Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1–3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.     

Developmentally regulated mannose 6-phosphate receptor-mediated transport of a lysosomal enzyme across the blood-brain barrier

Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of beta-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate/insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. (131)I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate (K(in)) of (131)I-P-GUS in 2-day-old mice was 0.21 microl/g.min and decreased with age. By 7 weeks of age, transport of (131)I-P-GUS was not significant. Capillary depletion revealed that 62% of the (131)I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of (131)I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner. In contrast, the K(in) of (131)I-NP-GUS (0.04 microl/g.min) was significantly lower than (131)I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither (131)I-P-GUS nor (131)I-NP-GUS crossed the BBB in adult mice. These results indicate that (131)I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice.     

Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation

Autophagy is a conserved cellular process to degrade and recycle cytoplasmic components. During autophagy, lysosomes fuse with an autophagosome to form an autolysosome. Sequestered components are degraded by lysosomal hydrolases and presumably released into the cytosol by lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the "target of rapamycin" (TOR) kinase. Spinster (Spin) encodes a putative lysosomal efflux permease with the hallmarks of a sugar transporter. Drosophila spin mutants accumulate lysosomal carbohydrates and enlarged lysosomes. Here we show that defects in spin lead to the accumulation of enlarged autolysosomes. We find that spin is essential for mTOR reactivation and lysosome reformation following prolonged starvation. Further, we demonstrate that the sugar transporter activity of Spin is essential for ALR.     

Senescent case of cholesterol ester storage disease that progressed to liver cirrhosis with a novel mutation (N250H) of lysosomal acid lipase gene

The patient, a 69‐year‐old man, had a chief complaint of hepatomegaly. The liver was palpated four fingerbreadths below the costal margin, and the spleen was three fingerbreadths below the costal margin. There were no other abnormal findings. Laparoscopy showed that the liver resembled an orange‐yellow crayon in appearance and was nodular. The pathological findings of the liver biopsy tissue were consistent with liver cirrhosis. Inside the fibrous septum was an apparent aggregation of enlarged macrophages that phagocytosed lipid components, as well as enlarged Kupffer cells that phagocytosed lipid droplets. Electron microscopy showed the lipid droplets to have a moth‐eaten appearance. Using monocytes extracted from the peripheral blood, acid lipase activity was measured by fluorescence spectrometry using 4‐methylumbelliferone palmitate as a substrate. This patient's human lysosomal acid lipase activity was 0.020 nM/min per 10⁶ cells, corresponding to 5.9% of that in healthy subjects (0.332 ± 0.066 nM/min per 10⁶ cells). Cholesterol ester storage disease was therefore diagnosed. The acid lipase A base sequence obtained from leukocytes by direct sequencing was compared with a library. This patient had a point mutation of N250H/N250H in exon 7, a novel gene abnormality that has not previously been reported.     

Reprogramming erythroid cells for lysosomal enzyme production leads to visceral and CNS cross-correction in mice with Hurler syndrome

Restricting transgene expression to maturing erythroid cells can reduce the risk for activating oncogenes in hematopoietic stem cells (HSCs) and their progeny, yet take advantage of their robust protein synthesis machinery for high-level protein production. This study sought to evaluate the feasibility and efficacy of reprogramming erythroid cells for production of a lysosomal enzyme, α-L-iduronidase (IDUA). An erythroid-specific hybrid promoter provided inducible IDUA expression and release during in vitro erythroid differentiation in murine erythroleukemia cells, resulting in phenotypical cross-correction in an enzyme-deficient lymphoblastoid cell line derived from patients with mucopolysaccharidosis type I (MPS I). Stable and higher than normal plasma IDUA levels were achieved in vivo in primary and secondary MPS I chimeras for at least 9 months after transplantation of HSCs transduced with the erythroid-specific IDUA-containing lentiviral vector (LV). Moreover, long-term metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in heart, liver, and spleen. Notably, neurological function and brain pathology were significantly improved in MPS I mice by erythroid-derived, higher than normal peripheral IDUA protein. These data demonstrate that late-stage erythroid cells, transduced with a tissue-specific LV, can deliver a lysosomal enzyme continuously at supraphysiological levels to the bloodstream and can correct the disease phenotype in both viscera and CNS of MPS I mice. This approach provides a paradigm for the utilization of RBC precursors as a depot for efficient and potentially safer systemic delivery of nonsecreted proteins by ex vivo HSC gene transfer.     

