BONE MARROW TRANSPLANTATION IN NEURONAL STORAGE DISEASES

神经元储存疾病是先天性代谢缺陷， 由于溶酶体水解酶活性不足。 所得 分解代谢不足导致劣化底物的积累 在细胞的消化泡（溶酶体）装置中，以及 不断扩大的级联事件最终会损害细胞功能。 虽然患有这些疾病的人在出生时通常表现正常， 神经退行性改变不可避免地随之而来。 精神缺陷 可能是严重的，可能包括智力迟钝，运动系统 功能障碍、感觉缺陷和癫痫。 虽然细胞内 在溶酶体疾病的非神经病形式中的储存已经被 通过骨髓移植（BMT）成功改善， 将BMT应用于具有神经元参与的胆积病（例如， Hurler's disease）一直备受争议。 操作某个 溶酶体α-D-甘露糖苷酶缺乏症（α- 甘露糖苷酶），我们已经明确表明，这不仅是 BMT后CNS中的酶活性增加，但神经元内 存储被反转和/或被阻止。 最重要的是，我们使用了 靛蓝组织化学底物，以证明酸性α- 甘露糖苷酶存在于CNS的神经元和其它细胞中。 这 一个显著的发现确立了治疗效果的原则 BMT在神经元储存疾病中的应用，并使我们评估治疗方法， 在不同类型的储存障碍-GM 2神经节苷脂沉积症-使用 BETA-D-N-乙酰氨基己糖苷酶缺乏的动物模型。 我们 初步研究揭示了一个截然不同的结果：尽管 脑中β-氨基己糖苷酶活性显著升高（30%的 正常），底物减少不明显，组织化学染色 证明该酶仅限于脑小胶质细胞/巨噬细胞。 我们认为，上述模型中的疗效差异可能是 在测试关于潜在机制的假设时使用 成功的治疗。 使用BMT的最常见理由 在儿童是供体血单核细胞进入大脑，分化为 小胶质细胞，并为酶缺乏的大脑提供酶源 细胞 替代假设包括摄取“游离”酶衍生物 从循环，和'代谢过滤'，这取决于 底物扩散出病变细胞，并通过 供体细胞 这些假设都没有被证明，我们建议测试 他们使用多学科的体内和体外研究。 移植 将在不同年龄的模型中进行，以评估 早期治疗的重要性，以及单核细胞侵袭的动力学 将对BMT后最初几周的大脑进行严格检查。 这些研究的疗效将通过临床， 生物化学、组织化学、免疫细胞化学和组织病理学 的搜索. 通过细胞培养，我们将确定是否假定的骨 来自正常动物的骨髓来源的细胞具有转移 溶酶体酶对受影响动物的脑细胞的影响，以及 α-甘露糖苷酶和β-甘露糖苷酶的差异分泌或摄取， 发生氨基己糖苷酶。 导致底物的替代机制 还将测试消耗。 综合起来，这些多学科 研究将提供有价值的洞察机制（S）的基础 BMT后神经元的代谢校正和实用问题 与BMT作为儿童神经元储存疾病的治疗相关。

Neuronal storage diseases are inborn errors of metabolism that result from deficient activity of lysosomal hydrolases. The resulting catabolic deficiency leads to an accumulation of undergraded substrates in the digestive-vacuolar (lysosomal) apparatus of cells, and to an expanding cascade of events that eventually compromises cell function. Although individuals with these diseases often appear normal at birth, neurodegenerative changes inevitable ensue. Psychomotor deficiencies can be severe and may include mental retardation, motor system dysfunction, sensory deficits, and seizures. Although intracellular storage in non-neuropathic forms of lysosomal disorders has been successfully ameliorated by bone marrow transplantation (BMT), the application of BMT to storage diseases with neuronal involvement (e.g, Hurler's disease) has been highly controversial. Working with an inherited model of lysosomal alpha-D-mannosidase deficiency (alpha- mannosidosis), we have unequivocally demonstrated not only that this enzyme increases in activity in the CNS post-BMT, but that intraneuronal storage is reversed and/or prevented. Most importantly, we have used an indigogenic histochemical substrate to demonstrate that acidic alpha- mannosidase is present with neurons and other cells of the CNS. This remarkable finding has established the principle of therapeutic efficacy for BMT in neuronal storage diseases and has led us to evaluate treatment in a different type of storage disorder - GM2 gangliosidosis - using an animal model of BETA-D-N-acetylhexosaminidase deficiency. Our preliminary studies reveal a dramatically different result: In spite of significant elevations of Beta-hexosaminidase activity in brain (30% of normal), substrate reduction was not evident and histochemical staining demonstrated that the enzyme was limited to brain microglia/macrophages. We believe that differences in efficacy in the above models can be exploited in the testing of hypotheses on the mechanism underlying successful treatment. The most commonly stated rationale for use of BMT in children is that donor blood monocytes enter brain, differentiate as microglia, and provide a source of enzyme to enzyme deficient brain cells. Alternative hypotheses include uptake of 'free' enzyme derived from the circulation, and 'metabolic filtration' which depends on substrate diffusion out of diseased cells with uptake and degradation by donor cells. None of these hypotheses is proven and we propose to test them using multidisciplinary in vivo and in vitro studies. Transplants will be carried out in out models at different ages to assess the importance of early treatment, and the dynamics of monocyte invasion of brain in the early weeks post-BMT will be critically examined. Therapeutic effectiveness for these studies will be assessed by clinical, biochemical, histochemical, immunocytochemical and histopathologic criteria. Using cell culture, we will determine whether putative bone marrow-derived cells from normal animals have the capacity to transfer lysosomal enzyme to brain cells from affected animals and whether differential secretion or uptake of alpha-mannosidase and Beta- hexosaminidase occurs. Alternative mechanisms leading to substrate depletion also will be tested. Taken together, these multidisciplinary studies will provide valuable insight into mechanism(s) underlying metabolic correction in neurons following BMT and into pragmatic issues related to BMT as therapy for neuronal storage diseases in children.