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: 10316179
• 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/10316179
• 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; 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.

克拉伯脑白质营养不良（KD）是一种致命的神经退行性溶酶体贮积症，由缺乏 半乳糖神经酰胺酶（GALC）影响中枢和周围神经系统。 KD 表现在婴儿身上 在生命的最初几个月内，会出现严重的烦躁、肌肉僵硬和运动能力下降， 几个月内迅速进展为总体临床衰退和死亡。不幸的是，KD 无法治愈。我们的 对发病机制的了解有限基于临床数据和自发抽搐小鼠 模型。只有进行造血干细胞移植 (HSCT) 才能部分减轻川崎病的病程 在症状出现之前，可能是因为干细胞衍生物会分泌 GALC，并被 通过甘露糖-6-磷酸受体调节髓鞘神经胶质细胞，即所谓的交叉校正。但目前尚不清楚如何 如果仅需要校正形成髓磷脂的神经胶质细胞，并且此时，有效的交叉校正会在体内发生 发育阶段。此外，由于 GALC 缺乏导致脂质精神嘧啶的积累，导致 KD 通过杀死形成髓磷脂的神经胶质细胞和神经元来实现，但精神碱的相对重要性、其起源和作用 致病事件的顺序尚不清楚。我们最近开发了一种有条件的 Galc floxed 小鼠并发现 1) 当普遍诱导 Galc 消融时，会获得与 twitcher 类似的 KD 样表型 [Galc-iKO] 在P4或之前。相反，在 P6 或之后的诱导显着延迟了表型并延长了生存期 （约 25 天）。 P4 之前的 Galc 缺失会导致严重的脑干发育问题，如果 P6后诱导缺失，表明脑干发育可能需要GALC； 2） 少突胶质细胞 (OL) 特异性 Galc 条件性敲除 [Galc-CKO] 会导致包括震颤、 消瘦、脊柱后凸、运动缺陷、脱髓鞘和轻度轴突变性，但不像 Galc- 那样严重 iKO 小鼠，表明 OL 中的 Galc 缺乏可能不足以触发完整的 KD 表型；和 3) 体外 Galc-null 细胞中 GALC 的摄取效率较低，周围的 WT 细胞提供的 GALC 极少 体内 OL 缺乏 Galc，表明 GALC 交叉校正效率低下。我们提出3个目标来确定； 1） 如果 GALC 在大脑发育中发挥早期作用并导致死亡率，2) 进展中的关键细胞 KD 病理学的影响，以及 3) GALC 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、时间特异性和组成型删除的实验，我们将 测试以下 3 个根据我们的初步数据和临床经验得出的假设：1) GALC 在关键时期具有特定的发育作用，包括 Krabbe 小鼠中的 P4-6； 2）任何脑细胞都可以 产生精神嘧啶或成为毒性目标； 3）由于交叉校正效率低下，HSCT未能治愈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， 将有助于开发出更好的治疗川崎病和类似溶酶体、神经退行性疾病和 脱髓鞘疾病。