Abstract
Purpose :
To investigate the requirement for dehydrodolichyl diphosphate synthase (Dhdds) gene expression in the early development of photoreceptor and bipolar cells.
Methods :
Dhdds was ablated in rod and cone photoreceptor and bipolar cells by crossing Dhddsflx/flx mice to CRX (cone-rod homeobox) promoter-driven Cre recombinase transgenic mice, to initiate Dhdds knockout (KO) at E12.5 days. Dhddsflx/flx CRX-Cre+ (KO) and Dhddsflx/flx CRX-Cre- (control) mice were selected by PCR for the presence of the loxP-modified Dhdds allele and CRX-Cre transgene. Cre recombinase expression and activity were tested by crossing CRX-Cre mice with ZsGreen reporter mice. Western blot (WB) analysis was performed, ±PNGase-F treatment, on retinas from PN 4-wk old KO and control mice, probing with 1D4 Mab to assess opsin N-glycosylation status. Eyes from PN 3-4-wk old KO and control mice were subjected to TUNEL labeling and immunohistochemistry (IHC), using cell type-specific antibodies, or probed with fluor-conjugated lectins (Con-A, PNA) or Cholera toxin-B (CTx). Retina whole mounts were probed with PNA to assess cone density. ERG analysis was performed on control and KO mice at PN 4-5 wk (N=3/group). Statistical analysis: Student’s t-test, P<0.05.
Results :
ZsGreen reporter analysis demonstrated Cre recombinase activity in rods, cones, and bipolar cells, as well as patchy expression in Mueller glia. ERG analysis showed extinguished scotopic and photopic a- and b-waves in KO mice at PN 4-wk. Relative to controls, IHC of PN 4-wk KO retinas revealed significant (>90%) loss of cones and bipolar cells, shortened rod outer segments, and gliosis; TUNEL+ cells were observed only in the ONL, with a cone-rod degeneration pattern. CTx binding revealed aberrant ganglioside GM1 accumulation in KO retinas; however, no overt protein N-glycosylation defects were evident by WB or Con-A staining.
Conclusions :
Despite initiation of Dhdds ablation at E12.5 days in progenitors of photoreceptor and bipolar cells, retinal degeneration (not dysplasia) was observed. These findings suggest the presence of a long-lived, progenitor-derived dolichol pool that persists even after differentiation of photoreceptors and bipolar cells has occurred and after de novo synthesis has ceased in those cells. This may explain the absence of obvious glycosylation defects in other Dhdds mutant mouse models of RP59.
This is a 2021 ARVO Annual Meeting abstract.