F M Leweke

Identification of a biological signature for schizophrenia in serum

Biomarkers are now used in many areas of medicine but are still lacking for psychiatric conditions such as schizophrenia (SCZ). We have used a multiplex molecular profiling approach to measure serum concentrations of 181 proteins and small molecules in 250 first and recent onset SCZ, 35 major depressive disorder (MDD), 32 euthymic bipolar disorder (BPD), 45 Asperger syndrome and 280 control subjects. Preliminary analysis resulted in identification of a signature comprised of 34 analytes in a cohort of closely matched SCZ (n=71) and control (n=59) subjects. Partial least squares discriminant analysis using this signature gave a separation of 60–75% of SCZ subjects from controls across five independent cohorts. The same analysis also gave a separation of ∼50% of MDD patients and 10–20% of BPD and Asperger syndrome subjects from controls. These results demonstrate for the first time that a biological signature for SCZ can be identified in blood serum. This study lays the groundwork for development of a diagnostic test that can be used as an aid for distinguishing SCZ subjects from healthy controls and from those affected by related psychiatric illnesses with overlapping symptoms.

Increased levels of circulating insulin-related peptides in first-onset, antipsychotic naïve schizophrenia patients

Increased levels of circulating insulin-related peptides in first-onset, antipsychotic naive schizophrenia patients

High Throughput Lipidomic Profiling of Schizophrenia and Bipolar Disorder Brain Tissue Reveals Alterations of Free Fatty Acids, Phosphatidylcholines, and Ceramides

A mass spectrometry based high throughput approach was employed to profile white and gray matter lipid levels in the prefrontal cortex (Brodmann area 9) of 45 subjects including 15 schizophrenia and 15 bipolar disorder patients as well as 15 controls samples. We found statistically significant alterations in levels of free fatty acids and phosphatidylcholine in gray and white matter of both schizophrenia and bipolar disorder samples compared to controls. Also, ceramides were identified to be significantly increased in white matter of both neuropsychiatric disorders as compared to control levels. The patient cohort investigated in this study includes a number of drug naive as well as untreated patients, allowing the assessment of drug effects on lipid levels. Our findings indicate that while gray matter phosphatidylcholine levels were influenced by antipsychotic medication, this was not the case for phosphatidylcholine levels in white matter. Changes in free fatty acids or ceramides in either white or gray matter also did not appear to be influenced by antipsychotic treatment. To assess lipid profiles in the living patient, we also profiled lipids of 40 red blood cell samples, including 7 samples from drug naive first onset patients. We found significant alterations in the concentrations of free fatty acids as well as ceramide. Overall, our findings suggest that lipid abnormalities may be a disease intrinsic feature of both schizophrenia and bipolar disorder reflected by significant changes in the central nervous system as well as peripheral tissues.

Independent protein-profiling studies show a decrease in apolipoprotein A1 levels in schizophrenia CSF, brain and peripheral tissues

Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI–TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (−35%, P=0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (−30%, P=0.02) and RBCs (−60%, P=0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (−18%, P=0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (−35%, P=0.05; −51%, P=0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.

Global proteomic profiling reveals altered proteomic signature in schizophrenia serum

Schizophrenia is one of the most severe psychiatric disorders affecting 1% of the world population. There is yet no empirical method to validate the diagnosis of the disease. The identification of an underlying molecular alteration could lead to an improved disease understanding and may yield an objective panel of biomarkers to aid in the diagnosis of this devastating disease. Presented is the largest reported liquid chromatography-mass spectrometry-based proteomic profiling study investigating serum samples taken from first-onset drug-naive patients compared with samples collected from healthy volunteers. The results of this large-scale study are presented along with enzyme-linked immunosorbent assay-based validation data.

Impaired glycolytic response in peripheral blood mononuclear cells of first onset antipsychotic-naive schizophrenia patients

Little is known about the biological mechanisms underpinning the pathology of schizophrenia. We have analysed the proteome of stimulated and unstimulated peripheral blood mononuclear cells (PBMCs) from schizophrenia patients and controls as a potential model of altered cellular signaling using liquid-chromatography mass spectrometry proteomic profiling. PBMCs from patients and controls were stimulated for 72 h in vitro using staphylococcal enterotoxin B. In total, 18 differentially expressed proteins between first-onset, antipsychotic-naive patients and controls in the unstimulated and stimulated conditions were identified. Remarkably, eight of these proteins were associated with the glycolytic pathway and patient–control differences were more prominent in stimulated compared with unstimulated PBMCs. None of these proteins were altered in chronically ill antipsychotic-treated patients. Non-linear multivariate statistical analysis showed that small subsets of these proteins could be used as a signal for distinguishing first-onset patients from controls with high precision. Functional analysis of PBMCs did not reveal any difference in the glycolytic rate between patients and controls despite increased levels of lactate and the glucose transporter-1, and decreased levels of the insulin receptor in patients. In addition, subjects showed increased serum levels of insulin, consistent with the idea that some schizophrenia patients are insulin resistant. These results show that schizophrenia patients respond differently to PBMC activation and this is manifested at disease onset and may be modulated by antipsychotic treatment. The glycolytic protein signature associated with this effect could therefore be of diagnostic and prognostic value. Moreover, these results highlight the importance of using cells for functional discovery and show that it may not be sufficient to measure protein expression levels in static states.

