Altered GM1 catabolism affects NMDAR-mediated Ca2+ signaling at ER-PM junctions and increases synaptic spine formation in a GM1-gangliosidosis model.

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to ß-galactosidase deficiency drastically alters neuronal Ca2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated N-methyl D-aspartate receptor (NMDAR) Ca2+ channel, thereby increasing Ca2+ flux, activating extracellular signal-regulated kinase (ERK) signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and worsens the generalized neuronal cell death characteristic of GM1-gangliosidosis.

AAV-mediated gene therapy for Sialidosis.

Sialidosis is a glycoprotein storage disease caused by deficiency of the lysosomal sialidase NEU1, which leads to pathogenic accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids. The disease belongs to the group of orphan disorders with no therapy currently available. Here, we have tested the therapeutic potential of AAV-mediated gene therapy for the treatment of sialidosis in a mouse model of the disease. One-month-old Neu1 -/- mice were co-injected with two scAAV2/8 vectors, expressing NEU1 and its chaperone PPCA, and sacrificed at 3 months post-injection. Treated mice were phenotypically indistinguishable from their WT controls. Histopathologically, they showed diminished or absent vacuolization in cells of visceral organs, including the kidney, as well as the choroid plexus and other areas of the brain. This was accompanied by restoration of NEU1 activity in most tissues, reversal of sialyl-oligosacchariduria, and normalization of lysosomal exocytosis in the CSF and serum of treated mice. AAV injection prevented the occurrence of generalized fibrosis, which is a prominent contributor of disease pathogenesis in Neu1 -/- mice and likely in patients. Overall, this therapeutic strategy holds promise for the treatment of sialidosis and may be applicable to adult forms of human idiopathic fibrosis with low NEU1 expression.

Mitochondrial proteostasis mediated by CRL5 Ozz and Alix maintains skeletal muscle function.

High energy-demanding tissues, such as skeletal muscle, require mitochondrial proteostasis to function properly. Two quality-control mechanisms, the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles, safeguard mitochondrial proteostasis. However, whether these processes interact is unknown. Here we show that the E3 ligase CRL5 Ozz , a member of the UPS, and its substrate Alix control the mitochondrial concentration of Slc25A4, a solute carrier that is essential for ATP production. The mitochondria in Ozz -/- or Alix -/- skeletal muscle share overt morphologic alterations (they are supernumerary, swollen, and dysmorphic) and have abnormal metabolomic profiles. We found that CRL5 Ozz ubiquitinates Slc25A4 and promotes its proteasomal degradation, while Alix facilitates SLC25A4 loading into exosomes destined for lysosomal destruction. The loss of Ozz or Alix offsets steady-state levels of Slc25A4, which disturbs mitochondrial metabolism and alters muscle fiber composition. These findings reveal hitherto unknown regulatory functions of Ozz and Alix in mitochondrial proteostasis.

Altered GM1 catabolism affects NMDAR-mediated Ca 2+ signaling at ER-PM junctions and increases synaptic spine formation.

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca 2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca 2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to ß-galactosidase deficiency drastically alters neuronal Ca 2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated NMDAR Ca 2+ channel, thereby increasing Ca 2+ flux, activating ERK signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and exacerbates the generalized neuronal cell death characteristic of GM1-gangliosidosis.

Distinct functions of dimeric and monomeric scaffold protein Alix in regulating F-actin assembly and loading of exosomal cargo.