Platelets are efficient and protective depots for storage,distribution, and delivery of lysosomal enzyme in mice with Hurler Syndrome

Use of megakaryocytes/platelets for transgene expression may take advantage of their rapid turnover and protective storage in platelets and reduce the risk of activating oncogenes in hematopoietic stem and progenitor cells (HSCs). Here, we show that human megakaryocytic cells could overexpress the lysosomal enzyme, α-l-iduronidase (IDUA), which is deficient in patients with mucopolysaccharidosis type I (MPS I). Upon megakaryocytic differentiation, the amount of released enzyme increased rapidly and steadily by 30-fold. Using a murine MPS I model, we demonstrated that megakaryocyte/platelets were capable of producing, packaging, and storing large amounts of IDUA with proper catalytic activity, lysosomal trafficking, and receptor-mediated uptake. IDUA can be released directly into extracellular space or within microparticles during megakaryocyte maturation or platelet activation, while retaining the capacity for cross-correction in patient’s cells. Gene transfer into 1.7% of HSCs led to long-term normalization of plasma IDUA and preferential distribution of enzyme in liver and spleen with complete metabolic correction in MPS I mice. Detection of GFP (coexpressed with IDUA) in Kupffer cells and hepatocytes suggested liver delivery of platelet-derived IDUA possibly via the clearance pathway for senile platelets. These findings provide proof of concept that cells from megakaryocytic lineage and platelets are capable of generating and storing fully functional lysosomal enzymes and can also lead to efficient delivery of both the enzymes released into the circulation and those protected within platelets/microparticles. This study opens a door for use of the megakaryocytes/platelets as a depot for efficient production, delivery, and effective tissue distribution of lysosomal enzymes.The potential of therapeutic benefits from genetically modified hematopoietic stem cells (HSCs) has been supported in recent gene therapy clinical trials (1, 2). High transgene dosage or selective growth of genetically corrected HSCs appears to be necessary for achieving clinical efficacy. However, genotoxic risk caused by proviral integration-associated oncogenesis is directly concomitant with high numbers of integration events or clonal expansion (3, 4). New approaches are needed to balance the need for high transgene frequency while limiting the associated increased risk of oncogenesis.Platelets are anuclear, secretory particulate entities containing proteins stored in cytoplasmic granules that can be released upon activation (5). Healthy adults produce 2–5 × 10¹¹ platelets daily with a baseline activation rate of 1–5% (6). Use of megakaryocytes (MKs)/platelets for transgene expression may (i) take advantage of this immense cell mass and its rapid turnover (5–9 d); (ii) provide protective storage of the transgene product, which is essential for proteins sensitive to plasma pH; and (iii) continuously dispense proteins via degranulation from platelet activation at baseline (without detectable injury) and/or at sites of vascular injury. Highly efficient protein production and delivery could further reduce the need for high transgene frequency and the risk of activating oncogenes in HSCs and all their progeny. Although using platelets as a delivery system has been demonstrated for the expression of coagulation factors to treat inherited bleeding disorders in mice (7, 8), there has been no report of the feasibility of using MKs/platelets for the generation of nonhematologic proteins.Lysosomal storage diseases (LSDs) are a group of inherited disorders, often affecting multiple organs including the liver and spleen, with a cumulative incidence of 1 in 5,000–7,000 live births (9). Overexpressing lysosomal enzymes in platelets not only can provide the protection of pH-sensitive enzymes and continuous enzyme release via low physiological levels of platelet activation but may also offer the benefit of on-target delivery of platelet-derived enzymes to spleen and liver in the process of platelet clearance (10). However, maintaining proper posttranslational modifications for appropriate lysosomal trafficking and intercellular lysosomal enzyme transfer is essential for metabolic cross correction in treating these multiorgan diseases (11). It is not known whether lysosomal enzymes generated from the MK/platelet lineage would be fully functional and capable of correcting lysosomal deficits in diseased cells.In this study, we used a mouse model of Hurler syndrome, which is the severe form of mucopolysaccharidosis type I (MPS I), one of most common LSDs. It is caused by the deficiency of α-l-iduronidase (IDUA) and consequent accumulation of glycosaminoglycans (GAGs) (12, 13). We show that MKs are capable of producing large amounts of IDUA with proper catalytic function, lysosomal trafficking and receptor-mediated uptake, which could be sorted to and stored within platelets. The IDUA can be released directly into the extracellular space or within microparticles (MPs) during MK maturation or platelet activation, while retaining its ability to cross-correct cells derived from patients with MPS I.     

Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I

Patients with mucopolysaccharidosis type I (MPS I), a genetic deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), exhibit accumulation of glycosaminoglycans in tissues, with resulting diverse clinical manifestations including neurological, ocular, skeletal, and cardiac disease. MPS I is currently treated with hematopoietic stem cell transplantation or weekly enzyme infusions, but these therapies have significant drawbacks for patient safety and quality of life and do not effectively address some of the most critical clinical sequelae, such as life-threatening cardiac valve involvement. Using the naturally occurring feline model of MPS I, we tested liver-directed gene therapy as a means of achieving long-term systemic IDUA reconstitution. We treated four MPS I cats at 3–5 mo of age with an adeno-associated virus serotype 8 vector expressing feline IDUA from a liver-specific promoter. We observed sustained serum enzyme activity for 6 mo at ∼30% of normal levels in one animal, and in excess of normal levels in three animals. Remarkably, treated animals not only demonstrated reductions in glycosaminoglycan storage in most tissues, but most also exhibited complete resolution of aortic valve lesions, an effect that has not been previously observed in this animal model or in MPS I patients treated with current therapies. These data point to clinically meaningful benefits of the robust enzyme expression achieved with hepatic gene transfer that extend beyond the economic and quality of life advantages over lifelong enzyme infusions.Mucopolysaccharidosis type I (MPS I) is a recessive genetic disorder caused by deficiency of the lysosomal enzyme α-l-iduronidase (IDUA). In the absence of IDUA, cells are unable to catabolize the ubiquitous glycosaminoglycans (GAGs) heparan and dermatan sulfates. The resulting lysosomal GAG storage causes multisystem organ pathology and diverse clinical manifestations, including bone and joint deformity, upper airway obstruction, hepatosplenomegaly, corneal clouding, and cognitive impairment (1). Most patients also develop cardiac disease, which arises from the combined effects of GAG deposition in the myocardium, coronary arteries, and left-sided heart valves (2). Without treatment, median survival in patients with the severe form of the disease is less than 7 y (3).Care of MPS I patients has been vastly improved by the introduction of two disease-modifying therapies—hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). Both treatments are based on the principle of cross-correction: that cells can efficiently endocytose extracellular lysosomal enzymes bearing a mannose-6-phosphate residue, allowing IDUA secreted from donor-derived cells after HSCT or recombinant enzyme delivered i.v. to correct the metabolic defect in many tissues (4, 5). The introduction of HSCT has increased the survival of MPS I patients and has demonstrated improvements in growth, mobility, hepatosplenomegaly, and some aspects of cardiac disease such as left ventricular hypertrophy (2, 6–9). ERT has shown a similar capacity to improve many of the clinical features of MPS I (7, 10, 11). ERT is favored in patients with an attenuated disease phenotype because of the high mortality associated with HSCT, although HSCT remains the first-line intervention for patients less than 2 y of age owing to the beneficial effect of early transplantation on cognitive outcomes (7).Despite the enormous advances that have been made in the treatment of MPS I, significant shortcomings remain. Neurological symptoms do not improve with ERT and are highly variable after HSCT (7, 9). Skeletal disease is incompletely treated by both therapies. ERT and HSCT may improve heart disease but do not reverse valvular GAG deposition, often leaving treated patients with persistent aortic and mitral valve insufficiency or stenosis (2, 12). Additionally, the current treatment options are fundamentally limited by the morbidity and mortality associated with HSCT and the need for lifelong, expensive, weekly enzyme infusions in ERT.For diseases such as MPS I that require lifelong systemic enzyme replacement, liver-directed gene therapy has emerged as a potential therapeutic option. The high synthetic capacity of the liver, coupled with the discovery of adeno-associated virus (AAV) vectors capable of safe and efficient hepatic targeting, make this a feasible alternative to exogenous enzyme infusion (13, 14). The first clinical success of AAV-mediated liver gene therapy was recently demonstrated in a trial for hemophilia B, in which some patients were able to discontinue prophylactic factor IX injections (15). Apart from the potential safety and quality of life benefits over HSCT and ERT, respectively, we hypothesized that liver-directed gene therapy could have three potential benefits specific to MPS I. First, the liver is a therapeutic target in MPS I, making the high local concentrations of enzyme potentially useful for efficiently treating hepatomegaly due to GAG storage. Second, liver-mediated expression could theoretically result in circulating concentrations of IDUA higher than those achieved with HSCT, and more stable than those achieved with i.v. infusion of the enzyme, which has a serum half-life of less than 4 h (11). Maintaining high levels of serum IDUA could drive greater enzyme uptake and improve efficacy in difficult-to-treat tissues. Finally, antibody responses to IDUA, which develop in the vast majority of patients receiving ERT, seem to limit treatment efficacy (16, 17). Evidence from mouse models suggests that AAV-mediated hepatic expression of an enzyme is less immunogenic than i.v. delivery of the recombinant protein, indicating that this approach could exhibit improved efficacy by eliciting less-robust immune responses to IDUA (18).In the present study we tested liver-directed gene therapy in the naturally occurring feline model of MPS I, which recapitulates many of the clinical and pathological features of the disease, including progressive cardiac valve involvement (19–23). Four animals were treated at 3–5 mo of age with an i.v. injection of an AAV serotype 8 vector expressing feline IDUA from a liver-specific promoter. Three of the animals exhibited sustained supraphysiologic IDUA expression, with subsequent GAG clearance from all tissues examined. Remarkably, aortic valve lesions were reversed in these three animals, indicating the potential utility of this approach for targeting treatment refractory tissues in MPS I.     

