Expression of SH3 and Multiple Ankyrin Repeat Domains Protein 3 in Mouse Retina

Abstract

Synapse-associated gene mutations of SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) may lead to autism spectrum disorder (ASD). In some ASD cases, patients may also have vision disorders. However, the effects of SHANK3 in the retina are barely mentioned in the literature. In this study, we used wild-type mice to systematically map the distribution of SHANK3 expression in entire mouse retinas. Using Western blot analysis and immunofluorescence double labeling, we identified a large number of prominent cells expressing high levels of SHANK3 in the inner retina, in particular, the ganglion cell layer (GCL) and inner nucleus layer. The inner plexiform layer and outer nucleus layer were also exhibited positive SHANK3 signals. In the inner layer, GABAergic amacrine cells (ACs) labeled by glutamate decarboxylase were colocalized with SHANK3-positive cells. Dopaminergic ACs (labeled by tyrosine hydroxylase) and cholinergic ACs (labeled by choline acetyltransferase) were also found to contain SHANK3-positive signals. Additionally, most GCs (labeled by Brn3a) were also found to be SHANK3 positive. In the outer retina, bipolar cells (labeled by homeobox protein ChX10) and horizontal cells (labeled by calbindin) were SHANK3 positive. In the outer nucleus layers, the somata of blue cones (labeled by S-opsin) were weekly co-labeled with SHANK3. The inner segments of blue cones and the outer segments of red/green cones (labeled by L/M-opsin) were partially colocalized with SHANK3 and the outer segments of rods (labeled by Rho4D2) were partially SHANK3 positive too. Moreover, SHANK3-positive labeling was also observed in Müller cells (labeled by cellular retinaldehyde-binding protein). These wide expression patterns indicate that SHANK3 may play an important role in the visual signaling pathway.

Keywords: ASD; SHANK3; double-labeled immunohistochemistry; excitatory synapse; retina.

FIGURE 1

Expression of SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) in mice retina. (A) Western blot analysis confirmed that SHANK3 was specifically expressed in the retinal homogenates, with three SHANK3 isoforms at 150–250 kDa. No band was detected when the SHANK3 antibody was pre-absorbed with the immunizing antigen. The similar was find when the primary antibody was omitted. And when the shank3 was mutant, the bands of SHANK3 were reduced. (B) Confocal fluorescence microphotographs of a vertical section of the mouse retina, labeled by SHANK3. Fluorescence staining shows that the SHANK3 is widely distributed through the whole retina. (C) Micrographs of a retinal vertical section, showing that no signal was detectable when the primary antibody for SHANK3 was pre-absorbed with the immunizing antigen. (D) Micrographs of a retinal vertical section, showing that no signal was detectable when the primary antibody was omitted. (E) Micrographs of a retinal vertical section, showing that the shank3 was mutant, the signal of SHANK3 was reduced. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 20 μm.

FIGURE 2

Confocal microphotographs showing the distribution of SHANK3 in the photoreceptors. Double-labeled elements (green for SHANK3; red for L/M-opsin, S-opsin and Rho4D2) appear yellowish and some double-labeled cells are indicated by arrows. (a–a”) Colocalization of SHANK3 with L/M- opsin in a retinal vertical section. Fluorescence double stainings show that SHANK3 (a) and the L/M-opsin (a’), the merged image of L/M-opsin and SHANK3 (a”). L/M-opsin-labeled the outer segment (OS) of red/green cones (a’). Part of the L/M-opsin-positive IS (arrows) are co-labeled by SHANK3 (a,a”). (b–b”) Colocalization of SHANK3 with S-opsin in a retinal vertical section. Fluorescence double stainings show that SHANK3 (b) and the S-opsin (b’), and b” is the merged image of S-opsin and SHANK3. S-opsin is a marker for blue cone. Note that labeling for SHANK3 is detected in S-opsin-positive blue cones (arrows). (c–c”) Colocalization of SHANK3 with Rho4D2 in a retinal vertical section. Fluorescence double stainings show that SHANK3 (c) and the Rho4D2 (c’), the merged image of Rho4D2 and SHANK3 (c”). Rho4D2-labeled the outer segment (OS) of rods (c’). Rho4D2-positive OS are partially labeled by SHANK3 (c,c”). OS, outer segment; IS, inner segment. Scale bar = 20 μm.

