Lysosomal Enzyme Activity Research Articles

The endo-lysosomal regulatory protein BLOC1S1 modulates hepatic lysosomal content and lysosomal lipolysis

BLOC1S1 is a common component of BLOC and BORC multiprotein complexes which play distinct roles in endosome and lysosome biology. Recent human mutations in BLOC1S1 associate with juvenile leukodystrophy. As leukodystrophy is linked to perturbed lysosomal lipid storage we explored whether BLOC1S1 itself modulates this biology. Given the central role of the liver in lipid storage, our investigations were performed in hepatocyte specific liver bloc1s1 knockout (LKO) mice and in human hepatocyte-like lines (HLCs) derived from inducible pluripotential stem cells (iPSCs) from a juvenile leukodystrophy subject's with bloc1s1 mutations and from isogenic corrected iPSCs. Here we show that hepatocyte lipid stores are diminished in parallel with increased lysosomal content, increased lysosomal lipid uptake and lipolysis in LKO mice. The lysosomal lipolysis program was independent of macro- and chaperone-mediated lipophagy but dependent on cellular lysosome content. In parallel, genetic induction of lysosomal biogenesis in a transformed hepatocyte cell line replicated depletion of intracellular lipid stores. Interestingly bloc1s1 mutant and isogenic corrected HLCs both showed normal lysosomal enzyme activity. However, relative to the isogenic corrected HLCs, mutant bloc1s1 HLCs showed reduced lysosomal content and increased lipid storage. Together these data show distinct phenotypes in human mutant HLCs compared to murine knockout cells. At the same time, human blcs1s1 mutation and murine hepatocyte bloc1s1 depletion disrupt lysosome content and the cellular lipid storage. These data support that BLOC1S1 modulates lysosome content and lipid handling independent of autophagy and show that lysosomal lipolysis is dependent on the cellular content of functional lysosomes.

Depletion of the AD risk gene SORL1 causes endo‐lysosomal dysfunction in human microglia

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder which is currently incurable. Prior to well‐established neuropathological changes in AD, enlarged early endosomes are cytopathological hallmark of AD and strong genetic evidence links genes involved in intra‐cellular trafficking, mediated by endo‐lysosomal pathway, to increased AD risk, suggesting that this pathway is integral to the pathogenesis of AD. We recently identified SORL1, an intracellular sorting receptor as a potential regulator of this pathway. Using CRISPR/Cas9 to deplete SORL1 in human induced pluripotent stem cells (hiPSCs), we showed that loss of SORL1 results in enlarged early endosomes in (hiPSCs)‐derived neurons, but not microglia. This model replicates the earliest AD cytopathology and our data underscores important cell‐type specific differences in response to loss of SORL1. Building on this study, we further examined alterations in lysosomal function, with altered expression of SORL1 in hiPSC‐derived microglia.MethodIsogenic hiPSC lines that are deficient in SORL1 were generated and differentiated into microglia using previous published protocols. Lysosome function was assayed by examining lysosome morphology, lysosomal trafficking of fibrillar Aβ, lysosomal degradation, lysosome enzyme activity and lysosomal exocytosis. Lysosome morphology and number were measured using immunocytochemistry, confocal microscopy and Cell Profiler and Image J software. To assess Aβ transport into lysosomes, pH sensitive fluorescent dye, Phrodo, conjugated to Aβ, was used. Lysosomal degradation was measured using a proteolysis sensitive fluorogenic substrate, DQ‐BSA. Fluorescence intensity of both Phrodo and DQ‐BSA was measured using flow cytometry. Lysosome enzyme activity and exocytosis assays were performed using previously published protocols.ResultLoss of SORL1 results in increased lysosome size and decreased lysosome number in SORL1 KO microglia indicating altered lysosome function. In support of this idea, we observed decreased lysosomal degradation, trafficking of Aβ to lysosomes and lysosomal enzyme activity and also decreased lysosomal exocytosis in SORL1 KO microglia.ConclusionOverall, our results have unraveled a previously unexplored function of the AD risk gene SORL1 in human microglia and our work indicates that maintaining homeostatic balance in the endo‐lysosomal network impacts diverse CNS cell types. Our study will define the endo‐lysosomal network in general, and SORL1 in particular, as a novel therapeutic target for AD.

Ginsenoside protopanaxadiol protects adult retinal pigment epithelial-19 cells from chloroquine by modulating autophagy and apoptosis.

