Combined neuroprotection and metabolic correction to treat leukodystrophies

联合神经保护和代谢校正治疗脑白质营养不良

基本信息

批准号：
8131096
负责人：
Ernesto Roque Bongarzone
金额：
$ 33.16万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8131096
关键词：
Address Affect Alzheimer&apos s Disease Animal Model Anti-Arrhythmia Agents Axon Axonal Transport Binding Biochemical Biological Assay Birth Blood - brain barrier anatomy Bone Marrow Cell Transplantation Bone Marrow Transplantation Brain Calcium Cells Childhood Clinical Complement Complex Confocal Microscopy Consensus Defect Demyelinating Diseases Demyelinations Deterioration Development Diffusion Disease Ensure Enzymes Evaluation Flecainide Functional disorder Genetic Globoid cell leukodystrophy Hematogenous Homeostasis Human Imagery Individual Inherited Injection of therapeutic agent Intervention Intravenous Laboratories Life Light Link Lysosomal Storage Diseases Magnetic Resonance Imaging Mediating Metabolic Microscope Modeling Molecular Multiple Sclerosis Mus Myelin Nerve Nerve Degeneration Nervous system structure Neural Conduction Neurodegenerative Disorders Neuroglia Neurologic Neuronal Dysfunction Neurons Newborn Infant Nicotinic Receptors Outcome Parkinson Disease Pathogenesis Pathology Patients Pharmaceutical Preparations Protein phosphatase Psychosine Rabies virus Ranvier&apos s Nodes Role Small Interfering RNA Sodium Channel Structure Swelling Symptoms Testing Therapeutic Time Transgenic Organisms axonopathy base biological adaptation to stress design enzyme deficiency experience fast axonal transport galactosylceramidase improved in vivo insight leukodystrophy macrophage mutant myelination neuron loss neuroprotection novel novel therapeutics postnatal prevent public health relevance reconstitution research study therapeutic enzyme traditional therapy

项目摘要

DESCRIPTION (provided by applicant): Galactosylceramidase (GALC) deficiency in Krabbe disease (KD) causes toxic accumulation of galactosylsphingosine (psychosine) in myelin-forming cells, leading to demyelination of the nervous system. To reduce demyelination, current therapies seek to provide the missing enzyme to myelinating glia via infiltrating macrophages after the transplantation of bone marrow cells (BMT) from healthy donors into affected patients. Although the experience gained from this approach supports the use of BMT, KD patients suffer neurological sequelae. This suggests that the pathogenic mechanisms in KD are more complex than previously thought and that new therapeutic strategies are needed to cure KD. Experiments in our laboratory using the Twitcher mouse, a natural model for KD, indicate: 1) BMT- treated mice show neuronal and axonal damage by the time sufficient therapeutic enzyme accumulates in the nervous system [1]; 2) psychosine is also produced and accumulates in neurons in the absence of mutant glia, causing the blockage of fast axonal transport via the activity of protein phosphatase 1 (PP1); and 3) mutant neurons show abnormal intracellular levels of Ca linked to deregulated expression of the Na+ Ca exchanger (NCX1). These observations suggest that GALC-deficient neurons mount a stress response that contributes to the pathology and that PP1 and NCX1 are two potential key components in the mechanism that mediates axonal defects in KD. Thus, we hypothesize that the deficiency of GALC in KD not only affects myelination but also triggers intrinsic and contemporaneous defects in neurons. To test this hypothesis we propose specific experiments to modulate PP1 and NCX1 activities in Twitcher neurons. These experiments will provide proof-of-concept that neuroprotective strategies can synergize with/improve the therapeutic benefits of traditional BMT-based treatments. Specifically, we will: 1) determine whether controlled and specific reduction of neuronal PP1 activity using siRNA specific silencing protects axonal transport in mutant neurons; 2) determine whether flecainide, an antiarrhythmic drug with a proven ability to reduce sodium channel firing and NCX1 activity, improves NCX1-mediated influx of calcium in axons; and 3) determine whether these neuroprotective strategies combined with metabolic correction after BMT in newborn Twitcher mice improve clinical outcome. Results from these experiments will shed light on the molecular role of PP1 and NCX1 activity mediating neuronal dysfunction in KD and will provide a unique opportunity to improve the current BMT-based metabolic corrective strategies used to treat this leukodystrophy. The insight obtained will be relevant to other lysosomal storage disorders, which like KD are associated with aggressive neurological deterioration and for which there are no available cures. PUBLIC HEALTH RELEVANCE: Krabbe disease is a lysosomal storage disease that results in demyelination of the brain and nerves in affected individuals. Some Krabbe patients are treated with hematogenous cell replacement, which delays the onset of symptoms. However, a definitive and complete cure for this disease has not been achieved and treated patients continue to undergo deterioration and neurological deficits. The role of neuronal loss in Krabbe disease is not completely understood, but a consensus is emerging that dysfunction of axons and neurons leads to permanent neurological deficits in several neurodegenerative disorders, including multiple sclerosis, Alzheimer disease, Parkinson disease and others. Our preliminary studies provide evidence that Krabbe disease is compounded by axonal defects. In addition to the loss of myelin, neurodegeneration is likely a limiting factor in reducing the efficiency of traditional therapies. Thus, a combined therapy that provides not only enzyme replacement but also neuroprotection is likely to synergize or enhance the therapeutic benefits. Our objective is to examine whether two novel neuroprotective strategies targeting specific aspects of neurodegeneration in Krabbe disease can be combined with traditional bone marrow transplantation to fully prevent development of the disease. Results of the proposed experiments will provide proof-of-concept for the design of combined neuroprotective therapies to treat human Krabbe patients and the rational basis for studies of other leukodystrophies that involve degeneration of axons and myelin.

