Adjunctive albuterol enhances the response to enzyme replacement therapy in late-onset Pompe disease.

Effective dosages for enzyme replacement therapy (ERT) in Pompe disease are much higher than for other lysosomal storage disorders, which has been attributed to low cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle. We have previously demonstrated the benefit of increased CI-MPR-mediated uptake of recombinant human acid-α-glucosidase during ERT in mice with Pompe disease following addition of albuterol therapy. Currently we have completed a pilot study of albuterol in patients with late-onset Pompe disease already on ERT for >2 yr, who were not improving further. The 6-min walk test (6MWT) distance increased in all 7 subjects at wk 6 (30±13 m; P=0.002), wk 12 (34±14 m; P=0.004), and wk 24 (42±37 m; P=0.02), in comparison with baseline. Grip strength was improved significantly for both hands at wk 12. Furthermore, individual subjects reported benefits; e.g., a female patient could stand up from sitting on the floor much more easily (time for supine to standing position decreased from 30 to 11 s), and a male patient could readily swing his legs out of his van seat (hip abduction increased from 1 to 2+ on manual muscle testing). Finally, analysis of the quadriceps biopsies suggested increased CI-MPR at wk 12 (P=0.08), compared with baseline. With the exception of 1 patient who succumbed to respiratory complications of Pompe disease in the first week, only mild adverse events have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding). Therefore, this pilot study revealed initial safety and efficacy in an open label study of adjunctive albuterol therapy in patients with late-onset Pompe disease who had been stable on ERT with no improvements noted over the previous several years.

Adjunctive β2-agonists reverse neuromuscular involvement in murine Pompe disease.

Pompe disease has resisted enzyme replacement therapy with acid α-glucosidase (GAA), which has been attributed to inefficient cation-independent mannose-6-phosphate receptor (CI-MPR) mediated uptake. We evaluated β2-agonist drugs, which increased CI-MPR expression in GAA knockout (KO) mice. Clenbuterol along with a low-dose adeno-associated virus vector increased Rotarod latency by 75% at 4 wk, in comparison with vector alone (P<2×10(-5)). Glycogen content was lower in skeletal muscles, including soleus (P<0.01), extensor digitorum longus (EDL; P<0.001), and tibialis anterior (P<0.05) following combination therapy, in comparison with vector alone. Glycogen remained elevated in the muscles following clenbuterol alone, indicating an adjunctive effect with gene therapy. Elderly GAA-KO mice treated with combination therapy demonstrated 2-fold increased wirehang latency, in comparison with vector or clenbuterol alone (P<0.001). The glycogen content of skeletal muscle decreased following combination therapy in elderly mice (P<0.05). Finally, CI-MPR-KO/GAA-KO mice did not respond to combination therapy, indicating that clenbuterol's effect depended on CI-MPR expression. In summary, adjunctive β2-agonist treatment increased CI-MPR expression and enhanced efficacy from gene therapy in Pompe disease, which has implications for other lysosomal storage disorders that involve primarily the brain.

Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

EpsinR an AP1/clathrin interacting protein involved in vesicle trafficking

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

Fonction de la protéine Ceroid lipofuscinosis neuronal 5 (CLN5) dans le tri et le recyclage à l’endosome

