shRNA knockdown of guanylate cyclase 2e or cyclic nucleotide gated channel alpha 1 increases photoreceptor survival in a cGMP phosphodiesterase mouse model of retinitis pigmentosa

Abstract

In vertebrate rods, dark and light conditions produce changes in guanosine 3',5'-cyclic monophosphate (cGMP) and calcium (Ca(2+) ) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP-gated calcium sodium channels (CNG) to increase Na(+) and Ca(2+) influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na(+) and Ca(2+) levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca(2+) levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca(2+) to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6b(H620Q) photoreceptors prior to degeneration. Gucy2e- or Cnga1-shRNA lentiviral-mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6b(H620Q) mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP.

Fig 1

Immunoblotting of retinal lysates from P60 wild-type C57BL/6J mice transduced with shRNA-Gucy2e (A, +), shRNA-Cnga1 (B, +) or shRNA-GFP control lentivirus (A and B, −). The antibodies used were anti-GUCY2E, anti-GNAT1 and anti-CNGA1. GNAT1 is a rod-specific protein, which serves as a control for photoreceptor protein content. Bottom panel is α-tubulin (TUB), which controls for protein loading (25 μg per lane).

Fig 2

shRNA knockdown has no detectable gross effects on photoreceptor survival or structure. Lentiviral shRNA targeting of Gucy2e (A) and Cnga1 (C) in C57BL/6J mice exhibit identical retinal structure as control retinas (B, D) injected with saline. Eight to ten rows of photoreceptor nuclei were seen in shRNA transduced and saline injected retinae.

Fig 3

Lentiviral shRNA targeting of Gucy2e (A) and Cnga1 (C) in Pde6b^H620Q mice. At P56, the control retinae (B, D) show a single row of photoreceptors with scattered outer segments, as is typically observed in Pde6b^H620Q mutants [8] (yellow arrows). In contrast, surrounding the injection site, there are rod outer segments and eight rows of photoreceptor nuclei after shRNA-Cnga1 and five rows of nuclei after shRNA-Gucy2e transduction (yellow brackets).

Fig 4

Maximal dark-adapted ERG traces of C57BL/6J (wild-type) mice transduced with shRNA-Gucy2e (1) and shRNA-Cnga1 (2), and corresponding control eyes at postnatal days 35, 60 and 90. ERGs were performed on both eyes simultaneously; each colour trace represented recordings from an individual subject and corresponding control.

Fig 6

ERG b-wave enhancement of rod-specific, maximal and cone responses from shRNA-lentivirus transduced C57BL/6J and Pde6b^H620Q mice. The upper panels display rod, maximal and cone ERG b-wave enhancement from C57BL/6J mice aged 1–3 months after transduction with shRNA-Gucy2e (left) or shRNA-Cnga1 (right). Lower panels show rod-specific, maximal and cone ERG b-wave enhancement from Pde6b^H620Q mice aged 1–3 months after transduction with shRNA-Gucy2e (left) or shRNA-Cnga1 (right) (White = 30 days postnatal; light shaded = 60 days postnatal; Black solid = 90 days postnatal).

Fig 5

Functional rescue of neuronal signaling in Pde6b^H620Q retinas transduced with shRNA- Gucy2e (1) and shRNA-Cnga1 (2). Each mutant received a sub-retinal injection of shRNA-Gucy2e (1) and shRNA-Cnga1 (2) lentivirus in the right eye and saline in the left eye (control) at P5. Maximal dark-adapted ERGs were then performed simultaneously on both eyes of Pde6b^H620Q mice at postnatal days 35, 60 and 90. Significant a-wave improvement are seen in shRNA transduced eyes at day 35. Each colour trace depicted recordings from an individual subject and corresponding control.