Lysosomal Transport Research Articles

Knockout of the lysosomal membrane protein, LAMP2C, improves transient gene expression in HEK293 cells via increased intracellular plasmid availability.

Plasmid-based transfection can be used in many applications such as transient gene expression (TGE)-based therapeutic protein production. These applications preferentially require maximization of intracellular plasmid availability. Here, we applied a lysosome engineering approach to alleviate lysosome-mediated nucleic acid degradation and enhance the TGE in mammalian cells. By knocking out the lysosomal membrane protein LAMP2C, which is known to be the main player in RNautophagy/DNautophagy (RDA), we significantly improved transient fluorescent protein expression in HEK293 cells by improving the retention rate of transfected plasmids; however, this effect was not observed in CHO cells. Additional knockout of a lysosomal membrane transporter and another RDA player, SIDT2, was ineffective, regardless of the presence of LAMP2C. LAMP2C knockout enhanced TGE-based mAb production in HEK293 cells by up to 2.82-fold increase in specific mAb productivity. Taken together, these results demonstrate that HEK293 cells can be engineered to improve the usage of the transfected plasmid via knockout of the lysosomal membrane protein LAMP2C and provide efficient host cells in TGE systems for therapeutic protein production.

Macrolide and Ketolide Antibiotics Inhibit the Cytotoxic Effect of Trastuzumab Emtansine in HER2-Positive Breast Cancer Cells: Implication of a Potential Drug-ADC Interaction in Cancer Chemotherapy.

Macrolides are widely used for the long-term treatment of infections and chronic inflammatory diseases. The pharmacokinetic features of macrolides include extensive tissue distribution because of favorable membrane permeability and accumulation within lysosomes. Trastuzumab emtansine (T-DM1), a HER2-targeting antibody-drug conjugate (ADC), is catabolized in the lysosomes, where Lys-SMCC-DM1, a potent cytotoxic agent, is processed by proteinase degradation and subsequently released from the lysosomes to the cytoplasm through the lysosomal membrane transporter SLC46A3, resulting in an antitumor effect. We recently demonstrated that erythromycin and clarithromycin inhibit SLC46A3 and attenuate the cytotoxicity of T-DM1; however, the effect of other macrolides and ketolides has not been determined. In this study, we evaluated the effect of macrolide and ketolide antibiotics on T-DM1 cytotoxicity in a human breast cancer cell line, KPL-4. Macrolides used in the clinic, such as roxithromycin, azithromycin, and josamycin, as well as solithromycin, a ketolide under clinical development, significantly attenuated T-DM1 cytotoxicity in addition to erythromycin and clarithromycin. Of these, azithromycin was the most potent inhibitor of T-DM1 efficacy. These antibiotics significantly inhibited the transport function of SLC46A3 in a concentration-dependent manner. Moreover, these compounds extensively accumulated in the lysosomes at the levels estimated to be 0.41-13.6 mM when cells were incubated with them at a 2 μM concentration. The immunofluorescence staining of trastuzumab revealed that azithromycin and solithromycin inhibit the degradation of T-DM1 in the lysosomes. These results suggest that the attenuation of T-DM1 cytotoxicity by macrolide and ketolide antibiotics involves their lysosomal accumulation and results in their greater lysosomal concentrations to inhibit the SLC46A3 function and T-DM1 degradation. This suggests a potential drug-ADC interaction during cancer chemotherapy.

Multi-modal proteomic characterization of lysosomal function and proteostasis in progranulin-deficient neurons

BackgroundProgranulin (PGRN) is a lysosomal glycoprotein implicated in various neurodegenerative diseases, including frontotemporal dementia and neuronal ceroid lipofuscinosis. Over 70 mutations discovered in the GRN gene all result in reduced expression of the PGRN protein. Genetic and functional studies point toward a regulatory role for PGRN in lysosome functions. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomes remain unclear.MethodsWe developed multifaceted proteomic techniques to characterize the dynamic lysosomal biology in living human neurons and fixed mouse brain tissues. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactome in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in human i3Neurons for the first time.ResultsLeveraging the multi-modal proteomics and live-cell imaging techniques, we comprehensively characterized how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. We found that PGRN loss impairs the lysosome’s degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased hydrolases within the lysosome, altered protein regulations related to lysosomal transport, and elevated lysosomal pH. Consistent with impairments in lysosomal function, GRN-null i3Neurons and frontotemporal dementia patient-derived i3Neurons carrying GRN mutation showed pronounced alterations in protein turnover, such as cathepsins and proteins related to supramolecular polymerization and inherited neurodegenerative diseases.ConclusionThis study suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which influences global proteostasis in neurons. Beyond the study of progranulin deficiency, these newly developed proteomic methods in neurons and brain tissues provided useful tools and data resources for the field to study the highly dynamic neuronal lysosome biology.Graphical

