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1.
Int J Mol Sci ; 23(23)2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36499264

RESUMO

Historically, disease burden and treatment responses in patients with Gaucher disease (GD) was assessed by monitoring clinical data, laboratory, imaging, chitotriosidase (CHITO), and other biomarkers; however, these biomarkers lack specificity and CHITO is uninformative in patients heterozygous or homozygous for the CHIT1 c.1049_1072dup24 variant. Recently, glucosylsphingosine (lyso-Gb1), a sensitive and specific GD biomarker, has been recommended for patient monitoring. Furthermore, studies measuring lyso-Gb1 and CHITO in patients on long-term treatment with enzyme replacement therapy (ERT) and/or substrate reduction therapy (SRT) reported as group data show a reduction in both analytes, yet individualized patient data are generally unavailable. We describe seven patients on long-term treatment with longitudinal clinical data with monitoring based on current treatment guidelines. We present four patients who exhibit stable disease with normalized CHITO despite elevated lyso-Gb1. We present one patient who transitioned from ERT to SRT due to lack of a clinical response with life-threatening thrombocytopenia who responded with marked improvement in platelets, and normalized levels of both CHITO and lyso-Gb1. Finally, we present two ERT to SRT switch patients with stable disease on ERT who exhibited non-compliance on SRT, one with mirrored marked elevations of CHITO and lyso-Gb1; and another with normal CHITO and platelets, but increasing lyso-Gb1 levels and enlarged spleen. These clinical vignettes highlight the role of lyso-Gb1 as a sensitive biomarker in management of patients with GD, and its further value when CHITO is normal and thus uninformative. We highlight the personalized medicine approach needed to optimize treatment outcomes and recommendations for these patients.


Assuntos
Doença de Gaucher , Humanos , Doença de Gaucher/diagnóstico , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Psicosina , Terapia de Reposição de Enzimas , Biomarcadores
2.
PLoS One ; 17(11): e0277058, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36409725

RESUMO

Isomeric lysosphingolipids, galactosylsphingosine (GalSph) and glucosylsphingosine (GlcSph), are present in only minute levels in healthy cells. Due to defects in their lysosomal hydrolysis, they accumulate at high levels and cause cytotoxicity in patients with Krabbe and Gaucher diseases, respectively. Here, we show that GalSph and GlcSph induce lysosomal membrane permeabilization, a hallmark of lysosome-dependent cell death, in human breast cancer cells (MCF7) and primary fibroblasts. Supporting lysosomal leakage as a causative event in lysosphingolipid-induced cytotoxicity, treatment of MCF7 cells with lysosome-stabilizing cholesterol prevented GalSph- and GlcSph-induced cell death almost completely. In line with this, fibroblasts from a patient with Niemann-Pick type C disease, which is caused by defective lysosomal cholesterol efflux, were significantly less sensitive to lysosphingolipid-induced lysosomal leakage and cell death. Prompted by the data showing that MCF7 cells with acquired resistance to lysosome-destabilizing cationic amphiphilic drugs (CADs) were partially resistant to the cell death induced by GalSph and GlcSph, we compared these cell death pathways with each other. Like CADs, GalSph and GlcSph activated the cyclic AMP (cAMP) signalling pathway, and cAMP-inducing forskolin sensitized cells to cell death induced by low concentrations of lysosphingolipids. Contrary to CADs, lysosphingolipid-induced cell death was independent of lysosomal Ca2+ efflux through P2X purinerigic receptor 4. These data reveal GalSph and GlcSph as lysosome-destabilizing lipids, whose putative use in cancer therapy should be further investigated. Furthermore, the data supports the development of lysosome stabilizing drugs for the treatment of Krabbe and Gaucher diseases and possibly other sphingolipidoses.


