Functional Characterization of Spinocerebellar Ataxia Associated Dynorphin A Mutant Peptides

Mutations in the prodynorphin gene (PDYN) are associated with the development of spinocerebellar ataxia type 23 (SCA23). Pathogenic missense mutations are localized predominantly in the PDYN region coding for the dynorphin A (DynA) neuropeptide and lead to persistently elevated mutant peptide levels with neurotoxic properties. The main DynA target in the central nervous system is the kappa opioid receptor (KOR), a member of the G-protein coupled receptor family, which can elicit signaling cascades mediated by G-protein dissociation as well as β-arrestin recruitment. To date, a thorough analysis of the functional profile for the pathogenic SCA23 DynA mutants at KOR is still missing. To elucidate the role of DynA mutants, we used a combination of assays to investigate the differential activation of G-protein subunits and β-arrestin. In addition, we applied molecular modelling techniques to provide a rationale for the underlying mechanism. Our results demonstrate that DynA mutations, associated with a severe ataxic phenotype, decrease potency of KOR activation, both for G-protein dissociation as well as β-arrestin recruitment. Molecular modelling suggests that this loss of function is due to disruption of critical interactions between DynA and the receptor. In conclusion, this study advances our understanding of KOR signal transduction upon DynA wild type or mutant peptide binding.

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Clinical and functional characterization of a novel STUB1 frameshift mutation in autosomal dominant spinocerebellar ataxia type 48 (SCA48)

Journal of Biomedical Science ◽

10.1186/s12929-021-00763-1 ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Huan-Yun Chen ◽

Chia-Lang Hsu ◽

Han-Yi Lin ◽

Yung-Feng Lin ◽

Shih-Feng Tsai ◽

...

Keyword(s):

Spinocerebellar Ataxia ◽

Autosomal Dominant ◽

Frameshift Mutation ◽

Functional Characterization ◽

Dominant Negative ◽

Spinocerebellar Ataxia Type ◽

Cerebellar Granule Neuron ◽

Clinical Genetic ◽

Autosomal Dominant Spinocerebellar Ataxia

Abstract Background Heterozygous pathogenic variants in STUB1 are implicated in autosomal dominant spinocerebellar ataxia type 48 (SCA48), which is a rare familial ataxia disorder. We investigated the clinical, genetic and functional characteristics of STUB1 mutations identified from a Taiwanese ataxia cohort. Methods We performed whole genome sequencing in a genetically undiagnosed family with an autosomal dominant ataxia syndrome. Further Sanger sequencing of all exons and intron–exon boundary junctions of STUB1 in 249 unrelated patients with cerebellar ataxia was performed. The pathogenicity of the identified novel STUB1 variant was investigated. Results We identified a novel heterozygous frameshift variant, c.832del (p.Glu278fs), in STUB1 in two patients from the same family. This rare mutation is located in the U-box of the carboxyl terminus of the Hsc70-interacting protein (CHIP) protein, which is encoded by STUB1. Further in vitro experiments demonstrated that this novel heterozygous STUB1 frameshift variant impairs the CHIP protein’s activity and its interaction with the E2 ubiquitin ligase, UbE2D1, leading to neuronal accumulation of tau and α-synuclein, caspase-3 activation, and promoting cellular apoptosis through a dominant-negative pathogenic effect. The in vivo study revealed the influence of the CHIP expression level on the differentiation and migration of cerebellar granule neuron progenitors during cerebellar development. Conclusions Our findings provide clinical, genetic, and a mechanistic insight linking the novel heterozygous STUB1 frameshift mutation at the highly conserved U-box domain of CHIP as the cause of autosomal dominant SCA48. Our results further stress the importance of CHIP activity in neuronal protein homeostasis and cerebellar functions.

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Aberrant IP3 receptor activities revealed by comprehensive analysis of pathological mutations causing spinocerebellar ataxia 29

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811129115 ◽

2018 ◽

Vol 115 (48) ◽

pp. 12259-12264 ◽

Cited By ~ 18

Author(s):

Hideaki Ando ◽

Matsumi Hirose ◽

Katsuhiko Mikoshiba

Keyword(s):

Spinocerebellar Ataxia ◽

Molecular Mechanisms ◽

Spinocerebellar Ataxia Type ◽

Missense Mutations ◽

Channel Activity ◽

Ip3 Receptor ◽

Causative Gene ◽

Motor Delay ◽

Intracellular Ca ◽

Cerebellar Purkinje Cells

Spinocerebellar ataxia type 29 (SCA29) is autosomal dominant congenital ataxia characterized by early-onset motor delay, hypotonia, and gait ataxia. Recently, heterozygous missense mutations in an intracellular Ca2+ channel, inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1), were identified as a cause of SCA29. However, the functional impacts of these mutations remain largely unknown. Here, we determined the molecular mechanisms by which pathological mutations affect IP3R1 activity and Ca2+ dynamics. Ca2+ imaging using IP3R-null HeLa cells generated by genome editing revealed that all SCA29 mutations identified within or near the IP3-binding domain of IP3R1 completely abolished channel activity. Among these mutations, R241K, T267M, T267R, R269G, R269W, S277I, K279E, A280D, and E497K impaired IP3 binding to IP3R1, whereas the T579I and N587D mutations disrupted channel activity without affecting IP3 binding, suggesting that T579I and N587D compromise channel gating mechanisms. Carbonic anhydrase-related protein VIII (CA8) is an IP3R1-regulating protein abundantly expressed in cerebellar Purkinje cells and is a causative gene of congenital ataxia. The SCA29 mutation V1538M within the CA8-binding site of IP3R1 completely eliminated its interaction with CA8 and CA8-mediated IP3R1 inhibition. Furthermore, pathological mutations in CA8 decreased CA8-mediated suppression of IP3R1 by reducing protein stability and the interaction with IP3R1. These results demonstrated the mechanisms by which pathological mutations cause IP3R1 dysfunction, i.e., the disruption of IP3 binding, IP3-mediated gating, and regulation via the IP3R-modulatory protein. The resulting aberrant Ca2+ homeostasis may contribute to the pathogenesis of cerebellar ataxia.

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