Altered secondary structure of Dynorphin A associates with loss of opioid signalling and NMDA-mediated excitotoxicity in SCA23.
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.
Department of Genetics, University of Groningen, University Medical Centre GroningenGroningen, the Netherlands d.s.verbeek@umcg.nl.
Cleo J L M
CJ
Smeets
Department of Genetics, University of Groningen, University Medical Centre GroningenGroningen, the Netherlands d.s.verbeek@umcg.nl.
Justyna
J
Zmorzyńska
Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Life Sciences, University of Groningen, Groningen, The Netherlands d.s.verbeek@umcg.nl.
Manuel N
MN
Melo
Department of Cell Biology and Histology, Academic Medical Centre, Amsterdam, The Netherlands d.s.verbeek@umcg.nl.
Anita
A
Stargardt
Torrey Pines Institute for Molecular Studies, Port St Lucie, FL, USA d.s.verbeek@umcg.nl.
Colette
C
Dooley
Division of Biological Research on Drug Dependence, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden d.s.verbeek@umcg.nl.
Georgy
G
Bakalkin
Department of Pharmacodynamics, University of Florida, Gainesville, FL, USA d.s.verbeek@umcg.nl.
Jay
J
McLaughlin
Department of Genetics, University of Groningen, University Medical Centre GroningenGroningen, the Netherlands d.s.verbeek@umcg.nl.
Richard J
RJ
Sinke
Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Life Sciences, University of Groningen, Groningen, The Netherlands d.s.verbeek@umcg.nl.
Siewert-Jan
SJ
Marrink
Department of Cell Biology and Histology, Academic Medical Centre, Amsterdam, The Netherlands d.s.verbeek@umcg.nl.
Eric
E
Reits
Department of Genetics, University of Groningen, University Medical Centre GroningenGroningen, the Netherlands d.s.verbeek@umcg.nl.
Dineke S
DS
Verbeek
D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques; D003198:Computer Simulation; D004399:Dynorphins / Q000378:metabolism* / Q000502:physiology; D004723:Endorphins / Q000378:metabolism; D004745:Enkephalins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016202:N-Methylaspartate / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009498:Neurotoxins; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D015398:Signal Transduction; D013116:Spinal Cord / Q000378:metabolism; D013132:Spinocerebellar Degenerations / Q000235:genetics / Q000378:metabolism*
D004723:Endorphins; D004745:Enkephalins; D009498:Neurotoxins; D011498:Protein Precursors; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; C025681:alpha-neoendorphin; D004399:Dynorphins; C033660:preproenkephalin