描述（由申请人提供）：克拉伯病（KD）中的半乳糖神经酰胺酶（GALC）缺陷导致半乳糖基鞘氨醇（精神鞘氨醇）在髓磷脂形成细胞中毒性积累，导致神经系统脱髓鞘。为了减少脱髓鞘，当前的疗法试图在将健康捐献者的骨髓细胞（BMT）移植到受影响的患者体内后，通过浸润巨噬细胞向髓鞘神经胶质细胞提供缺失的酶。尽管从这种方法中获得的经验支持 BMT 的使用，但 KD 患者会遭受神经系统后遗症。这表明川崎病的发病机制比以前认为的更为复杂，需要新的治疗策略来治愈川崎病。我们实验室使用 Twitcher 小鼠（一种 KD 的天然模型）进行的实验表明：1) BMT 治疗的小鼠在神经系统中积累足够的治疗酶时表现出神经元和轴突损伤 [1]； 2) 在没有突变神经胶质细胞的情况下，精神嘧啶也会在神经元中产生和积累，通过蛋白磷酸酶 1 (PP1) 的活性导致快速轴突运输受阻； 3) 突变神经元表现出异常的细胞内 Ca 水平，这与 Na+ Ca 交换器 (NCX1) 的表达失调有关。这些观察结果表明，缺乏 GALC 的神经元会产生有助于病理学的应激反应，并且 PP1 和 NCX1 是介导 KD 轴突缺陷机制中的两个潜在关键成分。因此，我们假设 KD 中 GALC 的缺乏不仅会影响髓鞘形成，还会引发神经元的内在和同时发生的缺陷。为了验证这一假设，我们提出了调节 Twitcher 神经元中 PP1 和 NCX1 活性的具体实验。这些实验将提供概念证明，证明神经保护策略可以与传统的基于 BMT 的治疗相协同/提高其治疗效果。具体来说，我们将： 1) 确定使用 siRNA 特异性沉默控制和特异性降低神经元 PP1 活性是否可以保护突变神经元中的轴突运输； 2) 确定氟卡尼（一种抗心律失常药物，经证明能够减少钠通道放电和 NCX1 活性）是否可以改善 NCX1 介导的轴突钙流入； 3) 确定这些神经保护策略与新生 Twitcher 小鼠 BMT 后的代谢校正相结合是否可以改善临床结果。这些实验的结果将揭示 PP1 和 NCX1 活性介导 KD 神经元功能障碍的分子作用，并将提供独特的机会来改善目前用于治疗这种脑白质营养不良的基于 BMT 的代谢纠正策略。获得的见解将与其他溶酶体贮积症相关，这些疾病与川崎病一样与侵袭性神经功能恶化有关，并且没有可用的治疗方法。公共卫生相关性：克拉伯病是一种溶酶体贮积病，会导致受影响个体的大脑和神经脱髓鞘。一些克拉伯患者接受血源细胞替代治疗，这延迟了症状的出现。然而，这种疾病尚未得到明确和彻底的治愈，接受治疗的患者继续出现病情恶化和神经功能缺损。神经元丢失在克拉伯病中的作用尚不完全清楚，但人们正在形成共识，即轴突和神经元功能障碍会导致多种神经退行性疾病的永久性神经缺陷，包括多发性硬化症、阿尔茨海默病、帕金森病等。我们的初步研究提供证据表明克拉伯病是由轴突缺陷引起的。除了髓磷脂的损失之外，神经退行性变可能是降低传统疗法效率的限制因素。因此，不仅提供酶替代而且提供神经保护的联合疗法可能会协同或增强治疗效果。我们的目标是研究针对克拉伯病神经变性特定方面的两种新型神经保护策略是否可以与传统骨髓移植相结合，以完全预防该疾病的发展。拟议实验的结果将为治疗人类克拉布患者的联合神经保护疗法的设计提供概念验证，并为涉及轴突和髓磷脂变性的其他脑白质营养不良的研究提供合理基础。