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/terapiaRESUMO
Despite recent advances in cancer research, glioblastoma multiforme (GBM) remains a highly aggressive brain tumor as its treatment options are limited. The current standard treatment includes surgery followed by radiotherapy and adjuvant chemotherapy. However, surgery without image guidance is often challenging to achieve maximal safe resection as it is difficult to precisely discern the lesion to be removed from surrounding brain tissue. In addition, the efficacy of adjuvant chemotherapy is limited by poor penetration of therapeutics through the blood-brain barrier (BBB) into brain tissues, and the lack of tumor targeting. In this regard, we utilized a tumor-targeting cell-penetration peptide, p28, as a therapeutic agent to improve the efficacy of a current chemotherapeutic agent for GBM, and as a carrier for a fluorescence imaging agent for a clear identification of GBM. Here, we show that a near-infrared (NIR) imaging agent, ICG-p28 (a chemical conjugate of an FDA-approved NIR dye, indocyanine green ICG, and tumor-targeting p28 peptide) can preferentially localize tumors in multiple GBM animal models. Moreover, xenograft studies show that p28, as a therapeutic agent, can enhance the cytotoxic activity of temozolomide (TMZ), one of the few effective drugs for brain tumors. Collectively, our findings highlight the important role of the tumor-targeting peptide, which has great potential for intraoperative image-guided surgery and the development of new therapeutic strategies for GBM.
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/metabolismoRESUMO
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 , RecidivaRESUMO
Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. Here, we hypothesize that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induce ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identify a critical period of vulnerability to GALC ablation between P4-6 in mice. Early Galc-iKO induction causes a worse KD phenotype, higher psychosine levels in the rodent brainstem and spinal cord, and a significantly shorter life-span of the mice. Intriguingly, GALC expression peaks during this critical developmental period in mice. Further analysis of this mouse model reveals a cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis.
Assuntos
Tronco Encefálico/enzimologia , Tronco Encefálico/crescimento & desenvolvimento , Tronco Encefálico/metabolismo , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Animais , Tronco Encefálico/embriologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Fenótipo , Psicosina/metabolismo , Tamoxifeno , TranscriptomaRESUMO
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/terapiaRESUMO
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/patologiaRESUMO
Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.
Assuntos
Ceramidase Ácida/antagonistas & inibidores , Benzoxazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Administração Oral , Animais , Benzoxazóis/administração & dosagem , Benzoxazóis/síntese química , Benzoxazóis/farmacocinética , Encéfalo/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Feminino , Doença de Gaucher/enzimologia , Doença de Gaucher/metabolismo , Humanos , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/metabolismo , Masculino , Camundongos , Estrutura Molecular , Psicosina/análogos & derivados , Psicosina/metabolismo , Relação Estrutura-AtividadeRESUMO
Glycosphingolipid (GSL) accumulation is implicated in the neuropathology of several lysosomal conditions, such as Krabbe disease, and may also contribute to neuronal and glial dysfunction in adult-onset conditions such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. GSLs accumulate in cellular membranes and disrupt their structure; however, how membrane disruption leads to cellular dysfunction remains unknown. Using authentic cellular and animal models for Krabbe disease, we provide a mechanism explaining the inactivation of lipid raft (LR)-associated IGF-1-PI3K-Akt-mTORC2, a pathway of crucial importance for neuronal function and survival. We show that psychosine, the GSL that accumulates in Krabbe disease, leads to a dose-dependent LR-mediated inhibition of this pathway by uncoupling IGF-1 receptor phosphorylation from downstream Akt activation. This occurs by interfering with the recruitment of PI3K and mTORC2 to LRs. Akt inhibition can be reversed by sustained IGF-1 stimulation, but only during a time window before psychosine accumulation reaches a threshold level. Our study shows a previously unknown connection between LR-dependent regulation of mTORC2 activity at the cell surface and a genetic neurodegenerative disease. Our results show that LR disruption by psychosine desensitizes cells to extracellular growth factors by inhibiting signal transmission from the plasma membrane to intracellular compartments. This mechanism serves also as a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of lipids undermines cell function, with potential implications in other genetic sphingolipidoses and adult neurodegenerative conditions. This article has an associated First Person interview with the first author of the paper.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingolipidoses/genética , Animais , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Lisossomos/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Psicosina/farmacologia , Receptor IGF Tipo 1/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingolipidoses/metabolismoRESUMO
We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.
