Diabetes induces changes in KIF1A, KIF5B and dynein distribution in the rat retina: Implications for axonal transport

Highlights

• Kinesin and dynein motor proteins are altered in the retinas of diabetic rats.

• High glucose per se did not lead to changes in motor proteins in retinal neurons.

• Other factors, like inflammation, may contribute for the alterations in motor proteins.

Abstract

Diabetic retinopathy is a leading cause of vision loss and blindness. Disruption of axonal transport is associated with many neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated the impact of type 1 diabetes (2 and 8 weeks duration) on KIF1A, KIF5B and dynein motor proteins in the retina. Additionally, since hyperglycemia is considered the main trigger of diabetic complications, we investigated whether prolonged exposure to elevated glucose could affect the content and distribution of motor proteins in retinal cultures. The immunoreactivity of motor proteins was evaluated by immunohistochemistry in retinal sections and by immunoblotting in total retinal extracts from streptozotocin-induced diabetic and age-matched control animals. Primary retinal cultures were exposed to high glucose (30 mM) or mannitol (osmotic control; 24.5 mM plus 5.5 mM glucose), for seven days. Diabetes decreased the content of KIF1A at 8 weeks of diabetes as well as KIF1A immunoreactivity in the majority of retinal layers, except for the photoreceptor and outer nuclear layer. Changes in KIF5B immunoreactivity were also detected by immunohistochemistry in the retina at 8 weeks of diabetes, being increased at the photoreceptor and outer nuclear layer, and decreased in the ganglion cell layer. Regarding dynein immunoreactivity there was an increase in the ganglion cell layer after 8 weeks of diabetes. No changes were detected in retinal cultures. These alterations suggest that axonal transport may be impaired under diabetes, which might contribute to early signs of neural dysfunction in the retina of diabetic patients and animal models.

Introduction

Diabetic retinopathy is the most common microvascular complication of diabetes mellitus and is a leading cause of vision loss and blindness among working-age adults in Western countries. However, increasing evidence has shown that the neural components of the retina are also affected (Antonetti et al., 2006). Alterations in electroretinograms in diabetic patients and animals, and loss of color and contrast sensitivity are early signs of neural dysfunction in the retina (Roy et al., 1986, Daley et al., 1987, Sakai et al., 1995), demonstrating that the neural retina can be also affected by this disease.

Neurons are highly polarized cells, with long axons, which constitute a major challenge to the movement of proteins, vesicles, and organelles between cell bodies and presynaptic sites. To overcome this, neurons possess specialized transport machinery consisting of cytoskeletal motor proteins (kinesins and dynein) generating directed movements along cytoskeletal tracks. Axonal transport motor proteins require ATP demands, which implies the localization of functional mitochondria along the axons. Mobile mitochondria can become stationary or pause in regions that have a high metabolic demand and can move again rapidly in response to physiological changes. Defects in mitochondrial transport are implicated in the pathogenesis of several major neurological disorders (Sheng and Cai, 2012). Axonal transport is therefore crucial to maintain neuronal viability, and any impairment in this transport may play a role in the development or progression of several diseases (De Vos et al., 2008).

A decrease in the levels of mRNAs encoding for neurofilament proteins was found in the dorsal root ganglia of streptozocin-induced diabetic rats (Mohiuddin et al., 1995). Additionally, slow axonal transport of neurofilament and microtubule components is reduced, leading to a decrease in axonal caliber (Medori et al., 1988). These evidences suggest that deficits in axonal transport may contribute to neuronal changes observed in diabetes in neural tissues. To our knowledge, only a few studies have evaluated the effect of diabetes on axonal transport in the retina and most of them have focused in studying fluoro-gold labeling in retinal ganglion cells (RGCs) (Zhang et al., 1998, Ino-Ue et al., 2000, Zhang et al., 2000). Despite these evidences, the impact of diabetes in motor proteins (kinesins and dynein) in the retina has not been addressed. Nevertheless, potential changes in their content and distribution might underlie some changes already observed in axonal transport in the retina and visual pathway under diabetic conditions (Zhang et al., 2000, Fernandez et al., 2012).

Previously, we found that diabetes changes the levels of several synaptic proteins in retinal nerve terminals, with no changes in total retinal extracts, suggesting that axonal transport of those proteins may be impaired in diabetes (Gaspar et al., 2010a). Hyperglycemia is considered the main pathogenic factor for the development of diabetic complications. We found that high glucose leads to an accumulation of vesicular glutamate transporter-1, syntaxin-1 and synaptotagmin-1 at the cell body in hippocampal cell cultures, further suggesting that axonal transport of these proteins to nerve terminals might be affected under hyperglycemic conditions (Gaspar et al., 2010b). Recently, we showed that mRNA levels and the content of kinesin motor proteins are altered in the hippocampus of diabetic rats (Baptista et al., 2013). We also demonstrated that high glucose leads to changes in the immunoreactivity of motor proteins and synaptic proteins specifically in the axons of hippocampal neurons further suggesting that anterograde axonal transport may be impaired in the hippocampus (Baptista et al., 2013). These changes detected in the hippocampus of diabetic rats lead us to check whether similar changes could also be occurring in the retina under diabetes. Therefore, in this work, we aimed to study the effect of diabetes and also high glucose per se (prolonged exposure for 7 days), mimicking hyperglycemic conditions, on the content and distribution of the motor proteins KIF1A (kinesin that transports synaptic vesicle precursors), KIF5B (kinesin involved in mitochondrial transport and in the transport of synaptic vesicle precursors and membrane organelles) and dynein (motor protein for retrograde axonal transport) in diabetic animals and primary rat retinal cell cultures. Since motor proteins need ATP to carry cargoes along the axons, the distribution of mitochondria was also analyzed in retinal neural cell cultures exposed to high glucose.