Assuntos
Doença de Gaucher , Neoplasias , Humanos , Psicosina/metabolismo , Lisossomos/metabolismo , Morte Celular , Doença de Gaucher/metabolismo , AMP Cíclico/metabolismo , Colesterol/metabolismo , Neoplasias/metabolismo
3.
Ecotoxicol Environ Saf ; 245: 114125, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36183426

RESUMO

There is limited knowledge of the ecotoxicological impacts of MPs at the environmentally relevant concentration on freshwater animals, even though numerous studies have demonstrated the toxic effects of MPs on living organisms. In this study, zebrafish (Danio rerio) was used as a model organism to investigate the ecotoxicological effects of acute exposure of virgin MPs on changes in metabolome and gut microbiota. High-throughput untargeted metabolomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS) provided comprehensive insights into the metabolic responses of zebrafish exposed to PE (polyethylene) and PES (polyester) MPs. Statistical analysis of metabolomics data indicated that 39 and 27 metabolites, such as lysophosphatidylcholine, phosphocholine, phosphatidylserine, triglyceride, glycosphingolipid, psychosine, 8-amino-7-oxononanoate, cholesterol fatty acid ester, phosphatidylinositol, n-Triacontanol, were significantly altered in PE- and PES-exposed zebrafish, respectively. Furthermore, the enrichment pathway analysis unveiled the synthesis of the structural and functional lipids, signaling molecules, fatty alcohol metabolism, and amino acid metabolism, which was considerably perturbated in MPs-exposed zebrafish. In addition, high-throughput DNA sequencing was conducted to examine changes in gut microbiota in the MPs-treated zebrafish. The MPs exposure increased in the relative abundance of Fusobacteria and Proteobacteria, while the relative abundance of Firmicutes declined in MPs-treated zebrafish. Also, microbial diversity and linear discriminant analyses indicated microbiota dysbiosis, metabolomic dysregulation, and oxidative stress. Taken together, the acute exposure of MPs at environmentally relevant concentrations could disrupt the metabolic interaction via the microbiota-gut-liver-brain relationship, implying gastrointestinal and neurological/immune disorders in zebrafish.


Assuntos
Disbiose , Poluentes Químicos da Água , Aminoácidos/metabolismo , Animais , Cromatografia Líquida , Disbiose/induzido quimicamente , Ésteres , Ácidos Graxos/metabolismo , Álcoois Graxos , Lisofosfatidilcolinas/metabolismo , Microplásticos , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Fosforilcolina/metabolismo , Plásticos/metabolismo , Poliésteres , Polietileno/metabolismo , Psicosina/metabolismo , Espectrometria de Massas em Tandem , Triglicerídeos/metabolismo , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo
4.
Neurobiol Dis ; 174: 105862, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36113749

RESUMO

Krabbe Disease (KD) is an autosomal recessive disorder that results from loss-of-function mutations in the GALC gene, which encodes lysosomal enzyme galactosylceramidase (GALC). Functional deficiency of GALC is toxic to myelin-producing cells, which leads to progressive demyelination in both the central and peripheral nervous systems. It is hypothesized that accumulation of psychosine, which can only be degraded by GALC, is a primary initiator of pathologic cascades. Despite the central role of GALC in KD pathomechanism, investigations of GALC deficiency at a protein level are largely absent, due in part, to the lack of sensitive antibodies in the field. Leveraging two custom antibodies that can detect GALC at endogenous levels, we demonstrated that GALC protein is predominantly localized to oligodendrocytes in cerebral white matter of an infant brain, consistent with its functional role in myelination. Mature GALC could also be quantitatively detected as a 26 kDa band by western blotting and correlated to enzyme activity in brain tissues. The p.Ile562Thr polymorphic variant, which is over-represented in the KD population, was associated with reduced mature GALC protein and activity. In three infantile KD cases, homozygous null mutations in GALC lead to deficiency in total GALC protein and activity. Interestingly, although GALC activity was absent, normal levels of total GALC protein were detected by a sandwich ELISA using our custom antibodies in a later-onset KD brain, which suggests that the assay has the potential to differentiate infantile- and later-onset KD cases. Among the infantile KD cases, we quantified a 5-fold increase in psychosine levels, and observed increased levels of acid ceramidase, a key enzyme for psychosine production, and hyperglycosylated lysosomal-associated membrane protein 1, a marker for lysosomal activation, in periventricular white matter, a major pathological brain region, when compared with age-matched normal controls. While near complete demyelination was observed in these cases, we quantified that an early-infantile case (age of death at 10 months) had about 3-fold increases in both globoid cells, a pathological hallmark for KD, and CD8-positive T lymphocytes, a pathological marker for multiple sclerosis, in the white matter when compared with a slower progressing infantile case (age of death at 21 months), which suggests a positive correlation between clinical severity and neuropathology. Taken together, our findings have advanced the understanding of GALC protein biology in the context of normal and KD brain white matter. We also revealed new neuropathological changes that may provide insights to understand KD pathogenesis.


