BCM-95 and (2-hydroxypropyl)-ß-cyclodextrin reverse autophagy dysfunction and deplete stored lipids in Sap C-deficient fibroblasts.

Saposin (Sap) C deficiency is a rare variant form of Gaucher disease caused by impaired Sap C expression or accelerated degradation, and associated with accumulation of glucosylceramide and other lipids in the endo/lysosomal compartment. No effective therapies are currently available for the treatment of Sap C deficiency. We previously reported that a reduced amount and enzymatic activity of cathepsin (Cath) B and Cath D, and defective autophagy occur in Sap C-deficient fibroblasts. Here, we explored the use of two compounds, BCM-95, a curcumin derivative, and (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD), to improve lysosomal function of Sap C-deficient fibroblasts. Immunofluorescence and biochemical studies documented that each compound promotes an increase of the expression levels and activities of Cath B and Cath D, and efficient clearance of cholesterol (Chol) and ceramide (Cer) in lysosomes. We provide evidence that BCM-95 and HP-ß-CD enhance lysosomal function promoting autophagic clearance capacity and lysosome reformation. Our findings suggest a novel pharmacological approach to Sap C deficiency directed to treat major secondary pathological aspects in this disorder.

Gaucher disease due to saposin C deficiency is an inherited lysosomal disease caused by rapidly degraded mutant proteins.

Saposin (Sap) C is an essential cofactor for the lysosomal degradation of glucosylceramide (GC) by glucosylceramidase (GCase) and its functional impairment underlies a rare variant form of Gaucher disease (GD). Sap C promotes rearrangement of lipid organization in lysosomal membranes favoring substrate accessibility to GCase. It is characterized by six invariantly conserved cysteine residues involved in three intramolecular disulfide bonds, which make the protein remarkably stable to acid environment and degradation. Five different mutations (i.e. p.C315S, p.342_348FDKMCSKdel, p.L349P, p.C382G and p.C382F) have been identified to underlie Sap C deficiency. The molecular mechanism by which these mutations affect Sap C function, however, has not been delineated in detail. Here, we characterized biochemically and functionally four of these gene lesions. We show that all Sap C mutants are efficiently produced, and exhibit lipid-binding properties, modulatory behavior on GCase activity and subcellular localization comparable with those of the wild-type protein. We then delineated the structural rearrangement of these mutants, documenting that most proteins assume diverse aberrant disulfide bridge arrangements, which result in a substantial diminished half-life, and rapid degradation via autophagy. These findings further document the paramount importance of disulfide bridges in the stability of Sap C and provide evidence that accelerated degradation of the Sap C mutants is the underlying pathogenetic mechanism of Sap C deficiency.

Cathepsin-mediated regulation of autophagy in saposin C deficiency.

Saposin C deficiency, a rare variant form of Gaucher disease, is due to mutations in the prosaposin gene (PSAP) affecting saposin C expression and/or function. We previously reported that saposin C mutations affecting one cysteine residue result in autophagy dysfunction. We further demonstrated that the accumulation of autophagosomes, observed in saposin C-deficient fibroblasts, is due to an impairment of autolysosome degradation, partially caused by the reduced amount and enzymatic activity of CTSB (cathepsin B) and CTSD (cathepsin D). The restoration of both proteases in pathological fibroblasts results in almost completely recovery of autophagic flux and lysosome homeostasis.

Reduced cathepsins B and D cause impaired autophagic degradation that can be almost completely restored by overexpression of these two proteases in Sap C-deficient fibroblasts.

Saposin (Sap) C deficiency, a rare variant form of Gaucher disease, is due to mutations in the Sap C coding region of the prosaposin (PSAP) gene. Sap C is required as an activator of the lysosomal enzyme glucosylceramidase (GCase), which catalyzes glucosylceramide (GC) degradation. Deficit of either GCase or Sap C leads to the accumulation of undegraded GC and other lipids in lysosomes of monocyte/macrophage lineage. Recently, we reported that Sap C mutations affecting a cysteine residue result in increased autophagy. Here, we characterized the basis for the autophagic dysfunction. We analyzed Sap C-deficient and GCase-deficient fibroblasts and observed that autophagic disturbance was only associated with lack of Sap C. By a combined fluorescence microscopy and biochemical studies, we demonstrated that the accumulation of autophagosomes in Sap C-deficient fibroblasts is not due to enhanced autophagosome formation but to delayed degradation of autolysosomes caused, in part, to decreased amount and reduced enzymatic activity of cathepsins B and D. On the contrary, in GCase-deficient fibroblasts, the protein level and enzymatic activity of cathepsin D were comparable with control fibroblasts, whereas those of cathepsin B were almost doubled. Moreover, the enhanced expression of both these lysosomal proteases in Sap C-deficient fibroblasts resulted in close to functional autophagic degradation. Our data provide a novel example of altered autophagy as secondary event resulting from insufficient lysosomal function.