Assuntos
Metabolismo dos Carboidratos , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Terapia Genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Fenótipo , Animais , Vias Autônomas/metabolismo , Vias Autônomas/patologia , Vias Autônomas/ultraestrutura , Axônios/metabolismo , Axônios/patologia , Axônios/ultraestrutura , Comportamento Animal , Encéfalo/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Leucodistrofia de Células Globoides/diagnóstico , Leucodistrofia de Células Globoides/terapia , Masculino , Camundongos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Distribuição Tecidual , Transdução Genética , Resultado do TratamentoRESUMO
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/patologiaRESUMO
Ascorbic acid (AA), the reduced form of vitamin C, acts as a neuroprotector by eliminating free radicals in the brain. Sodium/vitamin C co-transporter isoform 2 (SVCT2) mediates uptake of AA by neurons. It has been reported that SVCT2 mRNA is induced in astrocytes under ischemic damage, suggesting that its expression is enhanced in pathological conditions. However, it remains to be established if SVCT expression is altered in the presence of reactive astrogliosis generated by different brain pathologies. In the present work, we demonstrate that SVCT2 expression is increased in astrocytes present at sites of neuroinflammation induced by intracerebroventricular injection of a GFP-adenovirus or the microbial enzyme, neuraminidase. A similar result was observed at 5 and 10 days after damage in a model of traumatic injury and in the hippocampus and cerebral cortex in the in vivo kindling model of epilepsy. Furthermore, we defined that cortical astrocytes maintained in culture for long periods acquire markers of reactive gliosis and express SVCT2, in a similar way as previously observed in situ. Finally, by means of second harmonic generation and 2-photon fluorescence imaging, we analyzed brain necropsied material from patients with Alzheimer's disease (AD), which presented with an accumulation of amyloid plaques. Strikingly, although AD is characterized by focalized astrogliosis surrounding amyloid plaques, SVCT2 expression at the astroglial level was not detected. We conclude that SVCT2 is heterogeneously induced in reactive astrogliosis generated in different pathologies affecting the central nervous system (CNS).
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Adenoviridae/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Gliose/metabolismo , Gliose/patologia , Proteínas de Fluorescência Verde/metabolismo , Neuraminidase/metabolismo , Ratos Sprague-DawleyRESUMO
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 TratamentoRESUMO
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/metabolismoRESUMO
Expression of the sodium and ascorbic acid (AA) cotransporter SVCT2 is induced during the period of cellular arborization and synaptic maturation of early postnatal (P1-P5) rat cerebral neurons. The physiological importance of the transporter for neurons is evidenced by the lethality and delayed neuronal differentiation detected in mice with ablation of SVCT2. The mechanism(s) involved in these defects and the role of SVCT2 in neuronal branching have not been determined yet. To address this, we used lentiviral expression vectors to increase the levels of SVCT2 in N2a cells and analyzed the effects on neurite formation. Expression of a fusion protein containing the human SVCT2wt and EYFP induced an increase in the number of MAP2+ neurites and filopodia in N2a cells. Overexpression of SVCT2 and treatment with AA promoted ERK1/2 phosphorylation. Our data suggest that enhanced expression of the high affinity AA transporter SVCT2, which tightly regulates intracellular AA concentrations, induces neuronal branching that then activates key signaling pathways that are involved in the differentiation and maturation of cortical neurons during postnatal development.
