Abstract
Purpose :
Complete congenital stationary night blindness (cCSNB) is a genetically heterogeneous disorder of the retina characterized by impairment of low light vision and loss of the b-wave of the electroretinogram (ERG). Mutations in Grm6, Trpm1, Nyx, Gpr179, and Lrit3, cause cCSNB in humans. LRIT3 is a presynaptic leucine-rich repeat (LRR) protein and contains two additional extracellular protein binding domains, IG and FN3. The absence of LRIT3 causes loss of TRPM1 and Nyctalopin expression from the dendritic tips of rod bipolar cells (BCs), and in addition mGluR6, GPR179, and the RGS complex of proteins from dendritic tips of cone ON BCs. Here we investigate the role of each domain and elucidate the mechanism by which LRIT3 domains impact the BCs signalplex proteins expression and retinal function.
Methods :
To express full-length and ΔLRR, ΔIG, or ΔFN3 versions of LRIT3 in photoreceptors we used rhodopsin and GNAT2 promoters to limit expression in rods and cones, respectively. The expression constructs were packaged in the rAAV8 capsid. rAAVs were injected into adult (>P35) Lrit3-/- mice subretinally, and we analyzed retinal function using the ERG 4 weeks post injections. Expression and localization of signalplex proteins were examined by immunohistochemistry.
Results :
We show that rAAVs expressing ΔLRR or ΔIG forms of LRIT3 in rods and cones of Lrit3-/- mice (n=6 for each construct) did not restore the scotopic or photopic b-wave of ERG. In rods, the ΔLRR LRIT3 localized at rod terminals but it did not restore nyctalopin and TRPM1, however, it failed to traffic in cones. The ΔIG LRIT3 localized normally in rods and cones and restored nyctalopin in rod BCs dendrites and in addition, mGluR6, and GPR179 in ON cone BCs dendrites, but interestingly, it failed to restore TRPM1. Deletion of the FN3 domain did not affect LRIT3 localization and function.
Conclusions :
Our data show that the LRR and IG domains of LRIT3 are indispensable for TRPM1 localization and retinal function and indicate that the LRR domain plays the main role in the differential effects of LRIT3 in rods and cones. They show for the first time that nyctalopin is not required for TRPM1 localization in ON BCs dendrites. Based on our findings, we propose a model in which the LRR domain trans-synaptically binds with nyctalopin, and the IG domain interacts with TRPM1.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.