Combined neuroprotection and metabolic correction to treat leukodystrophies

描述（由申请人提供）：Krabbe病（KD）的半乳糖神经酰胺酶（GALC）缺乏导致髓鞘形成细胞中半乳糖神经酰胺（psychosine）的毒性积累，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供者的骨髓细胞（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BMT的使用，但KD患者有神经系统后遗症。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。本实验室使用天然KD模型Twitcher小鼠进行的实验表明：1)BMT治疗小鼠在神经系统中积累足够的治疗酶时出现神经元和轴突损伤；2)在没有突变的神经胶质的情况下，神经磷脂也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)突变神经元细胞内Ca水平异常，这与Na+ Ca交换器（NCX1）的表达失调有关。这些观察结果表明，galc缺乏的神经元产生应激反应，导致病理，PP1和NCX1是介导KD轴突缺陷机制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步缺陷。为了验证这一假设，我们提出了特定的实验来调节抽搐神经元中PP1和NCX1的活性。这些实验将提供概念证明，神经保护策略可以与传统的基于bmt的治疗方法协同/提高治疗效果。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活性是否保护突变神经元的轴突运输；2)确定抗心律失常药物氟屈胺（flecainide）是否能改善NCX1介导的轴突钙内流，该药物已被证实能够减少钠通道放电和NCX1活性；3)确定这些神经保护策略结合新生Twitcher小鼠BMT后的代谢纠正是否能改善临床结果。这些实验的结果将揭示PP1和NCX1活性在KD中介导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进目前用于治疗这种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性疾病相关，如KD与侵袭性神经退化有关，并且没有可用的治疗方法。

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.