Disorders of Copper Transport

我们的转化研究侧重于铜代谢障碍的诊断，治疗和理解，这是一种最近扩大的影响中枢和外周神经系统不同组成部分的人类疾病集合。 获得性和遗传性形式的异常铜稳态与神经功能障碍有关。门克斯病（MD）是一种典型的铜转运缺陷遗传综合征，其特征是脑铜含量低、婴儿神经变性和过早死亡。MD是由X连锁铜转运蛋白基因ATP 7A突变引起的。 枕角综合征（OHS）是一种与ATP 7A剪接点突变相关的轻度等位基因变异，其中自主神经功能障碍占主导地位。最近，我们确定了几个家庭的运动神经元疾病类似腓骨肌萎缩性侧索硬化症和腓骨肌萎缩性侧索硬化症，这是由几个新的错义突变的羧基一半的ATP 7A。 这些都是轻微的功能丧失缺陷，似乎影响ATP 7A的细胞内运输。因此，ATP 7A突变的表型谱已经扩大;这些新的等位基因变体的发现表明ATP 7A在运动神经元维持和功能中的先前未被认识的作用。 此外，我们最近确定了突变的铜伴侣基因，CCS，与降低铜/锌SOD活性和肝脏疾病，这意味着一种新的途径，我们目前正在表征细胞铜代谢。 目前对MD的治疗限于皮下铜注射，并且在具有不完全废除ATP 7A功能的突变等位基因的患者中有效，并且在出生时被鉴定（N Engl J Med 2008;358：605-614）。然而，对于具有完全功能丧失突变的患者，外周递送的铜不能有效地穿过血脑屏障，并且需要替代治疗方法。 在我们最近对治疗替代方案的探索中，我们使用组合脑定向疗法拯救了严重Menkes病的小鼠模型：表达减小的人ATP 7A的重组腺相关病毒血清型5（AAV 5）载体，加上铜。两种单独治疗均无效，但联合治疗显著改变了Kaplan-Meier生存曲线，16例联合治疗突变体中有5例（31%）存活超过110日龄（p<0.0002）。组合处理的mo-br小鼠比未处理的mo-br小鼠重（在9日龄时5.2g对4.2g，p<0.02）并且比野生型对照轻（在27日龄时11.9g对15.1g，p<0.01）。联合治疗的幸存者看起来健康、活跃和有生育能力。这种明显的协同作用的机制有助于阐明铜在脑中的正常转运过程，以及ATP 7A在神经元和神经胶质细胞中的作用。此外，这一进展可能对患有严重ATP 7A突变的Menkes病患者以及ATP 7A相关运动神经元疾病患者具有未来的临床意义。 合成氨基酸L-苏型-3，4-二羟基苯基丝氨酸（L-DOPS）在逆转神经源性直立性低血压和纠正常染色体隐性遗传先天性缺乏多巴胺-β-羟化酶（DBH）患者的神经化学异常方面非常有效。L-DOPS通过酶多巴脱羧酶代谢以产生去甲肾上腺素，从而绕过DBH酶缺陷。枕角综合征（OHS）患者存在铜依赖性DBH的部分缺乏，并表现出独特的神经化学异常和常见的自主神经功能障碍症状，如晕厥、头晕、直立性低血压、异常窦房传导、夜间心动过缓和肠或膀胱功能障碍。与先天性无DBH的患者一样，自主神经系统功能的这些问题似乎对恢复正常的神经化学水平有潜在的反应。我们假设，左旋多巴治疗OHS患者的自主神经功能障碍将纠正或改善血液神经化学水平，并产生并发症状的改善。最近IRB批准的一项初步研究将在6名有自主神经功能障碍症状的患者中检验这一假设，在接受研究药物的8周期间，通过提供L-DOPS治疗（5 mg/kg/d po qd）并评估神经化学反应（院内血浆儿茶酚水平）和对症状的长期影响（受试者和/或父母每周完成一次院外自主神经症状问卷）。

Our translational research focuses on the diagnosis, treatment, and understanding of copper metabolism disorders, a recently expanded collection of human diseases that impact varied components of the central and peripheral nervous systems. Acquired as well as inherited forms of abnormal copper homeostasis are implicated in neurologic dysfunction. Menkes disease (MD), a prototypical genetic syndrome of defective copper transport, is characterized by low brain copper, infantile neurodegeneration, and premature death. MD is caused by mutations in an X-linked copper transporter gene, ATP7A. The occipital horn syndrome (OHS) is a milder allelic variant associated with leaky ATP7A splice junction mutations, in which autonomic dysfunction predominates. Recently, we identified several families with motor neuron disease resembling Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, that are caused by several novel missense mutations in the carboxyl half of ATP7A. These are mild loss-of- function defects that appear to affect intracellular trafficking of ATP7A. Thus, the phenotypic spectrum of ATP7A mutations has expanded; discovery of these new allelic variants indicates a previously unappreciated role for the ATP7A in motor neuron maintenance and function. In addition, we recently identified mutations in a copper chaperon gene, CCS, associated with reduced Cu/Zn SOD activity and liver disease that implies a novel pathway in cellular copper metabolism which we are currently characterizing. Current treatment for MD is limited