Assuntos
Leucodistrofia de Células Globoides , Substância Branca , Humanos , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Psicosina/metabolismo , Substância Branca/patologia , Mutação
5.
Methods Mol Biol ; 2546: 271-284, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36127597

RESUMO

Plasma lysosphingolipids are highly elevated in patients with Gaucher, Krabbe, Fabry, and Niemann-Pick diseases and tend to accumulate to a greater extent than their respective primary sphingolipids in the plasma of affected patients. In this chapter, we describe two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to measure plasma concentrations of four lysosphingolipids species. The first method described measures glucosylsphingosine (lyso-GL1) and galactosylsphingosine (psychosine), biomarkers that accumulate in Gaucher and Krabbe diseases, respectively. The second method measures globotriaosylsphingosine (lyso-Gb3) and sphingosylphosphorylcholine (lyso-SPM), biomarkers for Fabry and Niemann-Pick diseases, respectively. Each method utilizes isotope-labeled internal standards and multipoint calibration curves to quantify the analytes of interest. Briefly, plasma samples are mixed with five volumes of LC-MS grade methanol containing internal standard, and protein is removed via centrifugation. Supernatant is dried and resuspended in initial mobile phase. Samples are separated by liquid chromatography using either a BEH amide column (lyso-GL1 + psychosine) or a C18 column (lyso-Gb3 + lyso-SPM). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. Using these methods, we have observed elevations of these lyso- species in Gaucher, Fabry, and Niemann-Pick and successfully distinguished different subtypes reflecting the disease severity.


Assuntos
Doença de Fabry , Doenças de Niemann-Pick , Amidas , Biomarcadores , Cromatografia Líquida/métodos , Humanos , Metanol , Psicosina , Esfingolipídeos/química , Espectrometria de Massas em Tandem/métodos
6.
Lung ; 200(5): 591-599, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35930050

RESUMO

PURPOSE: Extracellular acidification is a major component of tissue inflammation, including airway inflammation. The extracellular proton-sensing mechanisms are inherent in various cells including airway structural cells, although their physiological and pathophysiological roles in bronchial smooth muscles (BSMs) are not fully understood. In the present study, to explore the functional role of extracellular acidification on the BSM contraction, the isolated mouse BSMs were exposed to acidic pH under contractile stimulation. METHODS AND RESULTS: The RT-PCR analyses revealed that the proton-sensing G protein-coupled receptors were expressed both in mouse BSMs and cultured human BSM cells. In the mouse BSMs, change in the extracellular pH from 8.0 to 6.8 caused an augmentation of contraction induced by acetylcholine. Interestingly, the acidic pH-induced BSM hyper-contraction was further augmented in the mice that were sensitized and repeatedly challenged with ovalbumin antigen. In this animal model of asthma, upregulations of G protein-coupled receptor 68 (GPR68) and GPR65, that were believed to be coupled with Gq and Gs proteins respectively, were observed, indicating that the acidic pH could cause hyper-contraction probably via an activation of GPR68. However, psychosine, a putative antagonist for GPR68, failed to block the acidic pH-induced responses. CONCLUSION: These findings suggest that extracellular acidification contributes to the airway hyperresponsiveness, a characteristic feature of bronchial asthma. Further studies are required to identify the receptor(s) responsible for sensing extracellular protons in BSM cells.


