It has been shown previously that S-opsin aggregation causes rapid central/ventral cone degeneration in
Lrat–/– mice.
18 This study then examined by immunoblot the possibility that TUDCA exerted its protective effect on
Lrat–/– cones by reducing S-opsin aggregation. At P18, an early stage of cone degeneration, M-opsin was markedly reduced, whereas S-opsin accumulated in
Lrat–/– cones as previously described
18 (vehicle-injected
Lrat–/– versus vehicle-injected
WT, Fig. 6A). TUDCA treatment made no difference in the protein levels of M- and S-opsins in either
Lrat–/– or
WT mice, suggesting TUDCA did not reduce S-opsin aggregation.
As shown previously by study authors
18 and others,
41 S-opsin of
Lrat –/– cones (both vehicle and TUDCA treated) exhibited slower mobility in SDS-PAGE compared with WT S-opsin (
Fig. 6A), which was attributed to incomplete N-glycan processing.
41 Misfolded S-opsin aggregates in the ER, which may prevent the normal N-glycan trimming in the Golgi, resulted in higher molecular-weight products. However, the nature and significance of this “mobility shift” deserves further study. At P28, an advanced stage of cone degeneration, TUDCA treated
Lrat–/– retina contained far more S-opsin and cone arrestin than vehicle controls (
Fig. 6B), consistent with a significant increase of cone numbers in the central and ventral retina (
Fig. 1).
Surprisingly, an increase of other COS proteins (e.g., M-opsin, Gα
t2, and PDE6α′) was not observed, despite the large increase of cone numbers in TUDCA-treated
Lrat–/– retina. In fact, the level of Gα
t2 decreased in TUDCA-treated
Lrat–/– retina. This result suggests that TUDCA promotes the degradation of COS proteins (except S-opsin) in
Lrat–/– cones (see more in Discussion). In contrast to
Lrat–/– cones, TUDCA had no effect on protein stability of membrane-associated proteins in
Lrat–/– rods (i.e., Gα
t1 and rhodopsin) (
Fig. 6B). TUDCA may promote the degradation of COS proteins by enhancing the ER-associated degradation (ERAD) pathway. To examine this possibility, this study compared the expression level of valosin-containing protein (VCP/P97)—an important component of the ERAD machinery that provides the main driving force for extraction of poly-ubiquitinated ERAD substrates through the ER membrane
42,43 —in retinal extracts between TUDCA and vehicle-treated
Lrat–/– mice. Western blotting analysis showed that TUDCA increased the level of VCP (
Fig. 6B), indicating enhanced ERAD.