57.4 Discussion

Studies showed that some cells could not restore normal structure and physiological functions after retinal ischemia for 60 min, that is ischemia caused irreversible cell death, which was one of the cause of visual loss in patient with retinal ischemia.

Nitric oxide (NO) is a free radical that plays a variety of roles in the pathophysiology of many diseases (Stewart et al. 1994). It is synthesized by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) isoforms; the former two share enzymatic characteristic and are termed constitutive isoforms (cNOS) (Marietta 1994). All isoforms have been shown to be present or inducible in neural retina (Chakravarthy et al. 1995; Hangai et al. 1996). Hangai et al. (1996) suggested that the express of iNOS mRNA increased obviously during ischemic in rats and get to a peak at 2 h. iNOS mRNA mainly expressed in the heterophil granulocyte infiltrated in retina by hybridization in situ. It suggested that NO induced by iNOS in leucocyte could be one of the factors involving in retinal I/R injury. Masanori et al. (1999) found that after ischemia ended, eNOS mRNA initially decreased until 6 h, then increased to a peak at 12 h, and decreased progressively beyond 24 h until the final measurement at 96 h of reperfusion. While Cheon et al. (2003) suggest that an increase in eNOS expression could be associated with the degenerative changes in the ischemic retina.

Our study showed that serum NO content declined, serum iNOS activity increased and serum eNOS activity in I/R group, which was consistent with the reports of Masanori and Hangai. The results could be turned over by treating with PBNA.

This suggested that PBNA has a therapeutic effect on retina ischemia/reperfusion injury probability for the reason that it could increase the activity of T-NOS and eNOS, decrease the activity of iNOS, raise the content of NO, enhance the Antioxidation and expand the blood vessel.

Acknowledgments This work was supported by Nature Science Fund of Jiangxi Province (NO. 2008GZY0068) and Science research plan from department of Education of Jiangxi Province (NO. [2006] 252).

References

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Chapter 58

Recent Insights into the Mechanisms Underlying Light-Dependent Retinal Degeneration

from X. Laevis Models of Retinitis Pigmentosa

Orson L. Moritz and Beatrice M. Tam

Abstract We have recently developed transgenic X. laevis models of retinitis pigmentosa based on the rhodopsin P23H mutation in the context of rhodopsin cDNAs derived from several different species. The mutant rhodopsin in these animals is expressed at low levels, with levels of export from the endoplasmic reticulum to the outer segment that depend on the cDNA context. Retinal degeneration in these models demonstrates varying degrees of light dependence, with the highest light dependence coinciding with the highest ER export efficiency. Rescue of light dependent retinal degeneration by dark rearing is in turn dependent on the capacity of the mutant rhodopsin to bind chromophore. Our results indicate that rhodopsin chromophore can act in vivo as a pharmacological chaperone for P23H rhodopsin, and that light-dependent retinal degeneration caused by P23H rhodopsin is due to reduced chromophore binding.

Retinitis pigmentosa (RP) is a multigenic disorder involving progressive loss of visual function. Symptoms develop in childhood or early adulthood, including night blindness and progressive loss of peripheral vision (tunnel vision), and often progress to complete blindness (Berson 1993). Rhodopsin is the gene most frequently involved in autosomal dominant RP, accounting for a quarter of cases (Sohocki et al. 2001). Rhodopsin is comprised of the G-protein coupled receptor rod opsin and the covalently bound chromophore 11-cis retinal, and is the major protein of the rod outer segment (ROS). Light absorption causes chromophore isomerization, initiating visual transduction, which is quickly suppressed by rhodopsin phosphorylation, binding of arrestin, and dissociation of photoisomerized chromophore (Lamb and Pugh 2006).

O.L. Moritz (B)

Department of Ophthalmology and Visual Sciences, UBC/VGH Eye Care Centre, 2550 Willow Street, Vancouver, BC, V5Z 3N9, Canada

e-mail: olmoritz@interchange.ubc.ca

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_58,C Springer Science+Business Media, LLC 2010

There is significant anecdotal and experimental evidence indicating that light exposure modulates disease severity in some forms of RP. Several instances of variable phenotype in related patients suggest environmental influences, and several lines of evidence indicate that the retina is asymmetrically affected in patients with specific mutations, with the lower retina (which receives greater illumination from overhead lighting) more seriously affected. Furthermore, several animal models of RP harboring rhodopsin mutations similar to those found in RP patients have retinal degeneration (RD) with some degree of light dependence. This includes mouse models based on the rhodopsin mutation T17M, a dog model with a T4R mutation, rats harboring the P23H mutation, and drosophila expressing P23H rhodopsin. (For review of this literature, see Paskowitz et al. 2006). The P23H mutation is the most common cause of autosomal dominant RP in North America, where it is linked to 10% of cases (Sohocki et al. 2001).