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N- and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [¹⁴C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.Low-density lipoprotein-derived cholesterol is delivered to cells by receptor-mediated endocytosis and transport to lysosomes. Within lysosomes, cholesterol esters are hydrolyzed and cholesterol is exported to the cytoplasm for cellular use (1). Cholesterol export requires two lysosomal glycoproteins: NPC1 and NPC2 (2, 3). Patients carrying homozygous mutations in either of these proteins present with Niemann-Pick type C (NPC) disease, a neurological disorder that is associated with massive accumulation of unesterified cholesterol and glycosphingolipids in lysosomes (2–4).NPC2 is a small, soluble, cholesterol binding protein that is thought to pick up cholesterol both from lipoprotein lipase and from the abundant, intralumenal membranes present within lysosomes (5, 6). NPC1 is a much larger glycoprotein that contains 13 transmembrane domains and three, relatively large, lumenally oriented domains (2). NPC1’s first (N-terminal), luminal domain binds cholesterol directly (7, 8); the second domain can bind NPC2 in a cholesterol-dependent manner and has been proposed to facilitate transfer of cholesterol from NPC2 onto NPC1’s N-terminal domain (9).The requirement for NPC1 and NPC2 proteins for cholesterol export from lysosomes is somewhat enigmatic because cholesterol can generally partition freely into membrane bilayers (10). The limiting membrane of lysosomes is lined predominantly by densely packed, highly glycosylated, lysosomal membrane proteins; their oligosaccharides are thought to protect the phospholipid bilayer from hydrolytic destruction (11, 12). This glycoprotein coat can be visualized by electron microscopy and has an average thickness of about 8 nm (13). It has been proposed that NPC1 is needed to help cholesterol traverse this glycocalyx (8). A direct prediction of this model is that decreasing the lysosomal glycocalyx density should make cells less dependent on NPC1 function.In this paper, we have used two approaches to modify the glycocalyx. Alteration of glycosylation decreases lysosomal cholesterol levels in NPC1-deficient Chinese hamster ovary (CHO) and human cells. These experiments support a model in which NPC1 protein functions to help cholesterol traverse the glycocalyx that lines the interior of lysosome limiting membranes.     

Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice

Enzyme-replacement therapy is an established means of treating lysosomal storage diseases. Infused therapeutic enzymes are targeted to lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate, on the enzymes. We have tested an alternative, peptide-based targeting system for delivery of enzymes to lysosomes in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy depends on the interaction of a fragment of insulin-like growth factor II (IGF-II), with the IGF-II binding site on the bifunctional, IGF-II cation-independent mannose 6-phosphate receptor. A chimeric protein containing a portion of mature human IGF-II fused to the C terminus of human beta-glucuronidase was taken up by MPS VII fibroblasts in a mannose 6-phosphate-independent manner, and its uptake was inhibited by the addition of IGF-II. Furthermore, the tagged enzyme was delivered effectively to clinically significant tissues in MPS VII mice and was effective in reversing the storage pathology. The tagged enzyme was able to reduce storage in glomerular podocytes and osteoblasts at a dose at which untagged enzyme was much less effective. This peptide-based, glycosylation-independent lysosomal targeting system may enhance enzyme-replacement therapy for certain human lysosomal storage diseases.     

