Smeets Cleo J L M, Zmorzyńska Justyna, Melo Manuel N, Stargardt Anita, Dooley Colette, Bakalkin Georgy, McLaughlin Jay, Sinke Richard J, Marrink Siewert-Jan, Reits Eric, Verbeek Dineke S
Department of Genetics, University of Groningen, University Medical Centre GroningenGroningen, the Netherlands
Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Life Sciences, University of Groningen, Groningen, The Netherlands
Hum Mol Genet. 2016 Jul 1;25(13):2728-2737. doi: 10.1093/hmg/ddw130. Epub 2016 Jun 3.
Spinocerebellar ataxia type 23 (SCA23) is caused by missense mutations in prodynorphin, encoding the precursor protein for the opioid neuropeptides α-neoendorphin, Dynorphin (Dyn) A and Dyn B, leading to neurotoxic elevated mutant Dyn A levels. Dyn A acts on opioid receptors to reduce pain in the spinal cord, but its cerebellar function remains largely unknown. Increased concentration of or prolonged exposure to Dyn A is neurotoxic and these deleterious effects are very likely caused by an N-methyl-d-aspartate-mediated non-opioid mechanism as Dyn A peptides were shown to bind NMDA receptors and potentiate their glutamate-evoked currents. In the present study, we investigated the cellular mechanisms underlying SCA23-mutant Dyn A neurotoxicity. We show that SCA23 mutations in the Dyn A-coding region disrupted peptide secondary structure leading to a loss of the N-terminal α-helix associated with decreased κ-opioid receptor affinity. Additionally, the altered secondary structure led to increased peptide stability of R6W and R9C Dyn A, as these peptides showed marked degradation resistance, which coincided with decreased peptide solubility. Notably, L5S Dyn A displayed increased degradation and no aggregation. R6W and wt Dyn A peptides were most toxic to primary cerebellar neurons. For R6W Dyn A, this is likely because of a switch from opioid to NMDA- receptor signalling, while for wt Dyn A, this switch was not observed. We propose that the pathology of SCA23 results from converging mechanisms of loss of opioid-mediated neuroprotection and NMDA-mediated excitotoxicity.
23型脊髓小脑共济失调（SCA23）由强啡肽原的错义突变引起，该基因编码阿片类神经肽α-新内啡肽、强啡肽A（Dyn A）和强啡肽B的前体蛋白，导致具有神经毒性的突变型Dyn A水平升高。Dyn A作用于阿片受体以减轻脊髓疼痛，但其在小脑的功能在很大程度上仍不清楚。Dyn A浓度增加或暴露时间延长具有神经毒性，这些有害影响很可能是由N-甲基-D-天冬氨酸介导的非阿片类机制引起的，因为Dyn A肽已被证明可结合NMDA受体并增强其谷氨酸诱发的电流。在本研究中，我们调查了SCA23突变型Dyn A神经毒性的细胞机制。我们发现，Dyn A编码区的SCA23突变破坏了肽的二级结构，导致与κ-阿片受体亲和力降低相关的N端α-螺旋丢失。此外，二级结构的改变导致R6W和R9C Dyn A的肽稳定性增加，因为这些肽表现出明显的抗降解性，这与肽溶解度降低一致。值得注意的是，L5S Dyn A表现出降解增加且无聚集。R6W和野生型Dyn A肽对原代小脑神经元毒性最大。对于R6W Dyn A，这可能是由于从阿片受体信号传导转变为NMDA受体信号传导，而对于野生型Dyn A，则未观察到这种转变。我们认为，SCA23的病理学是由阿片类药物介导的神经保护作用丧失和NMDA介导的兴奋性毒性的共同机制导致的。
