27 November 2006. One of several holy grails of schizophrenia research is biological markers that could help distinguish schizophrenia from other psychotic disorders, tease out possible subtypes, help predict who might be at risk for developing the disorder, and guide drug development. A team led by Sabine Bahn of the University of Cambridge in the United Kingdom reports in the November PLoS Medicine that they have identified potential biomarkers of schizophrenia in the cerebrospinal fluid of never-medicated, first-episode patients: an upregulation of a peptide from the protein VGF and a downregulation of peptides from the protein transthyretin. They also report a surprising 95 percent sensitivity in distinguishing schizophrenia samples from controls, along with specificity nearly as high.
Screening for biological markers of disease
Biomarkers, such as high blood levels of LDL cholesterol in cardiovascular disease or high blood glucose levels in diabetes, are indicators of a disease state or an increased risk for disease. Even though they may not play a causative role, these molecules are usually linked to the ongoing pathophysiology and can provide information about the mechanisms of disease.
To identify biomarkers of schizophrenia, first author Jeffrey Huang of Cambridge, with collaborators at the University of Cologne in Germany, the Babraham Institute in Cambridge, U.K. and Ciphergen Biosystems in the U.K. relied on a technique called surface-enhanced laser desorption ionization (SELDI) mass spectrometry. SELDI mass spectrometry uses special surfaces to capture proteins and peptides in samples of body fluid while a time-of-flight mass spectrometer is used to quantitate and measure the molecular weights of these compounds. Hundreds of fluid samples can be run at once.
The range of detection is from peptides of less than 1,000 Da up to proteins of 500 kDa. (For a review of SELDI mass spectrometry and its applications, see Tang et al., 2004.) Huang and colleagues used this technology to analyze cerebrospinal fluid (CSF) samples from 41 first-onset, drug-naïve, paranoid schizophrenia patients and 40 demographically matched healthy volunteers. The researchers controlled for various demographic factors, including drugs of abuse.
VGF more abundant in the CSF of schizophrenics
The authors found that a 3,959-Da peptide was 2.8-fold more abundant in samples from schizophrenia patients than it was in samples from controls (P = 10-8); this peptide was subsequently eluted, sequenced, and matched to amino acids 23–62 of the VGF protein. VGF is a neurosecretory protein that is selectively expressed in neurons in brain, particularly in the hypothalamus, and it appears to regulate metabolism (Hahm et al., 1999) and synaptic plasticity (Alder et al., 2003). (For a review of VGF, see Levi et al., 2004; Salton et al., 2000.)
Of note, the researchers were able to elute and sequence a second peptide fragment from VGF with a molecular weight of 3,690 Da. Unlike the 3,959-Da peptide, the amount of the 3,690-Da peptide did not differ significantly between control samples and samples from schizophrenia patients. Both peptides were exactly homologous, save that three amino acids at the N-terminus were absent from the 3,690-Da peptide. According to the authors, “This
indicates that the 40-amino-acid VGF peptide with the ‘APP’ sequence in the amino terminus may have specific functions and/or may be linked to the pathophysiology of schizophrenia.”
To follow up on their VGF findings, Huang and colleagues examined VGF protein expression in schizophrenic postmortem brain tissue—fresh-frozen prefrontal cortex tissue (Brodmann area 9) from gray matter of eight schizophrenics and eight matched controls. A
Western blot analysis with an antibody that recognized the C-terminal sequences of VGF showed strong expression in half of the schizophrenic patients, while no expression of VGF was detected in control brains.
In thinking about the clinical relevance of the VGF findings, Huang and colleagues point out that knocking out the VGF gene in mice produces a lean, hypermetabolic, hyperactive phenotype, implicating VGF in the regulation of energy balance (Hahm et al., 1999). They suggest that “the observation of an increase of the VGF peptide in CSF from patients with schizophrenia, therefore, may point to a hypometabolic state in the schizophrenia brain.” This may or may not be linked to decreased metabolic activity in the prefrontal cortex, or “hypofrontality,” during cognitive activation in patients with schizophrenia (Volz et al., 1999; Meyer-Lindenberg, et al., 2002).
