Lysosomal Activity Research Articles

VPS13C regulates phospho-Rab10-mediated lysosomal function in human dopaminergic neurons.

Loss-of-function mutations in VPS13C are linked to early-onset Parkinson's disease (PD). While VPS13C has been previously studied in non-neuronal cells, the neuronal role of VPS13C in disease-relevant human dopaminergic neurons has not been elucidated. Using live-cell microscopy, we investigated the role of VPS13C in regulating lysosomal dynamics and function in human iPSC-derived dopaminergic neurons. Loss of VPS13C in dopaminergic neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal contacts, leading to impaired lysosomal motility and cellular distribution, as well as defective lysosomal hydrolytic activity and acidification. We identified Rab10 as a phospho-dependent interactor of VPS13C on lysosomes and observed a decreased phospho-Rab10-mediated lysosomal stress response upon loss of VPS13C. These findings highlight an important role of VPS13C in regulating lysosomal homeostasis in human dopaminergic neurons and suggest that disruptions in Rab10-mediated lysosomal stress response contribute to disease pathogenesis in VPS13C-linked PD.

Fluorofenidone attenuates renal fibrosis by inhibiting lysosomal cathepsin‑mediated NLRP3 inflammasome activation.

Currently, no antifibrotic drug in clinical use can effectively treat renal fibrosis. Fluorofenidone (AKFPD), a novel pyridone agent, significantly reduces renal fibrosis by inhibiting the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome; however, the underlying mechanism of this inhibition is not fully understood. The present study aimed to reveal the molecular mechanism underlying the suppression of NLRP3 inflammasome activation by AKFPD. It investigated the effect of AKFPD on NLRP3 activation and lysosomal cathepsins in a unilateral ureteral obstruction (UUO) rat model, and hypoxia/reoxygenation (H/R)-treated HK-2 cells and murine peritoneal-derived macrophages (PDMs) stimulated with lipopolysaccharide (LPS) and ATP. The results confirmed that AKFPD suppressed renal interstitial fibrosis and inflammation by inhibiting NLRP3 inflammasome activation in UUO rat kidney tissues. In addition, AKFPD reduced the production of activated caspase-1 and maturation of IL-1β by suppressing NLRP3 inflammasome activation in H/R-treated HK-2 cells and murine PDMs stimulated with LPS and ATP. AKFPD also decreased the activities of cathepsins B, L and S both in vivo and in vitro. Notably, AKFPD downregulated cathepsin B expression and NLRP3 colocalization in the cytoplasm after lysosomal disruptions. Overall, the results suggested that AKFPD attenuates renal fibrosis by inhibiting lysosomal cathepsin-mediated activation of the NLRP3 inflammasome.

Fluctuation of lysosomal protein degradation in neural stem cells of the postnatal mouse brain.

Lysosomes are intracellular organelles responsible for degrading diverse macromolecules delivered from several pathways, including the endo-lysosomal and autophagic pathways. Recent reports have suggested that lysosomes are essential for regulating neural stem cells in developing, adult and aged brains. However, the activity of these lysosomes has yet to be monitored in these brain tissues. Here, we report the development of a new probe to measure lysosomal protein degradation in brain tissue by immunostaining. Our results indicate that lysosomal protein degradation fluctuates in neural stem cells of the hippocampal dentate gyrus, depending on age and brain disorders. Neural stem cells increase their lysosomal activity during hippocampal development in the dentate gyrus, but aging and aging-related disease reduce lysosomal activity. In addition, physical exercise increases lysosomal activity in neural stem cells and astrocytes in the dentate gyrus. We therefore propose that three different stages of lysosomal activity exist: the state of increase during development, the stable state during adulthood and the state of reduction due to damage caused by either age or disease.

The P4' Peptide-Carrying Bacillus subtilis in Cottonseed Meal Improves the Chinese Mitten Crab Eriocheir sinensis Innate Immunity, Redox Defense, and Growth Performance.