Omic Studies on In Vitro Cystinosis Model: siRNA-Mediated CTNS Gene Silencing in HK-2 Cells

Cystinosis is an autosomal recessive disease caused by mutations in the CTNS gene encoding a protein called cystinosine, which is a lysosomal cystine transporter. Disease-causing mutations lead to accumulation of cystine crystals in the lysosomes, thereby causing dysfunction of vital organs. Determination of the increased leukocyte cystine level is one of the most used methods for diagnosis. However, this method is expensive, difficult to perform, and may yield different results in different laboratories. In this study, a disease model was created with CTNS gene-silenced HK2 cells, which can mimic cystinosis in cell culture, and multiomics methods (ie, proteomics, metabolomics, and fluxomics) were implemented at this cell culture to investigate new biomarkers for the diagnosis. CTNS-silenced cell line exhibited distinct metabolic profiles compared with the control cell line. Pathway analysis highlighted significant alterations in various metabolic pathways, including alanine, aspartate, and glutamate metabolism; glutathione metabolism; aminoacyl-tRNA biosynthesis; arginine and proline metabolism; beta-alanine metabolism; ascorbate and aldarate metabolism; and histidine metabolism upon CTNS silencing. Fluxomics analysis revealed increased cycle rates of Krebs cycle intermediates such as fumarate, malate, and citrate, accompanied by enhanced activation of inorganic phosphate and ATP production. Furthermore, proteomic analysis unveiled differential expression levels of key proteins involved in crucial cellular processes. Notably, peptidyl-prolyl cis–trans isomerase A, translation elongation factor 1-beta (EF-1beta), and 60S acidic ribosomal protein decreased in CTNS-silenced cells. Additionally, levels of P0 and tubulin α-1A chain were reduced, whereas levels of 40S ribosomal protein S8 and Midasin increased. Overall, our study, through the utilization of an in vitro cystinosis model and comprehensive multiomics approach, led to the way toward the identification of potential new biomarkers while offering valuable insights into the pathogenesis of cystinosis.

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis.

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.

TMEM241 is a UDP-N-acetylglucosamine transporter required for M6P modification of NPC2 and cholesterol transport

Accurate intracellular cholesterol traffic plays crucial roles. Niemann Pick type C (NPC) proteins NPC1 and NPC2, are two lysosomal cholesterol transporters that mediate the cholesterol exit from lysosomes. However, other proteins involved in this process remain poorly defined. Here, we find that the previously unannotated protein TMEM241 is required for cholesterol egressing from lysosomes through amphotericin B-based genome-wide CRISPR-Cas9 KO screening. Ablation of TMEM241 caused impaired sorting of NPC2, a protein utilizes the mannose-6-phosphate (M6P) modification for lysosomal targeting, resulting in cholesterol accumulation in the lysosomes. TMEM241 is a member of solute transporters 35 nucleotide sugar transporters family and localizes on the cis-Golgi network. Our data indicate that TMEM241 transports UDP-N-acetylglucosamine (UDP-GlcNAc) into Golgi lumen and UDP-GlcNAc is used for the M6P modification of proteins including NPC2. Furthermore, Tmem241-deficient mice display cholesterol accumulation in pulmonary cells and behave pulmonary injury and hypokinesia. Taken together, we demonstrate that TMEM241 is a Golgi-localized UDP-GlcNAc transporter and loss of TMEM241 causes cholesterol accumulation in lysosomes because of the impaired M6P-dependent lysosomal targeting of NPC2.

