June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Alternative splicing and nonsense-mediated mRNA decay contribute to regulation of LOXL1 expression in response to cellular stress in pseudoexfoliation
Author Affiliations & Notes
  • Daniel Berner
    Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Matthias Zenkel
    Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Francesca Pasutto
    Institute of Human Genetics, University of Erlangen-Nürnberg, Erlangen, Germany
  • Johannes Schödel
    Department of Nephrology and Hypertension, University of Erlangen-Nürnberg, Erlangen, Germany
  • André Reis
    Institute of Human Genetics, University of Erlangen-Nürnberg, Erlangen, Germany
  • Friedrich E Kruse
    Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Ursula Schlotzer-Schrehardt
    Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Footnotes
    Commercial Relationships   Daniel Berner, None; Matthias Zenkel, None; Francesca Pasutto, None; Johannes Schödel, None; André Reis, None; Friedrich Kruse, None; Ursula Schlotzer-Schrehardt, None
  • Footnotes
    Support  Interdisciplinary Center for Clinical Research (IZKF), Erlangen, Germany
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4909. doi:
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      Daniel Berner, Matthias Zenkel, Francesca Pasutto, Johannes Schödel, André Reis, Friedrich E Kruse, Ursula Schlotzer-Schrehardt; Alternative splicing and nonsense-mediated mRNA decay contribute to regulation of LOXL1 expression in response to cellular stress in pseudoexfoliation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4909.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Alternative mRNA splicing coupled to nonsense-mediated decay (NMD) is a common mRNA surveillance pathway also known to dynamically modulate gene expression in response to cellular stress. Here, we investigated the involvement of this pathway in regulation of lysyl oxidase-like 1 (LOXL1) expression in response to pseudoexfoliation (PEX)-associated stress factors.

Methods : Transcript levels of LOXL1 isoforms were determined by qPCR using specific primers in ocular tissues obtained from donor eyes without and with PEX syndrome. Human Tenon’s capsule fibroblasts (hTCF), trabecular meshwork cells (hTMC), lens epithelial cells (hLEPC), and optic nerve head astrocytes (hONHA) were exposed to puromycin, emetine, caffeine, TGF-ß1, homocysteine, interleukin-6, retinoic acid, UVB-radiation, oxidative and mechanical stress for up to 48 hours. siRNA mediated silencing of UPF1 (regulator of nonsense transcripts 1) was performed in hTCF.

Results : ENSEMBL database analysis indicated the presence of an alternative LOXL1 transcript (LOXL1-002) characterized by inclusion of an additional exon X between exon 1 and 2 introducing a premature termination codon in exon 2. LOXL1-002 transcripts were detected in all ocular tissues showing highest expression levels in trabecular meshwork, lens and lamina cribrosa, with significant differences between PEX and control tissues. Treatment of hTCF, hTMC, hLEPC and hONHA with NMD inhibitors puromycin, emetine and caffeine significantly increased LOXL1-002 mRNA levels, but reduced levels of wild-type LOXL1 mRNA. Significantly enhanced LOXL1-002 expression upon knockdown of UPF1, a key regulator of NMD pathway, further confirmed that LOXL1-002 is a direct target of NMD. Exposure of cells to various PEX-associated (stress) factors, including TGF-ß1, retinoic acid, UV light, oxidative and mechanical stress, resulted in upregulated LOXL1-002 expression in a cell type-specific manner and altered steady state levels of wild-type LOXL1 expression.

Conclusions : These findings indicate that alternative splicing coupled to NMD is dynamically modulated by cellular stress and contributes to post-transcriptional regulation of LOXL1 expression in the pathogenesis of PEX syndrome.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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