Multi-Modal Proteomic Analysis of Retinal Protein Expression Alterations in a Rat Model of Diabetic Retinopathy

As a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.A multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated.These results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies

Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts [version 1; referees: 2 approved]

REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1-/- mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin

Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding

In vivo-determined rates of protein synthesis in gastrocnemius, heart and liver of male and female WT and 4E-BP1/BP2 DKO mice.

<p>Values are means ± SEM; n = 9–12 per group. For all bar graphs, values having a different superscript letter (a versus b) are statistically different (<i>P</i><0.05); values with the same letter are not significantly different. For each tissue, female mice had a smaller sepsis-induced decrease in protein synthesis (<i>P</i><0.05).</p

Amount of eIF4E•eIF4G complex as well as eIF4B and eEF2 phosphorylation in muscle from male WT and 4E-BP1/BP2 DKO mice under basal and septic conditions.

<p>Bar graphs are means ± SEM; n = 5 male mice per group (panel A) and 8–9 mice per group (panels B and C). For all bar graphs, values having a different superscript letter (a versus b) are statistically different (<i>P</i><0.05); values with the same letter are not significantly different.</p

Representative Western blots for various proteins important in the control of muscle protein synthesis.

<p>Immunoblots for total and/or phosphorylated AKT, 4E-BP1, 4E-BP2, and S6K1 are presented for both male and female WT and 4E-BP1/BP2 DKO mice. Each lane represents a sample from a different mouse. Western blots were quantitated for male mice only, but data for female mice were qualitatively similar (data not shown). Bar graphs are means ± SEM; n = 8 male mice per group. For all bar graphs, values having a different superscript letter (a versus b) are statistically different (<i>P</i><0.05); values with the same letter are not significantly different. ND  =  not detectable.</p

Sepsis-induced changes in the insulin-like growth factor (IGF)-I mRNA in gastrocnemius from male and female WT and 4E-BP1/BP2 DKO mice.

<p>Values are means ± SEM; n = 9–12 per group. Values are expressed as percent of control normalized for L32, where the male WT control value is arbitrarily set at 100%. For all bar graphs, values having a different superscript letter (a versus b) are statistically different (<i>P</i><0.05); values with the same letter are not significantly different.</p

Sepsis-induced body weight loss in male and female WT and 4E-BP1/BP2 DKO mice.

<p>Values are means ± SEM; n = 9–12 per group. For all bar graphs, values having a different superscript letter (a versus b) are statistically different (<i>P</i><0.05); values with the same letter are not significantly different.</p