This study developed a recombinant Bacillus subtilis to carry the LGSPDVIVIR peptide (cmP4) isolated from the hydrolyzed products of cottonseed meal with excellent antioxidant and immune-enhancing properties in vitro. It was carried as a tandem of five cmP4 peptides (cmP4') to be stably expressed on a large scale. Then, its effectiveness was evaluated in Chinese mitten crab (Eriocheir sinensis) based on growth performance, redox defense, and innate immunity. A total of 280 crabs (mean body weight: 41.40 ± 0.14) were randomly assigned to seven diets including a control one (without B. subtilis) and six experimental ones with different doses (107,108, and 109 CFU/kg) of unmodified and recombinant B. subtilis, respectively, for 12 weeks. Each diet was tested in four tanks of crabs (10/tank). In terms of bacterial dosages, the final weight (FW), weight gain (WG), hemolymph and hepatopancreatic activities of superoxide dismutase (SOD), catalase (CAT), lysosome (LZM), acid phosphatase (ACP) and alkaline phosphatase (AKP), and hepatopancreatic transcriptions of cat, mitochondrial manganese superoxide dismutase (mtmnsod), thioredoxin-1 (trx1), and prophenoloxidase (propo) all increased significantly with increasing B. subtilis dosages, while hemolymph and hepatopancreatic malondialdehyde (MDA) content and the transcriptions of toll like receptors (tlrs), NF-κB-like transcription factor (relish), and lipopolysaccharide-induced TNF-α factor (litaf) all decreased remarkably. In terms of bacterial species, the recombinant B. subtilis group obtained significantly high values of FW, WG, hemolymph, and hepatopancreatic activities of SOD, CAT, LZM, ACP, and AKP, and the transcriptions of mtmnsod, peroxiredoxin 6 (prx6), and propo compared with the unmodified B. subtilis, while opposite results were noted in hemolymph and hepatopancreatic MDA content and the transcriptions of tlrs, relish, and litaf. These results indicated that dietary supplementation with 109 CFU/kg of recombinant B. subtilis can improve the growth performance, redox defense, and nonspecific immunity of E. sinensis.

Spatial transcriptomics reveals alterations in perivascular macrophage lipid metabolism in the onset of Wooden Breast myopathy in broiler chickens

This study aims to use spatial transcriptomics to characterize the cell-type-specific expression profile associated with the microscopic features observed in Wooden Breast myopathy. 1 cm3 muscle sample was dissected from the cranial part of the right pectoralis major muscle from three randomly sampled broiler chickens at 23 days post-hatch and processed with Visium Spatial Gene Expression kits (10X Genomics), followed by high-resolution imaging and sequencing on the Illumina Nextseq 2000 system. WB classification was based on histopathologic features identified. Sequence reads were aligned to the chicken reference genome (Galgal6) and mapped to histological images. Unsupervised K-means clustering and Seurat integrative analysis differentiated histologic features and their specific gene expression pattern, including lipid laden macrophages (LLM), unaffected myofibers, myositis and vasculature. In particular, LLM exhibited reprogramming of lipid metabolism with up-regulated lipid transporters and genes in peroxisome proliferator-activated receptors pathway, possibly through P. Moreover, overexpression of fatty acid binding protein 5 could enhance fatty acid uptake in adjacent veins. In myositis regions, increased expression of cathepsins may play a role in muscle homeostasis and repair by mediating lysosomal activity and apoptosis. A better knowledge of different cell-type interactions at early stages of WB is essential in developing a comprehensive understanding.

Amyloid-β aggregates activate peripheral monocytes in mild cognitive impairment

The peripheral immune system is important in neurodegenerative diseases, both in protecting and inflaming the brain, but the underlying mechanisms remain elusive. Alzheimer’s Disease is commonly preceded by a prodromal period. Here, we report the presence of large Aβ aggregates in plasma from patients with mild cognitive impairment (n = 38). The aggregates are associated with low level Alzheimer’s Disease-like brain pathology as observed by 11C-PiB PET and 18F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aβ aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aβ. Hydrodynamic calculations suggest Aβ aggregates associate with vessel walls of the cortical capillaries. In turn, we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis.

Inhibition of cysteine protease cathepsin L increases the level and activity of lysosomal glucocerebrosidase.

The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L-KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L-mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.

The potential of Rhodobacter sphaeroides extract as an alternative supplement for cell culture systems.

It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.

Ratiometric Fluorescent Probe for Real-Time Detection of β-Galactosidase Activity in Lysosomes and Its Application in Drug-Induced Senescence Imaging.

