Functional study of two biochemically unusual mutations in GUCY2D Leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas

Abstract

Purpose: To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1).

Methods: GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e^-/-Gucy2f^-/- knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinography (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining.

Results: Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts.

Conclusions: The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele.

Figure 1

Schematic representation of the GUCY2D (RetGC1) primary structure [5,6]. The S248W missense mutation is located in the extracellular domain, and the R1091x truncation is located near the C-terminus, downstream of the catalytic domain. LP = leader peptide; ECD = extracellular domain; TM = transmembrane domain; KHD = kinase homology domain; DD = dimerization domain; CAT = catalytic domain.

Figure 2

ERG responses to a bright flash imparted by AAV-mediated expression of human wild-type (hWT), S248W, and R1091x RetGC1. A: Representative scotopic electroretinography (ERG) in (top to bottom) S248W, R1091x, or wild-type GUCY2D adenoassociated virus (AAV)-injected eyes, each shown above the traces from the non-injected eyes; the small vertical arrows indicate timing of a 505 nm 1.2 × 10⁶ photon µm⁻² flash. B: Representative photopic ERG in (top to bottom) S248W, R1091x, or wild-type GUCY2D AAV-injected eyes; each group of three traces shows responses to 505 nm (upper trace) or 365 nm (middle trace) 6 × 10⁵ photon µm⁻² flash (small vertical arrows) delivered in the injected eyes and the 365 nm flash delivered in a non-injected eye, all in a constant background light equivalent of 33 × 10³ photoisomerizations rod⁻¹ s⁻¹. C: Maximal voltage deflection amplitudes (mean ± standard deviation, SD) of the scotopic ERG a-wave in the injected eyes among the hWT (136 ± 60 µV, n = 9), S248W (0.4 ± 4 µV, n = 9), and R1091x (105 ± 28 µV, n = 10) treated mice. Note that because the a-wave could not be identified in the ERG traces recorded from the S248W-injected mice, the amplitude of the voltage deflection at 8 ms after the flash was arbitrarily taken for comparison with the two other groups. The differences between the ERG responses in the S248W-injected eyes and the other two groups were statistically highly significant (p≤0.0001; here and from Student t test using unpaired data). There was no statistically significant difference between the hWT and R1091x-injected mice (p = 0.177). D: Maximal voltage deflection amplitudes of the scotopic b-wave in the same mouse group as panel C (305 ± 119, 229 ± 50, and 38 ± 14 µV for the hWT, R1091x, and S248W- injected eyes, respectively). The differences between the ERG responses in the S248W-injected eyes and the other two groups were highly statistically significant (p≤0.00013), but the difference between the hWT and the R1091x-injected mice was not (p = 0.102).

Figure 3

RetGC activity and AAV-mediated GUCY2D expression in GCdKO retinas. A: RetGC1 activity (mean ± standard deviation, SD) in retinal homogenates extracted from non-injected (-) eyes compared to those injected (+) with adenoassociated virus (AAV) vectors harboring wild-type (hWT), S248W, or R1091x GUCY2D cDNA. Results are the average of two independent experiments, each using two different mouse eyes. The difference in retinal membrane guanylyl cyclase 1 (RetGC1) activity between the eyes treated with AAV-S248W and the two other groups was highly statistically significant (p≤0.0002). There was no statistically significant difference between the hWT and R1091x-injected mice (p = 0.126). B: Western immunoblotting of the retinal proteins extracted from the injected and non-injected eyes probed with anti-RetGC1 antibody. Note the faint signal in the S248W lane compared to the two other GUCY2D variants. C: Quantitative real-time (QRT) PCR detection of GUCY2D-specific transcripts in the retinas from three wild-type (hWT) and two S248W GUCY2D-injected Gucy2e^−/−Gucy2f^−/− knockout (GCdKO) eyes plotted as fold-increase (mean ± standard error, SE) in the GUCY2D transcript signal relative to the non-injected (Log 0) GCdKO retinas.

Figure 4

Localization of GUCY2D LCA1 mutants in mouse retinas. A–D: Confocal retinal membrane guanylyl cyclase 1 (RetGC1) immunofluorescence images of the GUCY2D-injected human wild-type (A), S248W (B), and R1091x (C) or non-injected Gucy2e^−/−Gucy2 knockout (GCdKO) (D) retina sections probed with anti-RetGC1 antibody (red), cone outer segment sheath-staining peanut agglutinin (green), and TOPRO3 iodide (pseudoblue). The yellow arrowheads point at the cone outer segments surrounding the RetGC1 immunofluorescence. In all panels, the anti-RetGC1 fluorescence was recorded in the same experiment using identical laser settings. E–G: RetGC1 immunostaining in wild-type (E), S248W (F), and R1091x (G) GUCY2D-expressing retinas shown at lower magnification. Note the faint S248W GUCY2D immunofluorescence in the photoreceptor cell bodies and axons in the outer nuclear layer (gray arrowheads), along with sparse cone outer segments (yellow arrowheads) in panel F. Bar length = 50 µm. COS = cone outer segments; ROS = rod outer segments; ONL = outer nuclear layer.

Figure 5

The S248W GUCY2D activated by GCAP1 binds human RD3 in vitro. Fractional guanylyl cyclase activity was measured in human embryonic kidney cells (HEK293) membranes containing recombinant wild-type (open square) or S248W (black circle) GUCY2D reconstituted with 1.5 µM guanylyl cyclase activating protein 1 (GCAP1) and variable concentrations of recombinant human retinal degeneration 3 (RD3) in the presence of 2 mM EGTA. The EC₅₀ for RD3-dependent inhibition from the fit assuming the Hill function was 1.9 ± 0.3 nM (n = 5) and 1.4 ± 0.1 nM (n = 3), respectively (p = 0.07). Error bars = standard deviation.