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1.
Cells ; 12(7)2023 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-37048066

RESUMO

We have shown in vivo and in vitro previously that psychosine causes dysfunction of autophagy and the ubiquitin-proteasome system underlying the pathogenesis of globoid cell leukodystrophy (GLD), a devastating lysosomal storage disease complicated by global demyelination. Here, we investigated the therapeutic efficacy of the mTOR inhibitor rapamycin in twitcher mice, a murine model of infantile GLD, in biochemical, histochemical, and clinical aspects. Administration of rapamycin to twitcher mice inhibited mTOR signaling in the brains, and significantly reduced the accumulation of insoluble ubiquitinated protein and the formation of ubiquitin aggregates. The astrocytes and microglia reactivity were attenuated in that reactive astrocytes, ameboid microglia, and globoid cells were reduced in the brains of rapamycin-treated twitcher mice. Furthermore, rapamycin improved the cortical myelination, neurite density, and rescued the network complexity in the cortex of twitcher mice. The therapeutic action of rapamycin on the pathology of the twitcher mice's brains prolonged the longevity of treated twitcher mice. Overall, these findings validate the therapeutic efficacy of rapamycin and highlight enhancing degradation of aggregates as a therapeutic strategy to modulate neuroinflammation, demyelination, and disease progression of GLD and other leukodystrophies associated with intracellular aggregates.


Assuntos
Doenças Desmielinizantes , Leucodistrofia de Células Globoides , Camundongos , Animais , Leucodistrofia de Células Globoides/tratamento farmacológico , Leucodistrofia de Células Globoides/patologia , Galactosilceramidase/metabolismo , Galactosilceramidase/uso terapêutico , Agregados Proteicos , Doenças Neuroinflamatórias , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Doenças Desmielinizantes/tratamento farmacológico , Ubiquitinas , Serina-Treonina Quinases TOR
2.
J Neurosci Res ; 94(11): 1138-51, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27638599

RESUMO

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an autosomal recessive neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients but is well short of a cure. Gene transfer experiments using viral vectors have shown some success in extending the survival in the mouse model of GLD, twitcher mice. The present study compares three single-stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), and a self-complementary (sc) AAV vector, all packaged with a codon-optimized murine GALC gene. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on PND10-11 at a dose of 2 × 10(11) vector genomes (vg) per mouse. The results showed a similar significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) as well as the scAAV9 vector, compared to control cohorts. The rAAV gene transfer facilitated GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild-type mice, there was significant improvement in survival for ssAAV9. Histopathological analysis of brain, spinal cord, and sciatic nerve showed significant improvement in preservation of myelin, with ssAAV9 providing the greatest benefit. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at PND10-11 and that BMT synergizes with this treatment to improve the survival further. © 2016 Wiley Periodicals, Inc.


Assuntos
Transplante de Medula Óssea/métodos , Galactosilceramidase/uso terapêutico , Terapia Genética/métodos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Animais , Animais Recém-Nascidos , Dependovirus/genética , Modelos Animais de Doenças , Galactosilceramidase/biossíntese , Galactosilceramidase/genética , Vetores Genéticos/fisiologia , Injeções Espinhais , Leucodistrofia de Células Globoides/mortalidade , Camundongos , Camundongos Mutantes , RNA Mensageiro , Análise de Sobrevida , Resultado do Tratamento
3.
Pediatr Endocrinol Rev ; 13 Suppl 1: 689-96, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27491217

RESUMO

Krabbe disease (globoid cell leukodystrophy, GLD) is an inherited disease caused by a deficiency in the lysosomal enzyme galactocerebrosidase (GALC). The major galactosylated lipid degraded by GALC is galactosylceramide. However, GALC is also responsible for the degradation of galactosylsphingosine (psychosine), a highly cytotoxic glycolipid. It has been hypothesized that GALC-deficiency leads to psychosine accumulation that preferentially kills oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Krabbe disease has traditionally been considered a white matter disease characterized by the loss and disorganization of myelin, infiltration of multinucleated monocytes/macrophages (globoid cells) and lymphocytes, and dysregulation of pro-inflammatory cytokines and chemokines. However, new studies have revealed unexpected neuronal deficiencies. Infantile Krabbe disease is believed to be the most common and aggressive form. However, juvenile and adult onset forms have been described. Children affected with infantile Krabbe disease present with motor dysfunction, cognitive decline, intractable seizures, and premature death between two to five years of age. Murine, canine, and primate models of GALC deficiency have been described and have played an important role in our understanding of this invariably fatal disease. Although there is no cure for Krabbe disease, hematopoietic stem cell transplantation can slow the progression of disease. Recent pre-clinical data indicate that simulataneously targeting multiple pathogenic mechanisms greatly increases efficacy in the murine model of Krabbe disease. A better understanding of the underlying pathogenesis will identify new therapeutic targets that may further increase efficacy.