Structural basis for recruitment of TASL by SLC15A4 in human endolysosomal TLR signaling

Toll-like receptors (TLRs) are a class of proteins that play critical roles in recognizing pathogens and initiating innate immune responses. TASL, a recently identified innate immune adaptor protein for endolysosomal TLR7/8/9 signaling, is recruited by the lysosomal proton-coupled amino-acid transporter SLC15A4, and then activates IRF5, which in turn triggers the transcription of type I interferons and cytokines. Here, we report three cryo-electron microscopy (cryo-EM) structures of human SLC15A4 in the apo monomeric and dimeric state and as a TASL-bound complex. The apo forms are in an outward-facing conformation, with the dimeric form showing an extensive interface involving four cholesterol molecules. The structure of the TASL-bound complex reveals an unprecedented interaction mode with solute carriers. During the recruitment of TASL, SLC15A4 undergoes a conformational change from an outward-facing, lysosomal lumen-exposed state to an inward-facing state to form a binding pocket, allowing the N-terminal helix of TASL to be inserted into. Our findings provide insights into the molecular basis of regulatory switch involving a human solute carrier and offers an important framework for structure-guided drug discovery targeting SLC15A4-TASL-related human autoimmune diseases.

Axonal Transport of Lysosomes Is Unaffected in Glucocerebrosidase-Inhibited iPSC-Derived Forebrain Neurons.

Lysosomes are acidic organelles that traffic throughout neurons delivering catabolic enzymes to distal regions of the cell and maintaining degradative demands. Loss of function mutations in the gene GBA encoding the lysosomal enzyme glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher's disease (GD) and are the most common genetic risk factor for synucleinopathies like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GCase degrades the membrane lipid glucosylceramide (GlcCer) and mutations in GBA, or inhibiting its activity, results in the accumulation of GlcCer and disturbs the composition of the lysosomal membrane. The lysosomal membrane serves as the platform to which intracellular trafficking complexes are recruited and activated. Here, we investigated whether lysosomal trafficking in axons was altered by inhibition of GCase with the pharmacological agent Conduritol B Epoxide (CBE). Using live cell imaging in human male induced pluripotent human stem cell (iPSC)-derived forebrain neurons, we demonstrated that lysosomal transport was similar in both control and CBE-treated neurons. Furthermore, we tested whether lysosomal rupture, a process implicated in various neurodegenerative disorders, was affected by inhibition of GCase. Using L-leucyl-L-leucine methyl ester (LLoME) to induce lysosomal membrane damage and immunocytochemical staining for markers of lysosomal rupture, we found no difference in susceptibility to rupture between control and CBE-treated neurons. These results suggest the loss of GCase activity does not contribute to neurodegenerative disease by disrupting either lysosomal transport or rupture.

Activity of Lysosomal ABCC3, ABCC5 and ABCC10 is Responsible for Lysosomal Sequestration of Doxorubicin and Paclitaxel-Oregongreen488 in Paclitaxel-Resistant Cancer Cell Lines.

Cancer cell multidrug resistance induced by paclitaxel contributes to the high failure rates of chemotherapy and relapse of the disease. Several mechanisms have been described that underlie the observed resistance, including the overexpression of ABCB1 (P-glycoprotein), which represents an ATP-binding cassette (ABC) transmembrane protein, and its functional occurrence in lysosomal membranes is linked to drug accumulation in these organelles. Using clinically-relevant models of paclitaxel-resistant triple-negative breast cancer and non-small cell lung cancer cell lines, we provide evidence for the role of ABCC subfamily members in the lysosomal sequestration of drugs in multidrug resistant phenotypes. Proteins expression level and its cellular localisation was measured using Western Blot and confocal microscopy. Drug accumulation was analysed by confocal microscopy and flow cytometry. Drug cytotoxicity was tested using resasurin assay and anexin V propidium iodide staining. Regardless of the alteration in gene expression, paclitaxel induced the intracellular redistribution of ABCC3, ABCC5 and ABCC10 and their enrichment in lysosomes. The use of ABCC inhibitors and transient silencing of these three genes limited the accumulation of doxorubicin and paclitaxel-OregonGreen488 in lysosomes, while having little impact on the total drug level inside cells. The cancer cells were also sensitized to various structurally unrelated chemotherapeutics of differing acidity. The results suggest that lysosome membranes anchored ABCC proteins which remained functionally active and were capable to load chemotherapeutics into lysosomes in paclitaxel-resistant cancer cells. Therefore, targeting of lysosomal ABCC transporters may help to overcome paclitaxel-induced resistance by reducing the accumulation of drugs in lysosomes.