Efficient one-step chromatographic purification and functional characterization of recombinant human Saposin C.

Saposin (Sap) C is a small lysosomal disulfide bridge-containing glycoprotein required for glucosylceramide (GC) hydrolysis by glucosylceramidase (GCase). Sap C deficiency causes a variant form of Gaucher disease (GD), a rare genetic disorder characterized by GC accumulation in lysosomes of monocyte/macrophage lineage. Efforts to develop fast and efficient methodologies to express and purify Sap C have been made in the last years. Here, human Sap C was expressed in a bacterial strain that greatly enhances disulfide bond formation, and the recombinant protein was purified in a single chromatographic step using an affinity tag-based protein purification system. Mass spectrometry analysis demonstrated that disulfide bridges required for Sap C stability and functionality were retained. Consistently, the recombinant protein was shown to interact with anionic phospholipids-containing vesicles, and reconstitute GCase activity in vitro. Recombinant Sap C was efficiently endocytosed by Sap C-deficient fibroblasts, and targeted to lysosomes. These findings document that the bacterially purified Sap C exerts biological properties functionally equivalent to those observed for the native protein, indicating its potential use in the development of therapeutic intervention.

Autophagy in Gaucher disease due to saposin C deficiency.

Gaucher disease, due to a deficit of glucosylceramidase or, rarely, of its activator saposin C, is characterized by accumulation of glucosylceramide in the lysosomes of monocyte/macrophage lineage. In our study we demonstrate that saposin C deficiency due to mutations involving a cysteine residue results in increased autophagy. Autophagy was monitored by LC3 analysis and confirmed by electron microscopy; we observed a correlation among saposin C mutation, Gaucher phenotype and increased autophagy.

Saposin C mutations in Gaucher disease patients resulting in lysosomal lipid accumulation, saposin C deficiency, but normal prosaposin processing and sorting.

Gaucher disease (GD) is characterized by accumulation of glucosylceramide (GC) in the cells of monocyte/macrophage system. The degradation of GC is controlled by glucosylceramidase (GCase) and saposin (Sap) C, a member of a family of four small glycoproteins (Saps A, B, C and D), all derived by proteolytic processing of a common precursor, prosaposin (PSAP). Saps contain six cysteine residues, forming three disulfide bridges, that affect their structure and function. Sap C is an essential activator of GCase and its deficit impairs the GCase activity causing GD. In the present study the biological properties of cells from four recently described GD patients carrying mutations in the Sap C domain of the PSAP gene have been characterized. Two patients had mutations involving a cysteine residue, whereas the other two had a L349P mutation. It was found that: (i) in the four Sap C-deficient cells PSAP was normally processed and sorted, the lack of Sap C being mainly due to the Sap C instability in late endosomal/lysosomal environment; (ii) the decrease/absence of Sap C affected the GCase intracellular localization; (iii) the lowest level of Sap C and enhanced autophagy were observed in the cells, which carried a Sap C mutation involving a cysteine residue; (iv) the four Sap C-deficient fibroblasts stored GC, ceramide and cholesterol, the last two lipids being clearly localized in lysosomes; (v) a correlation was observed between the type of Sap C mutation and the Gaucher phenotype: apparently, mutations involving cysteine residues lead to a neurological variant of GD.

The potential action of galactose as a "chemical chaperone": increase of beta galactosidase activity in fibroblasts from an adult GM1-gangliosidosis patient.

