Optic nerve dynein motor protein distribution changes with intraocular pressure elevation in a rat model of glaucoma

doi:10.1016/j.exer.2005.11.025

Experimental Eye Research

Volume 83, Issue 2, August 2006, Pages 255-262

https://doi.org/10.1016/j.exer.2005.11.025 Get rights and content

Abstract

Acute intraocular pressure (IOP) elevation causes accumulation of retrogradely-transported brain derived neurotrophic factor and its receptor at the optic nerve head (ONH) in rats and monkeys. Obstruction of axonal transport may therefore be involved in glaucoma pathogenesis, but it is unknown if obstruction is specific to certain transported factors or represents a generalized failure of retrograde axonal transport. The dynein motor complex mediates retrograde axonal transport in retinal ganglion cells (RGC). Our hypothesis was that elevated IOP interferes with dynein-mediated axonal transport. We studied the distribution of dynein subunits in the retina and optic nerve after acute and chronic experimental IOP elevation in the rat. IOP was elevated unilaterally in 54 rats. Dynein subunit distribution was compared in treated and control eyes by immunohistochemistry and Western blotting at 1 day (n = 12), 3 days (n = 4), 1 week (n = 15), 2 weeks (n = 12) and 4 weeks (n = 11). For immunohistochemistry, sections through the ONH were probed with an anti-dynein heavy chain (HC) antibody and graded semi-quantitatively by masked observers. Other freshly enucleated eyes were microdissected for separate Western blot quantification of dynein intermediate complex (IC) in myelinated and unmyelinated optic nerve, ONH and retina. Immunohistochemistry showed accumulation of dynein HC at the ONH in IOP elevation eyes compared to controls (P < 0.001, Wilcoxon paired sign-rank test, n = 29). ONH dynein IC was elevated by 46.5% in chronic IOP elevation eyes compared to controls by Western blotting (P < 0.001, 95% CI = 25.9% to 67.8%, n = 17). The maximum increase in ONH dynein IC was 78.7% after 1 week (P < 0.05, n = 5), but significant increases were also detected after 4 h and 4 weeks of IOP elevation (P < 0.05, n = 4 rats per group). Total retinal dynein IC was increased by 8.7% in chronic IOP elevation eyes compared to controls (P < 0.03, 95% CI 1.4% to 16.1%, n = 24). In the retina, IOP elevation particularly affected the 72 kD subunit of dynein IC, which was 100.7% higher in chronic IOP elevation eyes compared to controls (P < 0.00001, 95% CI 71.0% to 130.4%, n = 21). Dynein IC changes in myelinated and unmyelinated optic nerve were not significant (P > 0.05). We conclude that dynein accumulates at the ONH with experimental IOP elevation in the rat, supporting the hypothesis that disrupted axonal transport in RGC may be involved in the pathogenesis of glaucoma. The effect of IOP elevation on other motor proteins deserves further investigation in the future.

Introduction

Glaucoma is the second leading cause of blindness in the world (Quigley, 1996). Although elevated intraocular pressure (IOP) is a major risk factor for retinal ganglion cell (RGC) death in glaucoma (Anderson, 1989, Sommer et al., 1991, Gramer and Tausch, 1995), the mechanisms of RGC death in the condition remain incompletely understood. Interruption to the retrograde axonal transport of neurotrophic factors has been postulated as a mechanism contributing to glaucomatous RGC death. This hypothesis has been supported by the observation that retrograde transport of brain-derived neurotrophic factor (BDNF) in association with its receptor (trkB) is obstructed at the optic nerve head in monkeys and rats with acutely elevated IOP (Pease et al., 2000). In addition, intravitreal delivery of BDNF, either by multiple injections of the protein (Ko et al., 2001) or by adeno-associated virus mediated gene therapy (Martin et al., 2003) has been shown to enhance RGC survival in experimental rat glaucoma, supporting the idea that neurotrophin deprivation may be involved in the pathogenesis of glaucoma. Yet it is uncertain if obstruction is specific to certain transported factors or represents a generalized failure of retrograde axonal transport.