Assuntos
Transplante de Medula Óssea , Terapia de Reposição de Enzimas , Galactosilceramidase/uso terapêutico , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Leucodistrofia de Células Globoides/terapia , Acetilcisteína/uso terapêutico , Animais , Antimetabólitos/uso terapêutico , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Ciclosserina/uso terapêutico , Modelos Animais de Doenças , Sequestradores de Radicais Livres/uso terapêutico , Galactosilceramidase/genética , Humanos , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/fisiopatologia , Fenótipo , Inibidores de Fosfodiesterase/uso terapêutico , Piridinas/uso terapêutico
4.
Stem Cells ; 29(10): 1559-71, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21809420

RESUMO

Murine neural stem cells (mNSCs), either naive or genetically modified to express supranormal levels of ß-galactocerebrosidase (GALC), were transplanted into the brain of Twitcher mice, a murine model of globoid cell leukodystrophy, a severe sphingolipidosis. Cells engrafted long-term into the host cytoarchitecture, producing functional GALC. Levels of enzyme activity in brain and spinal cord tissues were enhanced when GALC-overexpressing NSC were used. Enzymatic correction correlated with reduced tissue storage, decreased activation of astroglia and microglia, delayed onset of symptoms, and longer lifespan. Mechanisms underlying the therapeutic effect of mNSC included widespread enzyme distribution, cross-correction of host cells, anti-inflammatory activity, and neuroprotection. Similar cell engraftment and metabolic correction were reproduced using human NSC. Thus, NSC gene therapy rapidly reconstitutes sustained and long-lasting enzyme activity in central nervous system tissues. Combining this approach with treatments targeting the systemic disease associated with leukodystrophies may provide significant therapeutic benefit.


Assuntos
Encéfalo/enzimologia , Galactosilceramidase/metabolismo , Terapia Genética/métodos , Leucodistrofia de Células Globoides/terapia , Células-Tronco Neurais/transplante , Medula Espinal/enzimologia , Animais , Encéfalo/patologia , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Ativação Enzimática , Galactosilceramidase/genética , Galactosilceramidase/uso terapêutico , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Medula Espinal/patologia , Transplante de Células-Tronco , Transgenes
5.
Mol Genet Metab ; 97(1): 27-34, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19217332

RESUMO

Krabbe disease or globoid cell leukodystrophy is an autosomal recessive disorder resulting from mutations in the galactocerebrosidase (GALC) gene. These mutations lead to deficient GALC activity, storage of substrates of the enzyme, including psychosine, death to oligodendrocytes, decreased myelination, production of globoid cells and eventually death to the individual. While most affected individuals are infants, late-onset forms are also recognized. In addition to human patients, several animal models have been well characterized, including the twitcher mouse. A spontaneously transformed progenitor cell line was isolated from an astrocyte-enriched fraction of normal mice, partially characterized and transduced with a retrovirus-containing mouse GALC cDNA to produce increased GALC activity (20-30-fold above baseline). These cells, called MAR-52, were injected into the brains of newborn affected twitcher mice. While there was only a modest increase in lifespan and body weight, there was clear evidence for the correction of the astrocytic gliosis, normal appearing oligodendrocytes and evidence for remyelination. We demonstrate that the exogenously supplied neural progenitor cells can donate GALC enzyme to oligodendrocytes in the brains of affected mice resulting in normal myelination in the area of donor cells. At this time, hematopoietic stem cell transplantation provides the best outcome in affected mice and is the only treatment available for human patients, but it does not result in a cure even when performed in asymptomatic newborns. Complete correction probably will require a combined approach to effectively treat patients with Krabbe disease. With developments in the isolation and characterization of stem cells, this approach may improve the outcome for individuals diagnosed in the future.