Chloroquine often causes serious eye and vision problems, which are mainly mediated by lysosomotropic alteration. In this study, we investigated whether the ginsenoside protopanaxadiol relieves chloroquine-induced retinopathy by restoring lysosomotropic abnormalities in human adult retinal pigment epithelial-19 cells. Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological alterations in autophagosomes of adult retinal pigment epithelial-19 cells was detected using confocal microscopy. Apoptosis was examined using flow cytometry, whereas cellular reactive oxygen species levels were determined by measuring the fluorescence intensity of 5-(and-6)-carboxy-2'-7'-dichlorohydrofluorescein diacetate. Lysosomal function was assessed by measuring lysosomal pH and enzyme activity. Immunoprecipitation and western blotting analyses were performed. Adult retinal pigment epithelial-19 cells accumulated autophagosomes with fusion defects in lysosomes and reactive oxygen species formation following exposure to chloroquine. This effect trapped Beclin-1 and B-cell lymphoma 2 interfering with autophagy initiation and autophagosome development. Protopanaxadiol alleviated chloroquine-induced toxicity by modulating the interaction between Beclin-1 and Bcl-2, which was mediated by the AMP-activated protein kinase-mammalian target of rapamycin signal axis. Furthermore, autophagy and apoptosis were simultaneously controlled by protopanaxadiol via upregulation of autophagy flux and decreased reactive oxygen species formation and apoptotic protein expression. These findings suggest that protopanaxadiol is a promising treatment strategy for chloroquine-mediated retinopathy.

A Novel In Vivo‐Active Small Molecule Inducer of Progranulin for the Treatment of Frontotemporal Dementia

AbstractBackgroundHeterozygous mutations in GRN that lead to haploinsufficiency of the progranulin (PGRN) protein cause the fatal neurodegenerative disease frontotemporal dementia (FTD‐GRN). Complete loss of PGRN, on the other hand, leads to an early onset lysosomal storage disorder, indicating a critical role for PGRN in the lysosome. Although its exact function is unknown, PGRN is both secreted and trafficked to the lysosome where it is processed into granulins. Lysosomal granulins are reported to modulate the activity of lysosomal enzymes. Currently there are no treatments for FTD‐GRN but restoring PGRN to normal levels is a promising therapeutic strategy.MethodsWe performed a small molecule screen in BV‐2 cells and identified novel compounds that increase progranulin secretion. Some of these molecules were further characterized in vivo to help determine an exposure‐response relationship.ResultsThrough structure activity relationship optimization, we improved the potency of compounds at increasing PGRN secretion from >2 mM down to <1 nM. Importantly, we found that lysosomal granulins are increased following compound treatment and that this effect is sustained beyond the secreted PGRN increase. Using an optimized brain‐penetrant compound with good pharmacokinetic properties, we established an exposure‐response relationship for PGRN changes in the CSF of non‐human primates, demonstrating an excellent in vitro‐in vivo correlation. Finally, we report that multi‐day dosing produced a sustained increase in PGRN in CSF over the course of the treatment period.ConclusionOverall, these data support further development of our novel small molecules for the treatment of FTD‐GRN.

An altered transcriptome underlies cln5-deficiency phenotypes in Dictyostelium discoideum.

Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of neurodegenerative lysosomal storage diseases that affect all ages and ethnicities globally. Previous research showed that CLN5 participates in a variety of cellular processes. However, the precise function of CLN5 in the cell and the pathway(s) regulating its function are not well understood. In the model organism Dictyostelium discoideum, loss of the CLN5 homolog, cln5, impacts various cellular and developmental processes including cell proliferation, cytokinesis, aggregation, cell adhesion, and terminal differentiation. In this study, we used comparative transcriptomics to identify differentially expressed genes underlying cln5-deficiency phenotypes during growth and the early stages of multicellular development. During growth, genes associated with protein ubiquitination/deubiquitination, cell cycle progression, and proteasomal degradation were affected, while genes linked to protein and carbohydrate catabolism were affected during early development. We followed up this analysis by showing that loss of cln5 alters the intracellular and extracellular amounts of proliferation repressors during growth and increases the extracellular amount of conditioned medium factor, which regulates cAMP signalling during the early stages of development. Additionally, cln5 - cells displayed increased intracellular and extracellular amounts of discoidin, which is involved in cell-substrate adhesion and migration. Previous work in mammalian models reported altered lysosomal enzyme activity due to mutation or loss of CLN5. Here, we detected altered intracellular activities of various carbohydrate enzymes and cathepsins during cln5 - growth and starvation. Notably, cln5 - cells displayed reduced β-hexosaminidase activity, which aligns with previous work showing that D. discoideum Cln5 and human CLN5 can cleave the substrate acted upon by β-hexosaminidase. Finally, consistent with the differential expression of genes associated with proteasomal degradation in cln5 - cells, we also observed elevated amounts of a proteasome subunit and reduced proteasome 20S activity during cln5 - growth and starvation. Overall, this study reveals the impact of cln5-deficiency on gene expression in D. discoideum, provides insight on the genes and proteins that play a role in regulating Cln5-dependent processes, and sheds light on the molecular mechanisms underlying CLN5 disease.

