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Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by SCARB2 gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, GBA1 . However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by PSAP and GBA1 mutations, whereas SCARB2 -linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in GBA1 - and SCARB2 -mutant fibroblasts than PSAP -mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than GBA1 -mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare PSAP and SCARB2 biallelic pathogenic variants in comparison with the profile observed in GBA1 -linked GCase deficiency.

Saposin A is a post-translation product of the prosaposin (PSAP) gene that serves as an activator protein of the galactocerebrosidase (GALC) enzyme, and is necessary for the degradation of certain glycosphingolipids. Deficiency of saposin A leads to a clinical picture identical to that of early-infantile Krabbe disease caused by GALC enzyme deficiency. Galactosylsphingosine, also known as psychosine, is a substrate of the GALC enzyme that is known to be elevated in classic Krabbe disease. We present the case of an 18-month-old male with clinical and radiological findings concerning for Krabbe disease who had preserved GALC enzyme activity and negative GALC gene sequencing, but was found to have a homozygous variant, c.257 T > A (p.I86N), in the saposin A peptide of PSAP. Psychosine determination on dried blood spot at 18 months of age was elevated to 12 nmol/L (normal <3 nmol/L). We present this case to add to the literature on the rare diagnosis of atypical Krabbe disease due to saposin A deficiency, to report a novel presumed pathogenic variant within PSAP, and to suggest that individuals with saposin A deficiency may have elevated levels of psychosine, similar to children with classic Krabbe disease due to GALC deficiency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Metachromatic leukodystrophy (MLD) is a rare sphingolipid storage disorder caused by arylsulfatase A (ARSA) deficiency, resulting in central and peripheral demyelination. However, an uncommon form of MLD caused by saposin B deficiency is also described (around 10 mutations reported till date). MLD is a systemic disorder affecting the central and peripheral nervous system, gall bladder, and kidneys. Acute flaccid paralysis as the initial clinical presentation is previously known in ARSA-deficient MLD. Hereby, we report a child with acute flaccid paralysis with brain magnetic resonance imaging showing nonspecific periventricular leukodystrophy. He had progressive cognitive decline with gall bladder polyposis. ARSA levels were within normal limits. Leukodystrophy gene panel revealed a homozygous pathogenic deletion (Lys227del variant) in prosaposin ( PSAP ) gene. Hence, a final diagnosis of saposin B-deficient MLD was established. The index case highlights the importance of clinical and electrophysiological clues in the diagnosis of such atypical presentations of MLD., Competing Interests: None., (Georg Thieme Verlag KG Stuttgart · New York.)

Gaucher disease is caused by mutations in GBA1 encoding acid β-glucosidase (GCase). Saposin C enhances GCase activity and protects GCase from intracellular proteolysis. Structure simulations indicated that the mutant GCases, N370S (0 S), V394L (4L) and D409V(9V)/H(9H), had altered function. To investigate the in vivo function of Gba1 mutants, mouse models were generated by backcrossing the above homozygous mutant GCase mice into Saposin C deficient (C*) mice. Without saposin C, the mutant GCase activities in the resultant mouse tissues were reduced by ~50% compared with those in the presence of Saposin C. In contrast to 9H and 4L mice that have normal histology and life span, the 9H;C* and 4L;C* mice had shorter life spans. 9H;C* mice developed significant visceral glucosylceramide (GC) and glucosylsphingosine (GS) accumulation (GC»GS) and storage macrophages, but lesser GC in the brain, compared to 4L;C* mice that presents with a severe neuronopathic phenotype and accumulated GC and GS primarily in the brain. Unlike 9V mice that developed normally for over a year, 9V;C* pups had a lethal skin defect as did 0S;C* mice resembled that of 0S mice. These variant Gaucher disease mouse models presented a mutation specific phenotype and underscored the in vivo role of Saposin C in the modulation of Gaucher disease.

