Alice Polchi

Signaling Pathways in Exosomes Biogenesis, Secretion and Fate

Exosomes are small extracellular vesicles (30–100 nm) derived from the endosomal system, which have raised considerable interest in the last decade. Several studies have shown that they mediate cell-to-cell communication in a variety of biological processes. Thus, in addition to cell-to-cell direct interaction or secretion of active molecules, they are now considered another class of signal mediators. Exosomes can be secreted by several cell types and retrieved in many body fluids, such as blood, urine, saliva and cerebrospinal fluid. In addition to proteins and lipids, they also contain nucleic acids, namely mRNA and miRNA. These features have prompted extensive research to exploit them as a source of biomarkers for several pathologies, such as cancer and neurodegenerative disorders. In this context, exosomes also appear attractive as gene delivery vehicles. Furthermore, exosome immunomodulatory and regenerative properties are also encouraging their application for further therapeutic purposes. Nevertheless, several issues remain to be addressed: exosome biogenesis and secretion mechanisms have not been clearly understood, and physiological functions, as well as pathological roles, are far from being satisfactorily elucidated.

Recent developments in therapeutic approaches for lysosomal storage diseases.

Genetic mutations that cause specific lysosomal protein deficiencies account for more than 45 Lysosomal Storage Diseases (LSDs), mostly pre-adult disorders which are associated with neurological symptoms and mental retardation. Interestingly, such diseases are often characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases. During the past twenty years, different approaches have been introduced for the treatment of these disorders, several of which are now in routine clinical use or clinical trials. Among them, enzyme replacement therapy (ERT) represented a major progress. However, the usefulness of ERT is limited due to the fact that enzyme distribution is insufficient and treatment costs are very high. A further novel therapeutic option for LSDs is based on the use of small molecules, that can either inhibit a key enzyme which is responsible for substrate synthesis (substrate reduction) or act as a chaperone to increase the residual activity of the lysosomal enzyme (pharmacological chaperones). In addition, recently various gene therapy approaches have been developed, mostly based on adeno-associated and lentiviral vectors, and strategies based on stem cells administration are beginning their route. This review provides an update of the status of research on LSDs therapeutic approaches, including recent patents in the field.

Abnormal cortical lysosomal β-hexosaminidase and β-galactosidase activity at post-synaptic sites during Alzheimer's disease progression

A critical role of endosomal-lysosomal system alteration in neurodegeneration is supported by several studies. Dysfunction of the lysosomal compartment is a common feature also in Alzheimers disease. Altered expression of lysosomal glycohydrolases has been demonstrated not only in the brain and peripheral tissues of Alzheimers disease patients, but also in presymptomatic subjects before degenerative phenomenon becomes evident. Moreover, the presence of glycohydrolases associated to the plasma membrane have been widely demonstrated and their alteration in pathological conditions has been documented. In particular, lipid microdomains-associated glycohydrolases can be functional to the maintenance of the proper glycosphingolipids pattern, especially at cell surface level, where they are crucial for the function of cell types such as neurons. In this study we investigated the localization of β-hexosaminidase and β-galactosidase glycohydrolases, both involved in step by step degradation of the GM1 to GM3 gangliosides, in lipid microdomains from the cortex of both an early and advanced TgCRND8 mouse model of Alzheimers disease. Throughout immunoprecipitation experiments of purified cortical lipid microdomains, we demonstrated for the first time that β-hexosaminidase and β-galactosidase are associated with post-synaptic vesicles and that their activities are increased at both the early and the advanced stage of Alzheimers disease. The early increase of lipid microdomain-associated β-hexosaminidase and β-galactosidase activities could have relevant implications for the pathophysiology of the disease since their possible pharmacological manipulation could shed light on new reliable targets and biological markers of Alzheimers disease.

TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

Glycohydrolases β-hexosaminidase and β-galactosidase are associated with lipid microdomains of Jurkat T-lymphocytes

Growing evidence suggests the presence of active lysosomal enzymes in extra-lysosomal compartments, such as the plasma membrane. Although in the past little attention was paid to glycohydrolases acting on cellular compartments different from lysosomes, there is now increasing interest on plasma membrane-associated glycohydrolases because they should be involved, together with glycosyltransferases, in glycosphingolipids oligosaccharide modification processes regulating cell-to-cell and/or cell-environment interactions in both physiological and pathological conditions. Starting from the previous evidence of the presence of β-hexosaminidase and β-galactosidase at the plasma membrane of cultured fibroblasts, we here investigated the association of these glycohydrolases with lipid microdomains of Jurkat T-lymphocytes. Monosialoganglioside GM3 represents the major glycosphingolipid constituent of T-cell plasma membrane and its amount largely increases after T-cell stimulation. β-hexosaminidase and β-galactosidase cleave specific β-linked terminal residues from a wide range of glycoconjugates and in particular are involved in the stepwise degradation of GM1 to GM3 ganglioside. Here we demonstrated that fully processed plasma membrane-associated β-hexosaminidase and β-galactosidase co-distribute with the lipid microdomain markers and co-immunoprecipitate with the signalling protein lck in Jurkat T-cell. Furthermore, Jurkat cell stimulation up-regulates the expression and activity of lysosomal β-hexosaminidase and β-galactosidase and increases their targeting to lipid microdomains. The non-random distribution of plasma membrane-associated β-hexosaminidase and β-galactosidase and their localization within lipid microdomains, suggest a role of these enzymes in the local reorganization of glycosphingolipid-based signalling units.

Chaperone Therapy for GM2 Gangliosidosis: Effects of Pyrimethamine on β-Hexosaminidase Activity in Sandhoff Fibroblasts

Sphingolipidoses are inherited genetic diseases due to mutations in genes encoding proteins involved in the lysosomal catabolism of sphingolipids. Despite a low incidence of each individual disease, altogether, the number of patients involved is relatively high and resolutive approaches for treatment are still lacking. The chaperone therapy is one of the latest pharmacological approaches to these storage diseases. This therapy allows the mutated protein to escape its natural removal and to increase its quantity in lysosomes, thus partially restoring the metabolic functions. Sandhoff disease is an autosomal recessive inherited disorder resulting from β-hexosaminidase deficiency and characterized by large accumulation of GM2 ganglioside in brain. No enzymatic replacement therapy is currently available, and the use of inhibitors of glycosphingolipid biosynthesis for substrate reduction therapy, although very promising, is associated with serious side effects. The chaperone pyrimethamine has been proposed as a very promising drug in those cases characterized by a residual enzyme activity. In this review, we report the effect of pyrimethamine on the recovery of β-hexosaminidase activity in cultured fibroblasts from Sandhoff patients.

Rapamycin Loaded Solid Lipid Nanoparticles as a New Tool to Deliver mTOR Inhibitors: Formulation and in Vitro Characterization

Recently, the use of mammalian target of rapamycin (mTOR) inhibitors, in particular rapamycin (Rp), has been suggested to improve the treatment of neurodegenerative diseases. However, as Rp is a strong immunosuppressant, specific delivery to the brain has been postulated to avoid systemic exposure. In this work, we fabricated new Rp loaded solid lipid nanoparticles (Rp-SLN) stabilized with polysorbate 80 (PS80), comparing two different methods and lipids. The formulations were characterized by differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), wide angle X-ray scattering (WAXS), cryo-transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS) and particle tracking. In vitro release and short-term stability were assessed. Biological behavior of Rp-SLN was tested in SH-SY5Y neuroblastoma cells. The inhibition of mTOR complex 1 (mTORC1) was evaluated over time by a pulse-chase study compared to free Rp and Rp nanocrystals. Compritol Rp-SLN resulted more stable and possessing proper size and surface properties with respect to cetyl palmitate Rp-SLN. Rapamycin was entrapped in an amorphous form in the solid lipid matrix that showed partial crystallinity with stable Lβ, sub-Lα and Lβ′ arrangements. PS80 was stably anchored on particle surface. No drug release was observed over 24 h and Rp-SLN had a higher cell uptake and a more sustained effect over a week. The mTORC1 inhibition was higher with Rp-SLN. Overall, compritol Rp-SLN show suitable characteristics and stability to be considered for further investigation as Rp brain delivery system.

