Early alteration of retinal neurons in Aipl1-/- animals

Abstract

Purpose: Mutations in the photoreceptor cell-specific gene encoding aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) lead to Leber congenital amaurosis (LCA4), retinitis pigmentosa, and cone-rod dystrophy. Gene therapy appears to be promising in the treatment for AIPL1-mediated vision loss in humans. Prior to initiating these treatments, however, it is crucial to understand how the retinal neurons remodel themselves in response to photoreceptor cell degeneration. In this study, using an animal model for AIPL1-LCA, Aipl1(-/-) mice, we investigate the changes in postreceptoral retinal neurons during the course of photoreceptor cell loss.

Methods: Morphology of the Aipl1(-/-) retina from postnatal day 8 to 150 was compared to that of age-matched, wild-type C57Bl6/J retina (WT) by immunocytochemistry using cell-specific markers.

Results: Expression of postsynaptic proteins in bipolar cells is reduced prior to photoreceptor cell degeneration at postnatal day 8. Bipolar and horizontal cells retract their dendrites. Cell bodies and axons of bipolar and horizontal cells are disorganized during the course of degeneration. Müller cell processes become hypertrophic and form a dense fibrotic layer outside the inner nuclear layer.

Conclusions: An early defect in photoreceptor cells in the AIPL1-LCA mouse model affects the expression of postsynaptic markers, suggesting abnormal development of bipolar synapses. Once degeneration of photoreceptor cells is initiated, remodeling of retinal neurons in the Aipl1(-/-) animal is rapid.

Keywords: LCA; childhood blindness; photoreceptor degeneration; remodeling; retina.

Figure 1

Reduced expression of mGluR6 prior to photoreceptor loss in Aipl1^−/− animals. Immunostaining for CtBP2 (A–J) and mGluR6 (K–T) in WT and Aipl1^−/− retina at specified ages. Arrow indicates abnormal staining of CtBP2 (H, I) and mGluR6 (R, S) in the outer plexiform layer of retina from Aipl1^−/− animals. Scale bar: 10 μm. Images were obtained using confocal microscopy at 63× magnification with 2× zoom. Average number (±SD) of CtBP2 (U) and mGluR6 (V) puncta in a retinal cross window of 66.8 × 66.8 μm in Aipl1^−/− and WT retina at P8. ns, nonsignificant; ****P < 0.0001.

Figure 2

Reduction in VGLUT1 expression as photoreceptors degenerate. Labeling for VGLUT1 antibody at specified ages in WT (A–E) and Aipl1^−/− (F–J) retina. Arrows in WT retina indicate cone terminals (C) and arrowheads indicate rod terminals (D, E). Cone and rod terminals are also marked in (E). Arrows in Aipl1^−/− retina indicate reduction in VGLUT1 expression at P14 and P18 in the OPL (G, H). VGLUT1 expression is abolished at P30 in the OPL (I). Scale bar: 20 μm.

Figure 3

Loss of synaptophysin (SYP) from photoreceptor synaptic terminals in Aipl1^−/− retina. Synaptophysin labeling reveals changes in the synapse at indicated ages in WT (A–E) and Aipl1^−/− (F–J) retina. Arrows indicate discrete labeling of SYP in OPL in the Aipl1^−/− retina (G, H). Arrowhead (I, J) indicates SYP labeling in retinal pigment epithelium collapsed with OPL. Scale bar: 20 μm.

Figure 4

Loss of rod bipolar neurons in Aipl1^−/− retina. Rod bipolar cell morphology was assessed in retina from WT (A–F; A’–F’) and Aipl1^−/− (G–L; G’–L’) animals at indicated ages using an antibody against PKCα. Arrows at top indicate loss of dendrites (H), radial orientation of rod bipolar cell soma (J, K) in Aipl1^−/− animals. Arrowhead at bottom indicates rod bipolar axonal endings that show stunted axonal arborization and appear as rudimentary dots in Aipl1^−/− animals (J'–L'). Scale bar: 20 μm. Average number (±SD) of rod bipolar cells in a retinal cross window of 133 × 133 μm as a function of age (M). ***P < 0.001 and ****P < 0.0001.

Figure 5

Dendritic changes in cone bipolar cells. Cone bipolar cells labeled with an antibody against secretagogin in WT (A–F) and Aipl1^−/− (G–L) retina. Dendrites are disorganized and retract in the Aipl1^−/− retina from P60 (J, arrows). At P100 and P150, arrows indicate disorganized cell bodies in the Aipl1^−/− retina (K, L). Scale bar: 20 μm. (M) Average number (±SD) of cone bipolar cells in a retinal cross window of 133 × 133 μm as a function of age. ***P < 0.001 and ****P < 0.0001.

Figure 6

Altered horizontal cell morphology in the degenerating retina. Calbindin staining reveals changes in the horizontal cell morphology in WT (A–F) and Aipl1^−/− (G–L) retina. Arrows point to the loss of puncta from OPL at P14 in retina from animals lacking Aipl1^−/− compared to controls (G). (J–L) Arrows indicate thinning of horizontal cell processes and abnormal orientation of cell bodies as animal ages. Scale bar: 20 μm.

Figure 7

Sprouting of horizontal cell axons in response to photoreceptor degeneration. Horizontal cell axon morphology was assessed at indicated ages using an antibody against neurofilament 200 in WT (A–F) and Aipl1^−/− (G–L) retina. Arrow points to sprouting of horizontal cell axonal branches in the Aipl1^−/− retina at P14 (G). Sprouting of horizontal cell axons was extensive at P30 to P60 ([I, J], arrow). At P100 to P150, arrowhead indicates axonal complexes that are sparse with uneven and large gaps in NF-200 staining pattern (K, L). Scale bar: 20 μm.

Figure 8

Formation of Müller glial seal in the Aipl1^−/− retina. Müller cells in the WT (A–F) and Aipl1^−/− (G–L) retina were identified with an antibody against glutamine synthetase (GS). Arrows indicate glial seal formation in the Aipl1^−/− retina at P30 (I). Uneven distribution of GS-stained cell bodies is indicated at P60 to P150 in the Aipl1^−/− retina (J–L). Scale bar: 20 μm.

Figure 9

Activation of Müller glia in the degenerating retina. Müller glial cell morphology and activation were assessed at indicated ages using an antibody against glial fibrillary acidic protein (GFAP; green) in WT (A–F) and Aipl1^−/− (G–L) retina. In Aipl1^−/− retina, GFAP immunoreactivity is found from nerve fiber layer to the outer retina. DAPI nuclear staining is shown in blue. Scale bar: 20 μm.

Figure 10

Morphology of amacrine cells in WT and Aipl1^−/− retina. Amacrine cell morphology was assessed in WT (A–F) and Aipl1^−/− (G–L) at indicated ages using an antibody against calretinin. Scale bar: 20 μm.

Figure 11

A scheme summarizing changes observed in the Aipl1^−/− retina. See text for details. PR, photoreceptors; CBC, cone bipolar cell; HC, horizontal cell.