Alix is a ubiquitously expressed scaffold protein that participates in numerous cellular processes related to the remodeling/repair of membranes and the actin cytoskeleton. Alix exists in monomeric and dimeric/multimeric configurations, but how dimer formation occurs and what role the dimer has in Alix-mediated processes are still largely elusive. Here, we reveal a mechanism for Alix homodimerization mediated by disulfide bonds under physiological conditions and demonstrate that the Alix dimer is enriched in exosomes and F-actin cytoskeleton subcellular fractions. Proteomic analysis of exosomes derived from Alix-/- primary cells underlined the indispensable role of Alix in loading syntenin into exosomes, thereby regulating the cellular levels of this protein. Using a set of deletion mutants, we define the function of Alix Bro1 domain, which is solely required for its exosomal localization, and that of the V domain, which is needed for recruiting syntenin into exosomes. We reveal an essential role for Cys814 within the disordered proline-rich domain for Alix dimerization. By mutating this residue, we show that Alix remains exclusively monomeric and, in this configuration, is effective in loading syntenin into exosomes. In contrast, loss of dimerization affects the ability of Alix to associate with F-actin, thereby compromising Alix-mediated cytoskeleton remodeling. We propose that dimeric and monomeric forms of Alix selectively execute two of the protein's main functions: exosomal cargo loading and cytoskeleton remodeling.

Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex.

Ozz, a member of the SOCS-box family of proteins, is the substrate-binding component of CRL5Ozz, a muscle-specific Cullin-RING ubiquitin ligase complex composed of Elongin B/C, Cullin 5 and Rbx1. CRL5Ozz targets for proteasomal degradation selected pools of substrates, including sarcolemma-associated ß-catenin, sarcomeric MyHCemb and Alix/PDCD6IP, which all interact with the actin cytoskeleton. Ubiquitination and degradation of these substrates are required for the remodeling of the contractile sarcomeric apparatus. However, how CRL5Ozz assembles into an active E3 complex and interacts with its substrates remain unexplored. Here, we applied a baculovirus-based expression system to produce large quantities of two subcomplexes, Ozz-EloBC and Cul5-Rbx1. We show that these subcomplexes mixed in a 1:1 ratio reconstitutes a five-components CRL5Ozz monomer and dimer, but that the reconstituted complex interacts with its substrates only as monomer. The in vitro assembled CRL5Ozz complex maintains the capacity to polyubiquitinate each of its substrates, indicating that the protein production method used in these studies is well-suited to generate large amounts of a functional CRL5Ozz. Our findings highlight a mode of assembly of the CRL5Ozz that differs in presence or absence of its cognate substrates and grant further structural studies.

AAV-mediated gene therapy for galactosialidosis: A long-term safety and efficacy study.

AAV-mediated gene therapy holds promise for the treatment of lysosomal storage diseases (LSDs), some of which are already in clinical trials. Yet, ultra-rare subtypes of LSDs, such as some glycoproteinoses, have lagged. Here, we report on a long-term safety and efficacy preclinical study conducted in the murine model of galactosialidosis, a glycoproteinosis caused by a deficiency of protective protein/cathepsin A (PPCA). One-month-old Ctsa -/- mice were injected intravenously with a high dose of a self-complementary AAV2/8 vector expressing human CTSA in the liver. Treated mice, examined up to 12 months post injection, appeared grossly indistinguishable from their wild-type littermates. Sustained expression of scAAV2/8-CTSA in the liver resulted in the release of the therapeutic precursor protein in circulation and its widespread uptake by cells in visceral organs and the brain. Increased cathepsin A activity resolved lysosomal vacuolation throughout the affected organs and sialyl-oligosacchariduria. No signs of hyperplasia or inflammation were detected in the liver up to a year of age. Clinical chemistry panels, blood cell counts, and T cell immune responses were normal in all treated animals. These results warrant a close consideration of this gene therapy approach for the treatment of galactosialidosis, an orphan disease with no cure in sight.

Generation of human induced pluripotent stem cells (hIPSCs) from sialidosis types I and II patients with pathogenic neuraminidase 1 mutations.

