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

• 批准号: 8525467
• 负责人: Ernesto Roque Bongarzone
• 金额: $ 32万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525467
• 关键词: Address; Affect; Alzheimer' 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' 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

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)：Krabbe病(KD)的半乳糖基神经酰胺酶(GALC)缺乏会导致半乳糖神经鞘氨醇(精神病)在髓鞘形成细胞中有毒积聚，导致神经系统脱髓鞘。为了减少脱髓鞘，目前的治疗方法是在将健康捐赠者的骨髓细胞(BMT)移植到受影响的患者体内后，通过渗透巨噬细胞向髓鞘胶质细胞提供缺失的酶。尽管从这种方法获得的经验支持骨髓移植的使用，KD患者仍有神经后遗症。这表明KD的发病机制比以前认为的更复杂，需要新的治疗策略来治愈KD。我们实验室使用KD的天然模型Twitcher小鼠的实验表明：1)经过BMT处理的小鼠在神经系统中积累足够的治疗性酶时，就会显示出神经元和轴突的损伤[1]；2)在没有突变胶质细胞的情况下，神经细胞中也会产生并积累精神苷，通过蛋白磷酸酶1(PP1)的活性导致轴突的快速运输受阻；3)突变的神经元显示出与Na+Ca交换器(NCX1)的表达失控有关的细胞内钙异常水平。这些观察结果表明，GALC缺陷神经元启动的应激反应参与了KD的病理过程，PP1和NCX1是KD轴突缺陷机制中的两个潜在的关键成分。因此，我们假设KD患者中GALC的缺失不仅影响髓鞘形成，而且还触发神经元的固有和同期缺陷。为了验证这一假设，我们提出了调节抽动神经元中PP1和NCX1活动的具体实验。这些实验将提供概念证明，即神经保护策略可以与传统的基于骨髓移植的治疗方法协同/提高治疗效益。具体地说，我们将：1)确定使用siRNA特异性沉默来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的轴突运输；2)确定氟卡胺是一种抗心律失常药物，具有减少钠通道放电和NCX1活性的能力；以及3)确定这些神经保护策略结合新生的Twitcher小鼠骨髓移植后的代谢纠正是否会改善临床结果。这些实验的结果将阐明PP1和NCX1活性在KD神经功能障碍中的分子作用，并将为改进目前用于治疗这种脑白质营养不良的基于BMT的代谢纠正策略提供一个独特的机会。所获得的洞察力将与其他溶酶体储存障碍相关，这些疾病如KD与侵袭性神经恶化有关，并且没有可用的治疗方法。 公共卫生相关性：Krabbe病是一种溶酶体储存性疾病，会导致受影响个人的大脑和神经脱髓鞘。一些Krabbe患者接受造血细胞替代治疗，这会推迟症状的出现。然而，这种疾病还没有得到明确和彻底的治愈，接受治疗的患者继续经历恶化和神经功能障碍。神经元丢失在Krabbe病中的作用尚不完全清楚，但正在形成一个共识，即轴突和神经元功能障碍会导致几种神经退行性疾病的永久性神经功能障碍，包括多发性硬化症、阿尔茨海默病、帕金森病和其他疾病。我们的初步研究提供了证据，证明Krabbe病是由轴突缺陷引起的。除了髓鞘的丢失，神经退行性变可能是降低传统疗法效率的一个限制因素。因此，一种不仅提供酶替代而且提供神经保护的综合疗法可能会协同或增强治疗效果。我们的目标是研究两种针对Krabbe病神经变性特定方面的新的神经保护策略是否可以与传统的骨髓移植相结合，以充分防止疾病的发展。拟议的实验结果将为设计治疗人类Krabbe患者的联合神经保护疗法提供概念验证，并为涉及轴突和髓鞘变性的其他脑白质营养不良的研究提供合理的基础。