Effectiveness of enzyme replacement therapy in 1028 patients with type 1 Gaucher disease after 2 to 5 years of treatment: a report from the Gaucher Registry

PURPOSE: Gaucher disease is the first lysosomal storage disorder to be treated with macrophage-targeted enzyme replacement therapy. Previous studies in relatively small numbers of patients demonstrated short-term efficacy of this treatment. This study describes the effects of 2 to 5 years of treatment on specific manifestations of type 1 Gaucher disease. SUBJECTS AND METHODS: Physicians reported data from 1028 patients to the Gaucher Registry. Assessment of response included serial measurements of hemoglobin concentration, platelet count, liver and spleen volumes, and the occurrence of bone pain and bone crises. RESULTS: Among anemic patients, hemoglobin concentration increased to normal or near normal within 6 to 12 months, with a sustained response through 5 years. In thrombocytopenic patients with intact spleens, the most rapid response occurred during the first 2 years, with slower improvement thereafter. The likelihood of achieving a normal platelet count decreased with increasing severity of baseline thrombocytopenia. In patients who had undergone splenectomy, platelet counts returned to normal within 6 to 12 months. Hepatomegaly decreased by 30% to 40% during follow-up; splenomegaly decreased 50% to 60%, but rarely to volumes below five times normal size. In patients with pretreatment bone pain or bone crises, 52% (67/128) were pain free after 2 years and 94% (48/51) reported no additional crises. CONCLUSION: Enzyme replacement therapy prevents progressive manifestations of Gaucher disease, and ameliorates Gaucher disease-associated anemia, thrombocytopenia, organomegaly, bone pain, and bone crises.     

Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.     

Gaucher disease gene GBA functions in immune regulation

Inherited deficiency of acid β-glucosidase (GCase) due to biallelic mutations in the GBA (glucosidase, β, acid) gene causes the classic manifestations of Gaucher disease (GD) involving the viscera, the skeleton, and the lungs. Clinical observations point to immune defects in GD beyond the accumulation of activated macrophages engorged with lysosomal glucosylceramide. Here, we show a plethora of immune cell aberrations in mice in which the GBA gene is deleted conditionally in hematopoietic stem cells (HSCs). The thymus exhibited the earliest and most striking alterations reminiscent of impaired T-cell maturation, aberrant B-cell recruitment, enhanced antigen presentation, and impaired egress of mature thymocytes. These changes correlated strongly with disease severity. In contrast to the profound defects in the thymus, there were only limited cellular defects in peripheral lymphoid organs, mainly restricted to mice with severe disease. The cellular changes in GCase deficiency were accompanied by elevated T-helper (Th)1 and Th2 cytokines that also tracked with disease severity. Finally, the proliferation of GCase-deficient HSCs was inhibited significantly by both GL1 and Lyso-GL1, suggesting that the "supply" of early thymic progenitors from bone marrow may, in fact, be reduced in GBA deficiency. The results not only point to a fundamental role for GBA in immune regulation but also suggest that GBA mutations in GD may cause widespread immune dysregulation through the accumulation of substrates.     

Rheumatologic aspects of lysosomal storage diseases

Lysosomal storage diseases are rare metabolic disorders, some of which can now be treated using enzyme replacement therapies. Because the time point of treatment initiation significantly influences the outcome in Gaucher disease, Fabry disease, and mucopolysaccharidosis type I, early diagnosis is of utmost importance. All three disorders can present with musculoskeletal symptoms in early stages, therefore, the rheumatologist may be the first to be contacted by these patients. Here, we present three characteristic lysosomal storage disease cases to increase awareness in the rheumatological community of the typical symptom constellations associated with these rare but treatable disorders.     

The influence of endotoxin in vitro on hepatic macrophage lysosomal enzyme release in different rat models of hepatic injury

ABSTRACT— Since bacterial endotoxin is known to be involved in the pathogenesis of hepatic injury, the influence of endotoxin on lysosomal enzyme production by hepatic macrophages has been investigated. Macrophages have been isolated from the livers of normal rats, from the livers of rats given stilboestrol subcutaneously 4 days previously and from the livers of rats given Corynebacterium parvum intravenously 6 days previously. Following isolation and overnight culture, the macrophages have been maintained in in vitro culture for a further 24 h and the production of N-acetyl-β-glucosaminidase (NAG) has been measured. Histological assessment has shown that in the stilboestrol model an approximate doubling of sinusoidal cell numbers occurs and in the C. parvum model a heavy mononuclear cell infiltrate is present, together with granuloma formation. These changes are reflected in the numbers of macrophages isolated from the respective models. Levels of NAG production by resident macrophages from normal livers are low (0.25±0.05 nmol substrate hydrolysed/μg cell protein/h) and unchanged following endotoxin exposure (0.25±0.05 units). Macrophages isolated from the stilboestrol model show levels of NAG production similar to normal (0.34±0.06 units), but this increases significantly following exposure to endotoxin (0.42±0.07 units). Macrophages from the C. parvum model demonstrate markedly enhanced production (0.61±0.09 units), but this does not increase significantly following endotoxin exposure (0.65±0.09 units). In contrast to macrophages from normal rat livers, macrophages recently recruited in the stilboestrol model demonstrate enhanced lysosomal enzyme production following endotoxin exposure. It is suggested that endotoxin, as well as other mediators of macrophage activation, may promote hepatic damage through this influence on newly recruited macrophages.     