Loss of function of ENT3 drives histiocytosis and inflammation through TLR-MAPK signaling

AbstractHistiocytoses are inflammatory myeloid neoplasms often driven by somatic activating mutations in mitogen-activated protein kinase (MAPK) cascade genes. H syndrome is an inflammatory genetic disorder caused by germ line loss-of-function mutations in SLC29A3, encoding the lysosomal equilibrative nucleoside transporter 3 (ENT3). Patients with H syndrome are predisposed to develop histiocytosis, yet the mechanism is unclear. Here, through phenotypic, molecular, and functional analysis of primary cells from a cohort of patients with H syndrome, we reveal the molecular pathway leading to histiocytosis and inflammation in this genetic disorder. We show that loss of function of ENT3 activates nucleoside-sensing toll-like receptors (TLR) and downstream MAPK signaling, inducing cytokine secretion and inflammation. Importantly, MEK inhibitor therapy led to resolution of histiocytosis and inflammation in a patient with H syndrome. These results demonstrate a yet-unrecognized link between a defect in a lysosomal transporter and pathological activation of MAPK signaling, establishing a novel pathway leading to histiocytosis and inflammation.

LYSET is a New Factor Critical for Lysosomal Enzyme Transport and Viral Infection

LYSET is a New Factor Critical for Lysosomal Enzyme Transport and Viral Infection

LYSET is a New Factor Critical for Lysosomal Enzyme Transport and Viral Infection

LYSET is a New Factor Critical for Lysosomal Enzyme Transport and Viral Infection

Omics-Based Approaches for the Characterization of Pompe Disease Metabolic Phenotypes.

Lysosomal storage disorders (LSDs) constitute a large group of rare, multisystemic, inherited disorders of metabolism, characterized by defects in lysosomal enzymes, accessory proteins, membrane transporters or trafficking proteins. Pompe disease (PD) is produced by mutations in the acid alpha-glucosidase (GAA) lysosomal enzyme. This enzymatic deficiency leads to the aberrant accumulation of glycogen in the lysosome. The onset of symptoms, including a variety of neurological and multiple-organ pathologies, can range from birth to adulthood, and disease severity can vary between individuals. Although very significant advances related to the development of new treatments, and also to the improvement of newborn screening programs and tools for a more accurate diagnosis and follow-up of patients, have occurred over recent years, there exists an unmet need for further understanding the molecular mechanisms underlying the progression of the disease. Also, the reason why currently available treatments lose effectiveness over time in some patients is not completely understood. In this scenario, characterization of the metabolic phenotype is a valuable approach to gain insights into the global impact of lysosomal dysfunction, and its potential correlation with clinical progression and response to therapies. These approaches represent a discovery tool for investigating disease-induced modifications in the complete metabolic profile, including large numbers of metabolites that are simultaneously analyzed, enabling the identification of novel potential biomarkers associated with these conditions. This review aims to highlight the most relevant findings of recently published omics-based studies with a particular focus on describing the clinical potential of the specific metabolic phenotypes associated to different subgroups of PD patients.

Combined analysis of mRNA and miRNA reveals the mechanism of pacific white shrimp (Litopenaeus vannamei) under acute alkalinity stress.

The pacific white shrimp (Litopenaeus vannamei) is now a more common aquaculture species in saline-alkali waters, while alkalinity stress is considered to be one of the stressors for shrimp. Thus, an understanding of the molecular response to alkalinity stress is critical for advancing the sustainability of culture in pacific white shrimp. In this study, we aimed to explore the response mechanism to acute high-alkaline stress by RNA-seq at low-alkaline (50 mg/L) and high-alkaline (350 mg/L). We identified 215 differentially expressed mRNAs (DEGs) and 35 differentially expressed miRNAs (DEMs), of which 180 DEGs and 28 DEMs were up-regulated, 35 DEGs and 7 DEMs were down-regulated, respectively. The DEGs were enriched in several pathways, including carbohydrate digestion and absorption, pancreatic secretion, starch and sucrose metabolism, antigen processing and presentation and glutathione metabolism. The DEMs involved in lysosome and ion transport related pathways were significantly up-regulated. We also achieved 42 DEGs, which were targeted by DEMs. miRNA-mRNA regulatory network was constructed by integrated analysis of miRNA-mRNA data. We detected several genes and miRNAs which were identified as candidate regulators of alkalinity stress, and expression patterns of key genes related to alkalinity stress in pacific white shrimp. Among these genes, the expression levels of most key genes enriched in ion regulation, digestion and immunity were increased, and the expression levels of genes enriched in metabolism were down-regulated. This research indicated that the homeostatic regulation and digestion changed significantly under acute alkaline stress, and the variations from metabolic and immunity can cope with the osmotic shock of alkalinity stress in pacific white shrimp. This study provides key clues for exploring the molecular mechanism of pacific white shrimp under acute alkalinity stress, and also gives scientific basis for the optimisation of saline-alkaline aquaculture technology.