Spatiotemporally mapping temperature dynamics of lysosomes and mitochondria using cascade organelle-targeting upconversion nanoparticles

The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.

Saposin C, Key Regulator in the Alpha-Synuclein Degradation Mediated by Lysosome.

Lysosomal dysfunction has been proposed as one of the most important pathogenic molecular mechanisms in Parkinson disease (PD). The most significant evidence lies in the GBA gene, which encodes for the lysosomal enzyme β-glucocerebrosidase (β-GCase), considered the main genetic risk factor for sporadic PD. The loss of β-GCase activity results in the formation of α-synuclein deposits. The present study was aimed to determine the activity of the main lysosomal enzymes and the cofactors Prosaposin (PSAP) and Saposin C in PD and healthy controls, and their contribution to α-synuclein (α-Syn) aggregation. 42 PD patients and 37 age-matched healthy controls were included in the study. We first analyzed the β-GCase, β-galactosidase (β-gal), β-hexosaminidase (Hex B) and Cathepsin D (CatD) activities in white blood cells. We also measured the GBA, β-GAL, β-HEX, CTSD, PSAP, Saposin C and α-Syn protein levels by Western-blot. We found a 20% reduced β-GCase and β-gal activities in PD patients compared to controls. PSAP and Saposin C protein levels were significantly lower in PD patients and correlated with increased levels of α-synuclein. CatD, in contrast, showed significantly increased activity and protein levels in PD patients compared to controls. Increased CTSD protein levels in PD patients correlated, intriguingly, with a higher concentration of α-Syn. Our findings suggest that lysosomal dysfunction in sporadic PD is due, at least in part, to an alteration in Saposin C derived from reduced PSAP levels. That would lead to a significant decrease in the β-GCase activity, resulting in the accumulation of α-syn. The accumulation of monohexosylceramides might act in favor of CTSD activation and, therefore, increase its enzymatic activity. The evaluation of lysosomal activity in the peripheral blood of patients is expected to be a promising approach to investigate pathological mechanisms and novel therapies aimed to restore the lysosomal function in sporadic PD.

Lysosomal lipid alterations caused by glucocerebrosidase deficiency promote lysosomal dysfunction, chaperone-mediated-autophagy deficiency, and alpha-synuclein pathology

Mutations in the GBA gene that encodes the lysosomal enzyme β-glucocerebrosidase (GCase) are a major genetic risk factor for Parkinson’s disease (PD). In this study, we generated a set of differentiated and stable human dopaminergic cell lines that express the two most prevalent GBA mutations as well as GBA knockout cell lines as a in vitro disease modeling system to study the relationship between mutant GBA and the abnormal accumulation of α-synuclein. We performed a deep analysis of the consequences triggered by the presence of mutant GBA protein and the loss of GCase activity in different cellular compartments, focusing primarily on the lysosomal compartment, and analyzed in detail the lysosomal activity, composition, and integrity. The loss of GCase activity generates extensive lysosomal dysfunction, promoting the loss of activity of other lysosomal enzymes, affecting lysosomal membrane stability, promoting intralysosomal pH changes, and favoring the intralysosomal accumulation of sphingolipids and cholesterol. These local events, occurring only at a subcellular level, lead to an impairment of autophagy pathways, particularly chaperone-mediated autophagy, the main α-synuclein degradative pathway. The findings of this study highlighted the role of lysosomal function and lipid metabolism in PD and allowed us to describe a molecular mechanism to understand how mutations in GBA can contribute to an abnormal accumulation of different α-synuclein neurotoxic species in PD pathology.