FIGURE 3

Confocal microphotographs showing the distribution of SHANK3 in the outer retina. Double-labeled elements (green for SHANK3; red for CB and ChX10) appear yellowish and some double-labeled cells are indicated by arrows. (a–a”) Colocalization of SHANK3 with CB in a retinal vertical section. Fluorescence double stainings show that SHANK3 (a) and the CB (a’), the merged image of CB and SHANK3 (a”). CB-labeled horizontal cells (a’). The CB-positive somata (arrows) are colabeled by SHANK3 (a,a”). (b–b”) Colocalization of SHANK3 with ChX10 in a retinal vertical section. Fluorescence double stainings show that SHANK3 (b) and the ChX10 (b’b”) is the merged image of ChX10 and SHANK3. ChX10 is a marker for bipolar cells. Note that labeling for SHANK3 is detected in ChX10-positive BCs (arrows). Scale bar = 20 μm.

FIGURE 4

Confocal microphotographs showing the distribution of SHANK3 in different AC subtypes. Double staining (green for SHANK3; red for GAD 65, TH, and ChAT) appear yellowish and arrows indicate some double-labeled cells. (a–a”) Colocalization of SHANK3 with GAD 65 in a retinal vertical section. Fluorescence double stainings show that SHANK3 (a) and the GAD 65 (a’). Note that the GABAergic ACs in the innermost part of the INL are GAD 65-labeled, and also strongly labeled numerous neuronal processes of these cells in the IPL (a’). All the GAD 65-positive GABAergic ACs somata are labeled by SHANK3 (a”). (b–b”) Colocalization of SHANK3 with TH in a retinal vertical section. Fluorescence double stainings show that SHANK3 (b) and the TH (b’). The soma and processes of the dopaminergic ACs are strongly labeled by TH (b’,b”), and the soma is clearly labeled by SHANK3 (b”). (c–c”) Colocalization of SHANK3 with ChAT in a retinal vertical section. Fluorescence double stainings show that SHANK3 (c) and the ChAT (c’). ChAT-labeled mirror-symmetric cholinergic ACs and their processes, forming two bands in the IPL. The merged image shows that the somata of cholinergic ACs in INL and GCL were both SHANK3-positive (c”). Scale bar = 20 μm.

FIGURE 5

Confocal microphotographs showing the distribution of SHANK3 in synaptic. Double staining (green for SHANK3; red for Synaptophysin, PSD95 and VGluT1) appear yellowish. (a–a”) Colocalization of SHANK3 with Synaptophysin in a retinal vertical section. Fluorescence double stainings show that SHANK3 (a) and the Synaptophysin (a’,a”) is the merged image of Synaptophysin and SHANK3. (b–b”) are the locally magnified images for (a–a”). The merged image shows that the presynaptic membrane is not labeled by SHANK3. (c–c”) Colocalization of SHANK3 with PSD95 in a retinal vertical section. Fluorescence double stainings show that SHANK3 (c) and the PSD95 (c’,c”) is the merged image of PSD95 and SHANK3. (d–d”) are the locally magnified images for (c–c”). The merged image shows that the postsynaptic membrane is SHANK3-positive. (e–e”) Colocalization of SHANK3 with VGluT1 in a retinal vertical section. Fluorescence double stainings show that SHANK3 (e) and the VGluT1 (e’,e”) is the merged image of VGluT1 and SHANK3. Scale bar = 20 μm.

FIGURE 6

Confocal microphotographs showing the distribution of SHANK3 in GCL and Müller cells. Double staining (green for SHANK3; red for Brn3a and CRALBP) appear yellowish, and arrows indicate some of the double-labeled cells. (a–a”) Colocalization of SHANK3 with Brn3a in a retinal vertical section. Fluorescence double stainings show that SHANK3 (a) and the Brn3a (a’), (a”) are the merged image of Brn3a and SHANK3. Almost all the ganglion cells are clearly labeled by Brn3a (a’). SHANK3 immunoreactivity is observed in all Brn3a (arrows). Meanwhile, some SHANK3-positive cells in the GCL are not immunoreactive for Brn3a (arrowheads in a–a”), which could be displaced ACs or Brn3a-negative GCs. (b–b”) Colocalization of SHANK3 with CRALBP in a retinal vertical section. Fluorescence double stainings show that SHANK3 (b) and the CRALBP (b’,b”) is the merged image of CRALBP and SHANK3. CRALBP is a specific Müller cell marker. Note that SHANK3 immunostaining is observed in somata of CRALBP-positive Müller cells. Scale bar = 20 μm.