Activity Of Lysosomal Hydrolases Research Articles

RIT2 regulates autophagy lysosomal pathway induction and protects against α-synuclein pathology in a cellular model of Parkinson's disease

Substantial work has been devoted to better understand the contribution of the myriad of genes that may underly the development of Parkinson's disease (PD) and their role in disease etiology. The small GTPase Ras-like without CAAX2 (RIT2) is one such genetic risk factor, with one single nucleotide polymorphism in the RIT2 locus, rs12456492, having been associated with PD risk in multiple populations. While RIT2 has previously been shown to influence signaling pathways, dopamine transporter trafficking, and LRRK2 activity, its cellular function remains unclear. In the current study, we have situated RIT2 to be upstream of various diverse processes associated with PD. In cellular models, we have shown that RIT2 is necessary for activity-dependent changes in the expression of genes related to the autophagy-lysosomal pathway (ALP) by regulating the nuclear translocation of MiT/TFE3-family transcription factors. RIT2 is also associated with lysosomes and can regulate autophagic flux and clearance by regulating lysosomal hydrolase expression and activity. Interestingly, upregulation of RIT2 can augment ALP flux and protect against α-synuclein aggregation in cortical neurons. Taken together, the present study suggests that RIT2 can regulates gene expression upstream of ALP function and that enhancing RIT2 activity may provide therapeutic benefit in PD.

Optogenetic manipulation of lysosomal physiology and autophagy-dependent clearance of amyloid beta.

Lysosomes are degradation centers of cells and intracellular hubs of signal transduction, nutrient sensing, and autophagy regulation. Dysfunction of lysosomes contributes to a variety of diseases, such as lysosomal storage diseases (LSDs) and neurodegeneration, but the mechanisms are not well understood. Altering lysosomal activity and examining its impact on the occurrence and development of disease is an important strategy for studying lysosome-related diseases. However, methods to dynamically regulate lysosomal function in living cells or animals are still lacking. Here, we constructed lysosome-localized optogenetic actuators, named lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, to achieve optogenetic manipulation of lysosomes. These new actuators enable light-dependent control of lysosomal membrane potential, pH, hydrolase activity, degradation, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy through the mTOR pathway, promotes Aβ clearance in an autophagy-dependent manner in cellular models, and alleviates Aβ-induced paralysis in the Caenorhabditis elegans model of Alzheimer's disease. Our lysosomal optogenetic actuators supplement the optogenetic toolbox and provide a method to dynamically regulate lysosomal physiology and function in living cells and animals.

Late Effects of Ionizing Radiation on the Ultrastructure of Hepatocytes and Activity of Lysosomal Enzymes in Mouse Liver Irradiated In Vivo.

The study aimed to investigate late radiation-induced changes in the histology, ultrastructure, and activity of lysosomal enzymes in mouse liver exposed to ionizing radiation. The experiment was conducted on C57BL/6J male mice whose distal part of the liver was exposed occasionally to single doses of radiation (6 MV photons) during targeted heart irradiation; estimated doses delivered to analyzed tissue were 0.025 Gy, 0.25 Gy, 1 Gy, and 2 Gy. Tissues were collected 40 weeks after irradiation. We have observed that late effects of radiation have an adaptive nature and their intensity was dose-dependent. Morphological changes in hepatocytes included an increased number of primary lysosomes and autophagic vacuoles, which were visible in tissues irradiated with 0.25 Gy and higher doses. On the other hand, a significant increase in the activity of lysosomal hydrolases was observed only in tissues exposed to 2 Gy. The etiology of these changes may be multifactorial and result, among others, from unintentional irradiation of the distal part of the liver and/or functional interaction of the liver with an irradiated heart. In conclusion, we confirmed the presence of late dose-dependent ultrastructural and biochemical changes in mouse hepatocytes after liver irradiation in vivo.

Ginsenosides Rg1 and Rg2 Activate Autophagy and Attenuate Oxidative Stress in Neuroblastoma Cells Overexpressing Aβ(1-42).