Aberrant autophagy in lysosomal storage disorders marked by a lysosomal SNARE protein shortage due to suppression of endocytosis.

Lysosomal storage disorders (LSDs) are inherited metabolic diseases caused by genetic defects in lysosomal enzymes or related factors. LSDs are associated with excessive accumulation of natural substrates in lysosomes leading to central nervous system and peripheral tissue damage. Abnormal autophagy is also involved in pathogenesis, although the underlying mechanisms remain unclear. We demonstrated that impairment of lysosome-autophagosome fusion is due to suppressed endocytosis in LSDs. The fusion was reduced in several LSD cells and the brains of LSD model mice, suggesting that the completion of autophagy is suppressed by the accumulation of substrates. In this brain, the expression of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, VAMP8 and Syntaxin7, was decreased on the lysosomal surface but not intracellular. This aberrant autophagy preceded the development of pathological phenotypes in LSD-model mice. Furthermore, the enzyme deficiency leading to the substrate accumulation could suppress endocytosis, and the inhibited endocytosis decreased SNARE proteins localized on lysosomes. These findings suggest that the shortage of SNARE proteins on lysosomes is one of the reasons for the impairment of lysosome-autophagosome fusion in LSD cells. Defects in lysosomal enzyme activity suppress endocytosis and decrease the supply of intracellular SNARE proteins recruited to lysosomes. This shortage of lysosomal SNARE proteins impairs lysosome-autophagosome fusion in lysosomal storage disorders.

Effect of Dried Apple Pomace (DAP) as a Feed Additive on Antioxidant System in the Rumen Fluid.

The study aimed to evaluate the effect of dried apple pomace (DAP) as a feed additive on the enzymatic activity and non-enzymatic compounds belonging to the antioxidant system in cattle rumen fluid. The experiment included 4 Polish Holstein–Friesian cannulated dairy cows and lasted 52 days. The control group was fed with the standard diet, while in the experimental group, 6% of the feedstuff was replaced by dried apple pomace. After the feeding period, ruminal fluid was collected. The spectrophotometric technique for the activity of lysosomal enzymes, the content of vitamin C, polyphenols, and the potential to scavenge the free DPPH radical was used. The enzyme immunoassay tests (ELISA) were used to establish the activity of antioxidants enzymes and MDA. Among the rumen aminopeptidases, a significant reduction (p < 0.01) from 164.00 to 142.00 was observed for leucyl-aminopeptidase. The activity of glycosidases was decreased for HEX (from 231.00 to 194.00) and β-Glu (from 1294.00 to 1136.00), while a significant statistically increase was noticed for BGRD (from 31.10 to 42.40), α-Glu (from 245.00 to 327.00), and MAN (from 29.70 to 36.70). Furthermore, the activity of catalase and GSH (p < 0.01) was inhibited. In turn, the level of vitamin C (from 22.90 to 24.10) and MDA (from 0.36 to 0.45) was statistically higher (p < 0.01). The most positive correlations were observed between AlaAP and LeuAP (r = 0.897) in the aminopeptidases group and between β-Gal and MAN (r = 0.880) in the glycosidases group. Furthermore, one of the most significant correlations were perceived between SOD and AlaAP (r = 0.505) and AcP (r = 0.450). The most negative correlation was noticed between α-Gal and DPPH (r = −0.533) based on these observations. Apple pomace as a feed additive has an influence on lysosomal degradation processes and modifies oxidation–reduction potential in the rumen fluid. Polyphenols and other low-weight antioxidant compounds are sufficient to maintain redox balance in the rumen.

Mitochondria-lysosome crosstalk in GBA1-associated Parkinson's disease.

