Wildtype s1 receptor and the receptor agonist improve ALS-associated mutation-induced insolubility and toxicity

Yasuharu Shinoda, Yudai Haga, Koichiro Akagawa, Kohji Fukunaga

Research output: Contribution to journalArticlepeer-review

Abstract

Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding s-1 receptor (s1R). We previously reported that s1RE102Q elicits toxicity in cells. The s1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALS-related mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with s1R-mCherry (mCh), s1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photobleaching. The oligomeric state was determined using crosslinking procedure. s1Rs were soluble to detergents, whereas the mutants accumulated in the insoluble fraction. Within the soluble fraction, peak distribution of mutants appeared in higher sucrose density fractions. Mutants formed intracellular aggregates that were co-stained with p62, ubiquitin, and phosphorylated pancreatic eukaryotic translation initiation factor-2-a kinase in NSC-34 cells but not in HEK293T cells. The aggregates had significantly lower recovery in fluorescence recovery after photobleaching. Acute treatment with s1R agonist SA4503 failed to improve recovery, whereas prolonged treatment for 48 h significantly decreased s1RE102Q-mCh insolubility and inhibited apoptosis. Whereas s1R-mCh formed monomers and dimers, s1RE102Q-mCh also formed trimers and tetramers. SA4503 reduced accumulation of the four types in the insoluble fraction and increased monomers in the soluble fraction. The s1RE102Q insolubility was diminished by s1RmCh co-expression. These results suggest that the agonist and WT s1R modify the detergent insolubility, toxicity, and oligomeric state of s1RE102Q, which may lead to promising new treatments for s1R-related ALS.

Original languageEnglish
Pages (from-to)17573-17587
Number of pages15
JournalJournal of Biological Chemistry
Volume295
Issue number51
DOIs
Publication statusPublished - 2020 Dec 18

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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