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Selective Galactosylceramidase Ablation to Study the Pathogenesis of Krabbe Leukodystrophy

选择性半乳糖神经酰胺酶消融研究克拉伯脑白质营养不良的发病机制

基本信息

批准号：
10531586
负责人：
Daesung Shin
金额：
$ 34.89万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10531586
关键词：
2&apos ,3&apos -Cyclic-Nucleotide Phosphodiesterases 3 year old ARHGEF5 gene Ablation Affect Alleles Animal Model Astrocytes Attenuated Behavioral Biochemistry Brain Brain Stem CSPG4 gene Cause of Death Cell Death Cell secretion Cells Central Nervous System Cessation of life Clinical Clinical Data Data Defect Demyelinating Diseases Demyelinations Deterioration Development Disease Enzymes Event Extracellular Space Galactosylceramides Glial Fibrillary Acidic Protein Globoid cell leukodystrophy Hematopoietic Stem Cell Transplantation Hematopoietic stem cells IGF Type 2 Receptor Immunohistochemistry In Vitro Infant Knockout Mice Kyphosis deformity of spine Life Lipids LoxP-flanked allele Lysosomal Storage Diseases Lysosomes Measures Messenger RNA Microglia Morphology Motor Mus Muscle Rigidity Mutation Myelin Nerve Degeneration Neurodegenerative Disorders Neuroglia Neurologic Neurons Null Lymphocytes Oligodendroglia Pathogenesis Pathogenicity Pathologic Pathology Patient-Focused Outcomes Patients Peripheral Nervous System Phenotype Positioning Attribute Production Proteins Psychosine Role Series Survival Analysis Symptoms Tamoxifen Testing Therapeutic Time Toxic effect Transcript Tremor Wild Type Mouse autosome axonal degeneration behavioral phenotyping brain cell cell type conditional knockout critical period cytotoxic design effective therapy experience experimental study galactosylceramidase improved in vivo infancy mortality mouse model oligodendrocyte precursor postnatal stem cell therapy stem cells uptake wasting

项目摘要

Krabbe leukodystrophy (KD) is a fatal neurodegenerative lysosomal storage disorder caused by deficiency of galactosylceramidase (GALC) that affects both central and peripheral nervous systems. KD manifests in infants in the first few months of life and presents with severe irritability, muscle rigidity and motor deterioration, which quickly progresses to overall clinical decline and death within months. Unfortunately, there is no cure for KD. Our limited understanding of the pathogenesis is based on clinical data and on the spontaneous twitcher mouse model. Hematopoietic stem cell transplantation (HSCT) partially attenuates the course of KD only if performed before the onset of symptoms, presumably because stem cell derivatives secrete GALC that is uptaken by myelinating glia via the mannose-6-phosphate receptor, so called cross-correction. However, it is not clear how efficiently cross-correction happens in vivo, if only myelin-forming glia need to be corrected and at which developmental stage. Furthermore, accumulation of the lipid psychosine due to GALC deficiency contributes to KD by killing myelin-forming glia and neurons, but the relative importance of psychosine, its origin and the sequence of pathogenic events is unclear. We recently developed a conditional Galc floxed mouse and found that: 1) A KD-like phenotype similar to twitcher is obtained when Galc ablation is induced ubiquitously [Galc-iKO] at P4 or before. In contrast, induction at P6 or later significantly delayed the phenotype and prolonged survival (~25 days). Galc deletion before P4 caused severe developmental brainstem problems that were milder if deletion was induced after P6, indicating that GALC may be required for brainstem development; 2) Oligodendrocyte (OL)-specific Galc conditional knockout [Galc-CKO] results in a phenotype that includes tremor, wasting, kyphosis, motor defects, demyelination and mild axonal degeneration, but that is not as severe as Galc- iKO mice, suggesting that Galc deficiency in OLs may be not sufficient to trigger a complete KD phenotype; and 3) GALC uptake is less efficient in Galc-null cells in vitro, and surrounding WT cells provides minimal GALC to Galc-deficient OLs in vivo, indicating inefficient cross-correction of GALC. We propose 3 Aims to determine; 1) if GALC has an early role in brain development and contributes to mortality, 2) the key cells in the progression of KD pathology, and 3) the efficiency of cell-specific cross-correction of GALC. By combining a series of in vitro experiments with the comparison of cell-specific, time–specific and constitutive deletion of Galc in vivo, we will test the following 3 hypotheses that derived from our preliminary data and from the clinical experience: 1) GALC has specific developmental roles during critical periods including P4-6 in Krabbe mice; 2) any brain cell can produce psychosine or be the target of toxicity; 3) HSCT fails to cure KD due to inefficient cross-correction of GALC. Our results will help to understand the disease mechanisms of KD and the limitations of HSCT, which will allow the development of better therapies for KD and similar lysosomal, neurodegenerative and demyelinating diseases.

Krabbe脑白质营养不良（KD）是一种致命的神经退行性溶酶体储存障碍，半乳糖神经酰胺酶（GALC）影响中枢和外周神经系统。KD在婴儿中表现在生命的最初几个月，并表现出严重的易怒，肌肉僵硬和运动退化，在几个月内迅速发展为整体临床衰退和死亡。不幸的是，KD没有治愈方法。我们对发病机制的有限理解是基于临床数据和自发抽搐小鼠模型造血干细胞移植（HSCT）只有在进行时才能部分减轻KD的病程在症状发作之前，可能是因为干细胞衍生物分泌GALC，通过甘露糖-6-磷酸受体使神经胶质髓鞘化，所谓的交叉校正。然而，目前尚不清楚如何如果只有髓鞘形成神经胶质需要纠正，并且在这种情况下，发育阶段此外，由于GALC缺乏导致的脂质精神病肽的积累有助于 KD通过杀死髓鞘形成胶质细胞和神经元，但精神分裂素的相对重要性，其来源和致病事件的顺序尚不清楚。我们最近开发了一种条件性Galc floxed小鼠， 1）当Galc消融被普遍诱导时，获得类似于抽搐的KD样表型[Galc-iKO] P4或之前。相反，在P6或更晚的时间诱导显著延迟表型和延长存活（约25天）。P4前Galc缺失导致严重的发育性脑干问题，在P6后出现缺失，表明GALC可能是脑干发育所必需的; 2）少突胶质细胞（OL）特异性Galc条件性敲除[Galc-CKO]导致包括震颤的表型，消瘦、脊柱后凸、运动缺陷、脱髓鞘和轻度轴突变性，但不像Galc那么严重， iKO小鼠，表明OL中的Galc缺陷可能不足以触发完全KD表型;以及 3)GALC摄取在体外Galc缺失细胞中效率较低，并且周围的WT细胞提供最少的GALC以使GALC在细胞中的摄取减少。体内缺乏Galc的OL，表明GALC的交叉校正无效。我们提出了三个目标来确定; 1）如果GALC在大脑发育中起早期作用并导致死亡，2）进展中的关键细胞 KD病理学，和3）GALC的细胞特异性交叉校正的效率。通过结合一系列体外通过比较体内Galc的细胞特异性、时间特异性和组成性缺失的实验，根据我们的初步数据和临床经验，检验以下3个假设：1）GALC 在Krabbe小鼠的关键时期（包括P4-6）具有特定的发育作用; 2）任何脑细胞都可以产生精神分裂素或成为毒性靶点; 3）HSCT未能治愈KD，原因是交叉校正效率低， GALC。我们的研究结果将有助于了解KD的发病机制和HSCT的局限性，将允许开发更好的治疗KD和类似的溶酶体，神经退行性和脱髓鞘疾病