Organelle crosstalk is significant in regulating their respective functions and subsequent cell fate. Mitochondria and lysosomes are amongst the essential organelles in maintaining cellular homeostasis. Mitochondria-lysosome connections, which may develop dynamically in the human neurons, have been identified as sites of bidirectional communication. Aberrancies are often associated with neurodegenerative disorders like Parkinson's disease (PD), suggesting the physical and functional link between these two organelles. PD is often linked with genetic mutations of several mutations discovered in the familial forms of the disease; some are considered risk factors. Many of these genes are either associated with mitochondrial function or belong to endo-lysosomal pathways. The recent investigations have indicated that neurons with mutant glucosylceramidase beta (GBA1) exhibit extended mitochondria-lysosome connections in individuals with PD. This may be due to impaired control of the untethering protein, which aids in the hydrolysis of Rab7 GTP required for contact untethering. A GCase modulator may be used to augment the reduced GBA1 lysosomal enzyme activity in the neurons of PD patients. This review focuses on how GBA1 mutation in PD is interlinked with mitochondria-lysosome (ML) crosstalk, exploring the pathways governing these interactions and mechanistically comprehending the mitochondrial and lysosomal miscommunication in the pathophysiology of PD. This review is based on the limited literature available on the topic and hence may be subject to bias in its views. Our estimates may be conservative and limited due to the lack of studies under the said discipline due to its inherent complex nature. The current association of GBA1 to PD pathogenesis is based on the limited scope of study and further research is necessary to explore the risk factors further and identify the relationship with more detail.

Ratiometrically pH-Insensitive Upconversion Nanoprobe: Toward Simultaneously Quantifying Organellar Calcium and Chloride and Understanding the Interaction of the Two Ions in Lysosome Function.

Calcium and chloride levels are closely related to lysosome dysfunction. However, the simultaneous measurement of calcium (Ca2+) and chloride (Cl-) in acidic subcellular organelles, which is conducive to a deep understanding of lysosome-related biological events, remains a challenge. In this study, we developed a pH-insensitive, ratiometric NIR nanoprobe for the simultaneous detection of Ca2+ and Cl- in acidic lysosomes and determined the roles of the two ions in lysosome function. The upconversion nanoprobe with blue, green, and red emissions was modified with a Ca2+-sensitive dye (Rhod-5N) and Cl--responsive fluorophore (10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate, BAC). As a result of a dual-luminescence resonance energy transfer between upconversion nanoparticles (UCNPs) and Rhod-5N/BAC, the blue and green upconversion luminescence (UCL) of UCNPs were quenched and the red UCL was used as the reference signal. The ratiometric upconversion nanoprobe possesses a specific ability for the concurrent recognition of Ca2+ and Cl- ions independent of the influence of the environmental pH. To locate the probe in the lysosome, dextran was further modified with upconversion nanoparticles. Then, the nanoprobe with a high spatial resolution was constructed for the simultaneous monitoring of Ca2+ and Cl- in acidic lysosomes. Moreover, it was found that the reduction of lysosomal Cl- affects the release of lysosomal Ca2+, which further blocks the activities of specific lysosomal enzymes. The ratiometric NIR nanoprobe has great potential for decoding and evaluating lysosomal diseases.

Mechanism and Changes Associated with Edible Camphor Induced Neurotoxicity

ABSTRACT&#x0D; Background and Purpose: Camphor is an additive in certain remedies for the treatment of coryza, bronchitis, musculoskeletal pains and wounds. This study investigated the possible seizure-induction mechanism of edible camphor (EC) and the biochemical changes in brain and blood after its administration.&#x0D; Methods: Seizure characteristics were evaluated in twenty-five male Wistar rats randomly distributed into five groups and given intraperitoneal injections of 0.5 mL coconut oil; 100 mg/kg pentylenetetrazol (PTZ); 1000 mg/kg EC; 5 mg/kg diazepam + 1000 mg/kg EC; and 250 mg/kg carbamazepine + 1000 mg/kg EC respectively. Oral acute toxicity was evaluated using 15 male rats assigned to 3 groups (n=5) and orally administered a single sub-convulsive dose (300 mg/kg) of EC and monitored for 3, 24, and 168 hours respectively. Fasting blood sugar, blood lipid parameters and brain weight were evaluated. In a 28-day toxicity evaluation, 300 mg/kg/day EC was administered to test and control groups of male rats (n=10) and brains were excised for evaluation of weight, oxidative, inflammatory and cell component changes. &#x0D; Results: At 1000 mg/kg, EC had superior seizure-inducing potential, longer seizure latency but shorter duration than PTZ. Acute administration of EC increased blood sugar, decrease serum phospholipids and triglycerides. Daily treatment for 28 days increased brain weight, and lysosomal enzyme activities neuroinflammatory indicators (TNF?, IL-4, IL-8, IL-9 and IL-10) except IL-2. Treatment also resulted in the elevation of malondialdehyde (MDA) level but reduction in catalase (CAT) and reduced glutathione (GSH) levels decreased, decreased acetylcholinesterase activity. Photomicrographs of the cerebral cortex of treated animals showed degenerated axonal and dendritic projections as well as soma filled with neurofibrillary tangles.&#x0D; Conclusions: EC neurotoxicity may be due to inhibition of GABAergic neurotransmission resulting in oxidative, inflammatory and eventual neurodegenerative damages.