Gaucher disease is mainly caused by the deficiency of lysosomal acid β-glucosidase. Gaucher disease caused by the deficiency of saposin C is rare. Here we report a patient mainly presenting with hepatosplenomegaly, thrombocytopenia and anemia. EEG examination revealed increased theta waves. Gaucher cells identified in his bone marrow and the highly elevated plasma chitotriosidase activity and glucosylsphingosine supported a diagnosis of Gaucher disease. However, the leukocyte β-glucosidase activity was in a normal range. Sanger sequencing revealed a novel maternal exonic mutation c.1133C>G (p.Pro378Arg) in exon 10 of the PSAP gene, which codes the Sap C domain of PSAP protein. To search for other underlying mutations in this patient, whole genome sequencing was applied and revealed a deletion involving exon 2 to 7 of PSAP gene. The deletion appears as a de novo event on paternal chromosome. We concluded that biallelic mutations of PSAP gene were the cause of this patient's Gaucher disease. Our finding expands the mutation spectrum of Gaucher disease with saposin C deficiency., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Prosaposin (PSAP) deficiency is an ultra-rare, fatal infantile lysosomal storage disorder (LSD) caused by variants in the PSAP gene, with seven subjects reported so far. Here, we provide the clinical, biochemical and molecular characterization of two additional PSAP deficiency cases. Lysoplex, a targeted resequencing approach was utilized to identify the variant in the first patient, while quantification of plasma lysosphingolipids (lysoSLs), assessed by liquid chromatography mass spectrometry (LC-MS/MS) and brain magnetic resonance imaging (MRI), followed by Sanger sequencing allowed to attain diagnosis in the second case. Functional studies were carried out on patients' fibroblast lines to explore the functional impact of variants. The two patients were homozygous for two different truncating PSAP mutations (c.895G>T, p.Glu299*; c.834&#95;835delGA, p.Glu278Aspfs*27). Both variants led to a complete lack of processed transcript. LC-MS/MS and brain MRI analyses consistently provided a distinctive profile in the two children. Quantification of specific plasma lysoSLs revealed elevated levels of globotriaosylsphingosine (LysoGb3) and glucosylsphingosine (GlSph), and accumulation of autophagosomes, due to a decreased autophagic flux, was observed. This report documents the successful use of plasma lysoSLs profiling in the PSAP deficiency diagnosis, as a reliable and informative tool to obtain a preliminary information in infantile cases with complex traits displaying severe neurological signs and visceral involvement., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations., (© 2015 Zhou et al.)

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)-β-cyclodextrin (HP-β-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-β-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., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Saposin B (Sap B) is an essential activator protein for arylsulfatase A in the hydrolysis of sulfatide, a lipid component of myelin. To study Sap B's role in hearing and balance, a Sap B-deficient (B(-/-)) mouse was evaluated. At both light and electron microscopy (EM) levels, inclusion body accumulation was seen in satellite cells surrounding spiral ganglion (SG) neurons from postnatal month 1 onward, progressing into large vacuoles preceding satellite cell degeneration, and followed by SG degeneration. EM also revealed reduced or absent myelin sheaths in SG neurons from postnatal month 8 onwards. Hearing loss was initially seen at postnatal month 6 and progressed thereafter for frequency-specific stimuli, whereas click responses became abnormal from postnatal month 13 onward. The progressive hearing loss correlated with the accumulation of inclusion bodies in the satellite cells and their subsequent degeneration. Outer hair cell numbers and efferent function measures (distortion product otoacoustic emissions and contralateral suppression) were normal in the B(-/-) mice throughout this period. Alcian blue staining of SGs demonstrated that these inclusion bodies corresponded to sulfatide accumulation. In contrast, changes in the vestibular system were much milder, but caused severe physiologic deficits. These results demonstrate that loss of Sap B function leads to progressive sulfatide accumulation in satellite cells surrounding the SG neurons, leading to satellite cell degeneration and subsequent SG degeneration with a resultant loss of hearing. Relative sparing of the efferent auditory and vestibular neurons suggests that alternate glycosphingolipid metabolic pathways predominate in these other systems., (Copyright © 2015 the authors 0270-6474/15/353263-13$15.00/0.)

