Date Published: May 31, 2019
Publisher: Public Library of Science
Author(s): Masaaki Kageyama, Takashi Ota, Masaaki Sasaoka, Osamu Katsuta, Katsuhiko Shinomiya, Etsuro Ito.
Chemical proteasome inhibition has been a valuable animal model of neurodegeneration to uncover roles for the ubiquitin-proteasome system in the central nervous system. However, little is known about the effects of chemical proteasome inhibitors on retinal integrity. Therefore, we characterized the effects of structurally different chemical proteasome inhibitors on the retinal morphology and the mechanisms of their action in the normal adult rat eyes. Intravitreal injection of MG-262 and other proteasome inhibitors led to inner retinal degeneration. MG-262-induced inner retinal degeneration was accompanied by reduced proteasome activity, increased poly-ubiquitinated protein levels, and increased positive immunostaining of ubiquitin, 20S proteasome subunit and GADD153/CHOP in the retina. Its retinal degenerative effect was also associated with reduced retinal neurofilament light chain gene expression, reflecting retinal ganglion cell death. MG-262-induced neurofilament light chain downregulation was largely resistant to pharmacological modulation including endoplasmic reticulum stress, apoptosis or MAP kinase inhibitors. Thus, this study provides further evidence of roles for the ubiquitin-proteasome system in the maintenance of the retinal structural integrity. Chemical proteasome inhibition may be used as a novel animal model of inner retinal degeneration, including retinal ganglion cell loss, which warrants further analysis of the molecular mechanisms underlying its retinal degenerative effect.
The ubiquitin-proteasome system (UPS) is a crucial component of protein degradation processes, which contributes to protein quality control and proteome homeostasis in eukaryotic cells . UPS-dependent protein degradation is regulated by three sequential enzymatic reactions, namely, 1) an adenosine-triphosphate (ATP)-dependent activation of ubiquitin by the ubiquitin activating enzyme E1, 2) ubiquitin ligation of the target protein by the conjugating enzyme E2, and the protein-ubiquitin ligase E3, and 3) proteolytic degradation of the tagged protein by the 26S proteasome complex. The 26S proteasome is composed of two 19S regulatory particles capping the 20S core particle that is a major catalytic site of ubiquitin-dependent protein degradation. The 20S proteasome also mediates ubiquitin-independent degradation of unfolded proteins subjected to oxidative stress . UPS function declines with cellular senescence and aging in various organs and tissues including the central nervous system . Furthermore, UPS dysfunction is associated with accumulation of misfolded and/or damaged proteins in the brain, which is the hallmark of several age-related neurodegenerative diseases such as Alzheimer, Parkinson and Huntington diseases . Similarly, in the retina, proteasome activity declines in an age-dependent manner [4, 5]. Recent studies demonstrated that impaired proteasome function or proteasome overload caused retinal degeneration [6, 7], whereas increased proteasome activity prevented it . These results suggest that UPS dysfunction is also associated with age-related and inherited retinal degenerative diseases caused by misfolded and damaged proteins. Thus, the UPS can be a potential therapeutic target for these retinal diseases.
Our main finding in the present study is that intravitreal injection of the potent and selective proteasome inhibitor MG-262  led to time- and dose-dependent inner retinal degeneration accompanied by reduced proteasome activity and poly-ubiquitinated protein accumulation in the normal adult rat retina. The most prominent feature of MG-262-induced inner retinal degeneration was cell loss in GCL accompanied by downregulation of NFL gene expression. Two structurally different proteasome inhibitors, lactacystin  and bortezomib , caused exactly the same pattern of inner retinal degeneration as MG-262 did. Furthermore, we found that 17-DMAG, a presumed HSP90 inhibitor, also suppressed proteasome activity and caused the same pattern of inner retinal degeneration, suggesting that 17-DMAG behaves as a direct/indirect proteasome inhibitor under our experimental conditions. Interestingly, intravitreal injection of two authentic ER stress inducers, tunicamycin and thapsigargin , resulted in massive retinal degeneration predominantly in the photoreceptor layer, which was a distinguishable pattern from that observed with chemical proteasome inhibitors. To our best knowledge, this is the first report demonstrating the unique pattern of inner retinal degeneration induced by chemical proteasome inhibitors. Furthermore, our study provides in vivo evidence to further support that the UPS may play a crucial role in the maintenance of the structural integrity of the normal adult retina.