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German Team Uses Proteomic Methods to Identify Three Proteins Linked to Suicide

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Researchers in Germany have used proteomic methods to identy three proteins that may be linked to suicide in humans.
 
Previous efforts to establish a biological link to suicide have suggested that low levels of serotonin in the brain can lead to depression and suicidal behavior. But attempts at determining a genetic association with suicidal behavior have been inconsistent, especially in comparison to results looking at a connection between such behavior and the presence of certain proteins, the authors wrote in their study, published in the September issue of the Journal of Psychiatric Research.
 
“Therefore, an intensified investigation of suicide associated alterations in protein expression might be helpful to identify new candidate genes,” they wrote.
 
The researchers chose to study the proteome because it “is a highly dynamic and variable entity which is very vulnerable to various intra- and extracellular influences.” As a result, “the variety of factors that trigger suicidal behavior might predominantly affect the proteome rather than the relative[ly] stable genome and result in alterations of protein expression.”
 
The researchers compared postmortem brain tissue of 17 suicide victims with the brain tissue of nine controls who died from accidents or heart disease. Samples were taken from the prefrontal cortex, which governs an individual’s ability to differentiate among conflicting thoughts, determine good and bad, anticipate future consequences of current activities, predict outcomes, and control the ability to suppress urges that, if not suppressed, could lead to socially unacceptable outcomes, such as suicide.
 
The team also used that portion of the brain because other studies have shown the region to have “many important results associated with suicidality,” the researchers said. Cerebellum samples, commonly used in postmortem studies, were also used.
 
The researchers used 2D gel electrophoresis to separate proteins and used MALDI-TOF mass spectrometry to analyze identified proteins.
 
The authors identified six proteins that differed significantly between suicide victims and controls. Of them, three — a glial fibrillary acid protein, a manganese superoxide dismutase, and an alpha-crystallin chain B — were found in the tissue samples of the suicides but not in the controls.
 
In the paper, Brigitta Bondy at Psychiatric Hospital of the Ludwig-Maximilians University in Munich and the Max Planck Institute in Martinsried, Germany, and the lead researcher for the study, said that though the relationship between the proteins and suicidal behavior needs further research, “Our data further suggest that proteomic analyses may provide a useful tool to identify disease or symptom associated alterations on the protein level.”
 
Worldwide, about 1 million people committed suicide in 2000, according to the World Health Organization. In the US, suicide was the 11th leading cause of death in 2004, increasing 6 percent to 32,439 fatalities from 30,622 deaths in 2001, according to the National Institute of Mental Health. In addition, between eight and 25 suicides are attempted for every successful suicide.
 
Proteomic Pathway to Suicide?
 
The 17 samples from suicide victims consisted of 10 men and seven women, while the nine controls comprised seven men and two women. All samples were gathered from the University of Munich’s Institute of Legal Medicine.
 
Protein content was determined using Bio-Rad Laboratories’ protein assay and 2D gel electrophoresis was performed. Bio-Rad’s PDQuest software helped researchers to spot-edit, detect, quantify, and match the proteins.
 
Sample preparation for mass spec analysis was performed according to a method developed by Andreas Tebbe and others and described in a 2005 article in Proteomics. Spots of interest were excised, then transferred to filter microtiter plates and destained.
The destaining solution was removed and gel pieces were treated with 50 percent acetronitrile and 50 microMoles of ammonium hydrogen carbonate for 10 minutes each.
 

“The variety of factors that trigger suicidal behavior might predominantly affect the proteome rather than the relative[ly] stable genome and result in alterations of protein expression.”

The team used Bruker Daltonics’ Reflex III MALDI-TOF MS to measure mass spectra, and used the Mascot search engine to match peptide mass fingerprint against a mass spec database. They measured the validity of the protein-identification results using the probability-based Mowse score of Mascot.
 
In total, the researchers detected 1,200 protein spots per gel, of which a mean of 820 spots matched against the master gel. In the cerebellum tissue samples, researchers found no significant difference in the intensities of spots. But in the prefrontal cortex samples, six spots showed significant difference between the samples of suicide victims and controls. Three of them were found to be expressed only in suicide victims.
 
One spot, the glial fibrillary acid protein, or GFAP, is a protein associated with the regulation of neuronal function and survival. It has been linked to neurodegenerative disorders.
 
The authors detected two isoforms of GFAP and suggest one may be a phosphorylated isoform of the protein. Previous studies have suggested that the phosphorylation site of GFAP is associated with psychiatric disorders.
 
“The present findings may reveal a higher phosphorylation state of GFAP in the prefrontal cortices of suicide victims, and therefore might also have impact on the pathopsychology of suicidal behavior,” they said.
 
A second protein, manganese superoxide dismutase, is an antioxidant that protects against oxidative stress damage in cells. It has also been associated with schizophrenia and biopolar disorder. The authors said it is unclear whether the protein is an isoform or a post-translational modification of the enzyme. It is possible that in suicide victims, the protein was less stable or already affected by oxidative stress, they said.
 
The third protein, alpha-crystallin chain B, is a low-molecular-weight shock protein that functions as a molecular chaperone, the authors wrote in the study. Other research has suggested that prolonged exposure to mental stress could elevate levels of this protein. However, Bondy and her colleagues say it is unknown what relationship, if any, it may have with suicidal behavior.
 
Taken together, however, the presence of the three proteins in the tissue of suicide victims but not in controls suggests a neurobiological link to suicidal behavior, in particular in the serotonin system.
 
“Although the mechanisms of the interactions have to be elucidated, an astroglia-mediated alteration in serotonergic function might be proposed, referring on reports of previous animal studies,” the authors wrote.
 
Bondy did not respond to several requests for an interview.