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.

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.

Glycosphingolipids within membrane contact sites influence their function as signaling hubs in neurodegenerative diseases.

Intracellular organelles carry out many of their functions by engaging in extensive interorganellar communication through specialized membrane contact sites (MCSs) formed where two organelles tether to each other or to the plasma membrane (PM) without fusing. In recent years, these ubiquitous membrane structures have emerged as central signaling hubs that control a multitude of cellular pathways, ranging from lipid metabolism/transport to the exchange of metabolites and ions (i.e., Ca2+ ), and general organellar biogenesis. The functional crosstalk between juxtaposed membranes at MCSs relies on a defined composite of proteins and lipids that populate these microdomains in a dynamic fashion. This is particularly important in the nervous system, where alterations in the composition of MCSs have been shown to affect their functions and have been implicated in the pathogenesis of neurodegenerative diseases. In this review, we focus on the MCSs that are formed by the tethering of the endoplasmic reticulum (ER) to the mitochondria, the ER to the endo-lysosomes and the mitochondria to the lysosomes. We highlight how glycosphingolipids that are aberrantly processed/degraded and accumulate ectopically in intracellular membranes and the PM change the topology of MCSs, disrupting signaling pathways that lead to neuronal demise and neurodegeneration. In particular, we focus on neurodegenerative lysosomal storage diseases linked to altered glycosphingolipid catabolism.

Preclinical Enzyme Replacement Therapy with a Recombinant ß-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis.

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal ß-galactosidase (ß-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine ß-Gal fused to the plant lectin subunit B of ricin (mß-Gal:RTB). We show that long-term, bi-weekly systemic injection of mß-Gal:RTB in the ß-Gal-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, ß-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the ß-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.

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.

GM1 Gangliosidosis-A Mini-Review.

GM1 gangliosidosis is a progressive, neurosomatic, lysosomal storage disorder caused by mutations in the GLB1 gene encoding the enzyme ß-galactosidase. Absent or reduced ß-galactosidase activity leads to the accumulation of ß-linked galactose-containing glycoconjugates including the glycosphingolipid (GSL) GM1-ganglioside in neuronal tissue. GM1-gangliosidosis is classified into three forms [Type I (infantile), Type II (late-infantile and juvenile), and Type III (adult)], based on the age of onset of clinical symptoms, although the disorder is really a continuum that correlates only partially with the levels of residual enzyme activity. Severe neurocognitive decline is a feature of Type I and II disease and is associated with premature mortality. Most of the disease-causing ß-galactosidase mutations reported in the literature are clustered in exons 2, 6, 15, and 16 of the GLB1 gene. So far 261 pathogenic variants have been described, missense/nonsense mutations being the most prevalent. There are five mouse models of GM1-gangliosidosis reported in the literature generated using different targeting strategies of the Glb1 murine locus. Individual models differ in terms of age of onset of the clinical, biochemical, and pathological signs and symptoms, and overall lifespan. However, they do share the major abnormalities and neurological symptoms that are characteristic of the most severe forms of GM1-gangliosidosis. These mouse models have been used to study pathogenic mechanisms, to identify biomarkers, and to evaluate therapeutic strategies. Three GLB1 gene therapy trials are currently recruiting Type I and Type II patients (NCT04273269, NCT03952637, and NCT04713475) and Type II and Type III patients are being recruited for a trial utilizing the glucosylceramide synthase inhibitor, venglustat (NCT04221451).

Isolation of Mitochondria-Associated ER Membranes (MAMs), Synaptic MAMs, and Glycosphingolipid Enriched Microdomains (GEMs) from Brain Tissues and Neuronal Cells.

Subcellular fractionation is a valuable procedure in cell biology to separate and purify various subcellular constituents from one another, i.e., nucleus, cytosol, membranes/organelles, and cytoskeleton. The procedure relies on the use of differential centrifugation of cell and tissue homogenates. Fractionated subcellular organelles may be subjected to additional purification steps that enable the isolation of specific cellular sub-compartments, including interorganellar membrane contact sites. Here we outline a protocol tailored to the isolation of mitochondria, mitochondria-associated ER membranes (MAMs), and glycosphingolipid enriched microdomains (GEMs) from the adult mouse brain, primary neurospheres, and murine embryonic fibroblasts (MEFs). We also provide a detailed protocol for the purification of synaptosomes and their corresponding MAMs .

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.

Sialylation of host proteins as targetable risk factor for COVID-19 susceptibility and spreading: A hypothesis.

Individuals infected with the severe acute respiratory syndrome (SARS)-related coronavirus 2 (SARS-CoV-2) develop a critical and even fatal disease, called Coronavirus disease-19 (COVID-19), that eventually evolves into acute respiratory distress syndrome. The gravity of the SARS-CoV-2 pandemic, the escalating number of confirmed cases around the world, the many unknowns related to the virus mode of action, and the heterogenous outcome of COVID-19 disease in the population ask for the rapid development of alternative approaches, including repurposing of existing drugs, that may dampen virus infectivity. SARS-CoV-2 infects human cells through interaction with sialylated receptors at the surface of epithelial cells, such as angiotensin-converting enzyme 2 (ACE2). Glycan composition on virus entry receptors has been shown to influence the rate of infection of SARS-CoV-2 and spreading of virions has recently been linked to altered lysosomal exocytosis. These processes could concurrently involve the lysosomal system and its glycosidases. We hypothesize that modulating the activity of one of them, the lysosomal sialidase NEU1, could impinge on both the sialylation status of ACE2 and other host receptors as well as the extent of lysosomal exocytosis. Thus NEU1-controlled pathways may represent therapeutic targets, which could impact on SARS-CoV-2 susceptibility, infectivity, and spread.

