We used DIGE proteomics technology to investigate the differences in the protein expression pattern of suicide compared to control brain samples. We detected a total of 2,465 spots (after exclusion of false spots) from the prefrontal cortex and 2,115 from the amygdala on the master gels, defined to be the gel containing the most spots. Representative gel is shown in . We performed the spot gel-to-gel matching with the DeCyder 6.5 software (GE Healthcare) BVA module and after careful and rigours manual validation we matched 681 spots in the prefrontal cortex samples and 696 in the amygdala samples. From these matched spots with the t
test and 1.3 fold change as cut off we found 46 significant differences between the control and suicide prefrontal cortex samples from which we could identify 84 proteins (see Table S1
). Regarding the amygdala, 16 matched spots showed significant differences, and 20 proteins were identified from them (see Table S2
). After FDR adjustment we had 27 significant spots in the prefrontal cortex and 9 significant spots in the amygdala. This way the number of protein “hits” in the proposed profile reduced to 59 proteins in the prefrontal cortex and 11 proteins in the amygdala (see bold-italic gene names in and ).
Functionally clustered protein changes in the prefrontal cortex.
Functionally clustered changes in proteins of the amygdala.
Clustering of proteins in the prefrontal cortex revealed the following categories: cytoskeleton, signalling, metabolism, protein processing, development, synapse and neuron, proteolysis, RNA/DNA metabolism, redox system, and glia cell marker (see ). Changes in the protein expression pattern of the amygdala were smaller, but they formed almost the same clusters as the cortical protein changes (see ). The direction of change in the two brain structures was the opposite for several proteins. We identified several proteins in more than one spot of the 2D gel, most likely due to posttranslational or post mortem
processing. Thus, whenever more than one arrow is included, they represent the number of spots in which the protein was identified; the direction of each arrow shows the direction of change in a certain spot (see and ). The numerical values of changes and p
-values of significance are shown in the Supplementary material (see Table S1
Functional protein clusters of the amygdala and prefrontal cortex demonstrated both similarities and differences in the brains of suicide victims compared to controls. Of the nine proteins whose levels were altered in both the brain structures (), three (actin (ACTB), glial fibrillary acidic protein (GFAP) and cathepsin D (CTSD)) showed altered levels in opposing directions; elevated in the amygdala and lower in the cortex.
Altered proteins in the prefrontal cortex and amygdala.
In an attempt to validate our proteomic results, western blot analysis was carried out on the proteins that showed opposing directions of change in the two brain structures as these proteins are the most promising biomarker protein candidates, e.g. for brain imaging PET probe targets. Expressions of cathepsin (p
0.0321) and GFAP (p
0.0192) were significantly decreased in the suicide prefrontal cortex samples compared to the control samples, while in the amygdala, the expression of cathepsin (p
0.0164) and GFAP (p
0.0383) significantly increased in suicide samples (). In case of the actin we also observed decreased level in the cortex and increased level in the amygdala of suicide samples although these changes were not significant because of high SD and low n ().
Western blot validation of GFAP, cathepsin and actin expressions in the cortex and amygdala of suicide and control subjects.
Another set of proteins displayed parallel changes in both brain structures: creatin kinase B-type (CKB), alpha-internexin (INA), neurofilament light polypeptide (NEFL), neurofilament medium polypeptide (NEFM), tubulin alpha-1B chain (TUBA1A) and heat shock cognate 71 kDa protein (HSPA8). We did not find proteins change simultaneously in the prefrontal cortex and amygdala in functional categories: signalling, redox system and development.
Interestingly, nearly half of the altered proteins in the wider data pool had already been identified as indicative factors of suicide risk (see and ). In our study, we identified 35 proteins from the cortex and 16 proteins from the amygdala that had been previously linked to schizophrenia. We also identified 21 protein changes from the cortex and 9 from the amygdala that are related to depression as well as 5 proteins from the cortex and 2 proteins from the amygdala mentioned in the suicide literature (see and ). In this study, we identified 43 proteins from the cortex and 2 proteins from the amygdala that have never been connected to schizophrenia or depression.