Effects of Schisandrin B on Rod Photoreceptors in the pde6c Larval Retina
Degeneration of photoreceptors (PRs) in the retina is a common cellular consequence in inherited retinal degenerative diseases. In human patients and animal models, the loss of PRs expedites loss of vision. Some of the causal mutations for PR degeneration, result in the death of only one PR type leaving the other functionally intact; whereas some mutations result in death of both PR types. As such there is no definitive chemical therapy to preserve the function of PRs or to prevent them from dying in such degenerative conditions. Our lab previously showed that an active compound extracted from the five-flavored fruit (Fructus Schisandrae) called Schisandrin B (SchB) improves light sensation in the pde6cw59 zebrafish mutant model, as assessed by Visual Motor Response (VMR) assay. We also showed that the SchB treatment reduces the size of the morphologically abnormal rods usually observed in pde6cw59 larval retina, whereas no significant difference was found on cones. The pde6cw59 mutants harbor a mutation in a cone specific phosphodiesterase enzyme subunit gene which causes the cones to degenerate. Additionally, due to unknown reasons, the rods also degenerate non-autonomously thereby causing the pde6c w59 to lose vision starting 4 days post fertilisation (dpf). Thus, the pde6cw59 mutants provide a unique model for studying PRs degeneration. Based on these results, I hypothesized that SchB improves light sensation in dystrophic rods thereby enhancing VMR in pde6cw59 larvae. Moreover, I hypothesized that SchB reduces size of dystrophic rods thereby reducing apoptosis in larval pde6cw59. Thus, the overarching goal of my thesis was to build a mechanistic framework behind action of SchB treatment in pde6cw59 mutants. In order to examine the cellular changes caused by SchB treatment in rods of pde6c w59, I first sought to establish that rods are apoptotic in larval pde6cw59 and then test if SchB could reduce this apoptosis of rods in larval pde6cw59. Consistent with previous reports, I observed increased overall TUNEL staining in pde6cw59 larvae compared with WT between 4 dpf and 5 dpf. However, I found the higher apoptotic signals were observed in the Outer Nuclear Layer (ONL) and Inner Nuclear Layer (INL), indicating that photoreceptors and other second order neurons in INL (but not ganglion cells) were affected in these mutants. Using a transgenic reporter that expresses GFP in rods bred into the pde6cw59 mutants, I observed co-localisation of apoptotic signals with rods. This showed us that rods are indeed apoptotic in the pde6cw59 larvae. Having established that rods are apoptotic in pde6cw59 larval retinas, I sought to determine if treatment with SchB could reduce or prevent this. To test this, I subjected the pde6cw59 mutants to previously established concentrations of SchB from 3 dpf to 6 dpf and assayed apoptosis on 6 dpf. I observed that the SchB treatment seemed to cause a reduction in number of apoptotic rods in 6 dpf pde6c w59 larval retinas compared to untreated counterparts. In order to examine effects of SchB on rods directly, using visual behavior, I first sought to define conditions that stimulate only rods to drive VMR and then to use these conditions to directly test the effect of SchB in pde6c w59 larvae. By using light stimuli with different irradiance together with nof/gnat2w21 mutants (that lack functional cones) I defined the irradiance that could stimulate only the rods to drive the VMR. I reasoned that since nof/gnat2w21 mutants lack cone function, only rods could be stimulated to drive the VMR. As expected, I observed the light-on VMR of WT and the nof/gnat2 w21 mutant to be identical using a very low light stimulus. This systematic approach allowed me to define the conditions required to drive scotopic light-on VMR. Following this, the light intensity to drive the scotopic VMR was used to re-evaluate the VMR of untreated and SchB treated pde6c w59 mutants. I observed that SchB treatment caused an enhanced light-on scotopic VMR in SchB treated pde6cw59 but not in the untreated mutants. Taking together my results and observations, I show that SchB does exert a preserving effect on rods in the pde6c w59 mutants. Furthermore, I propose that SchB treatment reduces apoptotic rods in these mutants. To summarize, I propose that SchB treatment could induce enhanced phagocytosis of apoptotic debris in the pde6cw59 mutants which in turn causes an improvement in their vision as assayed by the VMR. Thus, my thesis study provides a foundation for a mechanistic framework of action of SchB in pde6c w59 larvae.
Leung, Purdue University.
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