Le tri et le transport efficace des hydrolases acides vers le lysosome jouent un rôle critique pour la fonction des cellules. Plus de 50 maladies humaines sont dues à des mutations des enzymes lysosomales, des protéines régulant des processus-clés du transport vers le lysosome ou des enzymes effectuant des modifications posttraductionnelles importantes pour la fonction du lysosome. L’objectif de cette thèse est d’identifier des protéines et des mécanismes permettant à la cellule de réguler le transport des enzymes vers le lysosome. Nous avons formulé l’hypothèse que des protéines mutées dans des maladies lysosomales et dont les fonctions étaient inconnues pouvaient jouer un rôle dans le transport vers le lysosome. Les céroïdes-lipofuscinoses neuronales forment une famille de maladies lysosomales rares mais sont aussi les maladies neurodégénératives infantiles les plus fréquentes. Plusieurs gènes impliqués dans les NCL encodent des protéines aux fonctions inconnues. Les travaux présentés dans cette thèse ont identifié la protéine « ceroid lipofuscinosis neuronal-5 » (CLN5) qui est localisée à l’endosome et au lysosome comme élément nécessaire au recrutement et à l’activation de rab7. Rab7 est une protéine Rab-clé qui contrôle le trafic à l’endosome tardif. Cette petite GTPase est impliquée dans le recrutement de retromer, un complexe protéique qui régule le trafic de l’endosome vers l’appareil de Golgi des récepteurs de tri lysosomal comme sortilin et le récepteur du mannose-6-phosphate. Dans les cellules où CLN5 est déplété, les récepteurs de tri lysosomal sont moins recyclés plus rapidement dégradés. En utilisant des expériences de photomarquage nous avons aussi pu démontrer que Rab7 est moins activées en l’absence de CLN5. Pour exécuter leur fonction les protéines rabs doivent être recrutée à la membrane et activées par l’échange d’une molécule de GDP pour une molécule de GTP. Le recrutement des Rabs à la membrane nécessite une modification posttraductionnelle lipidique pour être facilités. En utilisant un modèle de levures nous avons démontré que l’homologue de Rab7, Ypt7 est palmitoylée. Nous avons aussi démontré que la palmitoyltransférase Swif1 est nécessaire au recrutement de Ypt7 à la membrane. Nous avons aussi remarqué que les sous- unités de retromer chez la levure sont moins recrutées lorsque les palmitoyltransférases sont déplétées. Dans les cellules de mammifères nous avons démontré que Rab7 est également palmitoylé et que cette palmitoylation est possiblement effectuée par les palmitoyltransférases DHHC1 et DHHC8. La palmitoylation de Rab7 a lieu sur les cystéines en C-terminal qui sont nécessaires au recrutement membranaire et qui auparavant étaient uniquement décrites comme prénylées. En utilisant la méthode de « click chemistry » nous avons découvert que lorsque la prénylation de Rab7 est bloquée le niveau de palmitoylation augmente. Pour caractériser l’interaction entre CLN5 et Rab7 nous avons performé des expériences afin d’établir définitivement la topologie de cette protéine. Nous avons ainsi démontré que CLN5 est une protéine hautement glycosylée qui est initialement traduite en protéine transmembranaire et subséquemment clivée par un membre de la famille des peptidase de peptide signal (SPP). Cette protéine soluble peut alors possiblement interagir avec CLN3 qui est aussi palmitoylée pour recruter et activer Rab7. Nos études suggèrent pour la première fois que CLN5 pourrait être un recruteur et un activateur de Rab7 qui agirait avec la protéine CLN3 pour séquestrer Rab7 avec les autres récepteurs palmitoylés et permettre leur recyclage vers l’appareil de Golgi.

Polymorphisms, linkage and mapping of four enzyme loci in the fish genus Xiphophorus (Poeciliidae).

Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 to MPI region. No significant sex-specific differences in recombination were observed. This group (designated linkage group II) was shown to assort independently from the three loci of linkage group I (adenosine deaminase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) and from glyceraldehyde-3-phosphate dehydrogenase and two isocitrate dehydrogenase loci. Evidence for conservation of the linkage group, at least in part, in other vertebrate species is presented.

Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.

Effect of Carbohydrate Position on Lysosomal Transport of Procathepsin L

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30–50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH4Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure–function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP–CALM was targeted to the plasma membrane–coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP–CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP–CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin–CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60–80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Infusion of α-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb3; also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified α-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered α-gal A, (ii) to assess the pharmacokinetics of i.v.-administered α-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb3. α-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified α-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of α-gal A, nine of the 10 patients had significantly reduced Gb3 levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of α-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb3 burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

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.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).