Development of a blood-based molecular biomarker test for identification of schizophrenia before disease onset.

Recent research efforts have progressively shifted towards preventative psychiatry and prognostic identification of individuals before disease onset. We describe the development of a serum biomarker test for the identification of individuals at risk of developing schizophrenia based on multiplex immunoassay profiling analysis of 957 serum samples. First, we conducted a meta-analysis of five independent cohorts of 127 first-onset drug-naive schizophrenia patients and 204 controls. Using least absolute shrinkage and selection operator regression, we identified an optimal panel of 26 biomarkers that best discriminated patients and controls. Next, we successfully validated this biomarker panel using two independent validation cohorts of 93 patients and 88 controls, which yielded an area under the curve (AUC) of 0.97 (0.95–1.00) for schizophrenia detection. Finally, we tested its predictive performance for identifying patients before onset of psychosis using two cohorts of 445 pre-onset or at-risk individuals. The predictive performance achieved by the panel was excellent for identifying USA military personnel (AUC: 0.90 (0.86–0.95)) and help-seeking prodromal individuals (AUC: 0.82 (0.71–0.93)) who developed schizophrenia up to 2 years after baseline sampling. The performance increased further using the latter cohort following the incorporation of CAARMS (Comprehensive Assessment of At-Risk Mental State) positive subscale symptom scores into the model (AUC: 0.90 (0.82–0.98)). The current findings may represent the first successful step towards a test that could address the clinical need for early intervention in psychiatry. Further developments of a combined molecular/symptom-based test will aid clinicians in the identification of vulnerable patients early in the disease process, allowing more effective therapeutic intervention before overt disease onset.

Marked reduction of soluble superoxide dismutase-1 (SOD1) in cerebrospinal fluid of patients with recent-onset schizophrenia

Recent schizophrenia research supports a model wherein aberrant brain changes in late-adolescence contribute to the onset and early progression of disease.1 To further examine this model, longitudinal studies of genetic high-risk subjects, including those in prodromal stage, have recently been conducted.2 Parallel studies evaluating patients with recent-onset schizophrenia are growing in number.3 Nonetheless, almost all these studies are limited to clinical and neuropsychological characterization, and to brain imaging. Therefore, the molecular mechanisms underlying these dynamic changes remain elusive. In order to address this fundamental question with better molecular understanding, we have examined the cerebrospinal fluid (CSF) from patients with recent-onset schizophrenia (within the first five years of disease) and have compared these samples to those from age-matched healthy controls and patients with chronic schizophrenia (five or more years of schizophrenia) (Supplementary Table 1). Building on the hypothesis that oxidative stress and associated neuroinflammatory response may play a role in this dynamic process,4 we measured the levels of 14 selected molecules (Supplementary Table 2). Here we report the novel finding of a dramatically reduced level of soluble superoxide dismutase-1 (SOD1) in CSF from patients with recent-onset schizophrenia in contrast to samples from age-matched healthy controls (Table 1, Supplementary Figure 1). Furthermore, the level of CSF soluble SOD1 from recent-onset patients is lower than that from patients with chronic schizophrenia. To our knowledge, this is the first report of decreased CSF SOD1 in recent-onset schizophrenia. We also highlight that while the sample size of patients with one year or less disease is small, the absolute concentrations of soluble SOD1 are most robustly decreased in this cohort. We speculate that the decrease in soluble SOD1 between patients with one year or less disease and age-matched healthy controls is limited in significance by the small sample size. Table 1 Comparison of CSF soluble SOD1 concentration between patients with recent-onset schizophrenia (less than five years disease) grouped by disease duration and age-matched healthy controls. While our reported observations require replication in a larger sample, this study implies two innovative conceptual contributions. First, the reduction of soluble SOD1 may directly underlie oxidative stress at the onset of schizophrenia through decreased availability of this important antioxidant enzyme. We further note that this decrease in CSF soluble SOD1 concentration could be a secondary effect of another pro-oxidative process. Nevertheless, this ultimate decrease in soluble SOD1 is likely to facilitate overall oxidative stress. Thus, we provide further evidence of the “oxidative stress hypothesis” in schizophrenia. Second, we note the possibility that a decrease in CSF soluble SOD1 may reflect an increase in “insoluble” SOD1. This is analogous to decreased Aβ1-42 in CSF from patients with Alzheimer’s disease reflecting insoluble Aβ1-42 and senile plaques.5 Likewise, SOD1 is amyloidogenic and can be associated with brain disorders, such as amyotrophic lateral sclerosis (ALS).6 We wish to recall that aggregate formation in Huntington’s disease and other brain disorders has been discovered only after specific probing of target molecules, such as Huntingtin detection using a specific antibody.7 Therefore, it is crucial to now explore insoluble SOD1 in autopsied brains or biopsied cells (e.g. induced neurons from fibroblasts, olfactory neurons) from patients with recent-onset schizophrenia, and such future studies may utilize antibodies against insoluble forms of SOD1.8 A role of misfolded protein in schizophrenia has also been suggested by studies stemming from genetic susceptibility factors. Korth and associates9 reported the recruitment of Dysbindin protein to cell-invasive DISC1 aggresomes, suggesting the important convergence of two promising genetic risk factors and the significance of protein insolubility in cases of schizophrenia. The levels of CSF soluble SOD1 are not significantly different between chronic cases and matched healthy controls (data not shown). The possible contrast in SOD1 between recent-onset schizophrenia and chronic cases may be addressed in follow-up studies with larger sample size. It is possible that transient downregulation around the onset of schizophrenia might play an important pathophysiological role. Of note, some studies suggest that neuroleptic medication might increase plasma or serum SOD levels,10 although levels between peripheral blood and CSF are not necessarily correlated. In this report with one exception, all patients were on medication at the time of CSF acquisition. In summary, we report diminished CSF soluble SOD1 in cases of recent-onset schizophrenia and discuss this observation as supporting the previously proposed role of “oxidative stress” in the pathophysiology and onset of this disease. Increased oxidative stress can lead to synaptic deterioration and interneuron deficits relevant to the pathophysiology of schizophrenia. Furthermore, this observation may also be an entry point for other working hypotheses, such as those of SOD1 protein misfolding and aberrant control of proteolysis, which prompt further investigation.

