Altered secondary structure of Dynorphin A associates with loss of opioid signalling and NMDA-mediated excitotoxicity in SCA23

Abstract

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.

Functional Characterization of Spinocerebellar Ataxia Associated Dynorphin A Mutant Peptides

“…To date, the mechanisms underlying these effects remain unclear. An attempt to investigate how SCA23 mutants promote neurotoxicity was recently made by Smeets and colleagues [ 6 ]. Their study revealed an altered secondary structure of the peptides resulting in increased peptide stability, reduced KOR receptor affinity and a partial switch from opioid to NMDA-receptor signaling.…”

“…In addition, transgenic mice ubiquitously expressing DynA_R6W (the most toxic SCA23-associated DynA mutant) showed altered glutamatergic signaling, neuronal excitability, and motor performance [ 5 ], thus demonstrating the importance of this mutant for the initiation and progression of SCA23. To date, the cellular mechanisms underlying these neurotoxic effects have been attributed in part to a loss of affinity to kappa opioid receptors (KOR), increased peptide degradation resistance, and a switch from opioid to NMDA-receptor signaling [ 6 ]. Furthermore, increased peptide stability may result in protein aggregation and penetration of bilayer membrane leading to leakage and cellular dysfunctions [ 7 , 8 , 9 ].…”

Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23

“…It can cause cell death via the NMDA (N-methyl-D -aspartate) receptor (51), and elicit neurotoxic effects via the AMPA (α-amino-hydroxy-5-methylisoxazole-4propionate) receptor and acid-sensing ion 1a channels (44,46). Additionally, we have shown that variants causing SCA23 affect the secondary structure of Dyn A, reducing its affinity with its natural κ-opioid receptor as well as peptide stability, leading to peptide aggregation (49).…”

Structure and dynamics of dynorphin peptide and its receptor

“…Modelling structures of complexes formed by KOP and its agonists or antagonists has been attempted for more than 20 years (Alonso, Bliznyuk, & Gready, 2006;Bailey & Husbands, 2018;Benyhe, Zador, & Otvos, 2015;Bera, Marathe, Payghan, & Ghoshal, 2018;Gentilucci, Tolomelli, De Marco, & Artali, 2012;Johnson, 2017;Kane, Svensson, & Ferguson, 2006;Kaserer, Lantero, Schmidhammer, Spetea, & Schuster, 2016;Kolinski & Filipek, 2010;Lavecchia, Greco, Novellino, Vittorio, & Ronsisvalle, 2000;Martinez-Mayorga et al, 2013;Patra, Kumar, Pasha, & Chopra, 2012;Tessmer, Meyer, Hruby, & Kallick, 1997;Wu, Song, Graaf, & Stevens, 2017;Yongye & Martínez-Mayorga, 2012). Some of these models specifically focused on dynorphin (Bjorneras, et al, 2014;Charles Chavkin, 2013;Iadanza, Höltje, Ronsisvalle, & Höltje, 2002;Kang, et al, 2015;O'Connor, et al, 2015;Paterlini, Portoghese, & Ferguson, 1997;Sankararamakrishnan & Weinstein, 2000;Smeets et al, 2016;Vardy, et al, 2013). Early studies, performed before any experimentally determined receptor structures were available, were based entirely on modelling (Iadanza, et al, 2002;Paterlini, et al, 1997;Wan et al, 2000).…”