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Altered Cytoskeleton as a Mitochondrial Decay Signature in the Retinal Pigment Epithelium

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Abstract

Mitochondria mediate energy metabolism, apoptosis, and aging, while mitochondrial disruption leads to age-related diseases that include age-related macular degeneration. Descriptions of mitochondrial morphology have been non-systematic and qualitative, due to lack of knowledge on the molecular mechanism of mitochondrial dynamics. The current study analyzed mitochondrial size, shape, and position quantitatively in retinal pigment epithelial cells (RPE) using a systematic computational model to suggest mitochondrial trafficking under oxidative environment. Our previous proteomic study suggested that prohibitin is a mitochondrial decay biomarker in the RPE. The current study examined the prohibitin interactome map using immunoprecipitation data to determine the indirect signaling on cytoskeletal changes and transcriptional regulation by prohibitin. Immunocytochemistry and immunoprecipitation demonstrated that there is a positive correlation between mitochondrial changes and altered filaments as well as prohibitin interactions with kinesin and unknown proteins in the RPE. Specific cytoskeletal and nuclear protein-binding mechanisms may exist to regulate prohibitin-mediated reactions as key elements, including vimentin and p53, to control apoptosis in mitochondria and the nucleus. Prohibitin may regulate mitochondrial trafficking through unknown proteins that include 110 kDa protein with myosin head domain and 88 kDa protein with cadherin repeat domain. Altered cytoskeleton may represent a mitochondrial decay signature in the RPE. The current study suggests that mitochondrial dynamics and cytoskeletal changes are critical for controlling mitochondrial distribution and function. Further, imbalance of retrograde versus anterograde mitochondrial trafficking may initiate the pathogenic reaction in adult-onset neurodegenerative diseases.

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Abbreviations

AMD:

Age-related macular degeneration

DAPI:

4′,6-Diamidino-2-phenylindole

DIGE:

Differential gel electrophoresis

DMEM:

Dulbecco’s modified eagle medium

IP:

Immunoprecipitation

PP2A:

Protein phosphatase 2A

ROS:

Reactive oxygen species

RPE:

Retinal pigment epithelium

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

siRNA:

Small interfering RNA

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Acknowledgments

We thank Dr. Harold J. Sheedlo (University of North Texas Health Science Center) for providing HRP cells. We thank Dr. Cameron Atkinson, Trevor Moser, Hyunju Lee, Dr. Hilal Arnouk, Dr. Ruonan Zhang, and Alex Keim for their excellent technical assistance. This study was supported by the Century II Equipment Fund, Research Excellence Fund from Michigan Technological University, Equipment Fund from NSF, Research and Teaching assistantship from American University of Nigeria. This work was supported in part by an unrestricted grant from Research to Prevent Blindness to the University of Utah Department of Ophthalmology and Visual Sciences. The authors thank Dr. Tristan Purvis for his critical reading and suggestions.

Author information

Authors and Affiliations

  1. Department of Ophthalmology, Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Srinivas R. Sripathi

  2. Department of Biomedical Engineering, Michigan Technological University, Houghton, MI, USA

    Weilue He

  3. Retina Proteomics Laboratory, Department of Petroleum Chemistry, American University of Nigeria, Yola, Nigeria

    O’Donnell Sylvester & Wan Jin Jahng

  4. Department of Chemistry, Modibbo Adama University of Technology, Yola, Nigeria

    Musa Neksumi

  5. Department of Optometry, Seoul National University of Science and Technology, Seoul, Korea

    Ji-Yeon Um

  6. Department of Biochemistry, Modibbo Adama University of Technology, Yola, Nigeria

    Thagriki Dluya

  7. Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT, USA

    Paul S. Bernstein

Authors
  1. Srinivas R. Sripathi
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  2. Weilue He
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  8. Wan Jin Jahng
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Correspondence to Wan Jin Jahng.

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Figure 1

Decreased mitochondria and prohibitin translocalization under oxygen imbalance conditions. Proteins in RPE cells treated by H2O2 (100 μM) were detected by immunocytochemistry. MitoTracker Orange, DAPI, prohibitin-specific primary antibody and Alexa488-conjugated secondary antibody showed mitochondria, the nucleus and prohibitin, respectively (PDF 704 kb)

Table 1

Immunoprecipitation experiments demonstrated that prohibitin binds to mitochondrial trafficking proteins, including kinesin, unknown protein at 110 kDa (myosin head motor domain, SH3 domain, ATP binding domain, GenBank: BAG 65583, Accession: AK 304840), unknown protein at 88 kDa (cadherin repeat, Ca2+ binding, GenBank: BAC 28880, Accession: AK 034910) (DOCX 15 kb)

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Sripathi, S.R., He, W., Sylvester, O. et al. Altered Cytoskeleton as a Mitochondrial Decay Signature in the Retinal Pigment Epithelium. Protein J 35, 179–192 (2016). https://doi.org/10.1007/s10930-016-9659-9

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