AAV-mediated gene therapy for sialidosis.

Sialidosis (mucolipidosis I) is a glycoprotein storage disease, clinically characterized by a spectrum of systemic and neurological phenotypes. The primary cause of the disease is deficiency of the lysosomal sialidase NEU1, resulting in accumulation of sialylated glycoproteins/oligosaccharides in tissues and body fluids. Neu1-/- mice recapitulate the severe, early-onset forms of the disease, affecting visceral organs, muscles, and the nervous system, with widespread lysosomal vacuolization evident in most cell types. Sialidosis is considered an orphan disorder with no therapy currently available. Here, we assessed the therapeutic potential of AAV-mediated gene therapy for the treatment of sialidosis. Neu1-/- mice were co-injected with two scAAV2/8 vectors, expressing human NEU1 and its chaperone PPCA. Treated mice were phenotypically indistinguishable from their WT controls. NEU1 activity was restored to different extent in most tissues, including the brain, heart, muscle, and visceral organs. This resulted in diminished/absent lysosomal vacuolization in multiple cell types and reversal of sialyl-oligosacchariduria. Lastly, normalization of lysosomal exocytosis in the cerebrospinal fluids and serum of treated mice, coupled to diminished neuroinflammation, were measures of therapeutic efficacy. These findings point to AAV-mediated gene therapy as a suitable treatment for sialidosis and possibly other diseases, associated with low NEU1 expression.

Haploinsufficiency of the lysosomal sialidase NEU1 results in a model of pleomorphic rhabdomyosarcoma in mice.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/-/ETV7TG/+/- mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor's metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.

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.

Lysosomes and Cancer Progression: A Malignant Liaison.

During primary tumorigenesis isolated cancer cells may undergo genetic or epigenetic changes that render them responsive to additional intrinsic or extrinsic cues, so that they enter a transitional state and eventually acquire an aggressive, metastatic phenotype. Among these changes is the alteration of the cell metabolic/catabolic machinery that creates the most permissive conditions for invasion, dissemination, and survival. The lysosomal system has emerged as a crucial player in this malignant transformation, making this system a potential therapeutic target in cancer. By virtue of their ubiquitous distribution in mammalian cells, their multifaced activities that control catabolic and anabolic processes, and their interplay with other organelles and the plasma membrane (PM), lysosomes function as platforms for inter- and intracellular communication. This is due to their capacity to adapt and sense nutrient availability, to spatially segregate specific functions depending on their position, to fuse with other compartments and with the PM, and to engage in membrane contact sites (MCS) with other organelles. Here we review the latest advances in our understanding of the role of the lysosomal system in cancer progression. We focus on how changes in lysosomal nutrient sensing, as well as lysosomal positioning, exocytosis, and fusion perturb the communication between tumor cells themselves and between tumor cells and their microenvironment. Finally, we describe the potential impact of MCS between lysosomes and other organelles in propelling cancer growth and spread.

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.

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.

Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1.

Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection.

Chaperone-mediated gene therapy with recombinant AAV-PPCA in a new mouse model of type I sialidosis.

The lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-α-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation. We have generated a new mouse model of type I sialidosis that ubiquitously expresses a NEU1 variant carrying a V54M amino acid substitution identified in an adult patient with type I sialidosis. Mutant mice developed signs of lysosomal disease after 1year of age, predominantly in the kidney, albeit low residual NEU1 activity was detected in most organs and cell types. We demonstrate that the activity of the mutant enzyme could be effectively increased in all systemic tissues by chaperone-mediated gene therapy with a liver-tropic recombinant AAV2/8 vector expressing PPCA. This resulted in clear amelioration of the disease phenotype. These results suggest that at least some of the NEU1 mutations associated with type I sialidosis may respond to PPCA-chaperone-mediated gene therapy.

Neuraminidase 1 is a negative regulator of lysosomal exocytosis.

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.