BACKGROUND: The glycosphingolipid storage disorder GM1-gangliosidosis is a severe neurodegenerative condition for which no therapy is currently available. Protein misfolding in lysosomal defects may have the potential to be corrected by chemical chaperones: in vitro and clinical approaches are being investigated. AIMS: We investigated the in vitro effect of galactose on some lysosomal hydrolases, and its in vitro efficacy as a chemical chaperone in GM1-gangliosidosis. METHODS: Galactose was added to the culture medium of fibroblasts from patients, controls and transfected COS-1 cells. Enzyme assays of lysosomal hydrolases, beta galactosidase in particular, were performed. RESULTS: Our data show that galactose alters selectively alpha and beta galactosidases. A significant increase (2,5 fold) in beta galactosidase activity occurred when galactose was added to the cultured fibroblasts of an adult patient. Chemical chaperone therapy requires the presence of residual enzyme activity. The adult patient here reported is heterozygous for the p.T329A mutation that showed no beta galactosidase activity, and for the p.R442Q mutation with residual enzyme activity. The p.R442Q mutation was therefore selected as a potential target for the galactose chaperone; after the addition of galactose, COS-1 cells transfected with this mutation showed an increase in beta galactosidase activity from 6.9% to 12% of control values. CONCLUSIONS: These results suggest that galactose or its derivatives with potential chaperone properties could be used in the development of non-invasive therapies for GM1-gangliosidosis.

The secretion and maturation of prosaposin and procathepsin D are blocked in embryonic neural progenitor cells.

The notion that prosaposin (Prosap) is likely involved in brain development and regeneration led us to explore its expression in stem/progenitor neural cells and its fate after cell differentiation. The expression of procathepsin-cathepsin D (proCath-Cath D), an endoprotease that plays an important role in the processing and sorting of Prosap, has been concomitantly examined. Our data evidenced that in embryonic human neural progenitor cells (eHNPCs) intact and high molecular weight intermediate forms of Prosap and intermediate forms of Cath D accumulated inside the cells, while the formation of saposins and mature Cath D was impaired. Furthermore, neither Prosap nor proCath D were secreted from eHNPCs. The block of the processing and secretion shared by Prosap and proCath D was overcome during the course of differentiation of eHNPCs into a mixed population of astrocytes and neuronal cells. Upon differentiation, large amounts of Prosap and proCath D were secreted from the cells, while saposins and mature Cath D were produced inside the cells. The dramatic accumulation of Prosap (an antiapoptotic factor) and reduction of mature Cath D (a proapoptotic factor) in the undifferentiated eHNPCs most likely play a role in the molecular mechanisms regulating the resistance to apoptotic signals of these cells and might represent a critically important issue in HNPCs biology.

Saposin B binds and transfers phospholipids.

Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known. Using large unilamellar vesicles (LUVs) as membrane models, we have now found that Sap B simultaneously extracts from the lipid surface neutral [phosphatidylcholine (PC)] and anionic [phosphatidylinositol (PI)] phospholipids, fewer SLs [ganglioside GM1 (GM1) or cerebroside sulfate (CS)], and no Chol. More PI than SL (GM1 or CS) was solubilized from LUVs containing equal amounts of PI and SLs. An increase in PI level had a poor effect on the Sap B-induced solubilization of GM1 or CS but strongly inhibited that of PC. Sap B was able not only to bind, but also to transfer phospholipids between lipid surfaces. Both the phospholipid binding and transfer activities were optimal at low pH values. These results represent the first biochemical analysis of the Sap B interaction with phospholipids. The capacity of Sap B to bind and transfer phospholipids occurs under conditions mimicking the interior of the late endosomal/lysosomal compartment and thus might have physiological relevance.

The N370S (Asn370-->Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C.

The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370-->Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (approximately 20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo.

Glucosylceramidase mass and subcellular localization are modulated by cholesterol in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability.

Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids.

Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles. The Sap D-particle complexes formed in an acidic milieu can be dissociated by an increase in pH, suggesting a dynamic association of Sap D with membranes. The presence of anionic phospholipids is required also for the Sap D-induced perturbation and solubilization of membranes containing a neutral sphingolipid such as ceramide or a ganglioside such as G(M1). At appropriate Sap D concentrations Cer and G(M1) are solubilized as constituents of small phospholipid particles. Our findings imply that most functions of Sap D are dependent on its interaction with anionic phospholipids, which mediate the Sap D effect on other components of the membrane such as sphingolipids. On consideration of the properties of Sap D we propose that Sap D might have a role in the definition of the structure and function of membranes, such as the intra-endolysosomal membranes, that are rich in anionic phospholipids.