Serum proteomic profiling of major depressive disorder

Much has still to be learned about the molecular mechanisms of depression. This study aims to gain insight into contributing mechanisms by identifying serum proteins related to major depressive disorder (MDD) in a large psychiatric cohort study. Our sample consisted of 1589 participants of the Netherlands Study of Depression and Anxiety, comprising 687 individuals with current MDD (cMDD), 482 individuals with remitted MDD (rMDD) and 420 controls. We studied the relationship between MDD status and the levels of 171 serum proteins detected on a multi-analyte profiling platform using adjusted linear regression models. Pooled analyses of two independent validation cohorts (totaling 78 MDD cases and 156 controls) was carried out to validate our top markers. Twenty-eight analytes differed significantly between cMDD cases and controls (P<0.05), whereas 10 partly overlapping markers differed significantly between rMDD cases and controls. Antidepressant medication use and comorbid anxiety status did not substantially impact on these findings. Sixteen of the cMDD-related markers had been assayed in the pooled validation cohorts, of which seven were associated with MDD. The analytes prominently associated with cMDD related to diverse cell communication and signal transduction processes (pancreatic polypeptide, macrophage migration inhibitory factor, ENRAGE, interleukin-1 receptor antagonist and tenascin-C), immune response (growth-regulated alpha protein) and protein metabolism (von Willebrand factor). Several proteins were implicated in depression. Changes were more prominent in cMDD, suggesting that molecular alterations in serum are associated with acute depression symptomatology. These findings may help to establish serum-based biomarkers of depression and could improve our understanding of its pathophysiology.

Antipsychotic treatment of acute paranoid schizophrenia patients with olanzapine results in altered glycosylation of serum glycoproteins.

Atypical antipsychotic drugs, such as olanzapine, have been shown to alleviate the positive, negative and, to a lesser degree, the cognitive symptoms of schizophrenia in many patients. However, the detailed mechanisms of action of these drugs have yet to be elucidated. We have carried out the first investigation aimed at evaluating the effects of olanzapine treatment on the glycosylation of serum proteins in schizophrenia patients. Olanzapine treatment resulted in increased levels of a disialylated biantennary glycan and reduced levels of a number of disialylated bi- and triantennary glycans on whole serum glycoproteins. These changes were not observed on a low-abundance serum protein fraction. α1 acid glycoprotein was identified as a carrier of some of the detected altered oligosaccharides. In addition, glycan analysis of haptoglobin, transferrin, and α1 antitrypsin reported similar findings, although these changes did not reach significance. Exoglycosidase digestion analysis showed that olanzapine treatment increased galactosylation and sialylation of whole serum proteins, suggesting increased activity of specific galactosyltransferases and increased availability of galactose residues for sialylation. Taken together, these findings indicate that olanzapine treatment results in altered glycosylation of serum proteins.