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（由申请方提供）：克拉贝病（KD）中的半乳糖神经酰胺酶（GALC）缺乏导致半乳糖鞘氨醇（精神病）在髓鞘形成细胞中毒性蓄积，导致神经系统脱髓鞘。为了减少脱髓鞘，目前的疗法寻求在将来自健康供体的骨髓细胞（BMT）移植到受影响的患者中后，通过浸润巨噬细胞向髓鞘生成胶质细胞提供缺失的酶。虽然从这种方法中获得的经验支持使用BMT，但KD患者患有神经系统后遗症。这表明KD的致病机制比以前认为的更复杂，需要新的治疗策略来治愈KD。 在我们实验室中使用Twitch小鼠（KD的天然模型）的实验表明：1）BMT处理的小鼠在足够的治疗酶在神经系统中积累时显示神经元和轴突损伤[1]; 2）在不存在突变胶质细胞的情况下，也产生并积累在神经元中，导致通过蛋白磷酸酶1（PP 1）活性的快速轴突运输的阻断;和3）突变神经元显示与Na+ Ca交换器（NCX 1）的失调表达相关的异常细胞内Ca水平。这些观察结果表明，GALC缺陷的神经元安装的应激反应，有助于病理和PP 1和NCX 1是两个潜在的关键组成部分的机制，介导的轴突缺陷在KD。因此，我们假设KD中GALC的缺乏不仅影响髓鞘形成，而且还引发神经元的内在和同期缺陷。 为了验证这一假设，我们提出了具体的实验来调节Twitcher神经元中的PP 1和NCX 1活性。这些实验将提供概念验证，即神经保护策略可以协同/改善传统的基于BMT的治疗的治疗益处。具体而言，我们将：1）确定使用siRNA特异性沉默的神经元PP 1活性的受控和特异性降低是否保护突变神经元中的轴突运输; 2）确定具有降低钠通道放电和NCX 1活性的能力的抗癫痫药物氟卡尼是否改善轴突中NCX 1介导的钙内流;和3）确定这些神经保护策略与新生Twitch小鼠BMT后的代谢校正相结合是否改善临床结果。 这些实验的结果将揭示PP 1和NCX 1活性介导KD神经元功能障碍的分子作用，并将提供一个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的代谢矫正策略。所获得的见解将与其他溶酶体贮积症相关，这些溶酶体贮积症与KD一样与侵袭性神经系统恶化相关，并且没有可用的治愈方法。 公共卫生相关性：克拉布病是一种溶酶体贮积病，导致受影响个体的大脑和神经脱髓鞘。一些Krabbe患者接受造血细胞替代治疗，这会延迟症状的发作。然而，这种疾病尚未得到彻底彻底的治愈，接受治疗的患者继续出现病情恶化和神经功能障碍。 神经元丢失在克拉伯病中的作用尚未完全了解，但正在形成共识，即轴突和神经元功能障碍导致几种神经退行性疾病中的永久性神经功能缺损，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的证据表明，克拉布病是由轴突缺陷。除了髓磷脂的损失之外，神经变性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组合疗法可能协同或增强治疗益处。 我们的目的是研究两种针对Krabbe病神经变性特定方面的新神经保护策略是否可以与传统的骨髓移植相结合，以完全预防疾病的发展。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。