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1.
Neuropathology ; 44(4): 255-277, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38444347

RESUMO

Since its initial documentation by Knud Krabbe in 1916, numerous studies have scrutinized the characteristics of Krabbe disease (KD) until the identification of the mutation in the GALC gene. In alignment with that, we investigated the natural history of KD spanning eight decades to gain a deeper understanding of the evolutionary trajectory of its mechanisms. Through our comprehensive analysis, we unearthed additional novel elements in molecular biology involving the micropathological mechanism of the disease. This review offers an updated perspective on the metabolic disorder that defines KD. Recently, extracellular vesicles (EVs), autophagy impairment, and α-synuclein have emerged as pivotal players in the neuropathological processes. EVs might serve as a cellular mechanism to avoid or alleviate the detrimental impacts of excessive toxic psychosine levels, and extracting EVs could contribute to synapse dysfunction. Autophagy impairment was found to be independent of psychosine and reliant on AKT and B-cell lymphoma 2. Additionally, α-synuclein has been recognized for inducing cellular death and dysfunction in common biological pathways. Our objective is to assess the effectiveness of advanced therapies in addressing this particular condition. While hematopoietic stem cells have been a primary treatment, its administration proves challenging, particularly in the presymptomatic phase. In this review, we have compiled information from over 10 therapy trials, comparing them based on their benefits and disadvantage.


Assuntos
Leucodistrofia de Células Globoides , Humanos , Leucodistrofia de Células Globoides/terapia , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , alfa-Sinucleína/metabolismo , Animais , Autofagia/fisiologia , Vesículas Extracelulares/metabolismo
2.
Brain Dev ; 45(7): 408-412, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37080866

RESUMO

BACKGROUND: Late-onset Krabbe disease is a disorder with autosomal recessive inheritance caused by a deficiency in galactocerebrosidase (GALC) activity. Its late-onset form usually shows slow disease progression with atypical symptoms including spastic paresis. The efficacy of hematopoietic stem cell transplantation (HSCT) in late-onset Krabbe disease has not been fully established. CASE REPORT: We describe the case of a patient with late-onset Krabbe disease showing progressive spastic paraparesis. At the age of 18, one and a half years after the development of symptoms, the patient underwent HSCT. After HSCT, the patient's GALC activity returned to a normal level and the lesions in the brain and spinal cord became faint on images. Over two and a half years after the HSCT, the patient's gait remained spastic, however, an improvement in gait speed and modified Rankin Scale score was observed. No severe adverse events occurred during this period. CONCLUSION: Our experience reported herein provides additional evidence for a favorable course in HSCT conducted in the early course of late-onset Krabbe disease.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucodistrofia de Células Globoides , Humanos , Leucodistrofia de Células Globoides/terapia , Leucodistrofia de Células Globoides/diagnóstico , Leucodistrofia de Células Globoides/patologia , Espasticidade Muscular , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Síncope , Galactosilceramidase/genética
3.
Mol Ther ; 31(1): 7-23, 2023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36196048

RESUMO

Krabbe disease (KD) is a lysosomal storage disease (LSD) caused by mutations in the galc gene. There are over 50 monogenetic LSDs, which largely impede the normal development of children and often lead to premature death. At present, there are no cures for LSDs and the available treatments are generally insufficient, short acting, and not without co-morbidities or long-term side effects. The last 30 years have seen significant advances in our understanding of LSD pathology as well as treatment options. Two gene therapy-based clinical trials, NCT04693598 and NCT04771416, for KD were recently started based on those advances. This review will discuss how our knowledge of KD got to where it is today, focusing on preclinical investigations, and how what was discovered may prove beneficial for the treatment of other LSDs.


Assuntos
Leucodistrofia de Células Globoides , Doenças por Armazenamento dos Lisossomos , Criança , Humanos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Leucodistrofia de Células Globoides/patologia , Terapia Combinada , Mutação , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/terapia
4.
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
5.
Biochim Biophys Acta Rev Cancer ; 1877(1): 188675, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34974112

RESUMO

ß-galactosylceramidase (GALC) is a lysosomal enzyme that removes ß-galactose from ß-galactosylceramide, leading to the formation of the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth by acting as an oncosuppressive or oncogenic enzyme depending on the different experimental approaches, in vitro versus in vivo observations, preclinical versus clinical findings, and tumor type investigated. This review will recapitulate and discuss the contrasting experimental evidence related to the impact of GALC on the biological behavior of cancer and stromal cells and its contribution to tumor progression.