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&#95;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., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A-D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A-D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Duchenne muscular dystrophy caused by a mutation in the X-linked dystrophin gene induces metabolic and structural disorders in the brain. A lack of dystrophin in brain structures is involved in impaired cognitive function. Prosaposin (PS), a neurotrophic factor, is abundant in the choroid plexus and various brain regions. We investigated whether PS serves as a link between dystrophin loss and gross and/or ultrastructural brain abnormalities., Methodology/principal Findings: The distribution of PS in the brains of juvenile and adult mdx mice was investigated by immunochemistry, Western blotting, and in situ hybridization. Immunochemistry revealed lower levels of PS in the cytoplasm of neurons of the cerebral cortex, hippocampus, cerebellum, and choroid plexus in mdx mice. Western blotting confirmed that PS levels were lower in these brain regions in both juveniles and adults. Even with low PS production in the choroids plexus, there was no significant PS decrease in cerebrospinal fluid (CSF). In situ hybridization revealed that the primary form of PS mRNA in both normal and mdx mice was Pro+9, a secretory-type PS, and the hybridization signals for Pro+9 in the above-mentioned brain regions were weaker in mdx mice than in normal mice. We also investigated mitogen-activated protein kinase signalling. Stronger activation of ERK1/2 was observed in mdx mice, ERK1/2 activity was positively correlated with PS activity, and exogenous PS18 stimulated both p-ERK1/2 and PS in SH-SY5Y cells., Conclusions/significance: Low levels of PS and its receptors suggest the participation of PS in some pathological changes in the brains of mdx mice.

Combined saposin A and saposin B deficiency (AB(-/-)) was created in mice by knock-in of point mutations into the saposin A and B domains of the Psap (encoding prosaposin) locus. PSAP is the precursor of saposin A, saposin B and two other members, saposin C and saposin D. Those four saposins have multiple functions including their roles as glycosphingolipid activator proteins in a lysosomal glycosphingolipid degradation pathway. Saposin A participates in the removal of galactose from galactosylceramide and galactosylsphingosine by enhancing β-galactosylceramidase activity. Saposin B has lipid binding properties and is involved in glycosphingolipid metabolism by presenting the substrates to specific enzymes for degradation, i.e., sulfatide to ARSA/arylsulfatase A, lactosylceramide to GALC/GM-1-β-galactosylceramidase, and globotriaosylceramide to GLA/α-galactosidase. Galactosylceramide and sulfatide are myelin glycosphingolipids involved in carbohydrate interaction between synapses. The AB(-/-) mice develop accumulation of multiple glycosphingolipids in various organs. Sulfatide and galactosylsphingosine, a deacylated form of galactosylceramide, are the major substrates accumulated in the CNS of AB(-/-) mice. The latter is a toxic metabolite to oligodendrocytes and results in demyelination and cell death.

Individual saposin A (A-/-) and saposin B (B-/-)-deficient mice show unique phenotypes caused by insufficient degradation of myelin-related glycosphingolipids (GSLs): galactosylceramide and galactosylsphingosine and sulfatide, respectively. To gain insight into the interrelated functions of saposins A and B, combined saposin AB-deficient mice (AB-/-) were created by knock-in point mutations into the saposins A and B domains on the prosaposin locus. Saposin A and B proteins were undetectable in AB-/- mice, whereas prosaposin, saposin C and saposin D were expressed near wild-type (WT) levels. AB-/- mice developed neuromotor deterioration at >61 days and exhibited abnormal locomotor activity and enhanced tremor. AB-/- mice (~96 days) lived longer than A-/- mice (~85 days), but shorter than B-/- mice (~644 days). Storage materials were observed in Schwann cells and neuronal processes by electron microscopy. Accumulation of p62 and increased levels of LC3-II were detected in the brainstem suggesting altered autophagy. GSL analyses by (liquid chromatography) LC/MS identified substantial increases in lactosylceramide in AB-/- mouse livers. Sulfatide accumulated, but galactosylceramide remained at WT levels, in the AB-/- mouse brains and kidneys. Brain galactosylsphingosine in AB-/- mice was ~68% of that in A-/- mice. These findings indicate that combined saposins A and B deficiencies attenuated GalCer-β-galactosylceramidase and GM1-β-galactosidase functions in the degradation of lactosylceramide preferentially in the liver. Blocking sulfatide degradation from the saposin B deficiency diminished galactosylceramide accumulation in the brain and kidney and galctosylsphingosine in the brain. These analyses of AB-/- mice continue to delineate the tissue differential interactions of saposins in GSL metabolism.

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.

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.

