Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the β-subunit of β-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.

Microglial activation precedes acute neurodegeneration in Sandhoff disease and is suppressed by bone marrow transplantation

Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids in the central nervous system. The glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. In symptomatic Sandhoff disease mice, apoptotic neuronal cell death was prominent in the caudal regions of the brain. cDNA microarray analysis to monitor gene expression during neuronal cell death revealed an upregulation of genes related to an inflammatory process dominated by activated microglia. Activated microglial expansion, based on gene expression and histologic analysis, was found to precede massive neuronal death. Extensive microglia activation also was detected in a human case of Sandhoff disease. Bone marrow transplantation of Sandhoff disease mice suppressed both the explosive expansion of activated microglia and the neuronal cell death without detectable decreases in neuronal GM2 ganglioside storage. These results suggest a mechanism of neurodegeneration that includes a vigorous inflammatory response as an important component. Thus, this lysosomal storage disease has parallels to other neurodegenerative disorders, such as Alzheimer's and prion diseases, where inflammatory processes are believed to participate directly in neuronal cell death.

Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of β-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gusmps/mps) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human β-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gusmps/mps) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gusmps/mps) mouse. However, they are now tolerant to immune challenge with human β-glucuronidase. This “tolerant MPS VII mouse model” should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII.

Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography

The positional relationships among all of the visible organelles in a densely packed region of cytoplasm from an insulin secreting, cultured mammalian cell have been analyzed in three dimensions (3-D) at ≈6 nm resolution. Part of a fast frozen/freeze-substituted HIT-T15 cell that included a large portion of the Golgi ribbon was reconstructed in 3-D by electron tomography. The reconstructed volume (3.1 × 3.2 × 1.2 μm3) allowed sites of interaction between organelles, and between microtubules and organellar membranes, to be accurately defined in 3-D and quantitatively analyzed by spatial density analyses. Our data confirm that the Golgi in an interphase mammalian cell is a single, ribbon-like organelle composed of stacks of flattened cisternae punctuated by openings of various sizes [Rambourg, A., Clermont, Y., & Hermo, L. (1979) Am. J. Anat. 154, 455–476]. The data also show that the endoplasmic reticulum (ER) is a single continuous compartment that forms close contacts with mitochondria, multiple trans Golgi cisternae, and compartments of the endo-lysosomal system. This ER traverses the Golgi ribbon from one side to the other via cisternal openings. Microtubules form close, non-random associations with the cis Golgi, the ER, and endo-lysosomal compartments. Despite the dense packing of organelles in this Golgi region, ≈66% of the reconstructed volume is calculated to represent cytoplasmic matrix. We relate the intimacy of structural associations between organelles in the Golgi region, as quantified by spatial density analyses, to biochemical mechanisms for membrane trafficking and organellar communication in mammalian cells.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling

The high affinity receptor for IgE, FcɛRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcɛRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcɛRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcɛRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcɛRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcɛRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcɛRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.

Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic EventsV⃞

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.

Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice

Stimulation of endothelial cells by various inflammatory mediators leads to release of Weibel–Palade bodies and therefore to exocytosis of both P-selectin (adhesion receptor for leukocytes) and von Willebrand factor (vWf) (platelet ligand). The potential role of vWf in leukocyte recruitment was investigated with the use of vWf-deficient mice. We report a strong reduction of leukocyte rolling in venules of vWf-deficient mice. Similarly, vWf deficiency led to a decrease in neutrophil recruitment in a cytokine-induced meningitis model as well as in early skin wounds. In all instances with an antibody that preferentially recognizes plasma membrane P-selectin, we observed a dramatic reduction in P-selectin expression at the cell surface of vWf-deficient endothelium. With confocal microscopy, we found that the typical rodlike shape of the Weibel–Palade body is missing in vWf −/− endothelial cells and that part of the P-selectin content in the vWf −/− cells colocalized with LAMP-1, a lysosomal marker. However, intracellular P-selectin levels were similar in tumor necrosis factor α- and lipopolysaccharide-activated cells of both genotypes. We conclude that the absence of vWf, as found in severe von Willebrand disease, leads to a defect in Weibel–Palade body formation. This defect results in decreased P-selectin translocation to the cell surface and reduced leukocyte recruitment in early phases of inflammation.

Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for ≈25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease

Niemann–Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers1

Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers.

The structure of the human βII-tryptase tetramer: Fo(u)r better or worse

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-Å crystal structure of human βII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 Å × 15 Å govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrations in vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.

Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

Afipia felis induces uptake by macrophages directly into a nonendocytic compartment

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, β-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome–lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.