Laboratory of Neurogenetics and Molecular Medicine - IPER, Institut de Recerca Sant Joan de Déu, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Madrid, Spain.
Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, Madrid, Spain.
Neurobiol Dis. 2021 May;152:105300. doi: 10.1016/j.nbd.2021.105300. Epub 2021 Feb 11.
Ganglioside-induced differentiation associated protein 1 (GDAP1) gene encodes a protein of the mitochondrial outer membrane and of the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs) and lysosomes. Since mutations in GDAP1 cause Charcot-Marie-Tooth, an inherited motor and sensory neuropathy, its function is essential for peripheral nerve physiology. Our previous studies showed structural and functional defects in mitochondria and their contacts when GDAP1 is depleted. Nevertheless, the underlying axonal pathophysiological events remain unclear. Here, we have used embryonic motor neurons (eMNs) cultures from Gdap1 knockout (Gdap1) mice to investigate in vivo mitochondria and calcium homeostasis in the axons. We imaged mitochondrial axonal transport and we found a defective pattern in the Gdap1 eMNs. We also detected pathological and functional mitochondria membrane abnormalities with a drop in ATP production and a deteriorated bioenergetic status. Another consequence of the loss of GDAP1 in the soma and axons of eMNs was the in vivo increase calcium levels in both basal conditions and during recovery after neuronal stimulation with glutamate. Further, we found that glutamate-stimulation of respiration was lower in Gdap1 eMNs showing that the basal bioenergetics failure jeopardizes a full respiratory response and prevents a rapid return of calcium to basal levels. Together, our results demonstrate that the loss of GDAP1 critically compromises the morphology and function of mitochondria and its relationship with calcium homeostasis in the soma and axons, offering important insight into the cellular mechanisms associated with axonal degeneration of GDAP1-related CMT neuropathies and the relevance that axon length may have.
神经节苷脂诱导分化相关蛋白 1（GDAP1）基因编码一种位于线粒体外膜和线粒体与内质网（MAMs）和溶酶体的膜接触部位的蛋白质。由于 GDAP1 基因突变可引起遗传性运动感觉神经病，即 Charcot-Marie-Tooth 病，因此其功能对于周围神经生理学至关重要。我们之前的研究表明，当 GDAP1 耗尽时，线粒体及其与内质网的接触部位存在结构和功能缺陷。然而，潜在的轴索病理生理事件仍不清楚。在这里，我们使用 Gdap1 基因敲除（Gdap1）小鼠的胚胎运动神经元（eMNs）培养物来研究体内轴突中线粒体和钙离子的动态平衡。我们对线粒体轴突运输进行了成像，发现 Gdap1 eMNs 的运输模式存在缺陷。我们还检测到病理性和功能性线粒体膜异常，表现为 ATP 生成减少和生物能状态恶化。eMNs 体和轴突中 GDAP1 缺失的另一个后果是在基础状态下和谷氨酸神经元刺激后恢复期间钙水平在体内增加。此外，我们发现 Gdap1 eMNs 的谷氨酸刺激呼吸作用降低，表明基础生物能衰竭危及全面呼吸反应，并阻止钙迅速恢复到基础水平。总之，我们的研究结果表明，GDAP1 的缺失严重损害了线粒体的形态和功能，以及其在体和轴突中钙动态平衡的关系，为与 GDAP1 相关的 CMT 神经病轴索变性相关的细胞机制提供了重要的见解，以及轴突长度的相关性。