Assuntos
Leucodistrofia de Células Globoides , Neoplasias , Carcinogênese , Galactosilceramidase/metabolismo , Humanos , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Esfingolipídeos
6.
Trends Cancer ; 7(11): 974-977, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34456156

RESUMO

Lysosomal ß-galactosylceramidase (GALC) removes ß-galactose from ß-galactosylceramide, thus generating the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth and differentiation, questioning its contribution to the sphingolipid metabolism in cancer cells and its role in tumor progression.


Assuntos
Leucodistrofia de Células Globoides , Neoplasias , Diferenciação Celular , Galactosilceramidase/metabolismo , Galactosilceramidas/metabolismo , Humanos , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Neoplasias/metabolismo , Neoplasias/patologia
7.
Neurosci Lett ; 752: 135841, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33766733

RESUMO

Krabbe disease (globoid cell leukodystrophy) is a lysosomal storage disease (LSD) characterized by progressive and profound demyelination. Infantile, juvenile and adult-onset forms of Krabbe disease have been described, with infantile being the most common. Children with an infantile-onset generally appear normal at birth but begin to miss developmental milestones by six months of age and die by two to four years of age. Krabbe disease is caused by a deficiency of the acid hydrolase galactosylceramidase (GALC) which is responsible for the degradation of galactosylceramides and sphingolipids, which are abundant in myelin membranes. The absence of GALC leads to the toxic accumulation of galactosylsphingosine (psychosine), a lysoderivative of galactosylceramides, in oligodendrocytes and Schwann cells resulting in demyelination of the central and peripheral nervous systems, respectively. Treatment strategies such as enzyme replacement, substrate reduction, enzyme chaperones, and gene therapy have shown promise in LSDs. Unfortunately, Krabbe disease has been relatively refractory to most single-therapy interventions. Although hematopoietic stem cell transplantation can alter the course of Krabbe disease and is the current standard-of-care, it simply slows the progression, even when initiated in pre-symptomatic children. However, the recent success of combinatorial therapeutic approaches in small animal models of Krabbe disease and the identification of new pathogenic mechanisms provide hope for the development of effective treatments for this devastating disease. This review provides a brief history of Krabbe disease and the evolution of single and combination therapeutic approaches and discusses new pathogenic mechanisms and how they might impact the development of more effective treatment strategies.


Assuntos
Galactosilceramidase/deficiência , Leucodistrofia de Células Globoides/terapia , Animais , Terapia Combinada/métodos , Modelos Animais de Doenças , Terapia de Reposição de Enzimas/métodos , Galactosilceramidase/genética , Galactosilceramidas/metabolismo , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Esfingolipídeos/metabolismo
8.
Mol Ther ; 29(5): 1883-1902, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33508430

RESUMO

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.


Assuntos
Galactosilceramidase/genética , Terapia Genética/métodos , Leucodistrofia de Células Globoides/patologia , Substância Branca/patologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Fibrinogênio/metabolismo , Galactosilceramidase/metabolismo , Vetores Genéticos/administração & dosagem , Leucodistrofia de Células Globoides/sangue , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Masculino , Camundongos , Recidiva
9.
J Clin Invest ; 130(9): 4906-4920, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32773406

RESUMO

Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.


Assuntos
Dependovirus , Galactosilceramidase , Terapia Genética , Leucodistrofia de Células Globoides , Animais , Modelos Animais de Doenças , Cães , Galactosilceramidase/biossíntese , Galactosilceramidase/genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/terapia
10.
Neuron ; 107(1): 65-81.e9, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32375064

RESUMO

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.


Assuntos
Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/enzimologia , Macrófagos/enzimologia , Células de Schwann/enzimologia , Animais , Doenças Desmielinizantes/enzimologia , Doenças Desmielinizantes/patologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/enzimologia , Degeneração Neural/patologia
12.
Int J Mol Sci ; 21(1)2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31905906

RESUMO

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase ß-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31-/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.


Assuntos
Medula Óssea/metabolismo , Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Animais , Medula Óssea/patologia , Encéfalo/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Galactosilceramidase/genética , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
14.
Biol Blood Marrow Transplant ; 24(11): 2233-2238, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29933067

RESUMO

Allogeneic hematopoietic stem cell transplantation (HSCT) can retard the progression of early infantile Krabbe disease (EIKD). Superior outcomes are achieved if HSCT is performed before the onset of symptoms; however, little information is available about the long-term outcomes in surviving patients. We now describe functional outcomes in presymptomatic infants who underwent HSCT for EIKD at ≤ 2 months of age. Records of the 19 patients who underwent HSCT for EIKD at ≤ 2 months of age from 1996 to 2010 were reviewed. Long-term functional outcomes were compared between those transplanted at < 30 days and ≥ 30 days of life. Median age at transplant was 27 days (range, 19 to 61). Median follow-up of the cohort was 12.6 years. Overall survival at 5 and 10 years post-transplant was 84.2% (95% confidence interval, 58.7% to 94.6%) and 78.6% (95% confidence interval, 52.5% to 91.4%), respectively. More favorable outcomes were seen in patients who underwent HSCT at < 30 days of age, particularly in domains of mobility (P = .01), communication (P = .02), and feeding (P = .008). Improved functional outcomes were observed when HSCT was performed in the first month of life, defining a critical period for intervention. These results support the implementation of newborn screening to enable rapid diagnosis and early treatment of infants with EIKD.


Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Leucodistrofia de Células Globoides/terapia , Condicionamento Pré-Transplante/métodos , Feminino , Humanos , Recém-Nascido , Leucodistrofia de Células Globoides/mortalidade , Leucodistrofia de Células Globoides/patologia , Masculino , Análise de Sobrevida , Resultado do Tratamento
15.
Hum Gene Ther ; 29(7): 785-801, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29316812

RESUMO

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.


Assuntos
Galactosilceramidase/administração & dosagem , Terapia Genética , Leucodistrofia de Células Globoides/terapia , Doenças do Sistema Nervoso Periférico/terapia , Animais , Encéfalo/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Dependovirus/genética , Modelos Animais de Doenças , Cães , Galactosilceramidase/genética , Vetores Genéticos/administração & dosagem , Humanos , Lactente , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia
16.
Adv Neurobiol ; 15: 365-382, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28674989

RESUMO

Leukodystrophies are serious, progressive, genetic disorders of CNS myelin. They may result from abnormalities of the oligodendrocyte or any of the other of myriad of supporting cells or tissues. With recent developments in neuroimaging, their presence is becoming increasingly noted even in situations where they were not suspected. More importantly, new genetic tools have improved our ability to diagnose. An understanding of pathogenesis is still evolving, but it is expected that this will assist in developing targeted therapies for these devastating disorders.


Assuntos
Adrenoleucodistrofia/metabolismo , Doença de Alexander/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Adrenoleucodistrofia/diagnóstico por imagem , Adrenoleucodistrofia/patologia , Adrenoleucodistrofia/fisiopatologia , Doença de Alexander/diagnóstico por imagem , Doença de Alexander/patologia , Doença de Alexander/fisiopatologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucodistrofia de Células Globoides/diagnóstico por imagem , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/fisiopatologia , Imageamento por Ressonância Magnética , Bainha de Mielina/patologia , Oligodendroglia/patologia
17.
J Neurosci Res ; 94(11): 1152-68, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27638600

RESUMO

Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)-induced central nervous system damage. However, all HSPCT-treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor-derived cells and 2) insufficient uptake of donor cell-secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation-independent mannose 6-phosphate-receptor (CI-MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu-mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell-specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT-mediated correction of GALC deficiency in target cells expressing low levels of CI-MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose-6-phosphate-independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.


Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Leucodistrofia de Células Globoides/terapia , Animais , Antígenos CD/metabolismo , Antimetabólitos/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Bussulfano/farmacologia , Linhagem Celular Transformada , Ciclosserina/uso terapêutico , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Terapia Genética/tendências , Vetores Genéticos/fisiologia , Transplante de Células-Tronco Hematopoéticas/tendências , Humanos , Imunossupressores/uso terapêutico , Leucodistrofia de Células Globoides/tratamento farmacológico , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Receptor IGF Tipo 2/metabolismo , Receptores de Somatomedina/metabolismo
18.
J Neurosci Res ; 94(11): 1231-45, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27638606

RESUMO

Krabbe's disease, also known as globoid cell leukodystrophy (GLD), is a lysosomal storage disease caused by the deficiency of the lysosomal enzyme ß-galactocerebrosidase (GALC), resulting in severe neurological manifestations related to demyelination secondary to elevated galactosylsphingosine (psychosine) with its subsequent cytotoxicity. The only available treatment is hematopoietic stem cell transplantation, which delays disease onset but does not prevent long-term neurological manifestations. This article describes the identification of small molecules that enhance mutant GALC activity, identified by quantitative cell-based high-throughput screening (qHTS). Using a specific neurologically relevant murine cell line (145M-Twi) modified to express common human hGALC-G270D mutant, we were able to detect GALC activity in a 1,536-well microplate format. The qHTS of approximately 46,000 compounds identified three small molecules that showed significant enhancements of residual mutant GALC activity in primary cell lines from GLD patients. These compounds were shown to increase the levels of GALC-G270D mutant in the lysosomal compartment. In kinetic assessments, these small molecules failed to disturb the GALC kinetic profile under acidic conditions, which is highly desirable for folding-assisting molecules operating in the endoplasmic reticulum and not affecting GALC catalytic properties in the lysosomal compartment. In addition, these small molecules rescued the decreased GALC activity at neutral pH and partially stabilized GALC under heat-denaturating conditions. These drug-like compounds can be used as the starting point to develop novel small-molecule agents to treat the progressive neurodegenerative course of GLD. © 2016 Wiley Periodicals, Inc.


