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

• 批准号: 10057403
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057403
• 关键词: 2' ,3' -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; Cells; 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; Structure; Survival Analysis; Symptoms; Tamoxifen; Testing; Therapeutic; Time; Toxic effect; Transcript; Tremor; Wild Type Mouse; axonal degeneration; base; 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; ranpirnase; stem cell therapy; stem cells; uptake; wasting; 2' ,3' -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; Cells; 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; Structure; Survival Analysis; Symptoms; Tamoxifen; Testing; Therapeutic; Time; Toxic effect; Transcript; Tremor; Wild Type Mouse; axonal degeneration; base; 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; ranpirnase; stem cell therapy; stem cells; uptake; wasting

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白细胞营养不良（KD）是由于缺乏症而引起的致命神经退行性溶酶体储存障碍 影响中央和周围神经系统的半乳糖基酰胺酶（GALC）。 KD在婴儿中表现出来 在生命的头几个月中 在几个月内迅速发展到整体临床下降和死亡。不幸的是，KD无法治愈。我们的 对发病机理的有限理解是基于临床数据和赞助的抽搐小鼠 模型。造血干细胞移植（HSCT）仅在执行时会部分衰减KD的过程 在症状发作之前，大概是因为干细胞衍生物被秘密galc被 通过甘露糖-6-磷酸受体对髓质神经胶质的胶质化，所谓的交叉校正。但是，尚不清楚如何 如果只需要校正髓鞘形成胶质神经胶质，则有效地发生交叉校正在体内发生 发展阶段。此外，由于galc缺乏而导致脂质心理的积累有助于 KD通过杀死形成髓磷脂的神经元和神经元，但是心理，起源和起源的相对重要性 致病事件的序列尚不清楚。我们最近开发了一种条件的galc floxed小鼠，发现 那就是：1）当galc消融无处不在时，获得了类似于Twitcher的KD样表型[Galc-iko] 在P4或之前。相反，P6或更高版本的诱导显着延迟了表型并延长生存率 （〜25天）。 P4之前的GALC缺失引起严重的发育性脑干问题，如果 P6后诱导缺失，表明脑干发育可能需要加仑。 2） 少突胶质细胞（OL）特异性GALC条件敲除[GALC-CKO]导致表型，包括震颤， 浪费，脑膜病，运动缺陷，脱髓鞘和轻度轴突变性，但不如Galc- IKO小鼠，表明OLS的GALC缺乏可能不足以触发完整的KD表型。和 3）galc摄取在体外的galc-null细胞中的效率较低，周围的WT细胞可提供最小的galc至 GALC缺陷型OLS体内，表明GALC的效率低下。我们提出3旨在确定的目标； 1） 如果Galc在大脑发育中起着早期作用并导致死亡率，则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 与体内galc的细胞特异性，特异性和本构缺失进行比较的实验，我们将 测试以下从我们的初步数据和临床经验得出的3个假设：1）galc 在关键时期中具有特定的发育作用，包括Krabbe小鼠的P4-6； 2）任何脑细胞都可以 产生心理或成为毒性的靶标； 3）由于效率低下的跨校正，HSCT无法治愈KD galc。我们的结果将有助于了解KD的疾病机制和HSCT的局限性，这 将允许开发针对KD和类似溶酶体，神经退行性和 脱髓鞘疾病。