Prosaposin, a precursor of four glycoprotein activators (saposin A, B, C and D) for lysosomal hydrolases, has previously been shown to be important for normal adult cochlear innervation and the maintenance of normal hearing. In these studies, we now investigate prosaposin in normal vestibular epithelium and the functional impairment of balance caused by prosaposin ablation. In normal mice, prosaposin is localized to all 3 vestibular end-organs (ampullae, saccule, and utricle) and Scarpa's ganglion by RT-PCR, Western blot analysis and immunofluorescence. Ablation of prosaposin function caused severe vestibular dysfunction on a battery of behavioral tasks. Histologically, the KO mice demonstrated an exuberant cellular proliferation below the vestibular hair cells with disruption of the supporting cells. Electron microscopy further demonstrated inclusion bodies and cellular proliferation disturbing the normal neuroepithelial structure of the vestibular end-organs. Lastly, immunofluorescence (neurofilament 200 and synaptophysin) staining suggests that this cellular proliferation corresponds to afferent and efferent neuronal overgrowth. These data suggest that prosaposin plays a role not only in the maintenance of normal hearing but also an important role in the neuronal maturation processes of the vestibular sensory epithelium and the maintenance of normal vestibular system function., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Gaucher disease occurs mainly as a result of a deficiency of the lysosomal enzyme beta-glucocerebrosidase activity. A rare variant form of Gaucher disease is known in which saposin C required for glucosylceramide degradation is deficient. In an earlier paper we described the first cases of two siblings with the non-neuronopathic form of Gaucher disease caused by saposin C deficiency [Tylki-Szymańska et al., 2007]. In this article, we present a follow up of clinical and biochemical findings in one patient who has been treated with miglustat for two years. We observed that administration of miglustat failed to exert any favorable effect on the clinical condition, haematological parameters and glucosylceramide level in the serum. In two individuals (described in this article) very slow deterioration of the peripheral and central nervous systems was observed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Saposins A, B, C, and D are small amphiphatic glycoproteins that are encoded in tandem within a precursor protein (prosaposin, PSAP), and are required for in vivo degradation of sphingolipids. Humans with saposin C deficiency exhibit the clinical presentation of Gaucher-like disease. We generated two types of saposin C mutant mice, one carrying a homozygous missense mutation (C384S) in the saposin C domain of prosaposin (Sap-C(-/-)) and the other carrying the compound heterozygous mutation with a second null Psap allele (Psap(-/C384S)). During early life stages, both Sap-C(-/-) and Psap(-/C384S) mice grew normally; however, they developed progressive motor and behavioral deficits after 3 months of age and the majority of affected mice could scarcely move by about 15 months. They showed no signs of hepatosplenomegaly throughout their lives. No accumulation of glucosylceramide and glucosylsphingosine was detected in the brain or liver of both Sap-C(-/-) and Psap(-/C384S) mice. Neuropathological analyses revealed patterned loss of cerebellar Purkinje cells, widespread axonal spheroids filled with membrane-derived concentric or lamellar electron-dense bodies, and lipofuscin-like deposition in the neurons. Soap-bubble-like inclusion bodies were detected in the trigeminal ganglion cells and the vascular endothelial cells. Compound heterozygous Psap(-/C384S) mice showed qualitatively identical but faster progression of the neurological phenotypes than Sap-C(-/-) mice. These results suggest the in vivo role of saposin C in axonal membrane homeostasis, the disruption of which leads to neurodegeneration in lysosomal storage disease.

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.

Gaucher disease is caused by defective acid beta-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C-/-) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities approximately 30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred approximately 48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C-/- alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease.

Saposins A, B, C and D are derived from a common precursor, prosaposin (psap). The few patients with saposin C deficiency develop a Gaucher disease-like central nervous system (CNS) phenotype attributed to diminished glucosylceramide (GC) cleavage activity by acid beta-glucosidase (GCase). The in vivo effects of saposin C were examined by creating mice with selective absence of saposin C (C-/-) using a knock-in point mutation (cysteine-to-proline) in exon 11 of the psap gene. In C-/- mice, prosaposin and saposins A, B and D proteins were present at near wild-type levels, but the saposin C protein was absent. By 1 year, the C-/- mice exhibited weakness of the hind limbs and progressive ataxia. Decreased neuromotor activity and impaired hippocampal long-term potentiation were evident. Foamy storage cells were observed in dorsal root ganglion and there was progressive loss of cerebellar Purkinje cells and atrophy of cerebellar granule cells. Ultrastructural analyses revealed inclusions in axonal processes in the spinal cord, sciatic nerve and brain, but no excess of multivesicular bodies. Activated microglial cells and astrocytes were present in thalamus, brain stem, cerebellum and spinal cord, indicating regional pro-inflammatory responses. No storage cells were found in visceral organs of these mice. The absence of saposin C led to moderate increases in GC and lactosylceramide (LacCer) and their deacylated analogues. These results support the view that saposin C has multiple roles in glycosphingolipid (GSL) catabolism as well as a prominent function in CNS and axonal integrity independent of its role as an optimizer/stabilizer of GCase.

Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65-70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A-D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated.

Prosaposin is a precursor of saposins A, B, C, and D. Saposins are indispensable for lysosomal hydrolysis of sphingolipids. The notion that prosaposin itself is likely involved in brain development led us to generate an anti-mouse prosaposin-specific antibody that do not cross-react with any of the processed saposins. We have used it to study expression of prosaposin in the brain of wild-type (WT) and saposin D knockout mice (Sap-D(-/-)). Immunoblot studies indicated that prosaposin, already abundant in the brain of WT, was dramatically increased in Sap-D(-/-). By immunohistochemistry, the brain of WT was rich in prosaposin in hippocampal CA3 pyramidal neurons, tufted cells and mitral cells in olfactory bulb, and cerebellar Purkinje cells. In Sap-D(-/-), immunoreactivity of prosaposin was increased in these neurons, most notably in the CA3 pyramidal neurons which contained prosaposin immuno-positive inclusion bodies in the endoplasmic reticulum. Further characterization of these prosaposin-rich neurons may provide new insights into the physiological functions of prosaposin in the nervous system.

Background: Prosaposin encodes, in tandem, four small acidic activator proteins (saposins) with specificities for glycosphingolipid (GSL) hydrolases in lysosomes. Extensive GSL storage occurs in various central nervous system regions in mammalian prosaposin deficiencies., Results: Our hypomorphic prosaposin deficient mouse, PS-NA, exhibited 45% WT levels of brain saposins and showed neuropathology that included neuronal GSL storage and Purkinje cell loss. Impairment of neuronal function was observed as early as 6 wks as demonstrated by the narrow bridges tests. Temporal transcriptome microarray analyses of brain tissues were conducted with mRNA from three prosaposin deficient mouse models: PS-NA, prosaposin null (PS-/-) and a V394L/V394L glucocerebrosidase mutation combined with PS-NA (4L/PS-NA). Gene expression alterations in cerebrum and cerebellum were detectable at birth preceding the neuronal deficits. Differentially expressed genes encompassed a broad spectrum of cellular functions. The number of down-regulated genes was constant, but up-regulated gene numbers increased with age. CCAAT/enhancer-binding protein delta (CEBPD) was the only up-regulated transcription factor in these two brain regions of all three models. Network analyses revealed that CEBPD has functional relationships with genes in transcription, pro-inflammation, cell death, binding, myelin and transport., Conclusion: These results show that: 1) Regionally specific gene expression abnormalities precede the brain histological and neuronal function changes, 2) Temporal gene expression profiles provide insights into the molecular mechanism during the GSL storage disease course, and 3) CEBPD is a candidate regulator of brain disease in prosaposin deficiency to participate in modulating disease acceleration or progression.

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative lysosomal disease characterized by accumulation of sulfatides, extensive white matter damage and loss of both cognitive and motor functions. In vivo, the catabolism of sulfatide requires both the enzyme arylsulfatase A and a specific sphingolipid activator protein, saposin-B, encoded by the PSAP gene. Arylsulfatase A activity is deficient in the classical forms of MLD, but exceedingly rare cases of MLD are due to saposin-B deficiency. We report here a detailed clinical, radiological and histological description of a new case in a 2-year-old Italian girl, who presented as a late infantile case of MLD with normal arylsulfatase A activity, urinary excretion of sulfatides and mutations in the PSAP gene.