Assuntos
Asma , Hiper-Reatividade Brônquica , Acetilcolina/efeitos adversos , Acetilcolina/metabolismo , Animais , Brônquios , Hiper-Reatividade Brônquica/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/metabolismo , Ovalbumina , Prótons , Psicosina/efeitos adversos , Psicosina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
7.
PLoS One ; 17(8): e0271360, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35921286

RESUMO

Globoid cell leukodystrophy (Krabbe disease) is a fatal neurodegenerative, demyelinating disease caused by dysfunctional activity of galactosylceramidase (GALC), leading to the accumulation of glycosphingolipids including psychosine. While oligodendrocytes have been extensively studied due to their high levels of GALC, the contribution of astrocytes to disease pathogenesis remains to be fully elucidated. In the current study, we generated induced pluripotent stem cells (iPSCs) from two donors with infantile onset Krabbe disease and differentiated them into cultures of astrocytes. Krabbe astrocytes recapitulated many key findings observed in humans and rodent models of the disease, including the accumulation of psychosine and elevated expression of the pro-inflammatory cytokine IL-6. Unexpectedly, Krabbe astrocytes had higher levels of glucosylceramide and ceramide, and displayed compensatory changes in genes encoding glycosphingolipid biosynthetic enzymes, suggesting a shunting away from the galactosylceramide and psychosine pathway. In co-culture, Krabbe astrocytes negatively impacted the survival of iPSC-derived human neurons while enhancing survival of iPSC-derived human microglia. Substrate reduction approaches targeting either glucosylceramide synthase or serine palmitoyltransferase to reduce the sphingolipids elevated in Krabbe astrocytes failed to rescue their detrimental impact on neuron survival. Our results suggest that astrocytes may contribute to the progression of Krabbe disease and warrant further exploration into their role as therapeutic targets.


Assuntos
Células-Tronco Pluripotentes Induzidas , Leucodistrofia de Células Globoides , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fenótipo , Psicosina/metabolismo
8.
Int J Mol Sci ; 23(16)2022 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-36012705

RESUMO

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a neurodegenerative sphingolipidosis caused by genetic deficiency of lysosomal ß-galactosylceramidase (GALC), characterized by neuroinflammation and demyelination of the central (CNS) and peripheral nervous system. The acute phase protein long pentraxin-3 (PTX3) is a soluble pattern recognition receptor and a regulator of innate immunity. Growing evidence points to the involvement of PTX3 in neurodegeneration. However, the expression and role of PTX3 in the neurodegenerative/neuroinflammatory processes that characterize GLD remain unexplored. Here, immunohistochemical analysis of brain samples from Krabbe patients showed that macrophages and globoid cells are intensely immunoreactive for PTX3. Accordingly, Ptx3 expression increases throughout the course of the disease in the cerebrum, cerebellum, and spinal cord of GALC-deficient twitcher (Galctwi/twi) mice, an authentic animal model of GLD. This was paralleled by the upregulation of proinflammatory genes and M1-polarized macrophage/microglia markers and of the levels of PTX3 protein in CNS and plasma of twitcher animals. Crossing of Galctwi/twi mice with transgenic PTX3 overexpressing animals (hPTX3 mice) demonstrated that constitutive PTX3 overexpression reduced the severity of clinical signs and the upregulation of proinflammatory genes in the spinal cord of P35 hPTX3/Galctwi/twi mice when compared to Galctwi/twi littermates, leading to a limited increase of their life span. However, this occurred in the absence of a significant impact on the histopathological findings and on the accumulation of the neurotoxic metabolite psychosine when evaluated at this late time point of the disease. In conclusion, our results provide the first evidence that PTX3 is produced in the CNS of GALC-deficient Krabbe patients and twitcher mice. PTX3 may exert a protective role by reducing the neuroinflammatory response that occurs in the spinal cord of GALC-deficient animals.


Assuntos
Proteína C-Reativa , Galactosilceramidase , Leucodistrofia de Células Globoides , Proteínas do Tecido Nervoso , Animais , Proteína C-Reativa/genética , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Galactosilceramidase/deficiência , Galactosilceramidase/genética , Humanos , Leucodistrofia de Células Globoides/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Psicosina , Regulação para Cima
9.
Biochim Biophys Acta Biomembr ; 1864(11): 184014, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-35908608

