Translational analysis of the mechanisms underlying small nerve fiber pathology in Fabry disease as a genetically determined model for neuropathic pain

在这个项目中，我们将使用创新的和直接与患者相关的方法来研究神经性疼痛的病理生理学。在周围神经系统中，伤害感觉通过薄髓神经纤维和无髓神经纤维（A-delta和C）传导。获得性或遗传性疾病引起这些伤害感受器的病理生理改变可导致神经性疼痛。神经性疼痛是法布里病的主要症状，法布里病是一种由α -半乳糖苷酶a基因突变引起的x染色体遗传疾病，伴有鞘脂球三烷基神经酰胺3 （Gb3）的连续溶酶体积累。基于我们之前的工作，我们假设Gb3干扰神经元离子通道活性，同时外周伤害感受器被周围皮肤细胞分泌的炎症介质致敏，这两种介质共同导致法布里病典型的神经性疼痛发作。我们将在临床实验方法中检验这一假设，使用从法布里病患者和健康对照者的皮肤穿刺活检中获得的成纤维细胞和角化细胞。对于皮肤-神经-神经元相互作用的分子、组织学和电生理特征，我们将从患者来源的成纤维细胞和角质形成细胞中生成二维和三维皮肤模型。然后，这些模型将被从患者的成纤维细胞中通过诱导多能干细胞获得的神经细胞所支配。从这些完全来源于患者的体外模型中获得的数据不仅将从根本上提高我们对法布里病神经性疼痛病理生理的理解，而且还将有助于破译其他疾病疼痛产生的基本机制。此外，这些模型系统将为基于机制的体外研究开辟全新的可能性，包括局部活性潜在神经保护和镇痛化合物的药理学测试。

In this project we will investigate the pathophysiology of neuropathic pain using innovative and directly patient-associated methods. In the peripheral nervous system, nociception is transduced via thinly- and un-myelinated nerve fibers (A-delta and C). Pathophysiological alterations of these nociceptors by acquired or hereditary diseases may lead to neuropathic pain. Neuropathic pain is a cardinal symptom of Fabry disease, a X-chromosomally inherited disorder caused by mutations in the alpha-galactosidase A gene with consecutive lysosomal accumulation of the sphingolipid globotriaosylceramide 3 (Gb3). Based on our previous work we hypothesize that Gb3 interferes with the neuronal ion channel activity and that in parallel peripheral nociceptors are sensitized by inflammatory mediators secreted by surrounding cutaneous cells which together results in the typical neuropathic pain attacks in Fabry disease. We will test this hypothesis in a clinical-experimental approach using fibroblasts and keratinocytes obtained from skin punch biopsies of patients with Fabry disease and healthy controls. For the molecular, histological, and electrophysiological characterization of skin-nerve-neuron interactions we will generate two- and three-dimensional skin models from patient-derived fibroblasts and keratinocytes. These models will then be innervated with neuronal cells obtained from patients` fibroblasts via induced pluripotent stem cells. Data obtained from these completely patient-derived in vitro models will not only fundamentally improve our understanding of neuropathic pain pathophysiology in Fabry disease, but will also allow deciphering basic mechanisms of pain generation in other disorders. Furthermore, these model systems will open entirely novel possibilities for mechanism-based in vitro research including pharmacological testing of locally active potentially neuroprotective and analgesic compounds.