Molecular characterization of a cation-dependent mannose-6-phosphate receptor gene in Crassostrea hongkongensis and its responsiveness in Vibrio alginolyticus infection.

The cation-dependent mannose-6-phosphate receptor (CD-M6PR) is a P-type lectin that plays a crucial role in lysosomal enzyme transport, bacterial resistance, and viral entry. In this study, we cloned and analyzed the ORF of the CD-M6PR gene from Crassostrea hongkongensis and named it ChCD-M6PR. We analyzed the nucleotide and amino acid sequence of ChCD-M6PR, its tissue expression pattern and immune response to Vibrio alginolyticus. Our results showed that the ORF of ChCD-M6PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus, as well as Man-6-P_recep, ATG27 and transmembrane structural domains. Phylogenetic analysis indicated that Crassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6PR. The ChCD-M6PR gene was found to be expressed in various tissues, with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR. Furthermore, the expression of ChCD-M6PR gene was significantly up-regulated for a short time in response to Vibrio alginolyticus infection in the gill and hemocytes, while it was down-regulated in the gonads. The expression patterns of ChCD-M6PR also varied in the other tissues. The 96h cumulative mortality rate of Crassostrea hongkongensis infected with Vibrio alginolyticus after knockdown the ChCD-M6PR gene was significantly higher. Overall, our findings suggests that ChCD-M6PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection, and its tissue-specific expression patterns may be indicatitive of varied immune responses across tissues.

Starry-like Cornea: A Case of Ocular Cystinosis

Ocular cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene, which encodes a lysosomal cystine transporter protein. This results in the accumulation of cystine crystals in various ocular structures, leading to a range of ocular manifestations. The incidence of cystinosis is estimated to be 1 in 100,000 to 200,000 live births, with a higher prevalence in certain populations such as those of European descent. We report the case of a 5-year-old child with ocular cystinosis. The ophthalmological examination revealed a photophobic child with a visual acuity of 3/10 in both eyes (Pigassou scale), and diffuse stromal crystal deposits over the entire cornea in both eyes. The rest of the examination was unremarkable. The patient was referred to pediatrics for work-up of storage disease and was diagnosed with ocular and nephrological cystinosis. The patient was able to start general treatment with Mercaptamine with improvement in renal function, but was unable to obtain local treatment due to lack of funds. The patient is still being followed in our clinic with stable corneal involvement. Ocular cystinosis is a very rare genetic disorder. There are three main types of cystinosis: nephropathic cystinosis and non-nephropathic cystinosis. Nephropathic cystinosis divides further on infantile and intermediate. The most common ocular manifestation of cystinosis is corneal cystine crystal deposit, which typically presents in the first year of life and can lead to photophobia, tearing, and decreased visual acuity. The corneal crystals can also cause recurrent erosions, which can be very painful. The severity of corneal involvement can range from mild punctate deposition to severe confluent crystal accumulation that can lead to corneal scarring and vision loss. Cysteamine drops, which are a form of cysteamine hydrochloride, can help dissolve the cystine crystals and improve corneal clarity, prevent further vision loss, and reduce the frequency of recurrent erosions. Early diagnosis and treatment are crucial in preventing further ocular damage in individuals with cystinosis. Regular ophthalmologic examinations should be conducted to monitor for ocular manifestations and initiate treatment as early as possible. A multidisciplinary approach is necessary, involving ophthalmologists, nephrologists, and other specialists, to manage the systemic manifestations of cystinosis.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

The Npc2Gt(LST105)BygNya mouse signifies pathological changes comparable to human Niemann-Pick type C2 disease

