BONE MARROW TRANSPLANTATION IN NEURONAL STORAGE DISEASES

神经元储存疾病是一种先天的新陈代谢错误 由于溶酶体水解酶活性不足。由此产生的 分解代谢不足会导致底物的堆积 在细胞的消化-空泡(溶酶体)器中，以及对一个 不断扩大的事件级联，最终损害细胞功能。 尽管患有这些疾病的人在出生时通常看起来是正常的， 神经退行性变化不可避免地随之而来。精神运动缺陷 可能很严重，可能包括精神发育迟滞、运动系统 功能障碍、感觉障碍和癫痫发作。虽然在细胞内 在非神经性形式的溶酶体疾病中储存已经被 通过骨髓移植(BMT)成功地改善了 骨髓移植在伴有神经元受累的贮存性疾病中的应用(例如， 霍勒氏病)一直备受争议。使用 溶酶体α-D-甘露糖苷酶缺乏症的遗传性模型 甘露糖症)，我们不仅明确地证明了这一点 骨髓移植后中枢神经系统中的酶活性增加，但神经元内的 存储被反转和/或阻止。最重要的是，我们使用了一个 生药组织化学底物证明酸性α- 甘露糖苷酶存在于神经元和中枢神经系统的其他细胞中。这 非凡的发现确立了治疗效果的原则 用于神经元储存疾病的骨髓移植，并导致我们评估治疗方法 在一种不同类型的存储障碍中-GM2神经节苷脂沉着症-使用 β-D-N-乙酰氨基己糖苷酶缺乏症动物模型。我们的 初步研究显示了一个截然不同的结果：尽管 脑组织中β-氨基己糖苷酶活性显著升高(30% 正常)，底物还原不明显，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，上述模式的疗效差异可能是 被用来测试关于潜在机制的假说 治疗成功。使用BMT的最常见的理由是 在儿童中，供者的血液单核细胞进入大脑，分化为 小胶质细胞，为酶缺乏的大脑提供酶的来源 细胞。可供选择的假说包括摄取源自“游离”的酶 来自血液循环的代谢过滤，这取决于 底物在病细胞外的扩散与摄取和降解 捐献细胞。所有这些假设都没有得到证实，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Out模型中进行评估 早期治疗的重要性以及单核细胞侵袭的动力学 骨髓移植后最初几周的大脑将进行严格的检查。 这些研究的疗效将由临床评估， 生化、组织化学、免疫细胞化学和组织病理学 标准。通过细胞培养，我们将确定推定的骨骼 来自正常动物的骨髓来源的细胞具有转移能力 感染动物对脑细胞的溶酶体酶，以及 α-甘露糖苷酶和β-甘露糖苷酶的不同分泌或摄取 会产生己糖氨酸酶。导致底物的替代机制 耗尽也将进行测试。总而言之，这些多学科 研究将为(S)潜在的机制提供宝贵的见解 骨髓移植后神经元的新陈代谢校正及其实用化问题 与骨髓移植治疗儿童神经贮存性疾病有关。

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.