Gaucher disease is generally caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The degradation of glycosphingolipids requires also the participation of sphingolipid activator proteins. The prosaposin PSAP gene codes for a single protein which undergoes post-translational cleavage to yield four proteins named saposins A, B, C and D. Saposin (SAP-) C is required for glucosylceramide degradation, and its deficiency results in a variant form of Gaucher disease. In this report, we present clinical, biochemical, and molecular findings in a 36-year-old man and his 30-year-old sister with non-neuronopathic Gaucher disease due to SAP-C deficiency. Very high levels of chitotriosidase activity, chemokine CCL18, and increased concentration of glucosylceramide in plasma and normal beta-glucosidase activity in skin fibroblasts were observed in the patients. A molecular genetics study of the PSAP gene enabled the identification of one missense mutation, p.L349P, located in the SAP-C domain and another mutation, p.M1L, located in the initiation codon of the prosaposin precursor protein. The presented findings describe the first cases where the non-neuronopathic Gaucher disease has been definitely demonstrated to be a consequence of SAP-C deficiency. Three previously described cases in the literature displayed a Gaucher type 3 phenotype.

We have generated specific saposin A and D deficient mouse mutants by the gene targeting technology. Saposin A deficient mice showed the clinical, biochemical and pathological phenotype of a chronic form of globoid cell leukodystrophy (Krabbe disease) establishing that saposin A is essential for in vivo degradation of galactosylceramide. Saposin D deficient mice showed an accumulation of ceramides containing alpha-hydroxy fatty acids (HFA/d18:1) in the brain and kidney and showed renal tubular degeneration and cerebellar Purkinje cell loss. Here we review the current information which we have learnt from these mouse models of specific sphingolipid activator protein deficiencies. Collectively, the information provides support for the potential importance of sphingolipids in the function of the nervous system.

Gaucher disease management in France is facilitated by CETG (Comitee of Evaluation and Treatment of Gaucher disease). Four hundred and fifty-six patients are enrolled in 2006 in the French Gaucher registry of CETG. Two hundred and thirty patients had treatment. Eighty five % are type 1, 10.5% type 2 and 5.5% type 3. Only 1 patient had a saposin C deficiency. Information on CETG is available on www.cetl.net.

A neuropathologic study of three cases of prosaposin (pSap) deficiency (ages at death 27, 89 and 119 days), carried out in the standard autopsy tissues, revealed a neurolysosomal pathology different from that in the non-neuronal cells. Non-neuronal storage is represented by massive lysosomal accumulation of glycosphingolipids (glucosyl-, galactosyl-, lactosyl-, globotriaosylceramides, sulphatide, and ceramide). The lysosomes in the central and peripheral neurons were distended by pleomorphic non-lipid aggregates lacking specific staining and autofluorescence. Lipid storage was borderline in case 1, and at a low level in the other cases. Neurolysosomal storage was associated with massive ubiquitination, which was absent in the non-neuronal cells and which did not display any immunohistochemical aggresomal properties. Confocal microscopy and cross-correlation function analyses revealed a positive correlation between the ubiquitin signal and the late endosomal/lysosomal markers. We suppose that the neuropathology most probably reflects excessive influx of non-lipid material (either in bulk or as individual molecules) into the neurolysosomes. The cortical neurons appeared to be uniquely vulnerable to pSap deficiency. Whereas in case 1 they populated the cortex, in cases 2 and 3 they had been replaced by dense populations of both phagocytic microglia and astrocytes. We suggest that this massive neuronal loss reflects a cortical neuronal survival crisis precipitated by the lack of pSap. The results of our study may extend the knowledge of the neurotrophic function of pSap, which should be considered essential for the survival and maintenance of human cortical neurons.

After a yeast two-hybrid screen identified prosaposin as a potential interacting protein with the nicotinic acetylcholine receptor (nAChR) subunit alpha10, studies were performed to characterize prosaposin in the normal rodent inner ear. Prosaposin demonstrates diffuse organ of Corti expression at birth, with gradual localization to the inner hair cells (IHCs) and its supporting cells, inner pillar cells, and synaptic region of the outer hair cells (OHCs) and Deiters' cells (DCs) by postnatal day 21 (P21). Microdissected OHC and DC quantitative reverse transcriptase-PCR and immunohistology localizes prosaposin mRNA to DCs and OHCs, and protein predominantly to the apex of the DCs. Subsequent studies in a prosaposin knock-out (KO) (-/-) mouse showed intact but slightly reduced hearing through P19, but deafness by P25 and reduced distortion product otoacoustic emissions from P15 onward. Beginning at P12, the prosaposin KO mice showed histologic organ of Corti changes including cellular hypertrophy in the region of the IHC and greater epithelial ridge, a loss of OHCs from cochlear apex, and vacuolization of OHCs. Immunofluorescence revealed exuberant overgrowth of auditory afferent neurites in the region of the IHCs and proliferation of auditory efferent neurites in the region of the tunnel of Corti. IHC recordings from these KO mice showed normal I-V curves and responses to applied acetylcholine. Together, these results suggest that prosaposin helps maintain normal innervation patterns to the organ of Corti. Furthermore, prosaposin's overlapping developmental expression pattern and binding capacity toward the nAChR alpha10 suggest that alpha10 may also play a role in this function.