Mfsd8 Modulates Growth and the Early Stages of Multicellular Development in Dictyostelium discoideum.

MFSD8 is a transmembrane protein that has been reported to transport chloride ions across the lysosomal membrane. Mutations in MFSD8 are associated with a subtype of Batten disease called CLN7 disease. Batten disease encompasses a family of 13 inherited neurodegenerative lysosomal storage diseases collectively referred to as the neuronal ceroid lipofuscinoses (NCLs). Previous work identified an ortholog of human MFSD8 in the social amoeba D. discoideum (gene: mfsd8, protein: Mfsd8), reported its localization to endocytic compartments, and demonstrated its involvement in protein secretion. In this study, we further characterized the effects of mfsd8 loss during D. discoideum growth and early stages of multicellular development. During growth, mfsd8 − cells displayed increased rates of proliferation, pinocytosis, and expansion on bacterial lawns. Loss of mfsd8 also increased cell size, inhibited cytokinesis, affected the intracellular and extracellular levels of the quorum-sensing protein autocrine proliferation repressor A, and altered lysosomal enzyme activity. During the early stages of development, loss of mfsd8 delayed aggregation, which we determined was at least partly due to impaired cell-substrate adhesion, defects in protein secretion, and alterations in lysosomal enzyme activity. Overall, these results show that Mfsd8 plays an important role in modulating a variety of processes during the growth and early development of D. discoideum.

Застосування методів консервативної терапії в дітей з післяопіковими рубцевими деформаціями та їх вплив на метаболізм сполучної тканини

One of the factors of scarring is changes in the connective tissue (CT). Defeat of CT is accompanied by changes in the content of glycosaminoglycans (GAG), activity of lysosomal enzymes, levels of mineral metabolism. To eliminate the influence of inflammatory phenomena and disorders of CT metabolism on the formation of scar tissue, it is urgent to use conservative rehabilitation measures. Purpose - to determine the effectiveness of measures of conservative therapy of post-burn scars in children for the clinical course and metabolism of connective tissue. Material and methods. 27 children aged 5 to 18 were supervised. The group of healthy children without clinical manifestations of inflammatory reactions consisted of 17 people. Conservative treatment of children with scarring included local, drug, physiotherapy, balneological and efferent therapy methods. The impact of treatment measures was assessed on a modified scar score scale, which consisted of a patient (parent) scale and a researcher scale. Laboratory criteria of effectiveness were the amount of ceruloplasmin (CP), lactic (MC) and sialic (SC) acids, GAG content, lysosomal enzyme activity (bone and alkaline phosphatase), mineral metabolism (total calcium). Laboratory parameters in patients were studied in the period from 150 to 170 and 310 to 340 days after injury. SPSS v was used for statistical analysis. 17.0. Results. Improvements in clinical signs of scar tissue were 1.7-fold according to objective estimates and 1.6-fold according to patient estimates. Laboratory monitoring showed the effectiveness of conservative treatment of convalescents, which was manifested in a reduction in GAG levels 1.6 times after 6 months and 2.0 times after a year. There was a decrease in the activity of acid phosphatase in 1.6 times, alkaline phosphatase and its bone fraction in 1.4 times. In addition, after treatment, there was an increase in ceruloplasmin activity by 1.5 times at all times. A 1.5-fold increase in the number of MCs was determined at all observation times. After treatment, the amount of MC decreased by 1.2 times (P&lt;0.05). Significant confirmation of an increase in the level of SC in 1.4 times (P&lt;0.05) was observed only on the 160th day of the study. At other times, the increase in the IC was unreliable. Conclusions. Long-term studies have shown that in children after thermal trauma in the process of productive scarring there are deviations in the metabolism of CT in the direction of increasing its synthetic function, metabolic disorders and inflammation continue. The use of complex conservative therapy in children with post-burn scars during convalescence allowed to reduce the severity of inflammatory reactions, improve CT metabolism and reduce scarring. The research was carried out in accordance with the principles of the Helsinki declaration. The study protocol was approved by the Local ethics committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors. Key words: post-burn scarring in children, connective tissue, metabolism, inflammation, conservative therapy.

