%(P<0.05)和15.46%(P<0.01),G基因型细胞糖酵解能力显著降低(P<0.01),活性氧含量水平显著降低了34%,ATP含量明显上升,为T基因型的1.25倍(P<0.05),线粒体膜电位显著上升,为T基因型的1.24倍(P<0.01);G基因型细胞tRNA-Lys总量显著提高50%(P<0.01),氨酰化tRNA-Lys比例较T型细胞提高33.4%(P<0.01)。线粒体基因组编码的13个多肽(ND1、ND2、ND3、ND4、ND4L、ND5、ND6、CtyB、COⅠ、COⅡ、COⅢ、ATP6、ATP8)表达水平均显著提高(P<0.05)。初步解析了线粒体tRNA-Lys(T7719G)变异影响绵羊颗粒细胞凋亡的生理机制,即tRNA-Lys(T7719G)变异影响tRNA-Lys稳态和氨酰化水平,影响mtDNA编码的多肽翻译效率,同时引起拷贝数、ATP水平、ROS含量和部分酶活性变化,进而改变线粒体能量代谢功能和细胞凋亡。

The emerging role of autophagic-lysosomal dysfunction in Gaucher disease and Parkinson's disease

Gaucher disease（GD）,the commonest lysosomal storage disorder,results from the lack or functional deficiency of glucocerebrosidase（GCase） secondary to mutations in the GBA1 gene.There is an established association between GBA1 mutations and Parkinson＇s disease（PD）,and indeed GBA1 mutations are now considered to be the greatest genetic risk factor for PD.Impaired lysosomal-autophagic degradation of cellular proteins,including α-synuclein（α-syn）,is implicated in the pathogenesis of PD,and there is increasing evidence for this also in GD and GBA1-PD.Indeed we have recently shown in a Drosophila model lacking neuronal GCase,that there are clear lysosomal-autophagic defects in association with synaptic loss and neurodegeneration.In addition,we demonstrated alterations in mechanistic target of rapamycin complex 1（mTORC1） signaling and functional rescue of the lifespan,locomotor defects and hypersensitivity to oxidative stress on treatment of GCase-deficient flies with the mT OR inhibitor rapamycin.Moreover,a number of other recent studies have shown autophagy-lysosomal system（ALS） dysfunction,with specific defects in both chaperone-mediated autophagy（CMA）,as well as macroautophagy,in GD and GBA1-PD model systems.Lastly we discuss the possible therapeutic benefits of inhibiting mT OR using drugs such as rapamycin to reverse the autophagy defects in GD and PD.

Efficacy of boswellic acid on lysosomal acid hydrolases,lipid peroxidation and anti-oxidant status in gouty arthritic mice

Objective:To evaluate the efficacy of boswellic acid against monosodium urate crystal-induced inflammation in mice.Methods:The mice were divided into four experimental groups.GroupⅠserved as control;mice in groupⅡwere injected with monosodium urate crystal;groupⅢconsisted of monosodium urate crystal-induced mice who were treated with boswellic acid(30mg/kg/b.w.);groupⅣcomprised monosodium urate crystal-induced mice who were treated with indomethacin(3mg/kg/b.w.).Paw volume and levels/activities of lysosomal enzymes,lipid peroxidation,anti-oxidant status and inflammatory mediator TNF-αwere determined in control and monosodium urate crystal-induced mice.In addition,the levels ofβ-glucuronidase and lactate dehydrogenase were also measured in monosodium urate crystal-incubated polymorphonuclear leucocytes(PMNL)in vitro.Results:The activities of lysosomal enzymes,lipid peroxidation,and tumour necrosis factor-αlevels and paw volume were increased significantly in monosodium urate crystal-induced mice,whereas the activities of antioxidant status were in turn decreased.However,these changes were modulated to near normal levels upon boswellic acid administration.In vitro,boswellic acid reduced the level ofβ-glucuronidase and lactate dehydrogenase in monosodium urate crystal-incubated PMNL in concentration dependent manner when compared with control cells.Conclusions:The results obtained in this study further strengthen the anti-inflammatory/antiarthritic effect of boswellic acid,which was already well established by several investigators.

VL30 retrotransposition signals activation of a caspaseindependent and p53-dependent death pathway associated with mitochondrial and lysosomal damage

The impact of long terminal repeat （LTR） retrotransposition on cell fate is unknown. Here, we investigated the effect of VL30 retrotransposition on cell death in SV40-transformed mouse SVTT1 cells. Transfection of a VL30 retrotransposon decreased the clonogenicity of SVTT1 by 17-fold, as compared to parental NIH3T3 cells. Correlated levels of retrotransposition frequency and cell death rates were found in retrotransposition-positive SVTT1 cloned cells, exhibiting DNA fragmentation, nuclear condensation, multinucleation and cytoplasmic vacuolization. Analysis of activation of effector caspases revealed a caspase-independent cell death mechanism. However, cell death was associated with p53 induction and concomitant upregulation of PUMAa and Bax and downregulation of Bcl-2 and Hsp70 protein expression. Moreover, we found partial loss of colocalization of large T-antigen （LT）/p53 and p53 translocation to mitochondria, leading to mitochondrial outer membrane permeabilization （MOMP） accompanied by lysosomal membrane permeabilization （LMP）. Interestingly, treatment with the antioxidant N-acetylcysteiue abolished cell death, suggesting the involvement of mitochondrial-derived reactive oxygen species, and resulted in an increase of retrotransposition frequency. Importantly, the induction of cell death was VL30 retrotransposon-specilic as VL30 mobilization was induced; in contrast, mobilization of the non-LTR L1 （LINE-1, long interspersed nuclear element-1）, B2 and LTR MusD retrotransposons decreased. Our results provide, for the first time, strong evidence that VL30 retrotransposition mediates cell death via mitochondrial and lysosomal damage, uncovering the role of retrotransposition as a nuclear signal activating a mitochondrial-lysosomal crosstalk in triggering cell death.

Salvianolic acid B protects hepatocytes from H2O2 injury by stabilizing the lysosomal membrane

AIM To investigate the capability of salvianolic acid B(Sal B) to protect hepatocytes from hydrogen peroxide(H_2O_2)/carbon tetrachloride(CCl_4)-induced lysosomal membrane permeabilization. METHODS Cell Counting Kit-8 assay was used to measure cell viability. Apoptosis and death were assayed through flow cytometry. Brd U incorporation was used to detect cell proliferation. Serum alanine aminotransferase activity and liver malondialdehyde(MDA) content were measured. Liver histopathological changes were evaluated using hematoxylin-eosin staining. Lysosomal membrane permeability was detected with Lyso Tracker Green-labeled probes and acridine orange staining. The levels of protein carbonyl content(PCC), cathepsins(Cat)B/D, and lysosome-associated membrane protein 1(LAMP1) were evaluated through western blotting. Cytosol Cat B activity analysis was performed with chemiluminescence detection. The m RNA level ofLAMP1 was evaluated through quantitative real-time polymerase chain reaction. RESULTS Results indicated that H_2O_2 induced cell injury/death. Sal B attenuated H_2O_2-induced cell apoptosis and death, restored the inhibition of proliferation, decreased the amount of PCC, and stabilized the lysosome membrane by increasing the LAMP1 protein level and antagonizing Cat B/D leakage into the cytosol. CCl_4 also triggered hepatocyte death. Furthermore, Sal B effectively rescued hepatocytes by increasing LAMP1 expression and by reducing lysosomal enzyme translocation to the cytosol.CONCLUSION Sal B protected mouse embryonic hepatocytes from H_2O_2/CCl_4-induced injury/death by stabilizing the lysosomal membrane.