Galactosialidosis: preclinical enzyme replacement therapy in a mouse model of the disease, a proof of concept.

Galactosialidosis is a rare lysosomal storage disease caused by a congenital defect of protective protein/cathepsin A (PPCA) and secondary deficiency of neuraminidase-1 and ß-galactosidase. PPCA is a lysosomal serine carboxypeptidase that functions as a chaperone for neuraminidase-1 and ß-galactosidase within a lysosomal multi-protein complex. Combined deficiency of the three enzymes leads to accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids and manifests in a systemic disease pathology with severity mostly correlating with the type of mutation(s) and age of onset of the symptoms. Here, we describe a proof-of-concept, preclinical study toward the development of enzyme replacement therapy for galactosialidosis, using a recombinant human PPCA. We show that the recombinant enzyme, taken up by patient-derived fibroblasts, restored cathepsin A, neuraminidase-1, and ß-galactosidase activities. Long-term, bi-weekly injection of the recombinant enzyme in a cohort of mice with null mutation at the PPCA (CTSA) locus (PPCA -/- ), a faithful model of the disease, demonstrated a dose-dependent, systemic internalization of the enzyme by cells of various organs, including the brain. This resulted in restoration/normalization of the three enzyme activities, resolution of histopathology, and reduction of sialyloligosacchariduria. These positive results underscore the benefits of a PPCA-mediated enzyme replacement therapy for the treatment of galactosialidosis.

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

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.

Transcription factor competition regulates lysosomal biogenesis and autophagy.

"In the field of observation, chance favours only the prepared mind" (Louis Pasteur). This motto seems to have guided our unexpected results published recently in Nature Communications, where we describe an epigenetic rheostat that regulates expression of the constituents of the lysosomal and autophagic systems.

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.

Isolation, Purification and Characterization of Exosomes fromFibroblast Cultures of Skeletal Muscle.

Exosomes are dynamic nanovesicles secreted by virtually all cells and are present in all biological fluids. Given their highly heterogeneous content exosomes have been implicated in many physiological and pathological processes that they exert by influencing cell-cell and cell-ECM communication. In recent years an increasing number of methods have been established for the purification and characterization of exosomes. These include ultracentrifugation, ultrafiltration, size exclusion chromatography, immune capture and precipitation using a proprietary polymer. Here, we provide a protocol based on differential ultracentrifugation and sucrose density gradients tailored for the isolation of crude and ultra-pure exosomes from primary fibroblast cultures derived from adult mouse skeletal muscle. This protocol can be adapted and modified for the isolation and characterization of exosomes from a variety of tissues and bodily fluids.

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.

Mitochondria-associated ER membranes (MAMs) and lysosomal storage diseases.

Lysosomal storage diseases (LSDs) comprise a large group of disorders of catabolism, mostly due to deficiency of a single glycan-cleaving hydrolase. The consequent endo-lysosomal accumulation of undigested or partially digested substrates in cells of virtually all organs, including the nervous system, is diagnostic of these diseases and underlies pathogenesis. A subgroup of LSDs, the glycosphingolipidoses, are caused by deficiency of glycosidases that process/degrade sphingolipids and glycosphingolipids (GSLs). GSLs are among the lipid constituents of mammalian membranes, where they orderly distribute and, together with a plethora of membrane proteins, contribute to the formation of discrete membrane microdomains or lipid rafts. The composition of intracellular membranes enclosing organelles reflects that at the plasma membrane (PM). Organelles have the tendencies to tether to one another and to the PM at specific membrane contact sites that, owing to their lipid and protein content, resemble PM lipid rafts. The focus of this review is on the MAMs, mitochondria associated ER membranes, sites of juxtaposition between ER and mitochondria that function as biological hubs for the exchange of molecules and ions, and control the functional status of the reciprocal organelles. We will focus on the lipid components of the MAMs, and highlight how failure to digest or process the sialylated GSL, GM1 ganglioside, in lysosomes alters the lipid conformation and functional properties of the MAMs and leads to neuronal cell death and neurodegeneration.

Galactosialidosis: historic aspects and overview of investigated and emerging treatment options.

INTRODUCTION: Galactosialidosis is a glycoprotein storage disease caused by mutations in the CTSA gene, encoding lysosomal protective protein/cathepsin A (PPCA). The enzyme's catalytic activity is distinct from its protective function towards ß-galactosidase (ß-GAL) and neuraminidase 1 (NEU1), with which PPCA forms a complex. In this configuration the two glycosidases acquire their full activity and stability in lysosomes. Deficiency of PPCA results in combined NEU1/ß-GAL deficiency. Because of its low incidence, galactosialidosis is considered an orphan disorder with no therapy yet available. AREAS COVERED: This review gives a historic overview on the discovery of PPCA, which defined galactosialidosis as a new clinical entity; the evidence for the existence of the PPCA/NEU1/ß-GAL complex; the clinical forms of galactosialidosis and disease-causing CTSA mutations. Ppca-/- mice have proven to be a suitable model to test different therapeutic approaches, paving the way for the development of clinical trials for patients with galactosialidosis. EXPERT OPINION: Improved understanding of the molecular bases of disease has sparked renewed incentive from clinicians and scientists alike to develop therapies for rare conditions, like GS, and has increased the willingness of biotech companies to invest in the manufacturing of new therapeutics. Both ERT and gene therapy may become available to patients in the near future.