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Aussie Researchers ID Proteins That May Play Role in Schizophrenia

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Researchers in Australia have used 2D gel electrophoresis and mass spectrometry to identify proteins and pathways that may play a role in schizophrenia.
 
In a study published in the October issue of Proteomics Clinical Applications, the research team, consisting of scientists from the Schizophrenia Research Institute in Sydney and the University of Sydney, identified 34 proteins expressed in a certain portion of the brain of schizophrenia patients.
 
The study also describes three pathways that may also contribute to the condition. While the authors of the study did not explicitly state the long-term goals of their work, much of the genetics-based research into the disease has been aimed at trying to explain its origins and underlying factors, so that, ultimately, better treatments can be found.
 
To arrive at their results, the Australian team studied the genu, or anterior end of the corpus callosum portion of the brain, which connects the left and right hemispheres.
 
They chose this region because earlier research had identified abnormalities in the brains of schizophrenia patients. In particular, researchers have noticed that individuals with schizophrenia have less left-right genu asymmetry than those without a history of the disease.
 
Working on the premise that biochemical differences in the corpus callosum may contain clues to the origin of schizophrenia, the researchers studied freshly frozen corpus callosum tissue from post-mortem brains of 10 individuals who had been diagnosed with schizophrenia and 10 controls to see if they could see any differences in the protein expression profile between the two groups.
 
The aim of the work was two-fold: to identify proteins and molecular mechanisms that may shed light on the “subtle neuropathology and functional impairment” of the corpus callosum; and “to explore molecular asymmetry” underlying hemisphere asymmetry in the corpus callosum of healthy individuals and changes in the asymmetry of individuals with schizophrenia.
 
To do so, they based their work on 2D gel electrophoresis and mass spectrometry techniques. They chose a proteomic approach to their research because it “offers a sensitive and efficient technology to examine the abundance of a larger number of proteins simultaneously,” even though they acknowledge limitations in the technology such as “poor representation of low abundant, hydrophobic proteins, and proteins of low and high molecular weight.”
 
Proteins in the Brain
 
The researchers obtained tissue samples from the New South Wales Tissue Resource Center. All cases of schizophrenia met the guidelines of the Diagnostic and Statistical Manual of Mental Disorders of the American Psychiatric Association.
 
They extracted proteins according to methods described by Danielle Clark and her colleagues at the University of Sydney in their study “A proteome analysis of the anterior cingulate cortex gray matter in schizophrenia,” published in the May 2006 issue of  Molecular Psychiatry available here. They then used the Bradford method to determine the protein concentration, using a complex human brain protein mixture as the standard.
 
The team used 2D gel electrophoresis to separate the proteins, and relied on Nonlinear Dynamics’ Phoretix Powerscan and Phoretix 2D Expression programs to scan and analyze them. Spots detected automatically were checked manually for consistency across all gels.
 
The researchers used Applied Biosystems’ QSTAR XL with an oMALDI ion source mass spec to manually generate peptide mass data. The resulting data were used to search the mammalian SwissProt, NCBI, and TrEMBL databases using the Aldente and Mascot programs.
 
They then used Pearson correlation analysis to correlate protein-expression levels and post-mortem factors such as age, brain pH, post-mortem interval, duration of illness, and medication. To determine the false discovery rate, the researchers used QVALUE software.
 

“I am convinced proteins play a major role … [and] proteins are still a major focus of study as again there are no answers in schizophrenia.”

Their analysis resulted in 751 and 786 protein spots matched to the reference gel in control and case subgels, respectively, with 681 protein spots identified in both control and schizophrenia groups.
 
Sixty-four protein spots representing 34 different proteins were found to be differentially expressed in the genu of the corpus callosum of the schizophrenia patients. Of the 64 spots, 45 representing 21 different proteins were down-regulated, including 13 spots that represented four different proteins that were down-regulated by 1.5-fold and greater.
 
Nineteen protein spots representing 14 different proteins and one unidentified spot were up-regulated, of which five spots representing three proteins and one unidentified protein were up-regulated by 1.5-fold or greater.
 
Using the Pearson correlation test, the researchers assessed changes in protein expression levels that may have been due to post-mortem factors. While a few proteins showed strong correlations with duration of illness or medication, the researchers concluded “the 64 differentially expressed protein spots represent true changes.”
 
Additionally, they identified 21 proteins and four unidentified protein spots that showed different expression levels between the left and right hemispheres in controls. In schizophrenia brains, those numbers were reduced to 13 proteins and three unidentified protein spots.
 
The researchers acknowledged that false positives could skew the results, but determined the study had a low false-positive rate. They did caution, however, that while their study may link some proteins to schizophrenia, it may be specific isoforms or post-translation modifications of the proteins that play a role in the disease.
 
“Particular attention should also be paid to isoforms and PTM in a brain region-specific manner,” they write in their study.
 
Lastly, they determined three functional pathways that may be impaired and thus contribute to the “neuropathology and functional impairment of the [corpus callosum] in schizophrenia patients:” compromised cytoskeleton structure/function; impaired antioxidant system; and decreased energy production.
 
The three pathways may explain some of the structural and functional abnormalities found in the corpus callosum of schizophrenia patients, and “seem to be associated with the reported asymmetry between the left and right hemisphere in control brains and reduced asymmetry in schizophrenia brains.”
 
Mystery Unsolved
 
Alfredo Bellon a postdoc fellow at Hospital Saint Anne in Paris who has studied the genetics of schizophrenia, and who is not involved in the Proteomics Clinical Applications study, said he is “convinced proteins play a major role … [and] proteins are still a major focus of study as again there are no answers in schizophrenia.”
 
Still, the work of the Australian team falls far short of unraveling the complexity of schizophrenia. Bellon said that while interesting, the study left many questions unanswered. Schizophrenia is a mysterious illness and the study’s results, even if validated, would not explain the causes or mechanisms of the disease.
 
“Nobody has done it so far,” Bellon said.
 
Additionally, it was unclear to him why the researchers chose to study the corpus callosum “as opposed to other brain areas with a lot more significance to schizophrenia such as the hippocampus or the prefrontal cortex,” and how factors not mentioned in the paper, such as a patient’s right-handedness or left-handedness, education, and gender may have influenced the results.
 

Sinthuja Sivagnanasundaram, the lead author on the Proteomics Clinical Applications study, did not respond to an e-mail requesting comment. In the study, she and her colleagues acknowledge that their work is only the basis of further research, but that their research nonetheless “opens up avenues for further investigation of molecular mechanisms involving the [corpus callosum] in schizophrenia pathogenesis and symptoms.”

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