Assuntos
Sistema de Sinalização das MAP Quinases , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Animais , Ácido Ascórbico/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Forma Celular , Suplementos Nutricionais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Fenótipo , Fosforilação/efeitos dos fármacos , Transporte ProteicoRESUMO
Disease-cell models that recapitulate specific molecular phenotypes are essential for the investigation of molecular pathogenesis of neurodegenerative diseases including lysosomal storage diseases (LSDs) with predominant neurological manifestations. Herein we report the development and characterization of a cell model for a rapid neurodegenerative LSDs, globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease. GLD is caused by the deficiency of ß-galactocerebrosidase (GALC), a lysosomal enzyme that hydrolyzes two glycosphingolipids, psychosine and galactosylceramide. Unfortunately, the available culture fibroblasts from GLD patients consist of a limited research tool as these cells fail to accumulate psychosine, the central pathogenic glycosphingolipid in this LSD that results in severe demyelination. Firstly, we obtained brain samples from the Twitcher (Twi) mice (GALC(twi/twi)), the natural mouse model with GALC deficiency. We immortalized the primary neuroglial cultured cells with SV40 large T antigen, generating the 145M-Twi and the 145C-Wt cell lines from the Twi and control mice, respectively. Both cell lines expressed specific oligodendrocyte markers including A2B5 and GalC. The 145M-Twi cells showed biochemical and cellular disturbances related to GLD neuropathogenesis including remarkable caspase-3 activation, release of cytochrome C into the cytosol and expansion of the lysosomal compartment. Under treatment with glycosphingolipids, 145M-Twi cells showed increased LC3B levels, a marker of autophagy. Using the LC-MS/MS method that we developed, the 145M-Twi cells showed significantly higher levels of psychosine. The 145M-Twi and 145C-Wt lines allowed the development of a robust throughput LC-MS/MS assay to measure cellular psychosine levels. In this throughput assay, l-cycloserine showed to significantly reduce the 145M-Twi cellular levels of psychosine. The established 145M-Twi cells are powerful research tools to investigate the neurologically relevant pathogenic pathways as well as to develop primary screening assays for the identification of therapeutic agents for GLD and potentially other glycosphingolipid disorders.
Assuntos
Efeito Fundador , Galactosilceramidase/deficiência , Leucodistrofia de Células Globoides/patologia , Modelos Biológicos , Psicosina/biossíntese , Adulto , Animais , Antígenos Transformantes de Poliomavirus/genética , Autofagia , Biomarcadores/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Química Encefálica , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Transformada , Ciclosserina/farmacologia , Citocromos c/metabolismo , Galactosilceramidas/metabolismo , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Lactente , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/genética , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Psicosina/antagonistas & inibidores , Psicosina/metabolismoRESUMO
Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme ß-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-ß-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine ß-galactosylceramidase complementary DNA. Finally, RNA interference-mediated ß-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that ß-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.
Assuntos
Leucodistrofia de Células Globoides/complicações , Neovascularização Patológica/etiologia , Neovascularização Patológica/patologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aorta/patologia , Aorta/ultraestrutura , Materiais Biocompatíveis , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/ultraestrutura , Bovinos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Colágeno/toxicidade , Modelos Animais de Doenças , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Laminina/toxicidade , Leucodistrofia de Células Globoides/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Neovascularização Patológica/prevenção & controle , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteoglicanas/toxicidade , Psicosina/metabolismo , Psicosina/farmacologia , RNA Interferente Pequeno/administração & dosagem , Fatores de Tempo , Transfecção , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
Lysosomal beta-galactosylceramidase deficiency results in demyelination and inflammation in the nervous system causing the neurological Krabbe disease. In the Twitcher mouse model of this disease, we found that neurological symptoms parallel progressive and severe lymphopenia. Although lymphopoiesis is normal before disease onset, primary and secondary lymphoid organs progressively degenerate afterward. This occurs despite preserved erythropoiesis and leads to severe peripheral lymphopenia caused by reduced numbers of T cell precursors and mature lymphocytes. Hematopoietic cell replacement experiments support the existence of an epigenetic factor in mutant mice reconcilable with a progressive loss of autonomic axons that hampers thymic functionality. We propose that degeneration of autonomic nerves leads to the irreversible thymic atrophy and loss of immune-competence. Our study describes a new aspect of Krabbe disease, placing patients at risk of immune-related pathologies, and identifies a novel target for therapeutic interventions.