Senescence is an important biological process, which leads to the gradual degradation of its physiological function and increases morbidity and mortality. Herein, a novel ratiometric fluorescent probe (P1) was constructed by using benzothiazolyl acetonitrile dye as fluorophore, exhibiting significantly enhanced blue-shifted emission to indicate the activity of β-galactosidase (β-gal), a commonly used biomarker for the detection of senescent cells. After incubation with β-gal, the excimer emission of P1 at 620 nm was weakened, while the emission at 533 nm was significantly enhanced, forming an obvious ratiometric probe with high sensitivity and low detection limit (2.7 mU·mL-1). More importantly, probe P1 can locate lysosomes accurately, allowing us to monitor the emergence of living cell senescence in real time. P1 was successfully used to detect β-gal activity in PC-12 cells, Hep G2 cells, and RAW 264.7 cells. It showed strong green fluorescence signal in senescent cells and red fluorescence signal in normal cells, indicating that it can detect endogenous senescence-related β-gal content in living cells. For in vivo drug-induced senescence imaging, after 5 weeks of injection of D-galactose or hydroxyurea, the mice showed significant fluorescence enhancement in specific channels to indicate the activity of β-gal in vivo. At the same time, the senescence of cell-specific organs and skin tissues at the organ level were also detected, which proved that the drug-induced senescence of brain, skin, and muscle tissues was the most serious. These results supported the important application value of P1 in senescence biomedical research.

Cutaneous Redox Senescence.

Our current understanding of skin cell senescence involves the role of environmental stressors (UV, O3, cigarette smoke, particulate matter, etc.), lifestyle (diet, exercise, etc.) as well as genetic factors (metabolic changes, hormonal, etc.). The common mechanism of action of these stressors is the disturbance of cellular redox balance characterized by increased free radicals and reactive oxygen species (ROS), and when these overload the intrinsic antioxidant defense system, it can lead to an oxidative stress cellular condition. The main redox mechanisms that activate cellular senescence in the skin involve (1) the oxidative damage of telomeres causing their shortening; (2) the oxidation of proteomes and DNA damage; (3) an a in lysosomal mass through the increased activity of resident enzymes such as senescence-associated β-galactosidase (SA-β-gal) as well as other proteins that are products of lysosomal activity; (4) and the increased expression of SASP, in particular pro-inflammatory cytokines transcriptionally regulated by NF-κB. However, the main targets of ROS on the skin are the proteome (oxi-proteome), followed by telomeres, nucleic acids (DNAs), lipids, proteins, and cytoplasmic organelles. As a result, cell cycle arrest pathways, lipid peroxidation, increased lysosomal content and dysfunctional mitochondria, and SASP synthesis occur. Furthermore, oxidative stress in skin cells increases the activity of p16INK4A and p53 as inhibitors of Rb and CDks, which are important for maintaining the cell cycle. p53 also promotes the inactivation of mTOR-mediated autophagic and apoptotic pathways, leading to senescence. However, these markers alone cannot establish the state of cellular senescence, and multiple analyses are encouraged for confirmation. An updated and more comprehensive approach to investigating skin senescence should include further assays of ox-inflammatory molecular pathways that can consolidate the understanding of cutaneous redox senescence.

Intranasal Administration of Sugarcane Ash Causes Chronic Kidney Disease in Rats.

Background. Silica nanoparticles found in sugarcane ash have been postulated to be a toxicant contributing to chronic kidney disease of unknown etiology (CKDu). However, while the administration of manufactured silica nanoparticles is known to cause chronic tubulointerstitial disease in rats, the effect of administering sugarcane ash on kidney pathology remains unknown. Here we investigate whether sugarcane ash can induce CKD in rats. Methods. Sugarcane ash was administered for 13 weeks into the nares of rats (5 mg/day for 5d/week), and blood, urine and kidney tissues were collected at 13 weeks (at the end of ash administration) and in a separate group of rats at 24 weeks (11 weeks after stopping ash administration). Kidney histology was evaluated, and inflammation and fibrosis (collagen deposition) measured. Results. Sugarcane ash exposure led to the accumulation of silica in the kidneys, lungs, liver and spleen of rats. Mild proteinuria developed although renal function was largely maintained. However, biopsies showed focal glomeruli with segmental glomerulosclerosis, and tubulointerstitial inflammation and fibrosis that tended to worsen even after the ash administration had been stopped. Staining for the lysosomal marker, LAMP-1, showed decreased staining in ash administered rats consistent with lysosomal activation. Conclusion. Sugarcane ash containing silica nanoparticles can cause CKD in rats.

Wnt signaling in cell adhesion, development, and colon cancer.