Assuntos
Galactosilceramidase/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Leucodistrofia de Células Globoides/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/enzimologia , Galactosilceramidase/química , Galactosilceramidase/genética , Humanos , Leucodistrofia de Células Globoides/patologia , Mutação/genética , Polilisina/metabolismo , Transfecção
19.
Eur Radiol ; 26(10): 3377-82, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27137647

RESUMO

OBJECTIVES: Evaluation of Krabbe disease burden and eligibility for hematopoietic stem cell transplantation are often based on neuroimaging findings using the modified Loes scoring system, which encompasses central but not peripheral nervous system changes. We show that quantitative evaluation of thickened cauda equina nerve roots may improve the evaluation of Krabbe disease and therapeutic guidance. METHODS: Lumbar spine MRI scans of patients obtained between March 2013 and September 2013 were retrospectively evaluated and compared to those of controls. Quantitative evaluation of cauda equina roots was performed on the axial plane obtained approximately 5 mm below the conus medullaris. The largest nerves in the right and left anterior quadrants of the spinal canal were acquired. RESULTS: Fifteen symptomatic patients with Krabbe disease (5-44 months old) and eleven age-matched controls were evaluated. The average areas (mm(2)) of anterior right and left nerves were 1.40 and 1.23, respectively, for patients and 0.61 and 0.60 for controls (differences: 0.79 and 0.63; p < 0.001). CONCLUSIONS: Cauda equina nerve root thickening is associated with Krabbe disease in both treated and untreated patients. Adding lumbar spine MRI to the current neurodiagnostic protocols, which fails to account for peripheral nerve abnormalities, will likely facilitate the diagnosis of Krabbe disease. KEY POINTS: • Neuroimaging is valuable for evaluating cauda equina nerve abnormality in Krabbe disease • MRI can be used to quantitatively evaluate cauda equina nerve thickening • Lumbar MRI could be useful for diagnosis and treatment monitoring of Krabbe disease.


Assuntos
Cauda Equina/patologia , Leucodistrofia de Células Globoides/patologia , Cauda Equina/diagnóstico por imagem , Pré-Escolar , Feminino , Humanos , Lactente , Leucodistrofia de Células Globoides/diagnóstico por imagem , Vértebras Lombares/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Masculino , Estudos Retrospectivos
20.
Apoptosis ; 21(1): 25-35, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26459425

RESUMO

Krabbe disease is a genetic demyelinating syndrome characterized by deficiency of the enzyme ß-galactosylceramidase, lysosomal psychosine accumulation, and loss of myelin-forming cells. In this study, some apoptotic markers such as apoptotic index (AI), DNA fragmentation, caspase-3, PTEN, Bad, and PI3K were determined in oligodendrocyte precursors from wild type or twitcher mice untreated or treated with psychosine. Twitcher is a natural mouse model of Krabbe disease containing a premature stop codon (W339X) in the ß-galactosylceramidase gene. Moreover, a possible involvement of connexin (Cx)43 in cell death of oligodendrocyte precursors induced by psychosine was investigated with the final aim to provide a contribution to the knowledge of the molecular mechanisms and pathophysiological events that occur in Krabbe disease. Connexins are a multigene family of structurally related trans-membrane proteins able to modulate essential cellular processes such as proliferation, differentiation and migration. Among these, Cx43 is the predominant isoform in many cell types, including neural progenitor cells. Our results showed an increase of AI, DNA fragmentation, caspase-3, PTEN, Bad, and Cx43 associated to a decrease of PI3K, pAKT and pBad. Taken together, these findings suggest an involvement of Cx43 in the psychosine-mediated apoptosis of primary oligodendrocyte progenitors from wild type or twitcher mice, used for the first time as cell models in comparison. It could open unexplored perspective also for other demyelinating diseases.


Assuntos
Encéfalo/efeitos dos fármacos , Conexina 43/genética , Galactosilceramidase/deficiência , Leucodistrofia de Células Globoides/genética , Oligodendroglia/efeitos dos fármacos , Psicosina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Encéfalo/enzimologia , Encéfalo/patologia , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Conexina 43/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Galactosilceramidase/genética , Regulação da Expressão Gênica , Humanos , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/patologia , Camundongos , Camundongos Knockout , Oligodendroglia/enzimologia , Oligodendroglia/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Psicosina/metabolismo , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
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