Synaptoproteomic analysis of a rat gene-environment model of depression reveals involvement of energy metabolism and cellular remodeling pathways

Abstract

Background: Major depression is a severe mental illness that causes heavy social and economic burdens worldwide. A number of studies have shown that interaction between individual genetic vulnerability and environmental risk factors, such as stress, is crucial in psychiatric pathophysiology. In particular, the experience of stressful events in childhood, such as neglect, abuse, or parental loss, was found to increase the risk for development of depression in adult life. Here, to reproduce the gene x environment interaction, we employed an animal model that combines genetic vulnerability with early-life stress.

Methods: The Flinders Sensitive Line rats (FSL), a validated genetic animal model of depression, and the Flinders Resistant Line (FRL) rats, their controls, were subjected to a standard protocol of maternal separation (MS) from postnatal days 2 to 14. A basal comparison between the two lines for the outcome of the environmental manipulation was performed at postnatal day 73, when the rats were into adulthood. We carried out a global proteomic analysis of purified synaptic terminals (synaptosomes), in order to study a subcellular compartment enriched in proteins involved in synaptic function. Two-dimensional gel electrophoresis (2-DE), mass spectrometry, and bioinformatic analysis were used to analyze proteins and related functional networks that were modulated by genetic susceptibility (FSL vs. FRL) or by exposure to early-life stress (FRL + MS vs. FRL and FSL + MS vs.

Fsl) results: We found that, at a synaptic level, mainly proteins and molecular pathways related to energy metabolism and cellular remodeling were dysregulated.

Conclusions: The present results, in line with previous works, suggest that dysfunction of energy metabolism and cytoskeleton dynamics at a synaptic level could be features of stress-related pathologies, in particular major depression.

Keywords: early-life stress; frontal cortex; hippocampus; prefrontal cortex; proteomics; synaptosome.

Figure 1.

Schematic representation of the experimental design. Flinders Resistant Line (FRL) and Flinders Sensitive Line (FSL) rats were separated from the dam for 180min/day from postnatal day (PND) 2 to PND 14. Control FSL and FRL rats were not separated. All rats were weaned at PND 22. On PND 73 animals were sacrificed and prefrontal and frontal cortices (PFC/FC) and hippocampi (HPC) were removed.

Figure 2.

Representative two-dimensional gel electrophoresis gel images of Sypro-Ruby-stained synaptosomal proteins from (A) prefrontal and frontal cortices or (B) hippocampi in FSL rats. Circles and numbers indicate differently-regulated proteins spots (see also Tables 2 and 3).

Figure 3.

Bioinformatic analysis in the prefrontal and frontal cortices. Major biological functions and cellular processes most relevant to the proteins differently expressed in: (A) Flinders Sensitive Line (FSL) vs. Flinders Resistant Line (FRL), (C) FRL after maternal separation (MS), and (E) FSL after MS, as calculated by Ingenuity Pathways Analysis software. Canonical pathways most relevant to the proteins differently expressed in: (B) FSL vs. FRL, (D) FRL after MS, and (F) FSL after MS. Biological functions and canonical pathways are listed based on higher statistical significance [-log(p value)], with a threshold set at 1.3.

Figure 4.

Bioinformatic analysis in the hippocampus. Major biological functions and cellular processes most relevant to the proteins differently expressed in: (A) Flinders Sensitive Line (FSL) vs. Flinders Resistant Line (FRL), (C) FRL after maternal separation (MS), and (E) FSL after MS, as calculated by Ingenuity Pathways Analysis software. Canonical pathways most relevant to the proteins differently expressed in: (B) FSL vs. FRL, (D) FRL after MS, and (F) FSL after MS. Biological functions and canonical pathways are listed based on higher statistical significance [-log(p value)], with a threshold set at 1.3.

Figure 5.

Significant pathway networks based on the proteins differently expressed between (A) Flinders Sensitive Line (FSL) vs. Flinders Resistant Line (FRL), (B) FRL after maternal separation (MS), and (C and D) FSL after MS, in prefrontal and frontal cortices. Protein nodes with a colored background correspond to the identified proteins in the various comparisons, while other proteins were added from the Ingenuity database. The intensity of node colors indicates the degree of upregulation (red) or downregulation (green). Fischer’s exact test was used to calculate a p-value determining the probability that each biological function assigned to that network is due to chance alone.

Figure 6.

Significant pathway networks based on the differently expressed protein between (A) Flinders Sensitive Line (FSL) vs. Flinders Resistant Line (FRL), (B and C) FRL after maternal separation (MS), and (D) FSL after MS, in the hippocampus. Protein nodes with a colored background correspond to the identified proteins in the various comparisons, while other proteins were added from the Ingenuity database. The intensity of node colors indicates the degree of upregulation (red) or downregulation (green). Fischer’s exact test was used to calculate a p-value determining the probability that each biological function assigned to that network is due to chance alone.

Figure 7.

Validation of two-dimensional gel electrophoresis results. (A) N-ethylmaleimide sensitive factor (NSF), (B) ATP synthase α, (C) aconitate hydratase, and (D) Syntaxin-binding protein 1 protein levels were analyzed in prefrontal and frontal cortices synaptosomes of Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL) after maternal separation (MS) by Western blot. Protein levels were normalized to β-actin and expressed as % relative to FSL rats. Data are presented as mean ± standard error of the mean (SEM; n = 6–8). Mann-Whitney test, *p < 0.01; **p < 0.001. (E) Dihydropyrimidinase-related protein 2 (DRP-2) protein levels were analyzed in hippocampus (HPC) synaptosomes of FRL and FSL by Western blot. Protein levels were normalized to β-actin and expressed as % relative to FRL rats. Data are presented as mean ± SEM (n = 9–12). Mann-Whitney test, **p < 0.001. (F) Synaptosomal-associated protein 25 (SNAP-25) protein levels were analyzed in HPC synaptosomes of FRL and FRL after MS by Western blot. Protein levels were normalized to β-actin and expressed as % relative to FRL rats. Data are presented as mean ± SEM (n = 10–11). Mann-Whitney test.