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T. S. D'Cruz, Z. Li, S. R. Kimball, A. J. Barber; Proteasomes and Eukaryotic Initiation Factors in Synapses Isolated from the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1034.
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© ARVO (1962-2015); The Authors (2016-present)
The content of retinal synaptic proteins synapsin I, phosphorylated synapsin I, synaptophysin, VAMP2, SNAP25 and PSD95 is significantly decreased after 1 month of streptozotocin (STZ) - diabetes in rats. Potential mechanisms for the reduction could be a reduction in mRNA translation or increased proteasomal degradation. The aim of this study was to test the hypothesis that translation initiation factors (eIF2 alpha and eIF2B epsilon) and proteasomes are present in isolated synapses (synaptosomes) of rat retinas.
Synaptosomes were isolated by hand homogenizing extracted retinal tissue in a sucrose buffer followed by three centrifugation steps. The first centrifugation pellet (pP1) consisted of nuclei and cell debris while the second (P1) contained cytoplasm. The final centrifugation pellet (P2) was comprised of synaptosomes. The synaptosome isolation technique was validated by assessing the distribution of synaptic and non-synaptic markers in the synaptosome pellets and supernatants. The content of eIF2 alpha, eIF2B epsilon, and the proteasome alpha subunit was determined by immunoblotting whole retina lysates as well as retinal synaptosomes, standardized to beta-actin content and expressed as % average control.
Synapsin I protein was abundant in the synaptosome pellet (P2) but as reduced in the cytoplasmic fraction (P1) and absent from the nuclear pellet (pP1). eIF2 alpha, eIF2B epsilon, and the proteasome alpha subunit were detected in diabetic and healthy whole retina. eIF2 alpha and the proteasome alpha subunit were also detected in isolated retinal synaptosomes.
The data show that eIF2 alpha, eIF2B epsilon, and the proteasome alpha subunit are present in synapses of the rat retina. This suggests the possibility that mRNA translation and proteasomal degradation be active within retinal synapses, and may be part of the mechanism of synaptic protein depletion in diabetes.
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