Functional proteomic analysis of an animal model of depression combining genetic vulnerability and environmental stress.

Genetic studies found no evidence of classic Mendelian inheritance for depression in humans, but showed an important influence of stress factors, such as adverse life events, on an individually variable background of genetic vulnerability. The Flinders Sensitive Line (FSL) rats are a well validated model of depression carrying genetic vulnerability associated to distinct features of pathology. FSL and Flinders Resistant Line (FRL, the corresponding control animal) rats were subjected to a maternal separation protocol (180rain/day from posmatal days 2-14) in order to investigate the outcome of gene-environment interaction. Drug treatment with the antidepressant (AD) escitalopram (10mg/kg/day), a selective serotonin reuptake inhibitor, was carried out at weeks 11-14 of age. Many of the biological targets of AD are localized at synapses; thus to reduce the complexity of the proteome analyzed and to enrich for less abundant proteins, purified nerve terminals (synaptosomes) from prefrontal/frontal cortex (P/FC) of FSL and FRL rats were used. Synaptosomes, from 8 rats per group, were purified by Percoll gradients and analyzed by twodimensional polyacrylamide gel electrophoresis (2DE). Statistical analysis of 2DE maps from P/FC synaptosomes revealed 37 proteins differently regulated in FSL vs. FRL rats. Stress of maternal separation significantly dysregulated 48 proteins in FSL, and 24 proteins in FRL P/FC synaptosomes. Chronic escitalopram treatment differently regulated 33 protein spots in FSL, and 7 protein spots in FSL subjected to maternal separation. Interestingly, in FSL rats 3 of the protein spots downregulated by maternal separation, were up-regulated by escitalopram treatment. Protein spots differently regulated in FRL vs. FSL by either treatment or maternal separation were excised from gels and submitted to identification by mass spectrometry analysis. In addition, protein phosphorylation, the most frequent posttranslational modification of protein, was investigated in the same subcellular fractions by means of Western Blot and immunostaining. Ca2+/calmodulin-dependent kinase II (CaMKII) and extracellular signal-regulated kinases 1/2 (ERK1/2) are protein kinases that have been involved in molecular mechanisms regulating synaptic plasticity,including long term potentiation and memory. Therefore, we investigated how maternal separation and antidepressant treatment affect CaMKII and ERK1/2 expression and phosphorylation in synaptosomes from both hippocampus (HI) and P/FC of FSL and FRL rats. Our results show that in P/FC synaptosomes, phosphorylated CaMKII levels where significantly different in FSL vs. FRL rats and maternal separation regulated CaMKII activation in opposite directions. ERK1/2 were basally hyperactivated in FSL vs. FRL hippocampal synaptosomes. In the same brain region, maternal separation induced a marked increase in ERK1/2 phosphorylation in FRL, whereas it did not significantly modified phosphorylated ERK1/2 levels in FSL rats. FSL and FRL rats displayed an opposite profile of activation for the two kinases investigated, suggesting that basic vulnerability is associated with a signaling dysfunction and that postnatal early adverse events (maternal separation) aggravate this dysfunction.