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Uptake of the fluid-phase marker horseradish peroxidase. Normal (NF) and Sandhoff (SF) fibroblasts were incubated with 3 mg/ml of HRP in DMEM for 2 h at 37°C. The amount of HRP internalized by the cells was quantified by enzyme activity assay. Three different fibroblast samples from both SD patients and healthy controls were used and three independent experiments for each cell sample were performed. Results show the means ± SD of all the experiments performed with each genotype. ** P \ 0.01 (SF vs. NF) according to unpaired two-tailed Student's t test
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Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the beta-subunit of beta-hexosaminidase, that result in beta-hexosaminidase A (alphabeta) and beta-hexosaminidase B (betabeta) deficiency. This leads to the storage of GM2 ganglioside in endosomes and lysosomes, which ends in a progressive neurodegenerat...
Citations
... Human fibroblasts from an anonymous Sandhoff patient, taken before 2002, were offered for previous studies by Cell Line and DNA Biobank from patients affected by Genetic Disease of Istituto Giannina Gaslini of Genova (Italy) [48]. These particular Sandhoff cells feature a genotype characterized by a missense mutation in the gene coding for the β subunit of the Hex enzymes, which leads to the formation of an unstable mRNA [49,50]. ...
Lysosomal storage disorders (LSDs) are a set of metabolic diseases caused by mutations in genes that are in charge of the production of lysosomal enzymes, resulting in the buildup of non-degraded substrates and the consequent systemic damage that mainly involves the Central Nervous System (CNS). One of the most widely used and studied treatments is Enzyme Replacement Therapy, which is based on the administration of the recombinant deficient enzyme. This strategy has often proved fallacious due to the enzyme instability in body fluids and its inability to reach adequate levels in the CNS. In this work, we developed a system based on nanotechnology that allows a stable enzyme to be obtained by its covalent immobilization on nanoparticles (NPs) of polylactic acid, subsequently administered to a cellular model of LSDs, i.e., Sandhoff disease, caused by the absence or deficiency of the β-d-N-acetyl-hexosaminidase A (HexA) enzyme. The HexA enzymes, loaded onto the polymeric NPs through an immobilization procedure that has already been investigated and validated, were found to be stable over time, maintain optimal kinetic parameters, be able to permeate the plasma membrane, hydrolyze HexA’s natural substrate, and restore enzyme activity close to the levels of healthy cells. These results thus lay the foundation for testing the HexA-NPs in animal models of the disease and thus obtaining an efficient drug-delivery system.
... Primary lysosomal storage disorders (LSDs) is caused by the dysfunction of the lysosomal system (Tancini et al. 2009). LSDs such as GM1 and GM2 gangliosidoses (GSLs) are caused by the defects in β-galactosidase and βhexosaminidase which leads to the degeneration of neurons and disruption of lysosomal system (Mahuran 1999;Walkley 2009). ...
The endosomal-lysosomal system mediates the process of protein degradation through endocytic pathway. This system consists of early endosomes, late endosomes, recycling endosomes and lysosomes. Each component in the endosomal-lysosomal system plays individual crucial role and they work concordantly to ensure protein degradation can be carried out functionally. Dysregulation in the endosomal-lysosomal system can contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD). In AD endosomal-lysosomal abnormalities are the earliest pathological features to note and hence it is important to understand the involvement of endosomal-lysosomal dysfunction in the pathogenesis of AD. In-depth understanding of this dysfunction can allow development of new therapeutic intervention to prevent and treat AD.
... Cells were incubated for 16 h and then stimulated or not (resting) with PHA for 24 h. The culture medium was recovered, and HRP activity was determined as previously reported [45]. One unit (U) corresponds to the amount of enzyme that oxidizes 1 µmol of substrate/min at pH 5.0 at 25 • C. ...
The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of β-hexosaminidase (Hex, EC 3.2.1.52) and β-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.
... Even when a primary defi ciency in a lysosomal glycohydrolase, activator protein, or saposin directly involved in the degradation of GM2 or GM3 ( 52 ) is not present, the accumulation of GM2/GM3 appears to occur, at least in part, in the endosomal/lysosomal system ( 26,29,30,34,53 ) and/or in lipid rafts ( 24,27,33 ). As well as for defi ciencies in catabolic proteins not directly involved in degradation of these gangliosides, defects in traffi cking of gangliosides from endosomes to the Golgi apparatus ( 53,54 ) or from late endosomes to lysosomes ( 55,56 ) also observed in some lysosomal storage diseases can produce similar accumulation. In chronic diseases, such ganglioside accumulation is typically detected in neurons ( 29,30,34,57 ) and, in some cases, in activated microglia ( 51 ). ...
Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol but also DMSO induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide (LPS), which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2 or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... Both isoenzymes Hex A (ab-dimer) and Hex B (bb-dimer) are able to hydrolyze neutral substrates, but only Hex A is capable to convert GM2 to GM3, in the presence of the GM2 activator protein (Mahuran, 1999). Lack of b-subunit activity, as is the case of Sandhoff disease, lead to an alteration in the terminal endocytic organelles of Sandhoff fibroblasts (Tancini et al., 2010), whereas the overexpression of Hex a-subunit induces the coordinate increased expression of other glycohydrolases participating in the glycosphingolipid catabolism, a regulatory effect that particularly involves the plasma membrane-associated isoforms . ...
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.
... Evidence have been reported demonstrating an increase of lysosomal glycohydrolases activity in the brain and also in peripheral tissues of AD patients (Adamec et al., 2000; Cataldo et al., 1996 Cataldo et al., , 1995 Emiliani et al., 2003; Mathews et al., 2002; Nixon et al., 2001). A critical role of lysosomal system alteration in neurodegeneration is supported by studies of primary lysosomal storage disorders (LSDs) (Tancini et al. 2010). GM1 and GM2 gangliosidoses are LSDs caused by defective -galactosidase (Gal, EC 3.2.1.23) ...
... Recently, it was shown that the activation of TFEB, a transcription factor that controls lysosomal biogenesis and function, is accompanied by an increase of mature bhexosaminidase on cell surface [21]. From a pathological point of view, mutations in the aand b-subunit coding genes lead to the development of Tay-Sachs and Sandhoff diseases, respectively, which are severe lysosomal storage disorders associated with neurodegeneration [22]. In addition, b-hexosaminidase altered expression has been often associated with cancer [23,24] and namely the presence of Hex S has been observed in leukaemic cells but not in their normal counterparts [25]. ...
... Recently, it was shown that the activation of TFEB, a transcription factor that controls lysosomal biogenesis and function, is accompanied by an increase of mature bhexosaminidase on cell surface [21]. From a pathological point of view, mutations in the aand b-subunit coding genes lead to the development of Tay-Sachs and Sandhoff diseases, respectively, which are severe lysosomal storage disorders associated with neurodegeneration [22]. In addition, b-hexosaminidase altered expression has been often associated with cancer [23,24] and namely the presence of Hex S has been observed in leukaemic cells but not in their normal counterparts [25]. ...
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.
... In patient fibroblasts no significant change in lysosomal pH has been found for Krabbe [32], NPA [14], NPC [14,30,31], MPS II and IIIA [7,14] and Farber disease [14]. Whilst other studies have found increased pH in MSD [7], Fabry [32], Sandhoff fibroblasts [33] and in a cell culture model of NPC [38]) RAW cells were labelled with 5 μM lysosensor yellow/blue in 1 ml RPMI (10% FCS) for 5 min at 37 °C and viewed under the fluorescent microscope. Imino sugars (NB-DMJ and NB-DHJ) with no inhibitory activity did not alter endolysosomal pH (not shown). ...
... Fluid phase endocytosis assay was performed with WT and WR MEFs according to Tancini et al. [28]. Wild type and wobbler MEFs were incubated with horseradish peroxidase (HRP) in DMEM and resuspended in 40 mM potassium phosphate buffer containing 0.5% (v/v) Triton X-100, pH 6.8 on ice. ...
The identification of the mutation causing the phenotype of the amyotrophic lateral sclerosis (ALS) model mouse, wobbler, has linked motor neuron degeneration with retrograde vesicle traffic. The wobbler mutation affects protein stability of Vps54, a ubiquitously expressed vesicle-tethering factor and leads to partial loss of Vps54 function. Moreover, the Vps54 null mutation causes embryonic lethality, which is associated with extensive membrane blebbing in the neural tube and is most likely a consequence of impaired vesicle transport. Investigation of cells derived from wobbler and Vps54 null mutant embryos demonstrates impaired retrograde transport of the Cholera-toxin B subunit to the trans-Golgi network and mis-sorting of mannose-6-phosphate receptors and cargo proteins dependent on retrograde vesicle transport. Endocytosis assays demonstrate no difference between wobbler and wild type cells, indicating that the retrograde vesicle traffic to the trans-Golgi network, but not endocytosis, is affected in Vps54 mutant cells. The results obtained on wobbler cells were extended to test the use of cultured skin fibroblasts from human ALS patients to investigate the retrograde vesicle traffic. Analysis of skin fibroblasts of ALS patients will support the investigation of the critical role of the retrograde vesicle transport in ALS pathogenesis and might yield a diagnostic prospect.
