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Unconventional secretory pathway activation restores hair cell mechanotransduction in an USH3A model.

ABSTRACT: The pathogenic variant c.144T>G (p.N48K) in the clarin1 gene (CLRN1) results in progressive loss of vision and hearing in Usher syndrome IIIA (USH3A) patients. CLRN1 is predicted to be an essential protein in hair bundles, the mechanosensory structure of hair cells critical for hearing and balance. When expressed in animal models, CLRN1 localizes to the hair bundle, whereas glycosylation-deficient CLRN1^N48K aggregates in the endoplasmic reticulum, with only a fraction reaching the bundle. We hypothesized that the small amount of CLRN1^N48K that reaches the hair bundle does so via an unconventional secretory pathway and that activation of this pathway could be therapeutic. Using genetic and pharmacological approaches, we find that clarin1 knockout (clrn1 ^KO/KO ) zebrafish that express the CLRN1 ^c.144T>G pathogenic variant display progressive hair cell dysfunction, and that CLRN1^N48K is trafficked to the hair bundle via the GRASP55 cargo-dependent unconventional secretory pathway (GCUSP). On expression of GRASP55 mRNA, or on exposure to the drug artemisinin (which activates GCUSP), the localization of CLRN1^N48K to the hair bundles was enhanced. Artemisinin treatment also effectively restored hair cell mechanotransduction and attenuated progressive hair cell dysfunction in clrn1 ^KO/KO larvae that express CLRN1 ^c.144T>G , highlighting the potential of artemisinin to prevent sensory loss in CLRN1 ^c.144T>G patients.

SUBMITTER: Gopal SR

PROVIDER: S-EPMC6561152 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Unconventional secretory pathway activation restores hair cell mechanotransduction in an USH3A model.

Gopal Suhasini R SR Lee Yvonne T YT Stepanyan Ruben R McDermott Brian M BM Alagramam Kumar N KN

Proceedings of the National Academy of Sciences of the United States of America 20190516 22

The pathogenic variant c.144T>G (p.N48K) in the clarin1 gene (<i>CLRN1</i>) results in progressive loss of vision and hearing in Usher syndrome IIIA (USH3A) patients. CLRN1 is predicted to be an essential protein in hair bundles, the mechanosensory structure of hair cells critical for hearing and balance. When expressed in animal models, CLRN1 localizes to the hair bundle, whereas glycosylation-deficient CLRN1<sup>N48K</sup> aggregates in the endoplasmic reticulum, with only a fraction reaching ...[more]

PMID: 31097578

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Project description:PARK7/DJ-1 is a Parkinson disease- and cancer-associated protein that functions as a multifunctional protein involved in gene transcription regulation and anti-oxidative defense. Although PARK7 lacks the secretory signal sequence, it is secreted and plays important physiological and pathophysiological roles. Whereas secretory proteins that lack the endoplasmic reticulum-targeting signal sequence are secreted from cells by way of what is called the unconventional secretion mechanism, the specific processes responsible for causing PARK7 to be secreted across the plasma membrane have remained unclear. In the present study, we found that PARK7 secretion was increased by treatment with 6-OHDA via the unconventional secretory pathway in human neuroblastoma SH-SY5Y cells and MEF cells. We also found that 6-OHDA-induced PARK7 secretion was suppressed in Atg5-, Atg9-, or Atg16l1-deficient MEF cells or ATG16L1 knockdown SH-SY5Y cells, indicating that the autophagy-based unconventional secretory pathway is involved in PARK7 secretion. We moreover observed that 6-OHDA-derived electrophilic quinone induced oxidative stress as indicated by a decrease in glutathione levels, and that this was suppressed by pretreatment with antioxidant NAC. We further found that NAC treatment suppressed autophagy and PARK7 secretion. We also observed that 6-OHDA-induced autophagy was associated with activation of AMPK and ULK1 via a pathway which was independent of MTOR. Collectively these results suggest that electrophilic 6-OHDA quinone enhances oxidative stress, and that this is followed by AMPK-ULK1 pathway activation and induction of secretory autophagy to produce unconventional secretion of PARK7. ABBREVIATIONS:6-OHDA: 6-hydroxydopamine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CAV1: caveolin 1; ER: endoplasmic reticulum; FN1: fibronectin 1; GSH: glutathione; IDE: insulin degrading enzyme; IL: interleukin; LDH: lactate dehydrogenase; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetyl-L-cysteine; PARK7/DJ-1: Parkinsonism associated deglycase; PD: Parkinson disease; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; RPN1: ribophorin I; ROS: reactive oxygen species; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Dataset Information

Unconventional secretory pathway activation restores hair cell mechanotransduction in an USH3A model.

Publications

Unconventional secretory pathway activation restores hair cell mechanotransduction in an USH3A model.

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