Transthyretin less abundant in the CSF of schizophrenics
In contrast to the upregulation of VGF, the authors found that three peptides from the protein transthyretin were significantly less abundant in the CSF of schizophrenics. Transthyretin is a thyroid hormone-binding protein that transports thyroxine from the bloodstream to the brain (see Schreiber, 2002). The lower levels of the peptides in schizophrenia could not be explained by demographic variables, according to the authors. A possible effect of cannabis use as detected by urine-positive drug screen was ruled out after a two-way ANOVA analysis found no correlation.
Blood transthyretin, largely derived from the liver, contributes to the supply of brain transthyretin. Huang and colleagues also found a significant decrease in serum transthyretin levels; however, they note that no correlation was detected between absolute levels in CSF and those in serum (in opposition to the results of Reiber, 2001), “suggesting that the liver-derived transthyretin may not contribute to the downregulation in CSF.”
As with VGF, postmortem brain studies supported the SELDI mass spectrometry results. The authors found a 40 percent downregulation of transthyretin levels in postmortem prefrontal cortex from patients with schizophrenia by Western blot.
Citing evidence linking transthyretin to the pathophysiology of schizophrenia and other psychiatric diseases, Huang and colleagues note that the reduced levels of transthyretin in the CSF, serum, and brains of schizophrenia patients suggest a lower level of thyroxine transport. “It is noteworthy that thyroid dysfunction is relatively common in patients with schizophrenia (Morley and Shafer, 1982; Ryan et al., 1994) and indeed with other psychiatric disorders (Kirkegaard and Faber, 1998), possibly genetically linked to the disorders. In addition, in patients with severe forms of both hypo- and hyperthyroidism, psychotic symptoms may occur, and the clinical picture frequently resembles that of schizophrenia (Hall et al., 1986, see ref. below), which may imply that an increase in central nervous system thyroxine function may be linked, the authors write. They also point out that long-term administration of clozapine has been implicated in enhancing central nervous system thyroxine function (Chen and Chen, 2007). (In another recent paper, Wan and colleagues  report alterations similar to those seen by Huang and colleagues, though in a tetramer of transthyretin and in response to treatment with the first-generation antipsychotic chlorpromazine.)
To validate their results, the researchers replicated the SELDI mass spectrometry experiments with a second sample of 17 first-onset, drug-naïve schizophrenia patients and 40 demographically matched healthy volunteers, with similar results. “This suggests that these identified alterations in CSF proteins and peptides are a consistent finding and thus may reflect genuinely the early pathophysiology of schizophrenia.” They report that the sensitivity of the overall profile of peptide changes to distinguish schizophrenia from control samples is 80 percent and 88 percent for the original and replication samples, respectively; the sensitivity was found to be 95 percent in both samples.
Disease specificity of the biomarker panel
Lastly, Huang and colleagues made an effort to demonstrate the disease specificity of the VGF and transthyretin results by testing CSF samples from 16 patients with depression and five patients with obsessive-compulsive disorder (OCD) along with CSF samples from another 40 healthy volunteers. Among patients with depression, the VGF peptide (amino acids 23-62) was upregulated and a peptide from the protein secretogranin II was downregulated compared with controls. No significant difference in transthyretin levels was noted. Among the patients with OCD, no differences were seen compared with controls. Additionally, 10 patients with Alzheimer disease and 10 matched controls were tested, with no significant differences found.
“These results indicate that the VGF peptide alone may not be a specific marker for a given psychiatric disease (possibly due to overlapping disease processes, which are not least implied by the fact that patients with a family history of affective
disorder have an increased risk for developing schizophrenia, and vice versa),” the authors write.
To see if their biomarkers might indicate psychosis, not just schizophrenia, they divided their depression patients into those with (n = 3) and without (n = 13) psychotic symptoms. They found no significant differences in the CSF analysis, possibly suggesting that VGF upregulation plus transthyretin downregulation is a biomarker panel of schizophrenia, not just psychosis. They admitted, however, that the sample size of three patients with psychotic depression is too small for any conclusions to be made with real certainty.—Jillian Lokere.
Huang JT, Leweke FM, Oxley D, Wang L, Harris N, Koethe D, Gerth CW, Nolden BM, Gross S, Schreiber D, Reed B, Bahn S. Disease Biomarkers in Cerebrospinal Fluid of Patients with First-Onset Psychosis. PLoS Med. 2006;3(11) [Epub ahead of print]
Hall RCW, Stickney S, Beresford TP (1986) Endocrine disease and behavior.
Integr Psychiatry 4: 122–135. No abstract available.