RESUMO

Glucosylsphingosine (GS) is an endogenous sphingolipid that specifically accumulates in the skin of patients with atopic dermatitis (AD). Notably, it was recently found that GS can induce itch sensation by activating serotonin receptor 2A and TRPV4 ion channels. However, it is still uncertain whether other molecules are involved in GS-induced itch sensation. Therefore, by using the calcium imaging technique, we investigated whether serotonin receptor 2 - specifically 2A and 2B - can interact with TRPV1 and TRPA1, because these are representative ion channels in the transmission of itch. As a result, it was found that GS did not activate TRPV1 or TRPA1 per se. Moreover, cells expressing both serotonin receptor 2 and TRPV1 did not show any changes in calcium responses. However, enhanced calcium responses were observed in cells expressing serotonin receptor 2 and TRPA1, suggesting a possible interaction between these two molecules. Similar synergistic effects were also observed in cells expressing serotonin receptor 2 and TRPA1, but not TRPV1. Furthermore, a phospholipase C inhibitor (U73122) and a store-operated calcium entry blocker (SKF96365) significantly reduced GS-induced responses in cells expressing both serotonin receptor 2 and TRPA1, but not with pre-treatment with a Gßγ-complex blocker (gallein). Therefore, we propose a putative novel pathway for GS-induced itch sensation, such that serotonin receptor 2 could be coupled to TRPA1 but not TRPV1 in sensory neurons.


Assuntos
Canais de Potencial de Receptor Transitório , Cálcio/metabolismo , Humanos , Prurido/metabolismo , Psicosina/análogos & derivados , Receptores de Serotonina/metabolismo , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV , Canais de Potencial de Receptor Transitório/fisiologia
10.
PLoS Biol ; 20(7): e3001661, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35789331

RESUMO

Krabbe disease is caused by a deficiency of the lysosomal galactosylceramidase (GALC) enzyme, which results in the accumulation of galactosylceramide (GalCer) and psychosine. In Krabbe disease, the brunt of demyelination and neurodegeneration is believed to result from the dysfunction of myelinating glia. Recent studies have shown that neuronal axons are both structurally and functionally compromised in Krabbe disease, even before demyelination, suggesting a possible neuron-autonomous role of GALC. Using a novel neuron-specific Galc knockout (CKO) model, we show that neuronal Galc deletion is sufficient to cause growth and motor coordination defects and inflammatory gliosis in mice. Furthermore, psychosine accumulates significantly in the nervous system of neuron-specific Galc-CKO. Confocal and electron microscopic analyses show profound neuro-axonal degeneration with a mild effect on myelin structure. Thus, we prove for the first time that neuronal GALC is essential to maintain and protect neuronal function independently of myelin and may directly contribute to the pathogenesis of Krabbe disease.


Assuntos
Galactosilceramidase , Leucodistrofia de Células Globoides , Animais , Modelos Animais de Doenças , Galactosilceramidase/genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Camundongos , Neurônios/patologia , Psicosina
11.
J Pharm Biomed Anal ; 217: 114852, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35636011

RESUMO

Mutations in the GBA gene, encoding glucocerebrosidase (GCase), are linked to Gaucher disease (GD) and are the most common risk factors for Parkinson's disease (PD). The glucosylsphingosine (GlcSph) in cerebrospinal fluid (CSF) is used as a pharmacodynamic marker for GCase functionalizing therapy in GD patients. Its isobaric structural isomer, galactosylsphingosine (GalSph, psychosine), is also used as a diagnostic blood marker in Krabbe disease (KD) which is caused by a deficiency in ß-galactocerebrosidase (GALC). However, there are no reports of GlcSph quantification in the CSF of GBA-PD patients and normal healthy humans due to low concentrations. In this study, we successfully quantified GlcSph in healthy human CSF using a highly sensitive LC-MS/MS method with separation of GalSph. The lower limit of quantitation (LLOQ) was 0.1 pg/mL. Additionally, GlcSph and GalSph concentrations in the plasma and brain were determined using different LC-MS/MS methods. The mean concentrations of GlcSph and GalSph in normal human CSF were 1.07 and 9.44 pg/mL, respectively. The GalSph level in the CSF and brain was higher than that of GlcSph, whereas plasma GalSph was lower than GlcSph. Because GCase and GALC are expressed in the brain and the peripheral tissues, GlcSph and GalSph in CSF would be a good surrogate of concentration change in the brain by targeted therapies. This method measures normal levels of GlcSph and GalSph in healthy human CSF without accumulation of sphingolipids, and confirms whether abnormal CSF concentrations can be reduced to normal levels by therapy.


Assuntos
Doença de Gaucher , Doença de Parkinson , Cromatografia Líquida , Humanos , Psicosina/análogos & derivados , Psicosina/análise , Psicosina/genética , Espectrometria de Massas em Tandem
12.
Hum Gene Ther ; 33(9-10): 499-517, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35333110

RESUMO

Krabbe disease is a lysosomal storage disease caused by mutations in the gene that encodes galactosylceramidase, in which galactosylsphingosine (psychosine) accumulation drives demyelination in the central and peripheral nervous systems, ultimately progressing to death in early childhood. Gene therapy, alone or in combination with transplant, has been developed for almost two decades in mouse models, with increasing therapeutic benefit paralleling the improvement of next-generation adeno-associated virus (AAV) vectors. This effort has recently shown remarkable efficacy in the canine model of the disease by two different groups that used either systemic or cerebrospinal fluid (CSF) administration of AAVrh10 or AAV9. Building on our experience developing CSF-delivered, AAV-based drug products for a variety of neurodegenerative disorders, we conducted efficacy, pharmacology, and safety studies of AAVhu68 delivered to the CSF in two relevant natural Krabbe animal models, and in nonhuman primates. In newborn Twitcher mice, the highest dose (1 × 1011 genome copies [GC]) of AAVhu68.hGALC injected into the lateral ventricle led to a median survival of 130 days compared to 40.5 days in vehicle-treated mice. When this dose was administered intravenously, the median survival was 49 days. A single intracisterna magna injection of AAVhu68.cGALC at 3 × 1013 GC into presymptomatic Krabbe dogs increased survival for up to 85 weeks compared to 12 weeks in controls. It prevented psychosine accumulation in the CSF, preserved peripheral nerve myelination, ambulation, and decreased brain neuroinflammation and demyelination, although some regions remained abnormal. In a Good Laboratory Practice-compliant toxicology study, we administered the clinical candidate into the cisterna magna of 18 juvenile rhesus macaques at 3 doses that displayed efficacy in mice. We observed no dose-limiting toxicity and sporadic minimal degeneration of dorsal root ganglia (DRG) neurons. Our studies demonstrate the efficacy, scalability, and safety of a single cisterna magna AAVhu68 administration to treat Krabbe disease. ClinicalTrials.Gov ID: NCT04771416.


Assuntos
Leucodistrofia de Células Globoides , Animais , Pré-Escolar , Dependovirus/genética , Modelos Animais de Doenças , Cães , Terapia Genética , Humanos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Macaca mulatta/genética , Camundongos , Psicosina
13.
J Lipid Res ; 63(5): 100199, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35315333

RESUMO

In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson's disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1-/- fish) and without GlcSph (gba1-/-:asah1b-/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1-/-:asah1b-/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons.


Assuntos
Doença de Gaucher , Glucosilceramidase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Ceramidase Ácida , Animais , Doença de Gaucher/genética , Glucosilceramidase/genética , Glucosilceramidas , Humanos , Psicosina/análogos & derivados , Peixe-Zebra/genética
14.
Biomed Pharmacother ; 149: 112808, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35290889

RESUMO

Krabbe disease is a rare, inherited neurodegenerative disease due to impaired lysosomal ß-galactosylceramidase (GALC) activity and formation of neurotoxic ß-galactosylsphingosine ('psychosine'). We investigated substrate reduction therapy with a novel brain-penetrant inhibitor of galactosylceramide biosynthesis, RA 5557, in twitcher mice that lack GALC activity and model Krabbe disease. This thienopyridine derivative selectively inhibits uridine diphosphate-galactose glycosyltransferase 8 (UGT8), the final step in the generation of galactosylceramides which are precursors of sulphatide and, in the pathological lysosome, the immediate source of psychosine. Administration of RA 5557, reduced pathologically elevated psychosine concentrations (72-86%) in the midbrain and cerebral cortex in twitcher mice: the inhibitor decreased galactosylceramides by about 70% in midbrain and cerebral cortex in mutant and wild type animals. Exposure to the inhibitor significantly decreased several characteristic inflammatory response markers without causing apparent toxicity to myelin-producing cells in wild type and mutant mice; transcript abundance of oligodendrocyte markers MBP (myelin basic protein) and murine UGT8 was unchanged. Administration of the inhibitor before conception and during several breeding cycles to mice did not impair fertility and gave rise to healthy offspring. Nevertheless, given the unchanged lifespan, it appears that GALC has critical functions in the nervous system beyond the hydrolysis of galactosylceramide and galactosylsphingosine. Our findings support further therapeutic exploration of orally active UGT8 inhibitors in Krabbe disease and related galactosphingolipid disorders. The potent thienopyridine derivative with effective target engagement here studied appears to have an acceptable safety profile in vivo; judicious dose optimization will be needed to ensure efficacious clinical translation.


Assuntos
Leucodistrofia de Células Globoides , Doenças Neurodegenerativas , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Galactosilceramidas/metabolismo , Galactosilceramidas/farmacologia , Leucodistrofia de Células Globoides/tratamento farmacológico , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Camundongos , Doenças Neurodegenerativas/patologia , Psicosina/metabolismo , Tienopiridinas
15.
ASN Neuro ; 14: 17590914221087817, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35300522

RESUMO

Psychosine exerts most of its toxic effects by altering membrane dynamics with increased shedding of extracellular vesicles (EVs). In this study, we discovered that a fraction of psychosine produced in the brain of the Twitcher mouse, a model for Krabbe disease, is associated with secreted EVs. We evaluated the effects of attenuating EV secretion in the Twitcher brain by depleting ceramide production with an inhibitor of neutral sphingomyelinase 2, GW4869. Twitcher mice treated with GW4869 had decreased overall EV levels, reduced EV-associated psychosine and unexpectedly, correlated with increased disease severity. Notably, characterization of well-established, neuroanatomic hallmarks of disease pathology, such as demyelination and inflammatory gliosis, remained essentially unaltered in the brains of GW4869-treated Twitcher mice compared to vehicle-treated Twitcher controls. Further analysis of Twitcher brain pathophysiology is required to understand the mechanism behind early-onset disease severity in GW4869-treated mice. The results herein demonstrate that some pathogenic lipids like psychosine may be secreted using EV pathways. Our results highlight the relevance of this secretory mechanism as a possible contributor to spreading pathogenic lipids in neurological lipidoses.


Assuntos
Vesículas Extracelulares , Leucodistrofia de Células Globoides , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Camundongos , Psicosina/análise , Psicosina/metabolismo , Psicosina/farmacologia , Esfingolipídeos/metabolismo
16.
Int J Biochem Cell Biol ; 145: 106184, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35217188

RESUMO

Galactocerebrosidase (GALC) hydrolyses galactose residues from various substrates, including galactosylceramide, psychosine (galactosylsphingosine), and lactosylceramide. Its severe deficiency has been associated with the accumulation of psychosine, a toxic molecule with detergent-like features, which alters membrane structures and signalling pathways, inducing the death of oligodendrocytes and a sequence of events in the nervous system that explain the appearance of many clinical signs typical of Krabbe disease. Nevertheless, new evidence suggests the existence of other possible links among GALC action, myelination, and myelin stability, apart from psychosine release. In this study, we demonstrated that lactosylceramide metabolism is impaired in fibroblasts isolated from patients with Krabbe disease in the absence of psychosine accumulation. This event is responsible for the aberrant and constitutive activation of the AKT/prolin-rich AKT substrate of 40 kDa (PRAS40) signalling axis, inducing B cell lymphoma 2 (BCL2) overexpression and glycogen synthase kinase 3 beta (GSK-3ß) inhibition. In addition, nuclear factor E2-related factor 2 (NRF2) showed increased nuclear translocation. Due to the relevance of these molecular alterations in neurodegeneration, lactosylceramide increase should be evaluated as a novel marker of Krabbe disease, and because of its significant connections with signalling pathways.


Assuntos
Lactosilceramidas , Leucodistrofia de Células Globoides , Proteínas Adaptadoras de Transdução de Sinal , Glicogênio Sintase Quinase 3 beta , Humanos , Lactosilceramidas/metabolismo , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Fator 2 Relacionado a NF-E2 , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2 , Psicosina/metabolismo
17.
Int J Mol Sci ; 23(3)2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-35163551

RESUMO

For years, the gold standard for diagnosing Gaucher disease (GD) has been detecting reduced ß-glucocerebrosidase (GCase) activity in peripheral blood cells combined with GBA1 mutation analysis. The use of dried blood spot (DBS) specimens offers many advantages, including easy collection, the need for a small amount of blood, and simpler transportation. However, DBS has limitations for measuring GCase activity. In this paper, we recount our cross-sectional study and publish seven years of experience using DBS samples and levels of the deacylated form of glucocerebroside, glucosylsphingosine (lyso-Gb1), for GD diagnosis. Of 444 screened subjects, 99 (22.3%) were diagnosed with GD at a median (range) age of 21 (1-78) years. Lyso-Gb levels for genetically confirmed GD patients vs. subjects negative to GD diagnosis were 252 (9-1340) ng/mL and 5.4 (1.5-16) ng/mL, respectively. Patients diagnosed with GD1 and mild GBA1 variants had lower median (range) lyso-Gb1, 194 (9-1050), compared to GD1 and severe GBA1 variants, 447 (38-1340) ng/mL, and neuronopathic GD, 325 (116-1270) ng/mL (p = 0.001). Subjects with heterozygous GBA1 variants (carrier) had higher lyso-Gb1 levels, 5.8 (2.5-15.3) ng/mL, compared to wild-type GBA1, 4.9 (1.5-16), ng/mL (p = 0.001). Lyso-Gb1 levels, median (range), were 5 (2.7-10.7) in heterozygous GBA1 carriers with Parkinson's disease (PD), similar to lyso-Gb1 levels in subjects without PD. We call for a paradigm change for the diagnosis of GD based on lyso-Gb1 measurements and confirmatory GBA1 mutation analyses in DBS. Lyso-Gb1 levels could not be used to differentiate between heterozygous GBA1 carriers and wild type.


Assuntos
Biomarcadores/sangue , Doença de Gaucher/diagnóstico , Glucosilceramidase/genética , Psicosina/análogos & derivados , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos Transversais , Detecção Precoce de Câncer , Feminino , Doença de Gaucher/sangue , Doença de Gaucher/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Psicosina/sangue , Adulto Jovem
20.
Acta Pharmacol Sin ; 43(3): 552-562, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33935286

RESUMO

We previously show that fatty acid-binding protein 3 (FABP3) triggers α-synuclein (Syn) accumulation and induces dopamine neuronal cell death in Parkinson disease mouse model. But the role of fatty acid-binding protein 7 (FABP7) in the brain remains unclear. In this study we investigated whether FABP7 was involved in synucleinopathies. We showed that FABP7 was co-localized and formed a complex with Syn in Syn-transfected U251 human glioblastoma cells, and treatment with arachidonic acid (100 M) significantly promoted FABP7-induced Syn aggregation, which was associated with cell death. We demonstrated that synthetic FABP7 ligand 6 displayed a high affinity against FABP7 with Kd value of 209 nM assessed in 8-anilinonaphthalene-1-sulfonic acid (ANS) assay; ligand 6 improved U251 cell survival via disrupting the FABP7-Syn interaction. We showed that activation of phospholipase A2 (PLA2) by psychosine (10 M) triggered oligomerization of endogenous Syn and FABP7, and induced cell death in both KG-1C human oligodendroglia cells and oligodendrocyte precursor cells (OPCs). FABP7 ligand 6 (1 M) significantly decreased Syn oligomerization and aggregation thereby prevented KG-1C and OPC cell death. This study demonstrates that FABP7 triggers α-synuclein oligomerization through oxidative stress, while FABP7 ligand 6 can inhibit FABP7-induced Syn oligomerization and aggregation, thereby rescuing glial cells and oligodendrocytes from cell death.


Assuntos
Proteína 7 de Ligação a Ácidos Graxos/metabolismo , Neuroglia/metabolismo , Oligodendroglia/metabolismo , Estresse Oxidativo/fisiologia , alfa-Sinucleína/metabolismo , Animais , Ácido Araquidônico/farmacologia , Morte Celular/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Fosfolipases A2/efeitos dos fármacos , Ligação Proteica/fisiologia , Psicosina/farmacologia
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