The cell death pathways relevant to RP remain largely uncharacterized. However, in the case of light-dependent RD associated with rhodopsin mutations, it seems plausible that the mechanisms of visual transduction mechanisms would be involved. However, this is largely unsupported by cell culture studies. In cultured epithelial cells, the majority of RP-causing rhodopsin mutations cause defects in biosynthesis and transport (Kaushal and Khorana 1994; Sung et al. 1991). Most RP-causing mutations, including P23H, are partly or completely retained in the ER of cultured cells (Kaushal and Khorana 1994). These mutants bind chromophore and activate transducin poorly. Thus, it is difficult to resolve how a misfolded protein that is retained or degraded in the ER by quality control mechanisms could inappropriately activate visual transduction. The confusion is further complicated by the finding that mice are partially protected from RD by knockout of transducin (Samardzija et al. 2006), and that P23H rhodopsin is present in the ROS of transgenic mice and rats (Ablonczy et al. 2000; Olsson et al. 1992).

Recently, we and others have addressed these questions using amphibian models of RP (Tam and Moritz 2006, 2007; Zhang et al. 2008). Our results suggest that ER export of mutant P23H rhodopsin is a light sensitive process, thereby reconciling several apparent discrepancies between systems. We utilized certain properties of transgenic X. laevis to address questions that would be intractable in other transgenic models. Primary transgenic X. laevis are relatively easy to generate, and their retinal photoreceptors are large and easily imaged. Furthermore, many antibodies raised against mammalian rhodopsins do not cross-react with endogenous X. laevis rhodopsin. These properties allowed us to compare expression levels and intracellular localizations (particularly ER retention) between a variety of different mutant rhodopsins in-vivo, as well as the light sensitivities of the resulting RDs.

We compared P23H rhodopsin transgenes based on an epitope tagged X. laevis cDNA, and bovine, mouse and human cDNAs, while Zhang et al. examined untagged X. laevis P23H rhodopsin (Tam and Moritz 2006, 2007; Zhang et al. 2008). All forms of P23H rhodopsin express at low levels. Because RD occurs at expression levels significantly below endogenous rhodopsin (even at the level of mRNA) this argues for a gain-of-function mechanism of cell death (Zhang et al. 2008). Furthermore, examination of the intracellular distributions of the mutant

proteins revealed that they do not efficiently exit the ER, suggesting a folding or stability defect (Tam and Moritz 2006, 2007). In addition, there are subtle differences in localization between variants. In particular, bovine P23H rhodopsin has a greater propensity to escape the ER and arrive at the ROS (Tam and Moritz 2007), while X. laevis P23H rhodopsin is almost exclusively restricted to the ER (Tam and Moritz 2006). However, bovine P23H rhodopsin expression levels are still low when compared to wildtype bovine rhodopsin.

Interestingly, the propensity for small quantities of mutant rhodopsin to escape the ER correlates strongly with the propensity for RD to be rescued by dark rearing. RD caused by the bovine P23H rhodopsin transgene is extremely light dependent, while other RDs are less light dependent (human P23H) or light independent (epitope tagged X. laevis P23H) (Tam and Moritz 2006, 2007). This suggests that light acts directly on P23H rhodopsin, rather than causing a general change in cell physiology. Any effects of light such as up-regulation of pro-apoptotic genes are secondary to a direct effect on mutant rhodopsin. Interestingly, in contrast to our results, Zhang et al. (2008) found RD caused by X. laevis P23H rhodopsin to be light dependent. Our transgenes differed only in that we used conservative changes to introduce epitope tags in the rhodopsin sequence. These changes appear to be responsible for the differences in our results.

The correlation of light dependent RD with ROS localization of P23H rhodopsin suggests that visual transduction through mutant rhodopsin might contribute to cell death. To test this, we blocked signal transduction using a P23H/K296R double mutant (K296R is a non-signaling rhodopsin mutant (Cohen et al. 1993)). We did not find significant rescue of RD, although we did observe a trend (Tam and Moritz 2007). This is analogous to experiments in which transducin knockout background partially rescued RD in P23H transgenic mice (Samardzija et al. 2006), although in our case, only signal transduction through P23H rhodopsin was blocked. Thus, our results do not contradict those of Samardzija et al. (2006) rather, both experiments demonstrate that visual transduction is not responsible for cell death, but may modulate the process.

Although P23H/K296R-induced RD tends to be less severe than P23H-induced RD in bright cyclic light (1,700 lux), the K296R mutation blocks rescue of RD by dark rearing, i.e. in dim light or darkness K296R exacerbates RD (Tam and Moritz 2007). The K296R mutation not only prevents signal transduction, but also prevents chromophore binding (Cohen et al. 1993). Thus, the rescue mechanism associated with dark rearing is blocked by preventing chromophore binding. This suggests the experiments of Noorwez et al. (2004), in which biosynthesis of P23H rhodopsin is enhanced by 11-cis retinal.