Wnt signaling is essential for embryonic development, influencing processes such as axis formation, cell proliferation and differentiation, cell fate decisions, and axon guidance. It also plays a role in maintaining tissue homeostasis in adult organisms. The loss of normal cell polarity and adhesion caused by Wnt signaling activation is a fundamental step for tumor progression and metastasis. Activating the canonical Wnt pathway is a driving force in many human cancers, especially colorectal, hepatocellular, and mammary carcinomas. Wnt causes the stabilization and nuclear transport of newly synthesized transcriptional regulator β-catenin. The generally accepted view is that the canonical effects of Wnt growth factors are caused by the transcription of β-catenin target genes. Here, we review recent findings that indicate Wnt is a regulator of many other cellular physiological activities, such as macropinocytosis, endosome trafficking, protein stability, focal adhesions, and lysosomal activity. Some of these regulatory responses occur within minutes and do not require new protein synthesis, indicating that there is much more to Wnt beyond the well-established transcriptional role of β-catenin. The main conclusion that emerges from these studies is that in basal cell conditions, the activity of the key protein kinase GSK3, which is inhibited by Wnt pathway activation, normally represses the actin machinery that orchestrates macropinocytosis with implications in cancer. These contributions expand our understanding of the multifaceted roles of Wnt signaling in cellular processes, development, and cancer, providing insights into potential therapeutic targets and strategies.

The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

Carnosine regulation of intracellular pH homeostasis promotes lysosome-dependent tumor immunoevasion.

Tumor cells and surrounding immune cells undergo metabolic reprogramming, leading to an acidic tumor microenvironment. However, it is unclear how tumor cells adapt to this acidic stress during tumor progression. Here we show that carnosine, a mobile buffering metabolite that accumulates under hypoxia in tumor cells, regulates intracellular pH homeostasis and drives lysosome-dependent tumor immune evasion. A previously unrecognized isoform of carnosine synthase, CARNS2, promotes carnosine synthesis under hypoxia. Carnosine maintains intracellular pH (pHi) homeostasis by functioning as a mobile proton carrier to accelerate cytosolic H+ mobility and release, which in turn controls lysosomal subcellular distribution, acidification and activity. Furthermore, by maintaining lysosomal activity, carnosine facilitates nuclear transcription factor X-box binding 1 (NFX1) degradation, triggering galectin-9 and T-cell-mediated immune escape and tumorigenesis. These findings indicate an unconventional mechanism for pHi regulation in cancer cells and demonstrate how lysosome contributes to immune evasion, thus providing a basis for development of combined therapeutic strategies against hepatocellular carcinoma that exploit disrupted pHi homeostasis with immune checkpoint blockade.

The ubiquitin ligases Cbl and Cbl-b regulate macrophage growth by controlling CSF-1R import into macropinosomes.

The ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow-derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKO macrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling, and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNA-sequencing comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.

Lysosomal BK channels facilitate silica-induced inflammation in macrophages

Background Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1β release. Methods Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1β release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. Results PAX pretreatment significantly attenuated silica-induced cell death and IL-1β release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1β release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. Conclusion Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1β release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.

Vitamin B12 Deficiency and the Nervous System: Beyond Metabolic Decompensation-Comparing Biological Models and Gaining New Insights into Molecular and Cellular Mechanisms.

Vitamin B12 (VitB12) is a micronutrient and acts as a cofactor for fundamental biochemical reactions: the synthesis of succinyl-CoA from methylmalonyl-CoA and biotin, and the synthesis of methionine from folic acid and homocysteine. VitB12 deficiency can determine a wide range of diseases, including nervous system impairments. Although clinical evidence shows a direct role of VitB12 in neuronal homeostasis, the molecular mechanisms are yet to be characterized in depth. Earlier investigations focused on exploring the biochemical shifts resulting from a deficiency in the function of VitB12 as a coenzyme, while more recent studies propose a broader mechanism, encompassing changes at the molecular/cellular levels. Here, we explore existing study models employed to investigate the role of VitB12 in the nervous system, including the challenges inherent in replicating deficiency/supplementation in experimental settings. Moreover, we discuss the potential biochemical alterations and ensuing mechanisms that might be modified at the molecular/cellular level (such as epigenetic modifications or changes in lysosomal activity). We also address the role of VitB12 deficiency in initiating processes that contribute to nervous system deterioration, including ROS accumulation, inflammation, and demyelination. Consequently, a complex biological landscape emerges, requiring further investigative efforts to grasp the intricacies involved and identify potential therapeutic targets.

Lysosomal membrane integrity in fibroblasts derived from patients with Gaucher disease.

Gaucher disease (GD) is a recessively inherited lysosomal storage disorder characterized by a deficiency of lysosomal glucocerebrosidase (GBA1). This deficiency results in the accumulation of its substrate, glucosylceramide (GlcCer), within lysosomes. Here, we investigated lysosomal abnormalities in fibroblasts derived from patients with GD. It is noteworthy that the cellular distribution of lysosomes and lysosomal proteolytic activity remained largely unaffected in GD fibroblasts. However, we found that lysosomal membranes of GD fibroblasts were susceptible to damage when exposed to a lysosomotropic agent. Moreover, the susceptibility of lysosomal membranes to a lysosomotropic agent could be partly restored by exogenous expression of wild-type GBA1. Here, we report that the lysosomal membrane integrity is altered in GD fibroblasts, but lysosomal distribution and proteolytic activity is not significantly altered.Key words: glucosylceramide, lysosome, Gaucher disease, lysosomotropic agent.

