RESUMO
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-ß-galactosylceramidase and GM1-ß-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.
Assuntos
Glicoesfingolipídeos/metabolismo , Doenças do Sistema Nervoso/metabolismo , Saposinas/deficiência , Animais , Encéfalo/metabolismo , Feminino , Galactosilceramidase/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/psicologia , Especificidade de Órgãos , Fenótipo , Saposinas/genética , beta-Galactosidase/metabolismoRESUMO
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.
Assuntos
Sistema Nervoso Central/metabolismo , Doença de Gaucher/metabolismo , Glucosilceramidase/metabolismo , Saposinas/deficiência , Animais , Comportamento Animal , Sistema Nervoso Central/citologia , Sistema Nervoso Central/enzimologia , Modelos Animais de Doenças , Feminino , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Doença de Gaucher/fisiopatologia , Glucosilceramidase/genética , Glicoesfingolipídeos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células de Purkinje/metabolismoRESUMO
Saposin B derives from the multi-functional precursor, prosaposin, and functions as an activity enhancer for several glycosphingolipid (GSL) hydrolases. Mutations in saposin B present in humans with phenotypes resembling metachromatic leukodystrophy. To gain insight into saposin B's physiological functions, a specific deficiency was created in mice by a knock-in mutation of an essential cysteine in exon 7 of the prosaposin locus. No saposin B protein was detected in the homozygotes (B-/-) mice, whereas prosaposin, and saposins A, C and D were at normal levels. B-/- mice exhibited slowly progressive neuromotor deterioration and minor head tremor by 15 months. Excess hydroxy and non-hydroxy fatty acid sulfatide levels were present in brain and kidney. Alcian blue positive (sulfatide) storage cells were found in the brain, spinal cord and kidney. Ultrastructural analyses showed lamellar inclusion material in the kidney, sciatic nerve, brain and spinal cord tissues. Lactosylceramide (LacCer) and globotriaosylceramide (TriCer) were increased in various tissues of B-/- mice supporting the in vivo role of saposin B in the degradation of these lipids. CD68 positive microglial cells and activated GFAP positive astrocytes showed a proinflammatory response in the brains of B-/- mice. These findings delineate the roles of saposin B for the in vivo degradation of several GSLs and its primary function in maintenance of CNS function. B-/- provide a useful model for understanding the contributions of this saposin to GSL metabolism and homeostasis.
Assuntos
Encéfalo/metabolismo , Glucosilceramidase/metabolismo , Glicoesfingolipídeos/metabolismo , Saposinas/fisiologia , Medula Espinal/metabolismo , Animais , Encéfalo/ultraestrutura , Feminino , Homozigoto , Rim/metabolismo , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Mutação Puntual , Saposinas/genética , Medula Espinal/ultraestruturaRESUMO
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.
Assuntos
Encefalopatias/genética , Proteína delta de Ligação ao Facilitador CCAAT/genética , Perfilação da Expressão Gênica , Saposinas/deficiência , Fatores Etários , Animais , Encefalopatias/fisiopatologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/fisiologia , Cerebelo/patologia , Cerebelo/fisiopatologia , Cérebro/patologia , Cérebro/fisiopatologia , Análise por Conglomerados , Modelos Animais de Doenças , Regulação para Baixo/genética , Genes Reguladores , Glucosilceramidase/deficiência , Glucosilceramidase/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Análise em Microsséries/métodos , Análise em Microsséries/estatística & dados numéricos , Atividade Motora/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saposinas/genética , Regulação para Cima/genéticaRESUMO
The pharmacological chaperone, isofagomine (IFG), enhances acid ß-glucosidase (GCase) function by altering folding, trafficking, and activity in wild-type and Gaucher disease fibroblasts. The in vivo effects of IFG on GCase activity, its substrate levels, and phenotype were evaluated using a neuronopathic Gaucher disease mouse model, 4L;C* (V394L/V394L + saposin C-/-) that has CNS accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) as well as progressive neurological deterioration. IFG administration to 4L;C* mice at 20 or 600 mg/kg/day resulted in life span extensions of 10 or 20 days, respectively, and increases in GCase activity and protein levels in the brain and visceral tissues. Cerebral cortical GC and GS levels showed no significant reductions with IFG treatment. Increases of GC or GS levels were detected in the visceral tissues of IFG treated (600 mg/kg/day) mice. The attenuations of brain proinflammatory responses in the treated mice were evidenced by reductions in astrogliosis and microglial cell activation, and decreased p38 phosphorylation and TNFα levels. Terminally, axonal degeneration was present in the brain and spinal cord from untreated and treated 4L;C* mice. These data demonstrate that IFG exerts in vivo effects by enhancing V394L GCase protein and activity levels, and in mediating suppression of proinflammation, which led to delayed onset of neurological disease and extension of the life span of 4L;C* mice. However, this was not correlated with a reduction in the accumulation of lipid substrates.
Assuntos
Modelos Animais de Doenças , Doença de Gaucher/tratamento farmacológico , Imino Piranoses/uso terapêutico , Animais , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Piranoses/farmacologia , Inflamação/tratamento farmacológico , Camundongos , Fosforilação , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Saposins (A, B, C and D) are approximately 80 amino acid stimulators of glycosphingolipid (GSL) hydrolases that derive from a single precursor, prosaposin. In both humans and mice, prosaposin/saposin deficiencies lead to severe neurological deficits. The CD-/- mice with saposin C and D combined deficiencies were produced by introducing genomic point mutations into a critical cysteine in each of these saposins. These mice develop a severe neurological phenotype with ataxia, kyphotic posturing and hind limb paralysis. Relative to prosaposin null mice ( approximately 30 days), CD-/- mice had an extended life span ( approximately 56 days). Loss of Purkinje cells was evident after 6 weeks, and storage bodies were present in neurons of the spinal cord, brain and dorsal root ganglion. Electron microscopy showed well-myelinated fibers and axonal inclusions in the brain and sciatic nerve. Marked accumulations of glucosylceramides and alpha-hydroxy ceramides were present in brain and kidney. Minor storage of lactosylceramide (LacCer) was observed when compared with tissues from the prosaposin null mice, suggesting a compensation in LacCer degradation by saposin B for the saposin C deficiency. Skin fibroblasts and tissues from CD-/- mice showed an increase of intracellular prosaposin, impaired prosaposin secretion, deficiencies of saposins C and D and decreases in saposins A and B. In addition, the deficiency of saposin C in CD-/- mice resulted in cellular decreases of acid beta-glucosidase activity and protein. This CD null mouse model provides a tool to explore the in vivo functional interactions of saposins in GSL metabolism and lysosomal storage diseases, and prosaposin's physiological effects.