Under dark rearing conditions, the proportion of P23H rhodopsin delivered to the ROS increases significantly for animals expressing human and bovine P23H rhodopsin (Tam and Moritz 2007), suggesting that chromophore binding improves ER exit efficiency. In fact, for bovine P23H rhodopsin, the expression level under dark rearing conditions approached levels obtained with wildtype bovine rhodopsin.

Unexpectedly, western blots demonstrated precise removal of at least 23 amino acids from the N-terminus of bovine P23H rhodopsin (Tam and Moritz 2007).

Furthermore, antibodies directed at N- and C-terminal epitopes produced distinct labeling patterns for human P23H, but not wildtype, rhodopsins. The N-terminal epitope was abundant in the ER and dramatically absent from ROS, while the C- terminal epitope was abundant in ROS, and less prominent in ER (Fig. 58.1). This suggests that the N-terminus was cleaved from the majority of P23H rhodopsin transported from the ER to the ROS. In fact, similarly truncated products have been reported in cell culture studies (Noorwez et al. 2004), and transgenic mice (Jin, Heth and Roof 1995). Because this proteolysis occurs within the secretory pathway, it is distinct from ER quality control mechanisms for removing and degrading misfolded proteins, which involve retrotranslocation to the cytoplasm and proteolytic degradation by the proteosome. Truncated P23H rhodopsin clearly remains in the secretory pathway and is transported to the ROS. This activity therefore resembles ER resident proteases such as gamma secretase or signal peptidase.

Fig. 58.1 ER retention and N-terminal cleavage of P23H rhodopsin. Sections of X. laevis retina expressing human P23H rhodopsin were labeled with wheat germ agglutinin (WGA, left panels), or anti-mammalian rhodopsin antibodies (right panels). The antibody epitopes are located in the C- terminus (1D4, top right) or N-terminus (2B2, bottom right). Note the labeling of inner segments (IS), consistent with ER retention of the P23H rhodopsin, and the lack of outer segment (OS) labeling by 2B2, consistent with truncation of the N-terminus of P23H rhodopsin in the secretory pathway. Bar = 20 μM

Truncated P23H rhodopsin is found in greatest abundance in retinas expressing bovine P23H rhodopsin, and is essentially absent from retinas expressing X. laevis P23H rhodopsin. Thus, between gene constructs, the light dependence of RD also correlates with the extent of P23H rhodopsin truncation (Tam and Moritz 2007). However, for a given transgene, dark rearing does not significantly increase the ratio of cleaved to full length P23H rhodopsin. Thus, dark rearing does not rescue RD

Fig. 58.2 Proposed mechanisms by which light influences P23H rhodopsin biosynthesis. Newly synthesized unfolded P23H opsin may either fold or misfold, and misfolded P23H opsin is subject to ER associated degradation (ERAD). Folded P23H opsin is unstable, but is stabilized by chromophore binding (P23H rhodopsin). P23H rhodopsin is likely to exit the ER, while P23H opsin is likely to unfold, increasing the likelihood of misfolding and ERAD. ER exit is inefficient in the presence of light (hν), either due to a reduction in chromophore supply (top) or conversion of rhodopsin to opsin, resulting in increased unstable opsin, increased misfolded opsin, ER stress, and cell death. ER exit of P23H rhodopsin may be further enhanced by N-terminal cleavage. However, this step is not light dependent. (Not shown: presumably P23H opsin may also exit the ER)

by increasing the rate of N-terminal truncation, although differences in truncation efficiency could explain differences in the extent of rescue between different P23H rhodopsin genotypes (Fig. 58.2).

On the basis of these findings, we propose a mechanism for the light dependence of RD in which export of P23H rhodopsin from the ER is more efficient in the dark due to increased binding of light-sensitive chromophore (Fig. 58.2). 11-cis retinal likely stabilizes P23H against unfolding prior to ER exit, or may facilitate the kinetics of a rate limiting step in folding. Furthermore, ER exit may be further facilitated by N-terminal cleavage. Differences in the thermodynamic stabilities of P23H rhodopsins or the efficiency of N-terminal cleavage may underlie the speciesdependent variation in rescue of RD by dark rearing. In support of this hypothesis, it has long been known that rhodopsin is considerably more thermodynamically stable than opsin (Hubbard 1958).

These results support the hypothesis that certain RP phenotypes are influenced by light exposure, and suggest that it may be possible to restore rhodopsin biosynthesis (to some extent) by protection from light in these patients. Furthermore, our results also imply that therapy with 11-cis retinal analogues suggested by other authors (Govardhan and Oprian 1994; Noorwez et al. 2003) may be a means of treating certain forms of RP.

These results largely resolve the discrepancies between in-vivo and cell culture studies. The models provide complementary insight: a single rhodopsin mutant may have a defect in biosynthesis or be correctly folded and transported, with the tipping point dependent on the cDNA used to construct the transgene, chromophore availability, and light intensity. Cell culture studies will provide a useful system