Abstract
Purpose :
TMEM97 (Sigma-2 receptor) is a transmembrane protein that has been implicated in cell proliferation and death, cholesterol homeostasis and lipoprotein uptake. Its distribution and role in the retina are not well understood. Here, we examined TMEM97 distribution in the adult mouse retina.
Methods :
Eyes from adult C57BL/6J wildtype (WT) and Tmem97 knockout (KO; Shen et al., 2021) mice were fixed in 4% paraformaldehyde/PBS, and cryosections (O.C.T.) were collected on glass microscope slides for immunohistochemistry (IHC) and probed with rabbit anti-TMEM97 (Invitrogen #PA5-2300 or Novus Biologicals #NBP1-30437), followed by fluorochrome-conjugated secondary antibodies (Abs), and examined by confocal fluorescence microscopy. Non-immune (normal) rabbit IgG (Sigma/Aldrich) served as a negative control. Markers of rod outer segments (ID4 anti-opsin), Müller glia (anti-glutamine synthetase (GS); BD, Clone 6 #610517), mitochondria (COX4-I1; #AF5814, Bio-Techne), cilia (chicken anti-rootletin; Tiansin Li, NEI), ECM (anti-IMPG1, #SC-377366) also were probed, using suitable secondary fluorochrome-conjugated Abs, and DAPI counterstain. Alternatively, WT mouse eyes were fixed in buffered mixed aldehydes, then embedded in LR White resin, and processed for immunoelectron microscopy, applying anti-TMEM97 Abs in combination with Nanogold-conjugated anti-rabbit Ab and silver enhancement (Wolfrum & Schmitt, 2000). Western blots (WB) of mouse neural retinas were probed with anti-TMEM97 Abs (ECL detection).
Results :
WB analysis of retina lysates showed a single anti-TMEM97-positive band (Mr ~21 kDa), consistent with TMEM97. Prominent TMEM97 immunofluorescence labeling was found in the photoreceptor layer of the mouse retina, but no colocalization with rod opsin, COX4, GS, or IMPG1 was observed; occasional co-localization with rootletin in rods and RPE cells was observed, otherwise little or no RPE labeling. Focal and more diffuse labeling was observed in the OPL, IPL, and GCL. In both the INL and GCL we observed elongated staining that co-localized with rootletin. Tmem97 KO retinas exhibited no Tmem97 immunostaining. Immunoelectron microscopy confirmed TMEM97 localization in rod ciliary rootlets, and periciliary and centriolar material of retinal cells.
Conclusions :
These findings are consistent with TMEM97 having a role in the structure and function of primary cilia in neural retina (especially photoreceptors) and RPE centrosomes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.