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

Abstract

Mitochondria mediate energy metabolism, apoptosis, and aging, while mitochondrial disruption leads to age-related diseases that include age-related macular degeneration (AMD). 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 vs. anterograde mitochondrial trafficking may initiate the pathogenic reaction in adult-onset neurodegenerative diseases.

Interactome Mapping Guided by Tissue-Specific Phosphorylation in Age-Related Macular Degeneration

“…Our previous data suggested the potential cell death mechanisms in the retina and RPE under oxidative stress [8,9,12,13,16,28–30]. We demonstrated that oxidative stress may trigger induction of anti-apoptotic erythropoietin, JAK2, and BCL-xL, as well as pro-apoptotic caspases [28,29,31].…”

“…Early signaling molecules under oxidative stress, including erythropoietin (EPO), crystallins, vimentin, protein phosphatase 2A (PP2A), hypoxia inducible factor-1 (HIF1), JAK2, prohibitin, RPE65, and nitric oxide synthase (NOS), are modulated and hyperphosphorylated in vitro and in vivo [7–9]. For instance, phosphorylations of crystallin and vimentin may participate in the pathogenesis of AMD by forming soft drusen with longer chain phosphatidylcholine and cholesteryl esters [10,11].…”

Mechanistic Dissection of Macular Degeneration Using the Phosphorylation Interactome

“…Cells were grown on sterile glass cover slips using DMEM/F12 medium with 10% FBS and 1% penicillin/streptomycin (Hyclone) in 5% CO 2 incubator at 37°C. Cells were treated under oxidative stress or intense light as previously reported [9][10][11][18][19][20]. Cells were washed with PBS and incubated with MitoTracker Orange CMTMRos (100 nM, Molecular Probes, Carlsbad, CA) in serum-free culture medium (30 min, 37°C), followed by washing (PBS) and fixing (10% formaldehyde, 30 min, room temperature).…”

“…Previous data demonstrated that Hsp70 (c-Jun N-terminal kinase), crystallins (Akt), and the increased expression of VDAC might be involved in AMD progression [22][23][24][25][26]. Altered phosphorylations of mitochondrial heat shock protein mtHsp70, αA/aB crystalline, vimentin, and ATP synthase were observed in RPE cell death under oxidative stress [9,22,[27][28][29]. Retinoid-binding proteins, including CRABP, RPE65, and RLBP1, could be involved in the advanced stages of AMD [30][31][32].…”

Melatonin Modulates Prohibitin and Cytoskeleton in the Retinal Pigment Epithelium

“…Previously, we showed that the visual cycle might be coordinated in a circadian-dependent manner to effectively protect the retina from the detrimental effects of light-induced ROS damage [2], [3], [13]. We hypothesize that these conditions may regulate protection mechanisms in the retina by anti-apoptosis, and that melatonin-targeted molecules may play crucial roles in maintaining neuroretinal architecture.…”