Washington University in Saint Louis, Genetics, St. Louis, MO, USA.
Disarm Therapeutics, Cambridge, MA, USA.
Exp Neurol. 2020 Jul;329:113252. doi: 10.1016/j.expneurol.2020.113252. Epub 2020 Feb 19.
SARM1 is the central executioner of pathological axon degeneration, promoting axonal demise in response to axotomy, traumatic brain injury, and neurotoxic chemotherapeutics that induce peripheral neuropathy. SARM1 is an injury-activated NAD cleavage enzyme, and this NADase activity is required for the pro-degenerative function of SARM1. At present, SARM1 function is assayed by either analysis of axonal loss, which is far downstream of SARM1 enzymatic activity, or via NAD levels, which are regulated by many competing pathways. Here we explored the utility of measuring cADPR, a product of SARM1-dependent cleavage of NAD, as an in cell and in vivo biomarker of SARM1 enzymatic activity. We find that SARM1 is a major producer of cADPR in cultured dorsal root ganglion (DRG) neurons, sciatic nerve, and brain, demonstrating that SARM1 has basal activity in the absence of injury. Following injury, there is a dramatic SARM1-dependent increase in the levels of axonal cADPR that precedes morphological axon degeneration. In vivo, there is also a rapid and large injury-stimulated increase in cADPR in sciatic and optic nerves. The increase in cADPR after injury is proportional to SARM1 gene dosage, suggesting that SARM1 activity is the prime regulator of cADPR levels. The role of cADPR as an important calcium mobilizing agent prompted exploration of its functional contribution to axon degeneration. We used multiple bacterial and mammalian engineered enzymes to manipulate cADPR levels in neurons but found no changes in the time course of axonal degeneration, suggesting that cADPR is unlikely to be an important contributor to the degenerative mechanism. Using cADPR as a SARM1 biomarker, we find that SARM1 can be partially activated by a diverse array of mitochondrial toxins administered at doses that do not induce axon degeneration. Hence, the subcritical activation of SARM1 induced by mitochondrial dysfunction may contribute to the axonal vulnerability common to many neurodegenerative diseases. Finally, we assay levels of both nerve cADPR and plasma neurofilament light chain (NfL) following nerve injury in vivo, and demonstrate that both biomarkers are excellent readouts of SARM1 activity, with cADPR reporting the early molecular changes in the nerve and NfL reporting subsequent axonal breakdown. The identification and characterization of cADPR as a SARM1 biomarker will help identify neurodegenerative diseases in which SARM1 contributes to axonal loss and expedite target validation studies of SARM1-directed therapeutics.
SARM1 是病理性轴突退化的核心执行者，它在轴突切断、创伤性脑损伤和诱导周围神经病变的神经毒性化疗药物的作用下，促进轴突死亡。SARM1 是一种损伤激活的 NAD 裂解酶，这种 NADase 活性是 SARM1 促退化功能所必需的。目前，SARM1 的功能通过分析轴突丢失来评估，而轴突丢失是 SARM1 酶活性的下游事件，或者通过 NAD 水平来评估，而 NAD 水平受许多竞争途径的调节。在这里，我们探讨了测量 cADPR（SARM1 依赖性 NAD 裂解的产物）作为 SARM1 酶活性的细胞内和体内生物标志物的效用。我们发现，SARM1 是培养的背根神经节（DRG）神经元、坐骨神经和大脑中 cADPR 的主要产生者，这表明 SARM1 在没有损伤的情况下具有基础活性。损伤后，cADPR 的水平会出现显著的 SARM1 依赖性增加，这先于形态学轴突退化。在体内，坐骨神经和视神经也会迅速且大量地受到损伤刺激而增加 cADPR。损伤后 cADPR 的增加与 SARM1 基因剂量成正比，表明 SARM1 活性是 cADPR 水平的主要调节因子。cADPR 作为一种重要的钙动员剂的作用促使我们探索其对轴突退化的功能贡献。我们使用多种细菌和哺乳动物工程酶来操纵神经元中的 cADPR 水平，但未发现轴突退化时间过程的变化，这表明 cADPR 不太可能是退化机制的重要贡献者。使用 cADPR 作为 SARM1 生物标志物，我们发现 SARM1 可以被多种线粒体毒素部分激活，这些毒素的剂量不会诱导轴突退化。因此，线粒体功能障碍引起的 SARM1 亚临界激活可能导致许多神经退行性疾病中常见的轴突易损性。最后，我们在体内检测神经损伤后神经 cADPR 和血浆神经丝轻链（NfL）的水平，并证明这两种生物标志物都是 SARM1 活性的良好指标，cADPR 报告神经中的早期分子变化，而 NfL 报告随后的轴突断裂。将 cADPR 确定和表征为 SARM1 生物标志物将有助于识别 SARM1 导致轴突丢失的神经退行性疾病，并加速 SARM1 靶向治疗的靶标验证研究。