The ameliorative effect of Apium graveolens & curcumin against Non-alcoholic fatty liver disease induced by high fructose-high fat diet in rats

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a condition resulting from fat aggregates in liver cells and is associated with metabolic syndrome, obesity, and oxidative stress. The present work was designed to investigate the role of celery and curcumin against high-fructose–high-fat (HFHF) diet-induced NAFLD in rats. Thirty male rats were classified into five groups: GP1: control group (rats were fed a normal control diet), GP2: HFHF group as a positive control (rats were fed a HFHF diet) for 20 weeks, GP3: HFHF + sily group, GP4: HFHF + celery group, and GP5: HFHF + cur group (rats in 3, 4, and 5 were treated as in the HFHF group for 16 weeks, then combined treatment daily by gavage for 4 weeks with either silymarin (as a reference drug, 50 mg/kg bw) or celery (300 mg/kg bw) or curcumin (200 mg/kg bw), respectively. The progression of NAFLD was evaluated by estimating tissue serum liver enzymes, glycemic profile, lipid profile, oxidative stress markers in liver tissue, and histopathological examination. Moreover, DNA fragmentation and the released lysosomal enzymes (acid phosphatase, β-galactosidase, and N-acetyl-B-glucosaminidase) were estimated.ResultsOur results showed that HFHF administration for 16 weeks caused liver enzymes elevation, hyperglycemia, and hyperlipidemia. Furthermore, increased hepatic MDA levels along with a decline in GSH levels were observed in the HFHF group as compared to the control group. The results were confirmed by a histopathological study, which showed pathological changes in the HFHF group. DNA fragmentation was also observed, and the lysosomal enzyme activities were increased. On the other hand, oral supplementation of celery and cur improved all these changes compared with positive control groups and HFHF + sily (as a reference drug). Moreover, celery, as well as curcumin co-treatment, reduced HFHF-enhanced DNA fragmentation and inhibited elevated lysosomal enzymes. The celery combined treatment showed the most pronounced ameliorative impact, even more than silymarin did.ConclusionOur findings suggest that celery and curcumin consumption may exhibit ameliorative impacts against NALFD progression, while celery showed more ameliorative effect in all parameters.

Multiplex Mass Spectrometry for Enzymatic Activities Screening of Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) are a group of rare and inherited disorders of lysosome function and they are classified into more than 50 types (e.g., mucopolysaccharidoses, neuronal ceroid lipofuscinoses, Fabry’s Disease; Pompe Disease, etc.). The majority of LSDs result from defective lysosomal enzyme activity leading to accumulation of endogenous macromolecules in the lysosome causing severe disease symptoms and finally death. For some LSDs, treatment is available in the form of enzyme replacement therapy with good results if the therapy is started as soon as possible, making the diagnosis of crucial importance. State of art now in LSDs diagnostic employs in first stage, enzymatic determinations by fluorimetry or by mass spectrometry (in few cases).In this report, we describe a highly specific and sensitive diagnostics on dry blood spots (DBSs) for (i) molecular determinations of LSDs, particularly mucopolysaccharidoses (MPSs) and neuronal ceroid lipofuscinoses (NCLs), by simultaneous fluorimetric and mass spectrometric analysis using new types of substrate derivatives based on different alkyl‐umbelliferone; (ii) clinical diagnostics of LSDs by multiplex‐MS‐MRM analysis using new type of substrate derivatives and (iii) clinical validation of the new developed methods using DBS samples for patients with established LSDs. We have developed a high‐throughput single and multiplexing assay for rare diseases (e.g. MPS 1, MPS 2, MPS 6, NCL 2, NCL 10, Fabry Disease, etc.) in DBSs. A common substrate for the fluorimetric measurement of a lysosomal enzyme activity is a molecule with 2 different parts which are bound covalently: a group recognized by the target enzyme (e.g. sugar, lipid) and 4‐umbelliferone and the product resulted after the enzymatic reaction is always 4‐methyl umbelliferone. In our tests, the enzymatic activity levels in DBSs were determinate by fluorimetry or multiple reactions monitoring mass spectrometry in the presence of an internal standard showing a good statistical correlation in singles assays. Moreover, we developed duplex and triplex assays for the diagnosis of different LSDs from the muccopolysacharidoses family using modified substrates based on different coumarin derivates obtained through Pechman condensation. The new umbelliferone derivatives were synthesize by substituting groups in position 4 and 7 of the alpha‐ coumarin molecule with others e.g. 4‐ ethyl‐ umbelliferone, 4‐propyl‐umbelliferone, etc.In conclusion, by quantification of umbelliferone derivatives as products of enzymatic reactions either by fluorimetry or mass spectrometry, the newly proposed substrates can be easily use for clinical diagnosis of several LSDs.