Effect of antibacterial antibody on bactericidal activities of superoxide and lysosomal enzyme from alveolar macrophages in rabbits

Abstract Alveolar macrophages (AM) have many Fc receptors for IgG, but are less reactive to lymphokines. They have a well-developed oxidative metabolism and contain large amounts of lysosomal enzyme. This suggests that the antibacterial antibody plays an important role in early resistance by AM to intracellular bacterial infection and that a bactericidal agent, dependent on oxygen and lysosomal enzyme, participates in the intracellular killing of bacteria in AM. We studied the effects of the antibacterial antibody on bactericidal activities of superoxide (O₂⁻) and lysosomal enzyme from rabbit AM. The number of Listeria monocytogenes in AM increased after pretreatment with saline or normal IgG but decreased by 60% after pretreatment with anti-Listeria and 120 min incubation. Alveolar macrophage-phagocytized Listeria monocytogenes and Bacille Calmette Guérin (BCG) bound with antibacterial antibody enhanced release of O₂⁻, and nitroblue tetrazolium (NBT) formazan reduced by O₂⁻ was observed around the bacteria in the phagosomes of AM. We also confirmed that Listeria and BCG were killed extracellularly by O₂⁻ released by a superoxide-generating system in vitro and/or by lysosomal enzyme obtained from AM at a low pH. We concluded that the antibacterial antibody of the IgG class enhances the antibacterial activity of AM thereby increasing the production of O₂⁻ and lysosomal enzyme in the phagosome. This finding may be important in the early resistance to intracellular bacteria infection by AM in the alveolar spaces.     

Association of transferrin G258A and transferrin receptor A82G polymorphisms with the risk of Parkinson disease in certain area

Background:It has been reported that polymorphisms of transferrin (TF) G258A and transferrin receptor (TFR) A82G might be associated with susceptibility to Parkinson disease (PD).Objective:Owing to limitation of sample size and inconclusive results, we conducted a meta-analysis to clarify the association.Methods:By searching PubMed, Embase, Chinese National Knowledge Infrastructure, China Biological Medicine Database, and Wanfang Databases, the published articles about studies of the association of the TF G258A, TFR A82G gene polymorphisms with the risk of PD were collected. Q-statistics and I² statistics were calculated to examine heterogeneity and summary odds ratios (ORs) and 95% confidence intervals (95%CI) were evaluated the association.Results:Five studies assessed the relationship between TF G258A and risk of PD. A significant increased protective of A allele and AA genotype was observed in allele model and recessive model (the allele model A vs G: OR = 0.54, 95%CI 0.40–0.72, P < .001; the recessive model AA vs GA + GG: OR = 0.32, 95%CI 0.20–0.52, P < .001). The remaining models of the TF G258A genotype showed no significant association with PD risk, while the protective tendency were increased (the heterozygote model GA vs GG: OR = 0.93, 95%CI 0.61–1.43, P = .75; the homozygous model AA vs GG: OR = 0.47, 95%CI 0.21–1.04, P = .06; the dominant model GA + AA vs GG: OR = 0.75, 95%CI 0.50–1.11, P = .15). There was also a lack of association between TFR A82G polymorphism and PD (the allele model G vs A: OR = 0.92, 95%CI 0.75–1.13, P = .43; the heterozygote model AG vs AA: OR = 1.17, 95%CI 0.79–1.71, P = .43; the homozygous model GG vs AA: OR = 0.91, 95%CI 0.60–139, P = .66; the dominant model AG + GG vs AA: OR = 1.05, 95%CI 0.73–1.49, P = .81; the recessive model GG vs AG +AA: OR = 0.80, 95%CI 0.59–1.09, P = .16).Conclusion:Our study suggests that TF G258A polymorphism may be associated with PD, while TFR A82G polymorphism may not contribute to PD based on the current evidence.