The microtubule associated proteins (MAP) that serve as motor proteins for axonal transport within neuronal axons are dynein (retrograde transport), kinesin (predominantly orthograde transport), and other associated molecules (Melloni et al., 1995, Dillman et al., 1996, Muresan et al., 1996, Waterman-Storer et al., 1997). Alterations in axonal transport in both directions have been observed in experimental and human glaucoma (Minckler et al., 1977, Minckler et al., 1978). Ou et al. have reported qualitative alteration of labeling for unspecified microtubule-associated proteins in acute IOP elevation in guinea pig eyes (Ou et al., 1998). Hirokawa et al. found accumulation of dynein on either side of ligations made in axons (Hirokawa et al., 1990).

Cytoplasmic dynein is a large multi-subunit protein complex (Fig. 1). The 530 kD heavy chains contain the site of ATP hydrolysis and is the force-generating part of the protein. The 74 kDa intermediate chain subunit forms a bridge between the heavy chain and the dynactin subunits which bind microtubules (via dynamitin) and the cargo to be transported.

Our hypothesis was that disruption of dynein transport in glaucoma could contribute to a failure of retrograde axonal transport and thus contribute to RGC death. Furthermore, dynein is known to be associated with intracellular vesicles, such as those carrying activated trkB receptors within axons (Melloni et al., 1995, Dillman et al., 1996, Muresan et al., 1996, Waterman-Storer et al., 1997). Glaucoma could alter the ability of dynein to transport neurotrophin signalling molecules to the RGC. We compared dynein subunit distribution by immunohistochemistry and quantitative Western blotting in control eyes and eyes that had experimental IOP elevation for between 4 h and 4 weeks. We found significant accumulation of dynein in the optic nerve head and changes in the relative distribution of dynein subunits that may be relevant to the pathogenesis of glaucoma.

Section snippets

Animals used

A total of 54 male Wistar rats (375–425 g) were used for the experiments. All animals were treated in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research using protocols approved and monitored by the Animal Care Committee of the Johns Hopkins University School of Medicine. Animals were housed with a 14 h light/10 h dark cycle with standard chow and water ad libitum.

Intraocular pressure elevation

All procedures were carried out under anesthesia with animals were anesthetized with

Intraocular pressure exposure

IOP elevation was achieved in all laser-treated eyes. Peak IOP levels in experimental glaucoma eyes were approximately twice as high as in control eyes (Table 1). No eyes were excluded from analysis.

Immunohistochemistry

Significant changes in the localization of dynein HC were observed by immunohistochemistry (Fig. 2). Dynein HC labelling at the ONH was consistently more intense in eyes exposed to acute IOP elevation compared to control eyes (Figs. 2A,B). The increased labelling was seen throughout the ONH.

Discussion

In the ONH and retina of rat eyes exposed to elevated IOP, we found significant alterations in the distribution of the dynein motor protein complex by both immunohistochemistry and quantitative Western blotting. Changes were most marked four hours after acute IOP elevation to 25 mmHg above the mean ocular blood pressure but were also observed after chronic moderate IOP elevation in a well-verified experimental glaucoma model. These experiments demonstrate a potential cause of the retrograde

Acknowledgements

Supported in part by PHS Research Grants EY 02120 (Dr Quigley), EY 01765 (Core Facilities Grant, Wilmer Institute), The TFC Frost Trust, UK (Dr Martin), University College, Oxford UK (Dr Martin) and the GSK Clinical Fellowship Award (Dr Martin).

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Optic nerve dynein motor protein distribution changes with intraocular pressure elevation in a rat model of glaucoma

Abstract

Introduction

Section snippets

Animals used

Intraocular pressure elevation

Intraocular pressure exposure

Immunohistochemistry

Discussion

Acknowledgements

Am. J. Ophthalmol.

Anal. Biochem.

Methods

Neuron

Curr. Opin. Cell. Biol.

Neurosci. Lett.

J. Biol. Chem.

Cytoplasmic dynein is associated with slow axonal transport

Proc. Natl. Acad. Sci. U.S.A.

The risk profile of the glaucomatous patient

Curr. Opin. Ophthalmol.

Cargo-carrying motor vehicles on the neuronal highway: transport pathways and neurodegenerative disease