Assuntos
Doenças do Sistema Nervoso Autônomo/imunologia , Epigênese Genética , Leucodistrofia de Células Globoides/fisiopatologia , Linfopenia/fisiopatologia , Timo/inervação , Animais , Doenças do Sistema Nervoso Autônomo/genética , Doenças do Sistema Nervoso Autônomo/patologia , Axônios/patologia , Medula Óssea/patologia , Modelos Animais de Doenças , Progressão da Doença , Galactosilceramidase/deficiência , Galactosilceramidase/genética , Transplante de Células-Tronco Hematopoéticas , Contagem de Leucócitos , Leucócitos Mononucleares/patologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Linfopenia/genética , Linfopenia/patologia , Camundongos , Camundongos Mutantes Neurológicos , Psicosina/metabolismo , Baço/patologia , Taxa de Sobrevida , Timo/patologiaRESUMO
Irradiation is one way to condition Twitcher mice--a natural model of globoid cell leukodystrophy (GLD)--prior to receive bone marrow transplantation (BMT). BMT showed to delay but not to completely prevent GLD disease in treated mutants. The reasons why BMT is not completely preventive in Twitchers are unclear but we speculate that irradiation might contribute to worsen the neurological impairments generated by the disease by altering postnatal neurogenesis. To test this hypothesis, we examined proliferation, migration and differentiation of neural precursors in neurogenic areas of the Twitcher brain after exposure of 5 day-old mutant pups to 620 rad, a non-lethal dose that leads to 80-90% of bone-marrow engraftment in classic BMT. Twitchers showed to be sensitive to irradiation, leading to a severe retardation of body growth of irradiated mutants. Irradiated Twitchers had reduced proliferation of neural precursors and increased astrogliosis and microgliosis, with reduced numbers of migratory neuroblasts and significantly less brain myelination. These effects were accompanied by caspase-3 activation and appeared largely irreversible in the lifespan of the Twitcher. Our work confirms that exposure of the neonatal brain to irradiation conditions such as those performed prior to BMT, can lead to long-lasting alterations of postnatal neurogenesis and myelination, which might contribute to worsen the progression of disease in these myelin mutants and to reduce the success of BMT.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/efeitos da radiação , Raios gama , Leucodistrofia de Células Globoides/fisiopatologia , Animais , Apoptose/efeitos da radiação , Transplante de Medula Óssea , Encéfalo/citologia , Caspase 3/metabolismo , Proliferação de Células/efeitos da radiação , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Macrófagos/efeitos da radiação , Camundongos , Camundongos Mutantes Neurológicos , Proteína Proteolipídica de Mielina/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Neuroglia/fisiologia , Neuroglia/efeitos da radiação , Neurônios/fisiologia , Neurônios/efeitos da radiaçãoRESUMO
The postnatal subventricular zone (SVZ) is a niche for continuous neurogenesis in the adult brain and likely plays a fundamental role in self-repair responses in neurodegenerative conditions. Maintenance of the pool of neural stem cells within this area depends on cell-cell communication such as that provided by the Notch signaling pathway. Notch1 receptor mRNA has been found distributed in different areas of the postnatal brain including the SVZ. Although the identity of Notch1-expressing cells has been established in the majority of these areas, it is still unclear what cell types within the SVZ are expressing components of this pathway. Here we demonstrate that most of expression of Notch1 in the adult SVZ occurs in polysialylated neural cell adhesion molecule (PSA-NCAM)-positive neural precursors and in glial fibrillary acidic protein-positive SVZ astrocytes. Notch1 was also found in PSA-NCAM-positive neuroblasts located within the rostral migratory stream (RMS) but much less in those that have reached the olfactory bulb. We show that two of the naturally occurring Notch1 activators, Jagged1 and Delta1, are also expressed in the SVZ and within the RMS in the adult mouse brain. Finally, using a model of cortical stab wound, we show that the astrogliogenic response of the SVZ to injury is accompanied by activation of the Notch pathway.