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Marion Neuille, Catherine W Morgans, Elise Orhan, Christelle Michiels, Jose Alain Sahel, Isabelle S Audo, Robert M Duvoisin, Christina Zeitz; LRIT3 is essential to localize TRPM1 to the dendritic tips of ON-bipolar cells and may play a role in cone synapse formation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2612.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in LRIT3 lead to autosomal recessive cCSNB. The exact role of LRIT3 in the ON-bipolar cell signaling cascade remains to be elucidated. Recently, we have characterized a novel mouse model lacking Lrit3 (no b-wave 6 (nob6)), which displays similar abnormalities as patients with cCSNB due to LRIT3 mutations, most strikingly lacking the scotopic ERG b-wave. Here we compare the localization of many components of the ON-bipolar cell signaling cascade as well as the expression levels of Nyx and Trpm1 in wild-type and nob6 retinas.
An affinity purified anti-LRIT3 antibody was generated by immunizing a New Zealand White rabbit with a peptide corresponding to amino acids 180-195 of mouse LRIT3 (NP_001274153.1) (peptide sequence: AVTPSRSPDFPPRRII). Localization of LRIT3, TRPM1, mGluR6, GPR179, RGS7, RGS11, Gβ5, PKCα, ribeye, calbindin, Goα and PNA was analyzed on wild-type and nob6 retinal sections by immunofluorescence confocal microscopy. Expression levels of Nyx and Trpm1 were investigated by qPCR from wild-type and nob6 retinas.
Immunofluorescent staining of LRIT3 in wild-type revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in nob6 mice. LRIT3 staining did not colocalize with ribeye nor calbindin but it colocalized with mGluR6. Additionally, TRPM1 staining was severely decreased at the dendritic tips of ON-bipolar cells in nob6 mice. Interestingly, mGluR6, GPR179, RGS7, RGS11 and Gβ5 immunofluorescence was nearly absent at the dendritic tips of cone ON-bipolar cells in nob6, whereas their labeling at the dendritic tips of rod bipolar cells appeared normal. Furthermore, PNA and ribeye labeling was severely reduced at cone synaptic pedicles and ribbons, respectively, in nob6. qPCR studies are in progress at the time of submission.
Anti-mouse LRIT3 antibody confirmed the localization of the corresponding protein at the dendritic tips of rod bipolar cells and cone ON-bipolar cells in mouse retina. We also demonstrated that TRPM1 localization is dependent on the presence of LRIT3, reinforcing our hypothesis that LRIT3 may interact with scaffolding proteins to bring TRPM1 to the cell surface. Since most of the known components of the ON-bipolar cell signaling cascade, PNA and ribeye revealed disturbed localization in the OPL, an additional function of LRIT3 in synapse formation is suggested.
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