Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina

Abstract

Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague-Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for beta-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes.

A multistep validation process of biomarkers for preclinical drug development

“…We continue to confirm and validate further retinal gene and protein expression changes to gain deeper insight into DR disease processes. 29 In addition, this biomarker panel provides further support of the hypothesis that DR is a feed forward cycle of nutrient overload leading to chronic inflammation, neurodegeneration and compromised blood-retinal barrier function. 32 Future studies will use this biomarker panel in preclinical research of potential therapeutics to monitor disease models and gauge pharmacoefficacy, with the expectation that effective treatments will alter the DR gene expression biomarker panel signature.…”

“…At harvest, diabetic rats were hyperglycemic and underweight compared with controls as with previous assessments. 19,[27][28][29] Treatment with subcutaneous slow-release insulin pellets for the second half of the diabetic period reduced blood glucose levels, and diabetic animals receiving insulin weighed more than untreated diabetic animals. These data and the methods described are representative of experiments performed by the Penn State JDRF Diabetic Retinopathy Center Animal Models Core since 2003.…”

“…These data and the methods described are representative of experiments performed by the Penn State JDRF Diabetic Retinopathy Center Animal Models Core since 2003. 19,[27][28][29] Step 1: Defining pathological dysfunction The first step of the process links the biomarker panel with pathophysiological end points relevant to the disease model. We previously showed the time course of retinal tissue pathology (vascular permeability and apoptosis) in the STZ rat model of diabetes (Table 2).…”

Diabetic retinopathy: recent advances towards understanding neurodegeneration and vision loss

“…One explanation for these reductions is that a cumulative loss of amacrine or bipolar cells by apoptosis caused the reduction in tissue volume [1], but the loss of the inner plexiform layer suggests that there was also an accompanying reduction on the volume of dendrites and synaptic connections. To test this possibility the retinas of diabetic rats were labeled by immunofluorescence for several protein markers of synapses, including synaptophysin, which is an abundant presynaptic vesicle protein in retina and brain [43]. The results demonstrated that the protein content of synaptophysin and several other synaptic proteins was significantly reduced after 1 and 3 months of streptozotocin-induced diabetes in rats.…”

Neuropeptides, Trophic Factors, and Other Substances Providing Morphofunctional and Metabolic Protection in Experimental Models of Diabetic Retinopathy

“…The size and density of synaptophysin-immunoreactive puncta were reduced in the plexiform layers. The loss of synaptic proteins in retinal synaptosomes was accompanied by decreased mRNA content after 1 month of diabetes; therefore, diabetes may increase local degradation rate of presynaptic proteins at retinal synapses and also that of their mRNA (VanGuilder et al, 2008). Indeed, synaptophysin mRNA translation continues at a higher rate after the induction of diabetes as a compensatory mechanism of the reduction of the mature protein.…”