Assuntos
Encéfalo/patologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Neurônios/transplante , Transplante de Células-Tronco , Células-Tronco/metabolismo , Transformação Genética , Animais , Encéfalo/enzimologia , Modelos Animais de Doenças , Galactosilceramidase/genética , Galactosilceramidase/uso terapêutico , Humanos , Imuno-Histoquímica , Injeções Intraventriculares , Camundongos , Camundongos Mutantes Neurológicos , Neurônios/enzimologia , Fenótipo
6.
FASEB J ; 21(10): 2520-7, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17403939

RESUMO

Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a devastating, degenerative neurological disorder. It is inherited as an autosomal recessive trait caused by loss-of-function mutations in the galactocerebrosidase (GALC) gene. Previously, we have shown that peripheral injection of recombinant GALC, administered every other day, results in a substantial improvement in early clinical phenotype in the twitcher mouse model of GLD. While we did detect active enzyme in the brain following peripheral administration, most of the administered enzyme was localized to the periphery. Given the substantial central nervous system (CNS) involvement in this disease, we were interested in determining whether or not a single-dose administration of the recombinant enzyme directly to the CNS, which could potentially be achieved clinically, would result in any substantial improvement. Following intracerebroventricular (i.c.v.) administration of GALC we noted a significant, 16.5%, reduction in the GALC substrate psychosine, the abnormal accumulation of which is believed to play a pivotal role in the CNS pathology observed in this disease. Moreover, recombinant GALC was found not only in periventricular regions but also at sites distant to the injection such as the cerebral cortex and cerebellum. Most importantly, animals receiving a single i.c.v. dose of the enzyme at postnatal day 20 survived up to 51 days, which compares favorably to the control twitcher animals, which normally only live to postnatal day 40/42. These results indicate that even a single i.c.v. administration of the recombinant enzyme can have significant clinical impact and suggests that other lysosomal storage disorders with significant CNS involvement may similarly benefit.


Assuntos
Galactosilceramidase/uso terapêutico , Leucodistrofia de Células Globoides/tratamento farmacológico , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Galactosilceramidase/administração & dosagem , Galactosilceramidase/deficiência , Galactosilceramidase/genética , Galactosilceramidase/farmacocinética , Injeções Intraventriculares , Cinética , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Especificidade por Substrato , Distribuição Tecidual
7.
FASEB J ; 19(11): 1549-51, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15987783

RESUMO

Globoid cell leukodystrophy (GLD) or Krabbe disease is a devastating, degenerative neurological disorder caused by mutations in the galactosylceramidase (GALC) gene that severely affect enzyme activity. Currently, treatment options for this disorder are very limited. Enzyme replacement therapy (ERT) has been shown to be effective in lysosomal storage disorders with predominantly peripheral manifestations such as type I Gaucher's and Fabry's disease. Little however is known about the possible benefit of ERT in GLD, which has a substantial central nervous system component. In this study, we examined the effect of peripheral GALC injections in the twitcher mouse model of the disease. Although we were unable to block the precipitous decline that normally occurs just before death, we did observe significant early improvements in motor performance, a substantial attenuation in the initial failure to thrive, and an increase in life span. Immunohistochemical and activity analyses demonstrated GALC uptake in multiple tissues, including the brain. This was associated with a decrease in the abnormal accumulation of the GALC substrate psychosine, which is thought to play a pivotal role in disease pathology. These results indicate that peripheral ERT is likely to be beneficial in GLD.


Assuntos
Galactosilceramidase/uso terapêutico , Leucodistrofia de Células Globoides/tratamento farmacológico , Animais , Barreira Hematoencefálica , Linhagem Celular , Modelos Animais de Doenças , Insuficiência de Crescimento/tratamento farmacológico , Marcha/efeitos dos fármacos , Galactosilceramidase/análise , Humanos , Imuno-Histoquímica , Leucodistrofia de Células Globoides/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Psicosina/análise , Proteínas Recombinantes/uso terapêutico
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