Cardio-protective effects of terminalia catappa leaves and terminalia chebula fruit extract in doxorubicin-induced cardiomyopathy in rats

Background The cardio-protective effects of Terminalia catappa and Terminalia chebula are well-recognized in Ayurveda for its antimicrobial, antidiabetic and antioxidant potentials. The present study evaluates the effects of T. catappa leaves (Tct.LE) and T. chebula fruits (Tce.FE) against doxorubicin (DOX)-induced rats through analysis of the cardiac biomarkers, tricarboxylic acid (TCA) cycle enzymes and respiratory chain enzymes for their cardio-protective properties. Materials and methods This study includes 42 adult male Albino Wistar rats randomized into seven groups for 21-days. Groups were categorized as control; DOX (1.5 mg/kg) induced negative control; basal diet with 300 mg/kg of Tct.LE, with 300 mg/kg Tce.FE; DOX with 300 mg/kg of Tct.LE, Tce.FE, and propranolol (25 mg/kg). Results and discussion The doses of 300 mg/kg of both plants have a significant effect on the TCA cycle, respiratory and lysosomal enzymes activity. The troponin levels are significantly reduced in plant treated group than the DOX-treated rats when compared with the control and propranolol treated group. Likewise, the increased level of creatine kinase-muscle/MB, creatine kinase and lipid profile in the DOX-treated animals were significantly reduced upon being treated with extracts. Conclusion The cardio-protective activity of Tct.LE leaves and Tce.FE indicate its potential use in the management of cardiovascular diseases.

Lysosomal impairment-mediated autophagy dysfunction responsible for the vascular endothelial apoptosis caused by silica nanoparticle via ROS/PARP1/AIF signaling pathway

Understanding the underlying interactions of nanoparticles (NPs) with cells is crucial to the nanotoxicological research. Evidences suggested lysosomes as a vital target upon the accumulation of internalized NPs, and lysosomal damage and autophagy dysfunction are emerging molecular mechanisms for NPs-elicited toxicity. Nevertheless, the interaction with lysosomes, ensuing adverse effects and the underlying mechanisms are still largely obscure, especially in NPs-induced vascular toxicity. In this study, silica nanoparticles (SiNPs) were utilized to explore the adverse effects on lysosome in vascular endothelial cells by using in vitro cultured human endothelial cells (HUVECs), and in-depth investigated the mechanisms involved. Consequently, the internalized SiNPs accumulated explicitly in the lysosomes, and caused lysosomal dysfunction, which were prominent on the increased lysosomal membrane permeability, decline in lysosomal quantity, destruction of acidic environment of lysosome, and also disruption of lysosomal enzymes activities, resulting in autophagy flux blockage and autophagy dysfunction. More importantly, mechanistic results revealed the SiNPs-caused lysosomal impairments and resultant autophagy dysfunction could promote oxidative stress, DNA damage and the eventual cell apoptosis activated by ROS/PARP1/AIF signaling pathway. These findings improved the understanding of SiNPs-induced vascular injury, and may provide novel information and warnings for SiNPs applications in the fields of nanomedicine.

Ppt1-deficiency dysregulates lysosomal Ca++ homeostasis contributing to pathogenesis in a mouse model of CLN1 disease.

Inactivating mutations in the PPT1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S-palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1-deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl-protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1-deficiency may impair lysosomal degradative function leading to infantile neuronal ceroid lipofuscinosis pathogenesis. Here, we report that in Ppt1-/- mice, which mimic CLN1 disease, low level of inositol 3-phosphate receptor-1 (IP3R1) that mediates Ca++ transport from the endoplasmic reticulum to the lysosome dysregulated lysosomal Ca++ homeostasis. Intriguingly, the transcription factor nuclear factor of activated T-cells, cytoplasmic 4 (NFATC4), which regulates IP3R1-expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Ppt1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1-expression, thereby dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++ -dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Ppt1-/- mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca++ homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease.