Department of Medical Biosciences, Clinical Chemistry, Umeå University, SE-901 85 Umeå, Sweden.
Department of Clinical Sciences, Neuroscience, Umeå University, SE-901 85 Umeå, Sweden.
Neurobiol Dis. 2024 Nov;202:106718. doi: 10.1016/j.nbd.2024.106718. Epub 2024 Oct 26.
Mutations in superoxide dismutase-1 (SOD1) are a cause of hereditary amyotrophic lateral sclerosis (ALS) through a gain-of-function mechanism involving unfolded mutant SOD1. Intrathecal gene therapy using the antisense-oligo-nucleotide drug tofersen to reduce SOD1 expression delays disease progression and has recently been approved in the United States and the European Union. However, the discovery of children homozygous for inactivating SOD1 mutations developing the SOD1 Deficiency Syndrome (ISODDES) with injury to the motor system suggests that a too low SOD1 antioxidant activity may be deleterious in humans. Measuring SOD1 activity in cerebrospinal fluid (CSF) in tofersen-treated patients is recommended but difficult due to low concentration and the presence of the isoenzyme SOD3. We here present a sensitive method to assess SOD1 activity by removing SOD3 from CSF samples using highly specific immobilized antibodies and subsequent measurement of the SOD activity. We validated the method on 171 CSF samples from ALS patients with and without mutations and controls and used paired erythrocyte samples for comparison. We found that in ALS patients with wildtype SOD1, the SOD1 activity in CSF was equal to controls, but patients with mutant SOD1 show lower activity in CSF, even for patients with mutants previously reported to have full activity in erythrocytes. Activity variation in CSF was large among patients carrying the same SOD1 mutation and larger than in erythrocytes and in post-mortem nervous tissue. Additionally, we identified a discrepancy between the SOD1 activity and protein level measured with ELISA in both CSF and erythrocytes. Since antibodies used for SOD1 ELISA-quantification are raised against the natively folded wildtype SOD1, the concentration of mutant SOD1s may be underestimated. Analysis of SOD1 enzymatic activity in CSF is therefore a more reliable way to monitor the effect of SOD1-lowering drugs.
超氧化物歧化酶-1 (SOD1) 的突变是遗传性肌萎缩侧索硬化症 (ALS) 的一个原因，其通过涉及未折叠突变 SOD1 的功能获得机制。使用反义寡核苷酸药物 tofersen 进行鞘内基因治疗以降低 SOD1 表达，可延迟疾病进展，最近已在美国和欧盟获得批准。然而，发现纯合失活 SOD1 突变的儿童发展为 SOD1 缺乏综合征 (ISODDES) ，其运动系统受损，这表明人类 SOD1 抗氧化活性过低可能有害。建议测量鞘内注射 tofersen 的患者脑脊液 (CSF) 中的 SOD1 活性，但由于浓度低且存在同工酶 SOD3，因此测量困难。我们在此提出了一种通过使用高度特异性固定化抗体从 CSF 样本中去除 SOD3 并随后测量 SOD 活性来评估 SOD1 活性的敏感方法。我们在 171 例 ALS 患者和对照的 CSF 样本中验证了该方法，并使用配对的红细胞样本进行比较。我们发现，在 SOD1 野生型 ALS 患者中，CSF 中的 SOD1 活性与对照组相等，但携带突变 SOD1 的患者 CSF 中的活性较低，即使对于先前报道在红细胞中具有完全活性的突变体患者也是如此。携带相同 SOD1 突变的患者之间 CSF 中的活性变化很大，且大于红细胞和死后神经组织中的变化。此外，我们发现 CSF 和红细胞中 ELISA 测量的 SOD1 活性和蛋白水平之间存在差异。由于用于 SOD1 ELISA 定量的抗体是针对天然折叠的野生型 SOD1 产生的，因此突变 SOD1 的浓度可能被低估。因此，分析 CSF 中的 SOD1 酶活性是监测 SOD1 降低药物效果的更可靠方法。