Alzheimer's disease is a neurodegeneration with protein deposits, altered proteolysis, and inflammatory and oxidative processes as major hallmarks. Despite the continuous search for potential therapeutic treatments, no cure is available to date. The use of natural molecules as adjuvants in the treatment of Alzheimer's disease is a very promising strategy. In this regard, ginsenosides from ginseng root show a variety of biological effects. Here, we dissected the role of ginsenosides Rg1 and Rg2 in modulating autophagy and oxidative stress in neuroblastoma cells overexpressing Aβ(1-42). Key hallmarks of these cellular processes were detected through immunomethods and fluorometric assays. Our findings indicate that ginsenosides are able to upregulate autophagy in neuronal cells as demonstrated by increased levels of LC3II and Beclin-1 proteins and decreased amounts of p62. Simultaneously, an activation of lysosomal hydrolases was observed. Furthermore, autophagy activation promoted the clearance of Aβ(1-42). Rg1 and Rg2 also reduced oxidative stress sources and macromolecule oxidation, promoting NRF2 nuclear translocation and the expression of antioxidant enzymes. Our data further clarify the mechanisms of action of Rg1 and Rg2, indicating new insights into their role in the management of disorders like Alzheimer's disease.

The Effect of p.G2019S Mutation in the LRRK2 Gene on the Activity of Lysosomal Hydrolases and the Clinical Features of Parkinson's Disease Associated with p.N370S Mutation in the GBA1 Gene.

Mutations in the glucocerebrosidase (GBA1) and leucine-rich repeat kinase 2 (LRRK2) genes, encoding lysosomal enzyme glucocerebrosidase (GCase) and leucine-rich repeat kinase 2 (LRRK2), respectively, are the most common related to Parkinson's disease (PD). Recent data suggest a possible functional interaction between GCase and LRRK2 and their involvement in sphingolipid metabolism. The aim of the present study was to describe the clinical course and evaluate the lysosomal enzyme activities and sphingolipid concentrations in blood of patients with PD associated with dual mutations p.N370S GBA1 and p.G2019S LRRK2 (p.N370S/GBA-p.G2019S/LRRK2-PD) as well as in blood of asymptomatic mutation carriers (p.N370S/GBA1-p.G2019S/LRRK2-carrier). One patient with p.N370S/GBA1-p.G2019S/LRRK2-PD and one p.N370S/GBA1-p.G2019S/LRRK2-carrier were enrolled. GBA1-associated PD (GBA1-PD), LRRK2-associated PD (LRRK2-PD), sporadic PD (sPD) patients were described earlier by our research group. A neuropsychiatric examination of the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was carried out using scales (Montreal Cognitive Assessment scale (MoCA), Mini-mental State Examination scale (MMSE), Frontal Assessment Batter scale (FAB), Hospital Anxiety, and Depression Scale (HADS), etc). Lysosomal enzyme activity (GCase, alpha-galactosidase [GLA], acid sphingomyelinase [ASMase], galactosylcerebrosidase [GALC]) and sphingolipid concentrations (hexasylsphingosine [HexSph], lysoglobotriaosylsphingosine [LysoGb3], lysosphingomyelin [LysoSM]) were assessed with high-performance liquid chromatography-tandem mass spectrometry in blood. The following comparison with the previously described groups of GBA1-PD and sPD patients were conducted. Clinical features of p.N370S/GBA1-p.G2019S/LRRK2-PD included an early age of onset of the disease (46 years) and mild cognitive and affective disorders (MMSE = 29, MoCA = 23), despite a long (24 years) course of the disease. Interestingly, no differences were found in hydrolase activity and lysosphingolipid concentrations between the p.N370S/GBA1-p.G2019S/LRRK2-PD patient and GBA1-PD patients. However, GCase activity was lower in these groups than in LRRK2-PD, sPD, and controls. Additionally, the p.N370S/GBA1-p.G2019S/LRRK2-PD patient was characterized by a pronounced decreased in ASMase activity and increased LysoSM concentration compared to the p.N370S/GBA1-p.G2019S/LRRK2-carrier (p = 0.023, p = 0.027, respectively). Based on one patient, our results indicate a protective effect of the p.G2019S mutation in the LRRK2 gene on clinical course of p.N370S/GBA1-PD. The identified pronounced alteration of ASMase activity and LysoSM concentration in p.N370S/GBA1-p.G2019S/LRRK2-PD provide the basis for the further research.

