OPA1 expression in the normal rat retina and optic nerve - PubMed (original) (raw)

OPA1 expression in the normal rat retina and optic nerve

Won-Kyu Ju et al. J Comp Neurol. 2005.

Abstract

Autosomal dominant optic atrophy (DOA) is the most common form of hereditary optic neuropathy. DOA presents in the first decade of life and manifests as progressive vision loss. In DOA retinal ganglion cells and the optic nerve degenerate by an unknown mechanism. The gene mutated in DOA, Optic Atrophy Type 1 (OPA1), encodes a dynamin-related GTPase implicated in mitochondrial fusion and maintenance of the mitochondrial network and genome. Here, we determine which cell types in the normal retina and the optic nerve express OPA1. In the normal rat retina, OPA1 is expressed in the ganglion cell layer as well as in the outer plexiform layer, the inner nuclear layer, and the inner plexiform layer. In the ganglion cell layer, OPA1 is expressed predominantly in retinal ganglion cells. By contrast, OPA1 protein is low or undetectable in astrocytes and oligodendrocytes of the optic nerve. Additionally, OPA1 protein is present in axonal mitochondria. Last, OPA1 expression is present in mitochondria of processes and cell bodies of purified retinal ganglion cells and of the RGC-5 cell line. Thus, OPA1 is predominantly expressed in retinal ganglion cells of the normal rat retina and axons of the optic nerve. These findings may explain the selective vulnerability of retinal ganglion cells to OPA1 loss of function.

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Figures

Fig. 1

A: OPA1 expression in the normal retina and optic nerve. Immunoblot of extracts from mouse brain, rat retina, and optic nerve probed with OPA1 antibody. B: OPA1 expression in the normal retinal mixed cultures and RGC-5 cells shown by immunoblotting. The arrows show the positions, based on comparison with size standards, of the 90 and 80 kDa forms of OPA1. C: Immunoblotting of recombinant human OPA1 protein using OPA1 antibody. D: HeLa cells transfected with scramble II, control siRNA (a), or with OPA1 siRNA (b). Mitochondrial morphology and nuclei were identified by colabeling with MitoTracker Red and Hoechst. (c) OPA1 immunoblot of extracts from HeLa cells transfected with scramble II siRNA or OPA1 siRNA. The blot was stripped and reprobed with anti-actin antibody to ensure similar protein loading. Scale bar = 10 μm in a (applies to a,b).

Fig. 2

Cellular localization of OPA1 in the normal rat retina. A: Control. When the primary antibody was omitted, there was no binding of the secondary antibody. A1: Immunohistochemistry of rat retina using OPA1 antibody preabsorbed with OPA1 peptide. No OPA1 immunoreactivity was detected. B–G: OPA1 (B,E; green) and cytochrome c (C,F; red) double immunohistochemistry. OPA1 immunoreactivity (OPA1-IR) was seen in the OPL, INL, IPL, and GCL. Note that neurons were positive for OPA1 in the OPL (concave arrow) and GCL (arrow). Cytochrome c, a marker for the mitochondrial intermembrane space, colocalized with OPA1 (D). Higher magnification showed that OPA1-IR is present in horizontal cells (concave arrowhead) in the OPL (E) and OPA1-IR colocalized with cytochrome c-IR in cells with large (large arrowhead) or small (small arrowhead) sized soma (J). K,L: OPA1 (K, HRP-DAB) and Fluoro-Gold (L) double labeling. Neurons containing OPA1-IR were colabeled by Fluoro-Gold (arrowheads), indicating that retinal ganglion cells in the GCL contained OPA1 protein. HRP-DAB, horseradish peroxidase-diaminobenzidine; IR, immunoreactivity; PR, photoreceptor; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 20 μm in D (applies to A–D), G (applies to E–G), J (applies to H–J), L (applies to K,L).

Fig. 3

OPA1 immunoreactivity in cultured retinal ganglion cells and RGC-5 cells. A: OPA1 immunocytochemistry. OPA1-IR was present at high levels in the processes (arrowheads) and cell body (arrow) in a retinal ganglion cell. B–J: OPA1 (B,E,H) and DsRed2-Mito (C,F,I) double labeling. Higher magnification shows that OPA1 was present in DsRed-Mito-labeled mitochondria with long tubular forms in RGC-5 cells (merge, D,G,J; arrowheads). Scale bars = 100 μm in A; 20 μm in D (applies to B–D); 10 μm in G (applies to G–E), J (applies to H–J).

Fig. 4

Cellular localization of OPA1 in the normal rat optic nerve. A–F: OPA1 (A,D) and GFAP (B,E) double immunohistochemistry. Cells positive for OPA1 were not positive for GFAP, an astrocyte marker (F). G–I: OPA1 (G) and O4 (H) double immunohistochemistry. OPA1 cells were not positive for O4, an oligodendrocyte marker (I). J–L: OPA1 (J) and neurofilament (K) double immunohistochemistry. OPA1-positive cells were also positive for neurofilament, indicating that OPA1 was present in axons of the optic nerve (L). Scale bars = 100 μm in C (applies to A–C); 20 μm in L (applies to D–L).

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OPA1 expression in the normal rat retina and optic nerve - PubMed (original) (raw)