Sialidosis is an autosomal recessive lysosomal storage disease, belonging to the glycoproteinoses. The disease is caused by deficiency of the sialic acid-cleaving enzyme, sialidase 1 or neuraminidase 1 (NEU1). Patients with sialidosis are classified based on the age of onset and severity of the clinical symptoms into type I (normomorphic) and type II (dysmorphic). Patient-derived skin fibroblasts from both disease types were reprogrammed using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit. iPSCs were characterized for pluripotency, three germ-layer differentiation, normal karyotype and absence of viral components. These cell lines represent a valuable resource to model sialidosis and to screen for therapeutics.

Conventional and Unconventional Therapeutic Strategies for Sialidosis Type I.

Congenital deficiency of the lysosomal sialidase neuraminidase 1 (NEU1) causes the lysosomal storage disease, sialidosis, characterized by impaired processing/degradation of sialo-glycoproteins and sialo-oligosaccharides, and accumulation of sialylated metabolites in tissues and body fluids. Sialidosis is considered an ultra-rare clinical condition and falls into the category of the so-called orphan diseases, for which no therapy is currently available. In this study we aimed to identify potential therapeutic modalities, targeting primarily patients affected by type I sialidosis, the attenuated form of the disease. We tested the beneficial effects of a recombinant protective protein/cathepsin A (PPCA), the natural chaperone of NEU1, as well as pharmacological and dietary compounds on the residual activity of mutant NEU1 in a cohort of patients' primary fibroblasts. We observed a small, but consistent increase in NEU1 activity, following administration of all therapeutic agents in most of the fibroblasts tested. Interestingly, dietary supplementation of betaine, a natural amino acid derivative, in mouse models with residual NEU1 activity mimicking type I sialidosis, increased the levels of mutant NEU1 and resolved the oligosacchariduria. Overall these findings suggest that carefully balanced, unconventional dietary compounds in combination with conventional therapeutic approaches may prove to be beneficial for the treatment of sialidosis type I.

MYC competes with MiT/TFE in regulating lysosomal biogenesis and autophagy through an epigenetic rheostat.

Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.

Excessive exosome release is the pathogenic pathway linking a lysosomal deficiency to generalized fibrosis.

Lysosomal exocytosis is a ubiquitous process negatively regulated by neuraminidase 1 (NEU1), a sialidase mutated in the glycoprotein storage disease sialidosis. In Neu1-/- mice, excessive lysosomal exocytosis is at the basis of disease pathogenesis. Yet, the tissue-specific molecular consequences of this deregulated pathway are still unfolding. We now report that in muscle connective tissue, Neu1-/- fibroblasts have features of myofibroblasts and are proliferative, migratory, and exocytose large amounts of exosomes. These nanocarriers loaded with activated transforming growth factor-ß and wingless-related integration site (WNT)/ß-catenin signaling molecules propagate fibrotic signals to other cells, maintaining the tissue in a prolonged transitional status. Myofibroblast-derived exosomes fed to normal fibroblasts convert them into myofibroblasts, changing the recipient cells' proliferative and migratory properties. These findings reveal an unexpected exosome-mediated signaling pathway downstream of NEU1 deficiency that propagates a fibrotic disease and could be implicated in idiopathic forms of fibrosis in humans.

Palmitoylation is a post-translational modification of Alix regulating the membrane organization of exosome-like small extracellular vesicles.

BACKGROUND: Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions. METHODS: Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells. RESULTS: We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its protein-protein interaction with CD9, a member of the tetraspanin protein family. Furthermore, we showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. CONCLUSIONS: We propose that S-palmitoylation regulates the function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper structural organization of exosome-like extracellular vesicle membranes. GENERAL SIGNIFICANCE: Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs.

Alix-mediated assembly of the actomyosin-tight junction polarity complex preserves epithelial polarity and epithelial barrier.