IntroductionNiemann-Pick type C2 disease (NP-C2) is a fatal neurovisceral disorder caused by defects in the lysosomal cholesterol transporter protein NPC2. Consequently, cholesterol and other lipids accumulate within the lysosomes, causing a heterogeneous spectrum of clinical manifestations. Murine models are essential for increasing the understanding of the complex pathology of NP-C2. This study, therefore, aims to describe the neurovisceral pathology in the NPC2-deficient mouse model to evaluate its correlation to human NP-C2. MethodsNpc2−/− mice holding the LST105 mutation were used in the present study (Npc2Gt(LST105)BygNya). Body and organ weight and histopathological evaluations were carried out in six and 12-week-old Npc2−/− mice, with a special emphasis on neuropathology. The Purkinje cell (PC) marker calbindin, the astrocytic marker GFAP, and the microglia marker IBA1 were included to assess PC degeneration and neuroinflammation, respectively. In addition, the pathology of the liver, lungs, and spleen was assessed using hematoxylin and eosin staining. ResultsSix weeks old pre-symptomatic Npc2−/− mice showed splenomegaly and obvious neuropathological changes, especially in the cerebellum, where initial PC loss and neuroinflammation were evident. The Npc2−/− mice developed neurological symptoms at eight weeks of age, severely progressing until the end-stage of the disease at 12 weeks. At the end-stage of the disease, Npc2−/− mice were characterized by growth retardation, tremor, cerebellar ataxia, splenomegaly, foam cell accumulation in the lungs, liver, and spleen, brain atrophy, pronounced PC degeneration, and severe neuroinflammation. ConclusionThe Npc2Gt(LST105)BygNya mouse model resembles the pathology seen in NP-C2 patients and denotes a valuable model for increasing the understanding of the complex disease manifestation and is relevant for testing the efficacies of new treatment strategies.

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants.

The phosphatase FIG4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes. We therefore tested the ability of reduced CLCN7 expression to compensate for loss of FIG4 or VAC14. Knock-out of CLCN7 corrected lysosomal swelling and partially corrected lysosomal hyperacidification in FIG4 null cell cultures. Knockout of the related transporter CLCN6 (ClC-6) in FIG4 null cells did not affect the lysosome phenotype. In the Fig4 null mouse, reduction of ClC-7 by expression of the dominant negative CLCN7 variant p.Gly215Arg improved growth and neurological function and increased lifespan by 20%. These observations demonstrate a role for the CLCN7 chloride transporter in pathogenesis of FIG4 and VAC14 disorders. Reduction of CLCN7 provides a new target for treatment of FIG4 and VAC14 deficiencies that lack specific therapies, such as Charcot-Marie-Tooth Type 4J and Yunis-Varón syndrome.

Lysosomal calcium loading promotes spontaneous calcium release by potentiating ryanodine receptors

Spontaneous calcium release by ryanodine receptors (RyRs) due to intracellular calcium overload results in delayed afterdepolarizations, closely associated with life-threatening arrhythmias. In this regard, inhibiting lysosomal calcium release by two-pore channel 2 (TPC2) knockout has been shown to reduce the incidence of ventricular arrhythmias under β-adrenergic stimulation. However, mechanistic investigations into the role of lysosomal function on RyR spontaneous release remain missing. We investigate the calcium handling mechanisms by which lysosome function modulates RyR spontaneous release, and determine how lysosomes are able to mediate arrhythmias by its influence on calcium loading. Mechanistic studies were conducted using a population of biophysically detailed mouse ventricular models including for the first time modeling of lysosomal function, and calibrated by experimental calcium transients modulated by TPC2. We demonstrate that lysosomal calcium uptake and release can synergistically provide a pathway for fast calcium transport, by which lysosomal calcium release primarily modulates sarcoplasmic reticulum calcium reuptake and RyR release. Enhancement of this lysosomal transport pathway promoted RyR spontaneous release by elevating RyR open probability. In contrast, blocking either lysosomal calcium uptake or release revealed an antiarrhythmic impact. Under conditions of calcium overload, our results indicate that these responses are strongly modulated by intercellular variability in L-type calcium current, RyR release, and sarcoplasmic reticulum calcium-ATPase reuptake. Altogether, our investigations identify that lysosomal calcium handling directly influences RyR spontaneous release by regulating RyR open probability, suggesting antiarrhythmic strategies and identifying key modulators of lysosomal proarrhythmic action.