Krabbe disease is a genetic demyelinating disease caused by a deficiency of galactosylceramidase. The majority of cases are of infantile onset with rapid clinical course. A rare late onset form with milder clinical symptoms also exists. The latter form has been reported to respond well to the bone marrow transplantation (BMT) therapy. We tested whether the BMT could be an effective therapy for the mouse model of the late onset form, saposin-A-/- (SAP-A-/-) mice. We used green fluorescent protein transgenic mice as the donors. Chimeric SAP-A-/- mice that received BMT showed very little evidence of neurologic symptoms. At postnatal day 190 when severe demyelination was evident in naive SAP-A-/- mice, demyelination was virtually absent in the brain of chimeric SAP-A-/- mice. Presence of residual enzyme activity, at the time of rapid myelination in SAP-A-/- mice, appears to limit initial inflammatory responses and macrophage infiltration, thereby preventing progression of demyelination in the CNS in SAP-A-/- mice. In contrast, the peripheral nerves showed features of hypertrophic neuropathy with hypomyelination and onion bulb formation, suggesting that there are different cellular responses to the BMT in the CNS and PNS.

An infant presented with multifocal myoclonus and cyanotic hypoxemia immediately after birth, and severe feeding problems, a protein-losing enteropathy, massive ascites and grand-mal epilepsy marked his rapid downhill course, with death at 17 weeks. At 2 weeks, brain MRI revealed grey matter heterotopias in the parieto-occipital regions suggestive of a cortical morphogenetic disorder. In cultured skin fibroblasts, lipid storage and reduced activities of ceramidase, galactosylceramide beta-galactosidase and glucosylceramide beta-glucosidase were evident. Autopsy disclosed generalised lysosomal lipid storage with macrophages and adrenal cortex prominently affected. The pattern of stored lipids in cultured fibroblasts and in dewaxed spleen tissue blocks was compatible with a diagnosis of prosaposin (pSap) deficiency (pSap-d). Neuropathologically, there was a pronounced generalised neurolysosomal storage combined with a severe depletion of cortical neurons and extreme paucity of myelin and oligodendroglia. This pathology, in particular the massive neuronal loss, differed from that in other neurolipidoses and could be explained by the reduced hydrolysis of multiple sphingolipids and the loss of pSap's neurotrophic function. The absence of immunostainable saposins on tissue sections and the presence of a homozygous c.1 A > T mutation in the prosaposin gene confirmed the diagnosis. PSap-d may be an underdiagnosed condition in infants with severe neurological and dystrophic signs starting immediately after birth.

Acidic phospholipids and saposins associations are involved in the degradation process of glycosphingolipids/sphingolipids in late endosomes/lysosomes. In this report, we showed the colocalization of saposin C and lysobisphosphatidic acid (LBPA) in human fibroblasts by using cytoimmunofluorescence analysis. This colocalization pattern was not seen with other saposins. Large numbers of saposins A, B, and D illustrated the staining patterns that differ from LBPA. In addition, ingested anti-LBPA antibody altered the location of saposin C in human wild-type fibroblasts. In vitro assays demonstrated that saposin C at nM concentrations induced membrane fusion of LBPA containing phospholipid vesicles. Under the same condition, other saposins had no fusion induction on these vesicles. These results suggested a specific interaction between saposin C and LBPA. Total saposin-deficient fibroblasts showed a massive accumulation of multivesicular bodies (MVBs) by electron microscopic analysis. No significant increase of MVBs was found in saposins A and B deficient cells. Interestingly, the accumulated MVBs were significantly reduced by loading saposin C alone into the total saposin-deficient cells. Therefore, we propose that saposin C-LBPA interaction plays a role in the regulation of MVB formation in cells.