LRIT3 is required for nyctalopin expression and normal ON and OFF pathway signaling in the retina

Abstract

37At its first synapse, the retina establishes two parallel channels that encode light increments (ON) or 38 decrements (OFF). At the same synapse, changes in photoreceptor glutamate release are sensed by 39 ON bipolar cells (BCs) via the metabotropic glutamate receptor 6 (mGluR6), and OFF BCs via 40 ionotropic BCs, which differ in their synaptic configuration with the photoreceptor terminal. ON BCs 41 form invaginating synapses that bring them in close proximity to presynaptic ribbons and the presumed 42 sole source of glutamate release. OFF bipolar cells form flat contacts distal to the ribbon synapse. We 43 investigated the role of LRIT3 in normal assembly and function of the mGlur6 signaling cascade 44 present in ON BCs. We demonstrate that LRIT3 is required for nyctalopin expression and thus TRPM1 45 expression and function. Using glutamate imaging, whole-cell electrophysiology, and multi-electrode 46 array extracellular recordings we demonstrate that the loss of LRIT3 impacts both the ON and OFF 47 128 129 Generation of Lrit3 -/mice with Zinc Finger Nucleases (ZFN) 130 131 C3H/HeNTac/C57BL/6NTac hybrid embryos (363) were injected with 10 ng/µl Lrit3 ZFN mRNA and 132 254 viable embryos were implanted into 9 Swiss Webster recipient mothers. Tail biopsies from offspring 133 were collected and genomic DNA isolated using Direct Tail PCR solution (Thermo Scientific) 134 supplemented with 0.2 μg/ml proteinase K (Thermo Scientific). Primers (5'-135

Presynaptic Expression of LRIT3 Transsynaptically Organizes the Postsynaptic Glutamate Signaling Complex Containing TRPM1

“…Immunohistochemistry and confocal microscopy show that LRIT3 expression is confined to the retinal outer plexiform layer (OPL) and is co-localized with the ON BC signalplex proteins mGluR6, TRPM1, and GPR179 (Neuillé et al, 2017; Ray, 2013). In Lrit3 nob6 (Neuillé et al, 2015) and Lrit3 −/− (Hasan et al, 2018; Ray, 2013) mice, the loss of LRIT3 results in the loss of TRPM1 expression, suggesting that the two molecules may interact. To determine if LRIT3 is expressed pre- and/or postsynaptically, we used in situ hybridization.…”

“…We next examined if rescue of TRPM1 to the RBCs by expressing LRIT3 in rods restored function using the electroretinogram (ERG) and RGC recordings. Lrit3 mice retain a normal a-wave but lack b-waves under both scotopic and photopic conditions (Figure 3; Hasan et al, 2018; Neuillé et al, 2014; Ray, 2013). To assess the effect and efficiency of expression of LRIT3 in rods, we performed full-field ERGs in the same eyes in which we restored LRIT3 expression to ~50% of the rods in the Lrit3 −/− mice (Figure 1E).…”

“…We recorded from two pieces of retina from five eyes that had partial scotopic b-wave restoration and examined both rod- and cone-driven responses to flashes from 276 control, 377 Lrit3 −/− and 476 rAAV RHO::Lrit3-treated Lrit3 −/− RGCs. A unique feature of the Lrit3 −/− retina is that the majority of RGCs lack a visually evoked response (Hasan et al, 2018). Although they retain spontaneous activity, 92.6% and 61.3% of the RGCs are non-responsive (NR) under scotopic and photopic stimuli, respectively (Figure 4C; Table S1), compared with 28% and 2% in controls.…”

“…Here we show that presynaptic LRIT3 expression in rods is required for TRPM1 insertion into the postsynaptic RBCs membrane. In rods LRIT3 also is required for the postsynaptic expression of Nyctalopin (Hasan et al, 2018). In contrast, at the cone to cone bipolar synapse LRIT3 is required for not only for expression of Nyctalopin and TRPM1, but also for mGluR6, GPR179, and thus the RGS complex (Neuillé et al, 2015; Hasan et al, 2018; Ray, 2013).…”

“…The absence of LRIT3 disrupts trafficking of TRPM1 (Neuillé et al, 2015), which is the non-specific cation channel that leads to postsynaptic DBC depolarization. LRIT3 also is required for Nyctalopin expression at the dendritic tips of DBCs (Hasan et al, 2018). Taken together these results predicted that the LRR-containing proteins LRIT3 and Nyctalopin may interact postsynaptically and serve to organize postsynaptic receptor assembly in DBCs, but not synapse formation and/or maintenance.…”