Maintenance of epithelial cell polarity and epithelial barrier relies on the spatial organization of the actin cytoskeleton and proper positioning/assembly of intercellular junctions. However, how these processes are regulated is poorly understood. Here we reveal a key role for the multifunctional protein Alix in both processes. In a knockout mouse model of Alix, we identified overt structural changes in the epithelium of the choroid plexus and in the ependyma, such as asymmetrical cell shape and size, misplacement and abnormal beating of cilia, blebbing of the microvilli. These defects culminate in excessive cell extrusion, enlargement of the lateral ventricles and hydrocephalus. Mechanistically, we find that by interacting with F-actin, the Par complex and ZO-1, Alix ensures the formation and maintenance of the apically restricted actomyosin-tight junction complex. We propose that in this capacity Alix plays a role in the establishment of apical-basal polarity and in the maintenance of the epithelial barrier.

Regulated lysosomal exocytosis mediates cancer progression.

Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf (-/-) mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance.

Identification and characterization of the nano-sized vesicles released by muscle cells.

Several cell types secrete small membranous vesicles that contain cell-specific collections of proteins, lipids, and genetic material. The function of these vesicles is to allow cell-to-cell signaling and the horizontal transfer of their cargo molecules. Here, we demonstrate that muscle cells secrete nano-sized vesicles and that their release increases during muscle differentiation. Analysis of these nanovesicles allowed us to characterize them as exosome-like particles and to define the potential role of the multifunctional protein Alix in their biogenesis.

Emphysema in an adult with galactosialidosis linked to a defect in primary elastic fiber assembly.

Galactosialidosis is a lysosomal storage disorder caused by loss of function of protective protein cathepsin A, which leads to secondary deficiencies of ß-galactosidase and neuraminidase-1. Emphysema has not been previously reported as a possible complication of this disorder, but we now describe this condition in a 41-year-old, non-smoking male. Our patient did not display deficiency in α-1-antitrypsin, the most common cause of emphysema in non-smokers, which brings about disseminated elastolysis. We therefore hypothesized that loss of cathepsin A activity was responsible because of previously published evidence showing it is prerequisite for normal elastogenesis. We now present experimental evidence to support this theory by demonstrating impaired primary elastogenesis in cultures of dermal fibroblasts from our patient. The obtained data further endorse our previous finding that functional integrity of the cell surface-targeted molecular complex of cathepsin A, neuraminidase-1 and the elastin-binding protein (spliced variant of ß-galactosidase) is prerequisite for the normal assembly of elastic fibers. Importantly, we also found that elastic fiber production was increased after exposure either to losartan, spironolactone, or dexamethasone. Of immediate clinical relevance, our data suggest that surviving patients with galactosialidosis should have periodic assessment of their pulmonary function. We also encourage further experimental exploration of therapeutic potential of the afore-mentioned elastogenesis-stimulating drugs for the alleviation of pathological processes in galactosialidosis that could be mechanistically linked to impaired deposition of elastic fibers.

Alix protein is substrate of Ozz-E3 ligase and modulates actin remodeling in skeletal muscle.

Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz(-/-) myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling.

Ozz-E3 ubiquitin ligase targets sarcomeric embryonic myosin heavy chain during muscle development.

Muscle contractile proteins are expressed as a series of developmental isoforms that are in constant dynamic remodeling during embryogenesis, but how obsolete molecules are recognized and removed is not known. Ozz is a developmentally regulated protein that functions as the adaptor component of a RING-type ubiquitin ligase complex specific to striated muscle. Ozz(-/-) mutants exhibit defects in myofibrillogenesis and myofiber differentiation. Here we show that Ozz targets the rod portion of embryonic myosin heavy chain and preferentially recognizes the sarcomeric rather than the soluble pool of myosin. We present evidence that Ozz binding to the embryonic myosin isoform within sarcomeric thick filaments marks it for ubiquitination and proteolytic degradation, allowing its replacement with neonatal or adult isoforms. This unique function positions Ozz within a system that facilitates sarcomeric myosin remodeling during muscle maturation and regeneration. Our findings identify Ozz-E3 as the ubiquitin ligase complex that interacts with and regulates myosin within its fully assembled cytoskeletal structure.

Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with beta-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.