Oncogenic H-Ras Up-Regulates Acid β-Hexosaminidase by a Mechanism Dependent on the Autophagy Regulator TFEB

The expression of constitutively active H-RasV12 oncogene has been described to induce proliferative arrest and premature senescence in many cell models. There are a number of studies indicating an association between senescence and lysosomal enzyme alterations, e.g. lysosomal β-galactosidase is the most widely used biomarker to detect senescence in cultured cells and we previously reported that H-RasV12 up-regulates lysosomal glycohydrolases enzymatic activity in human fibroblasts. Here we investigated the molecular mechanisms underlying lysosomal glycohydrolase β-hexosaminidase up-regulation in human fibroblasts expressing the constitutively active H-RasV12. We demonstrated that H-Ras activation increases β-hexosaminidase expression and secretion by a Raf/extracellular signal-regulated protein kinase dependent pathway, through a mechanism that relies on the activity of the transcription factor EB (TFEB). Because of the pivotal role of TFEB in the regulation of lysosomal system biogenesis and function, our results suggest that this could be a general mechanism to enhance lysosomal enzymes activity during oncogene-induced senescence.

Methods to discriminate the distribution of acidic glycohydrolases between the endosomal-lysosomal systems and the plasma membrane.

The endosomal-lysosomal system plays important roles in cellular physiology. Beyond the well-known function as terminal degradative compartment, necessary to maintain the health of the cell, lysosomes are critical for many other cellular processes, such as termination of signaling mediated by cell surface receptors and processing of internalized peptides in antigen-presenting cells. Moreover, the intracellular membrane trafficking related to the endosomal-lysosomal system plays a pivotal role in diverse physiological and pathological processes, such as exocytosis, plasma membrane repair, and endocytosis. Increasing evidences suggest that several lysosomal glycohydrolases, together with nonlysosomal glycohydrolases, are associated with cell membranes in their active form, and they are localized into lipid microdomains. The role of these forms in physiological and pathological conditions, such as differentiation and aging, neurodegenerative diseases, and cancer spreading, is under investigation. Here we provide general methods to purify lipid microdomain proteins and to discriminate cell surface lipid microdomains-associated glycohydrolases from those not exposed on cell surface. The methods reported here have been developed to characterize the membrane-associated forms of the acidic glycohydrolases β-hexosaminidase and β-galactosidase, but they may be applied to any other protein of interest.

β-Hexosaminidase over-expression affects lysosomal glycohydrolases expression and glycosphingolipid metabolism in mammalian cells

Lysosomes are not only degrading organelles but also involved in other critical cellular processes. In addition, active lysosomal glycohydrolases have been detected in an extra-lysosomal compartment: the presence of glycohydrolases on the plasma membrane (PM) has been widely demonstrated, and a possible role on the modification of the cell surface glycosphingolipids (GSL) participating in the modulation of cell functions such as cell-to-cell interactions and signal transduction pathways has been proposed. On this basis, the coordinated expression of lysosomal glycohydrolases and their translocation to the PM appear to be crucial for many cellular events. In this paper, we report evidence for the existence of a coordinated mechanism regulating the expression/activity of both lysosomal and PM-associated glycohydrolases. We show that the over-expression of the acidic glycohydrolase β-hexosaminidase α-subunit in mouse NIH/3T3 fibroblasts induces the increased expression of the Hex β-subunit necessary to form the active isoenzyme dimers as well as of other glycohydrolases participating in the GSL catabolism, such as β-galactosidase and β-glucocerebrosidase. More interestingly, this regulatory effect was also extended to the PM-associated hydrolases. In addition, transfected cells displayed a rearrangement of the GSL expression pattern that cannot be simply explained by the increased activity of a single enzyme. These observations clearly indicate that the expression level of metabolically related glycohydrolases is regulated in a coordinated manner and this regulation mechanism also involves the PM-associated isoforms.