to subcutaneous copper injections and is effective in patients with mutant alleles that do not completely abrogate ATP7A function, and who are identified near birth (N Engl J Med 2008;358:605-614). For patients with complete loss-of-function mutations, however, copper delivered peripherally does not cross the blood-brain barrier efficiently, and alternative treatment approaches are needed. In our recent exploration of treatment alternatives, we rescued a mouse model of severe Menkes disease using combination brain-directed therapies: recombinant adeno-associated virus serotype 5 (AAV5) vector expressing a reduced size human ATP7A, plus copper. Neither treatment alone was effective, but combination therapy dramatically shifted the Kaplan-Meier survival curve, with 5 of 16 (31%) combination-treated mutants surviving beyond 110 days of age (p<0.0002). Combination-treated mo-br mice weighed more than untreated mo-br mice (5.2 g vs. 4.2 g at 9 days of age, p<0.02) and less than wild type controls (11.9 g vs. 15.1 grams at 27 days of age, p<0.01). The combination-treated survivors appear healthy, active, and fertile. The mechanisms underlying this pronounced synergistic effect help to illuminate the normal processes of copper transport in brain, and the role of ATP7A in neuronal and neuroglial cells. In addition, this advance may hold future clinical implications for Menkes disease patients with severe ATP7A mutations, as well as for patients with ATP7A-related motor neuron disease. The synthetic amino acid L-threo-3,4-dihydroxyphenylserine (L-DOPS) is highly effective in reversing neurogenic orthostatic hypotension and correcting neurochemical abnormalities in patients with autosomal recessive congenital absence of dopamine-beta-hydroxylase (DBH). L-DOPS is metabolized by the enzyme DOPA decarboxylase to produce norepinephrine, thus bypassing the DBH enzymatic defect. Individuals occipital horn syndrome (OHS), have partial deficiency of DBH, which is copper-dependent, and show distinctive neurochemical abnormalities and often symptoms of dysautonomia, such as syncope, dizziness, orthostatic hypotension, abnormal sinoatrial conduction, nocturnal bradycardia, and bowel or bladder dysfunction. As in patients with congenital absence of DBH, these problems in autonomic nervous system function seem potentially responsive to restoration of normal neurochemical levels. We hypothesize that L-DOPS treatment in OHS patients with dysautonomia will correct or improve blood neurochemical levels, and produce concurrent symptomatic improvement. A recently IRB-approved pilot study will test this hypothesis in six patients with dysautonomic symptoms, by providing L-DOPS treatment (5mg/kg/d po qd) and assessing the neurochemical response (in-hospital plasma catechol levels) and the longer-term effect on symptoms (out-of-hospital autonomic symptom questionnaire completed weekly by subject and/or parent), during an eight-week period while receiving the study drug.