Effects of Dietary Thymoquinone Inclusion on Antioxidative, Oxidative, Pro-Inflammatory Responses, Semen Attributes and Testicular Changes in Heat-Stressed Rabbit Bucks

Abstract Heat stress (HS) is considered a severe concern for rabbit buck’s health, welfare and reproductive features. Thymoquinone (THQ) is a natural molecule with robust antioxidant, antimicrobial, and anti-inflammatory properties. Thereafter, three months of feeding research was applied to assess the role of THQ-enriched diets in improving the health, semen attributes and testicular changes of stressed rabbit bucks. Forty rabbit bucks were used in this research, and animals were fed diets containing 0 (THQ0; serve as a heat stress group), 100 (THQ100), 200 (THQ200), and 400 (THQ400) mg/kg of THQ for three months continually during the summer season. Outcomes displayed that THQ significantly enhanced FBW (final body weight), DBWG (daily body weight gain), and FI (feed intake) but did not affect FCR (feed conversion ratio). Compared with the THQ0 group, dietary THQ addition significantly decreased creatinine, TB (total bilirubin), TG (triglycerides), and GGT (gamma-glutamyl transferase) levels. Bucks receiving 200 mg of THQ had higher TAC (total antioxidant capacity) and GSH (glutathione) than other groups. At the same time, MDA was significantly shrunk in THQ-treated groups relative to the THQ group. The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were significantly diminished (P <0.001), but nitric oxide and lysosome activity were significantly increased in bucks given diets containing THQ (200 and 400 mg/kg) as relative to the TH0 group. Libido score and reaction time were significantly improved by THQ dietary inclusion. Semen attributes, including volume, motility, concentration, and vitality, were significantly augmented in THQ100 and THQ200 groups. Histological screening of testicular tissues showed moderate to severe degenerative and necrotic changes in the testicular tissues of bucks exposed to HS. This was enhanced with the supplementation of THQ at different levels. Collectively, THQ (200 or 400 mg/kg diet) can improve health, semen attributes and restore testicular damage in rabbit bucks via boosting antioxidant and reducing inflammatory cytokines.

Immunological effects of ractopamine in rabbits receiving the viral inactivated rabbit hemorrhagic disease vaccine.

There is an obvious lack of information about the effects of ractopamine, a ß-adrenergic agonist, on the growth performance and immune responses of rabbits, particularly in those receiving the viral rabbit hemorrhagic disease (RHD) vaccine. The current study was undertaken to study the effects of ractopamine on growth performances and immunological parameters in rabbits inoculated with the viral RHD vaccine. Experimental rabbits were grouped into four groups, the first acted as a control and received distilled water, the second received ractopamine, the third received inactivated RHD vaccine, and the fourth received both ractopamine, and inactivated RHD vaccine. Then, blood analysis, histopathological, histomorphometric, and immunohistochemistry (IHC) examinations were followed. The obtained results demonstrated that ractopamine induced significant increases in body weight gain, neutrophils, monocytes, nitric oxide, lysosome, and improved feed conversion rate. A significant decrease in lymphocytes with insignificant decreases in eosinophils, phagocytic % and index, serum total protein, α, β, and γ globulin were observed. Vaccinated rabbits only showed a marked rise in WBCs, neutrophils, monocytes, eosinophils, basophils, phagocytic index and activity, nitric oxide, lysosome activity, total protein, albumin, γ globulin, and a decrease in lymphocytes. Rabbits that received ractopamine and then vaccinated had insignificant increases in body weight, weight gain, WBCs, neutrophils, monocyte, eosinophils, basophils, phagocytic activity, and index, globulins besides a significant decrease in lymphocytes. Pathologically, rabbits that received ractopamine alone, with a vaccine or vaccinated only showed an increase in villus length, villus width, and absorption surface area. IHC of rabbits' liver and kidneys of the control and vaccinated group showed negative expression for caspase-3, but rabbits received ractopamine only or rabbits vaccinated and received ractopamine showed diffuse positive moderate expression for caspase-3. Ractopamine induced several adverse effects on the immune responses of the rabbits inoculated with the viral HRD vaccine.