Commonality of Virulence-Promoting Function in Rhodococcus equi Virulence Associated Proteins (Vaps)

Rhodococcus equi is a Gram-positive facultative intracellular pathogen associated with life-threatening bronchopneumonial disease in foals. Key to R. equi’s intracellular survival in host macrophages is the production of virulence associated proteins (Vaps). Numerous vap genes are found on virulence plasmids isolated from different species, and the Vaps share a high degree of sequence identity. VapA has been extensively studied, and although vapK and vapN genes from other R. equi virulence plasmids have been shown to be essential for R. equi intracellular survival, their mode of action is less characterised. We, therefore, examined whether VapK and VapN worked mechanistically in the same way as VapA. Indeed, like VapA, VapK and VapN neutralised lysosomal pH and reduced lysosomal hydrolase activity. A loss of VapA and R. equi virulence could be regained by the presence of either VapK or VapN. The acid-neutralisation activity was also observed to a lesser extent with VapB. There was a differential activity across these virulence-promoting Vaps with the most “acid-neutralising” activity found with VapN, then VapA and K, and finally VapB. These data suggest that VapA production, which is often found in equine infections, can be substituted by VapK and B (produced by plasmids often found in porcine species) or VapN (produced by plasmids often isolated in bovine and human samples). These data imply that the molecular mechanism(s) that VapA uses to neutralise lysosomal acidity should also be seen in VapN and K which will help guide researchers in identifying their precise mode of action and aid the future development of targeted therapeutics.

The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity

ABSTRACT The mammalian endometrium is covered by the lumenal epithelium (Le), which directly interacts with the blastocyst and plays an important role in the establishment of reciprocal crosstalk between the embryo and receptive uterus during implantation. However, the effect of the blastocyst on uterine differentiation during the window of receptivity is far from well understood. Through transcriptomic profiling of the uterine Le isolated by laser capture microdissection (LCM), it was demonstrated that global gene expression changes occurred in Le between pseudopregnant mice without embryos and pregnant mice with embryos. Some differentially expressed genes, including upregulated Areg (amphiregulin), Ihh (Indian hedgehog), Lifr (leukemia inhibitory factor receptor) and downregulated Msx1 (msh homeobox 1), Pgr (progesterone receptor), and Gata2 (GATA binding protein 2) in pregnant mice, have been reported to regulate the establishment of uterine receptivity. Besides, we found that blastocysts induced an increase in both the number and acidification of lysosome, consistent with enhanced lysosomal hydrolase activity in uterine Le. Further exploration uncovered that blastocyst-derived IGF2 was involved into the activation of epithelial STAT3 to induce lysosomal hydrolase expression, and inhibition of lysosomal function derails both uterine receptive maker gene expressions and embryo implantation. Finally, based on the proteomic data of both epithelia and the separated lysosome, it was revealed that CLDN1 (claudin 1) and MUC1 (mucin 1, transmembrane), two well-known downregulated molecules for successful implantation, are degraded by epithelial lysosome. In brief, our data demonstrated that blastocysts induced normal epithelium differentiation with lysosome activation to promote the uterine epithelial differentiation for embryo implantation. Abbreviations ACTB: actin beta; AREG: amphiregulin; ATP6V0A4: ATPase, H+ transporting, lysosomal V0 subunit A4; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CLDN1: claudin 1; CTSB: cathepsin B; DEGs: differentially expressed genes; E2: 17β-estradiol; ESR: estrogen receptor; GATA2: GATA binding protein 2; GLA: galactosidase, alpha; GO: gene ontology; HBEGF: heparin-binding EGF-like growth factor; IGF1R: insulin-like growth factor 1 receptor; Ihh: Indian hedgehog; ISH: in situ hybridization; LAMP1: lysosomal-associated membrane protein 1; LCM: laser capture microdissection; Le: lumenal epithelium; LGMN: legumain; LIF: leukemia inhibitory factor; LIFR: LIF receptor alpha; MSX1: msh homeobox 1; MUC1: mucin 1, transmembrane; P4: progesterone; PBS: phosphate-buffered saline; PCA: principal component analysis; PPT1: palmitoyl-protein thioesterase 1; PGR: progesterone receptor; PSP: pseudopregnancy; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; qPCR: quantitative real-time polymerase chain reaction; SP: pregnancy; TFEB: transcription factor EB.

LRRK2 exonic variants are associated with lysosomal hydrolase activities and lysosphingolipid alterations in Parkinson's disease

Last data demonstrated that exonic variants of LRRK2 (p.G2019S, p.M1646T) may affect the catalytic activity of lysosomal enzyme glucocerebrosidase (GCase) probably through the phosphorylation of Rab10 protein. We aimed to evaluate an association of LRRK2 exonic variants previously associated with alteration of phosphorylation levels for Rab10Thr73 with PD risk in Russian population and analyze an impact of p.G2019S mutation and selected LRRK2 variants on lysosomal hydrolase activities. LRRK2 variants were determined by full sequencing of LRRK2 in 508 PD patients and 470 controls from Russian population. Activity of lysosomal enzymes (glucocerebrosidase (GCase), alpha-galactosidase A (GLA), acid sphingomyelinase (ASMase) and concentrations of their corresponded substrates (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM), respectively) were estimated in 211 PD patients and 179 controls by liquid chromatography with tandem mass spectrometry (LC-MS-MS) in dry blood spots. p.M1646T and p.N2081D were associated with PD (OR = 2.33, CI 95%: 1.1215 to 4.8253, p = 0.023; OR = 1.89, 95%CI: 1.0727 to 3.3313, p = 0.028, respectively) in Russian population. An increased LysoGb3 concentration was found in p.G2019S and p.N2081D LRRK2 carriers among PD patients compared to both PD patients and controls (p.G2019S: p = 0.00086, p = 0.0004, respectively; p.N2081D: p = 0.012, p = 0.0076, respectively). A decreased ASMase activity in p.G2019S LRRK2 carriers among PD patients (p = 0.014) was demonstrated as well. Our study supported possible involvement of LRRK2 dysfunction in an alteration of sphingolipid metabolism in PD.

The activity of lysosomal enzymes in organs of the White Sea mussel Mytilus edulis L. under crude oil impact in different salinity conditions

The effect of the toxicant on the activity of lysosomal hydrolases in organs of the White Sea mussels Mytilus edulis (Linnaeus 1758) was studied in aquatic experiments simulating a crude oil spill in the tidal zone. The mollusks were exposed for 1, 5 and 10 days to three pollutant concentrations (0.05; 0.25, and 2.50 ml/l). The impact of oil pollution was studied in combination with salinity normal for White Sea surface water (25 ‰) and low (15 ‰), which corresponds to reality during oil spills on the coast and in estuaries. We determined the activity of six lysosomal enzymes (acid phosphatase, RNase, DNase, β-glucosidase, β-galactosidase, and β-glucuronidase) in the gills and hepatopancreas. Both factors (sea water desalination and oil impact) had a significant effect on the activity of acid hydrolases. The most pronounced lysosomal reaction to oil appeared in the gills under normal salinity at 0.25 ml/l concentration and exposure for 10 days, as evidenced by a several-fold increase in the activity of acid phosphatase, DNase, β-galactosidase, and β-glucuronidase compared to the control. In the hepatopancreas, the same dependence in enzyme activity was noted, but to a lesser extent. Seawater desalination to 15 ‰ in the control caused a slight increase in the activity of DNase, β-galactosidase, and β-glucuronidase in the gills, while the effect for the hepatopancreas was insignificant. The combined effect of desalination and oil pollution somewhat reduced the protective functions of the lysosomal apparatus in mussels organs, which was especially noticeable at high oil concentrations and prolonged exposure of mollusks to experimental conditions. Thus, the results of the study demonstrate active participation of the lysosomal apparatus of the gills and hepatopancreas of mollusks in the adaptive responses to the combined effects of oil pollution and low water salinity. Compensatory changes in the activity of the enzyme complex of lysosomes are aimed at the utilization, transformation and excretion of oil components from the body, elimination of the structures and macromolecules damaged by the toxicant, as well as maintaining the vital activity of the organism under adverse conditions.

Drug Repositioning Applied to Cardiovascular Disease in Mucopolysaccharidosis.

Mucopolysaccharidoses (MPS) are genetic metabolic diseases characterized by defects in the activity of lysosomal hydrolases. In MPS, secondary cell disturbance affects pathways related to cardiovascular disorders. Hence, the study aimed to identify MPS-related drugs targeting cardiovascular disease and select a list of drugs for repositioning. We obtained a list of differentially expressed genes and pathways. To identify drug perturbation-driven gene expression and drug pathways interactions, we used the CMAP and LINCS databases. For molecular docking, we used the DockThor web server. Our results suggest that pirfenidone and colchicine are promising drugs to treat cardiovascular disease in MPS patients. We also provide a brief description of good practices for the repositioning analysis. Furthermore, the list of drugs and related MPS-enriched genes could be helpful to new treatments and considered for pathophysiological studies.

Secondary Mitochondrial Dysfunction as a Cause of Neurodegenerative Dysfunction in Lysosomal Storage Diseases and an Overview of Potential Therapies.

Mitochondrial dysfunction has been recognised a major contributory factor to the pathophysiology of a number of lysosomal storage disorders (LSDs). The cause of mitochondrial dysfunction in LSDs is as yet uncertain, but appears to be triggered by a number of different factors, although oxidative stress and impaired mitophagy appear to be common inhibitory mechanisms shared amongst this group of disorders, including Gaucher’s disease, Niemann–Pick disease, type C, and mucopolysaccharidosis. Many LSDs resulting from defects in lysosomal hydrolase activity show neurodegeneration, which remains challenging to treat. Currently available curative therapies are not sufficient to meet patients’ needs. In view of the documented evidence of mitochondrial dysfunction in the neurodegeneration of LSDs, along with the reciprocal interaction between the mitochondrion and the lysosome, novel therapeutic strategies that target the impairment in both of these organelles could be considered in the clinical management of the long-term neurodegenerative complications of these diseases. The purpose of this review is to outline the putative mechanisms that may be responsible for the reported mitochondrial dysfunction in LSDs and to discuss the new potential therapeutic developments.

Lysosome Interference Enabled by Proton‐Driven Dynamic Assembly of DNA Nanoframeworks inside Cells

Developing material chemistry systems coupled with biological processes is a promising way to rationally modulate lysosomal functions. Herein, we report a proton-driven dynamic assembly of DNA nanoframework inside cells coupled with the lysosome-mediated endocytosis pathways/lysosomal maturation, achieving the rational modulation of lysosomal functions, which we termed as lysosome interference. Through lysosome-mediated endocytosis, DNA nanoframework with acid-responsive semi-i-motif entered into lysosome and assembled into aggregate triggered by lysosomal acidity, which performed a long-term retention. Meanwhile, the consumption of protons resulted in lysosomal acidity reduction and hydrolase activity attenuation, thereby alleviating the degradation of nucleic acid drugs in interfered lysosomes and improving gene silencing effect. This study explores a new avenue to achieve lysosome interference by coupling subcellular microenvironment and precisely-programmable assembly system.

Lysosome Interference Enabled by Proton-Driven Dynamic Assembly of DNA Nanoframeworks inside Cells.

Coupling materials chemistry systems to biological processes is a promising way to rationally modulate lysosomal functions. A proton-driven dynamic assembly of a DNA nanoframework inside cells coupled with the lysosome-mediated endocytosis pathways/lysosomal maturation, gives the rational modulation of lysosomal functions, which we term "lysosome interference". Through lysosome-mediated endocytosis, the DNA nanoframework with acid-responsive semi-i-motif enters the lysosome and assembles into an aggregate in a process triggered by lysosomal acidity. The aggregate is suitable for long-term retention. The consumption of protons resulted in lysosomal acidity reduction and hydrolase activity attenuation, thus hindering the degradation of nucleic acid drugs in the lysosome and improving gene silencing effects. This study shows a new way to achieve lysosome interference by coupling the subcellular microenvironment with a precisely programmable assembly system.

Evaluation of Cardiovascular Experiment Animal

Abstract: In vivo models of myocardial infarction induced by coronary artery ligation in rats usually suffer from high early mortality and a low rate of induction. This study investigated the time course initiation of chronic myocardial infarction in albino rats and the possibility of reducing early mortality rate due to myocardial infarction by modification of the surgical technique. CAL was carried out by passing the suture through the pericardial layer around the midway of the left anterior descending coronary artery including a small area of the myocardium to avoid mechanical damage to the heart geometry. In addition, the role of endothelin-1 in rat heart with congestive heart failure was critically assessed. Time course initiation experiments were designed by sacrificing the animals at different time intervals and by carrying out physiological, biochemical, histopathological, electron microscopical and immunohistochemical studies. Specific markers of myocardial injury, viz. cardiac troponin-T, high sensitivity C-reactive protein, lactate dehydrogenase and fibrinogen were measured at different time points. Serum marker enzymes and activities of lysosomal hydrolases were found to be elevated on the eighth day post-ligation. Histopathological studies demonstrated focal areas showing fibrovascular tissue containing fibroblasts, collagenous ground substance and numerous small capillaries replacing cardiac muscle fibers. Transmission electron micrographs exhibited mitochondrial changes of well-developed irreversible cardiac injury, viz. swelling, disorganization of cristae, appearance of mitochondrial amorphous matrix densities, and significant distortion of muscle fibers and distinct disruption of the intercalated discs. Immune blotting studies confirmed the presence of alpha 2-macroglobulin which supported the inflammatory response. The severity of the CMI was inferred by the measurement of the level of ET-1 in plasma and left ventricle which was significantly higher in the CMI rats than in the sham-operated rats. Immunohistochemical studies at different time intervals showed that there was a significant immunoexpression of ET-1 on the eighth day post-ligation. This study conclusively showed that ligation of left anterior descending artery minimised mortality and ET-1 was expressed during CMI.

Ion Channels and Pumps in Autophagy: A Reciprocal Relationship.

Autophagy, the process of cellular self-degradation, is intrinsically tied to the degradative function of the lysosome. Several diseases have been linked to lysosomal degradative defects, including rare lysosomal storage disorders and neurodegenerative diseases. Ion channels and pumps play a major regulatory role in autophagy. Importantly, calcium signaling produced by TRPML1 (transient receptor potential cation channel, mucolipin subfamily) has been shown to regulate autophagic progression through biogenesis of autophagic-lysosomal organelles, activation of mTORC1 (mechanistic target of rapamycin complex 1) and degradation of autophagic cargo. ER calcium channels such as IP3Rs supply calcium for the lysosome, and lysosomal function is severely disrupted in the absence of lysosomal calcium replenishment by the ER. TRPML1 function is also regulated by LC3 (microtubule-associated protein light chain 3) and mTORC1, two critical components of the autophagic network. Here we provide an overview of the current knowledge about ion channels and pumps—including lysosomal V-ATPase (vacuolar proton-ATPase), which is required for acidification and hence proper enzymatic activity of lysosomal hydrolases—in the regulation of autophagy, and discuss how functional impairment of some of these leads to diseases.

Neuronetworks: Analysis of brain pathology in Mucopolysaccharidoses – A systems biology approach

Mucopolysaccharidoses (MPS) are rare lysosomal storage diseases characterized by defects in the activity of lysosomal hydrolases that degrade glycosaminoglycan, with progressive multisystemic involvement. Neurological damage is present in several MPS types. The relationship between the accumulation of glycosaminoglycans and the neurological disorder presented in these diseases remains unknown. For this purpose, we analyzed distinct types of MPS using publicly available transcriptomic data with a systems biology approach to search for clues about the pathophysiological mechanisms involved in the brain pathology of MPS. The most relevant proteins in the networks and ontology terms related to neurological damage in MPS were identified and compared among diseases. We performed the clustering analysis for GSE111906 (MPSI), GSE95224 (MPSII), GSE23075 (MPSIIIB), GSE15758 (MPSIIIB), and GSE76283 (MPSVII). Regarding biomarker discovery analysis, the top 10 genes were ranked according to the maximal clique centrality. Different ontologies were present in the different types of MPS. Ontologies were also present in all the MPS types, like axon guidance, Calcium signaling, PI3K-Akt signaling pathway, and Wnt signaling pathway. We hypothesize that these pathways are deranged because glycosaminoglycans play an essential role in the extracellular matrix composition, helping to regulate several processes. Systems biology approaches may help to understand the mechanisms of neuropathology in the different types of Mucopolysaccharidoses.

Ratiometric probe with optimized permeability for visualizing lysosomal acidification during autophagy

Lysosomal pH plays indispensable roles in regulating the activity of lysosomal hydrolases, and is involved in crucial biological procedures including autophagy. Fluorescent probes for lysosomal pH were significant molecular tools to investigate lysosomal pH regulation. In this work, we have designed a new fluorophore to respond to pH in a ratiometric manner based on intramolecular charge transfer (ICT) mechanism. By modifying different alkyl chains on the dye, we have obtained a novel ratiometric probe for lysosomal pH with optimized membrane permeability. The probe could stain lysosomes, and was capable of monitoring lysosomal pH in a ratiometric manner. Chloroquine (CQ) induced pH rise in lysosomes was successfully visualized with the probe. The down-regulation and subsequence up-regulation of lysosomal pH during starvation were also revealed in ratiometric manner. We firmly believe that the probe is a desirable tool to investigate lysosomal pH and promote the studies in autophagy and relative biological areas.

Measurement of GCase Activity in Cultured Cells.

Glucocerebrosidase (GCase), which is encoded by the GBA1 gene, has lysosomal glycoside hydrolase activity that hydrolyzes glucosylceramide. Defects in GCase lead to the accumulation of glucosylceramide, which causes the development of the lysosomal storage disease known as Gaucher's disease. Loss-of-function mutations in the GBA1 gene are the most important genetic risk factor for synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies. Recent studies on PD genes associated with lysosomal function suggest that GCase activity is decreased in cell models of PD and in neurons derived from PD patients. In this chapter, we describe a protocol to measure GCase activity in cultured cells.

Calciprotein particle-induced cytotoxicity via lysosomal dysfunction and altered cholesterol distribution in renal epithelial HK-2 cells

Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.