Hassan Rahmoune

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.

Validation of a Blood-Based Laboratory Test to Aid in the Confirmation of a Diagnosis of Schizophrenia

We describe the validation of a serum-based test developed by Rules-Based Medicine which can be used to help confirm the diagnosis of schizophrenia. In preliminary studies using multiplex immunoassay profiling technology, we identified a disease signature comprised of 51 analytes which could distinguish schizophrenia (n = 250) from control (n = 230) subjects. In the next stage, these analytes were developed as a refined 51-plex immunoassay panel for validation using a large independent cohort of schizophrenia (n = 577) and control (n = 229) subjects. The resulting test yielded an overall sensitivity of 83% and specificity of 83% with a receiver operating characteristic area under the curve (ROC-AUC) of 89%. These 51 immunoassays and the associated decision rule delivered a sensitive and specific prediction for the presence of schizophrenia in patients compared to matched healthy controls.

PPARγ activation enhances cell surface ENaCα via up-regulation of SGK1 in human collecting duct cells

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand‐dependent transcription factor that belongs to the nuclear receptor family that plays a critical role in adipocyte differentiation and lipid metabolism. Here we report for the first time that PPARγ is expressed in human renal cortical collecting ducts (CCD), segments of the nephor involved in regulation of sodium and water homeostasis via action of the epithelial sodium channel (ENaC). ENaC activity is regulated by the hormones aldosterone and insulin, primarily through co‐ordinate actions on serum and glucocorticoid regulated kinase 1 (SGK1). We show that SGK1 activity is stimulated by treatment of a human CCD cell line with PPARγ agonists, paralleled by an increase in SGK1 mRNA that is abolished by pretreatment with a specific PPARγ antagonist, and that this leads to increased levels of cell surface ENaCα. Electrophoretic mobility shift assays suggest that these effects are caused by binding of PPARγ to a specific response element in the SGK1 promoter. Our results identify SGK1 as a target for PPARγ and suggest a novel role for PPARγ in regulation of sodium re‐absorption in the CCD via stimulation of ENaC activity. This pathway may play a role in sodium retention caused by activation of PPARγ in man.

Altered levels of circulating insulin and other neuroendocrine hormones associated with the onset of schizophrenia

Recently, we showed that the circulating levels of insulin-related peptides and the secretory granule protein chromogranin A were increased in small cohorts of first onset schizophrenia patients. Assuming that this effect was associated with impaired insulin signalling, we investigated the possibility that secretion of other hormones is also affected in schizophrenia. Multiplex immunoassay analysis of 21 hormones and hormone-related molecules was carried out using sera from 236 first and recent onset schizophrenia patients and 230 matched controls. Serum concentrations of insulin and chromogranin A were increased in schizophrenia subjects, consistent with our previous study. In addition, we found elevated concentrations of pancreatic polypeptide, prolactin, progesterone and cortisol, and decreased levels of growth hormone. We also found that growth hormone levels were decreased in post-mortem pituitaries obtained from chronic schizophrenia patients. It will be important to determine whether any of these molecules are involved in the pathosphysiology of schizophrenia or if they reflect the associated insulin resistance. We conclude that function of multiple components of the hypothalamic-pituitary-adrenal-gonadal axis may be affected in schizophrenia. This could have important implications for future biomarker discovery efforts and personalized medicine strategies based on patient stratification for the treatment of this debilitating disorder.

Sex-specific serum biomarker patterns in adults with Asperger's syndrome

Autism spectrum conditions have been hypothesized to be an exaggeration of normal male low-empathizing and high-systemizing behaviors. We tested this hypothesis at the molecular level by performing comprehensive multi-analyte profiling of blood serum from adult subjects with Aspergers syndrome (AS) compared with controls. This led to identification of distinct sex-specific biomarker fingerprints for male and female subjects. Males with AS showed altered levels of 24 biomarkers including increased levels of cytokines and other inflammatory molecules. Multivariate statistical classification of males using this panel of 24 biomarkers revealed a marked separation between AS and controls with a sensitivity of 0.86 and specificity of 0.88. Testing this same panel in females did not result in a separation between the AS and control groups. In contrast, AS females showed altered levels of 17 biomarkers including growth factors and hormones such as androgens, growth hormone and insulin-related molecules. Classification of females using this biomarker panel resulted in a separation between AS and controls with sensitivities and specificities of 0.96 and 0.83, respectively, and testing this same panel in the male group did not result in a separation between the AS and control groups. The finding of elevated testosterone in AS females confirmed predictions from the ‘extreme male brain’ and androgen theories of autism spectrum conditions. We conclude that to understand the etiology and development of autism spectrum conditions, stratification by sex is essential.

Identification of proteomic signatures associated with depression and psychotic depression in post-mortem brains from major depression patients

Major depressive disorder (MDD) is a leading cause of disability worldwide and results tragically in the loss of almost one million lives in Western societies every year. This is due to poor understanding of the disease pathophysiology and lack of empirical medical tests for accurate diagnosis or for guiding antidepressant treatment strategies. Here, we have used shotgun proteomics in the analysis of post-mortem dorsolateral prefrontal cortex brain tissue from 24 MDD patients and 12 matched controls. Brain proteomes were pre-fractionated by gel electrophoresis and further analyzed by shotgun data-independent label-free liquid chromatography-mass spectrometry. This led to identification of distinct proteome fingerprints between MDD and control subjects. Some of these differences were validated by Western blot or selected reaction monitoring mass spectrometry. This included proteins associated with energy metabolism and synaptic function and we also found changes in the histidine triad nucleotide-binding protein 1 (HINT1), which has been implicated recently in regulation of mood and behavior. We also found differential proteome profiles in MDD with (n=11) and without (n=12) psychosis. Interestingly, the psychosis fingerprint showed a marked overlap to changes seen in the brain proteome of schizophrenia patients. These findings suggest that it may be possible to contribute to the disease understanding by distinguishing different subtypes of MDD based on distinct brain proteomic profiles.

Cytokine alterations in first-episode schizophrenia patients before and after antipsychotic treatment

Schizophrenia has been associated with central nervous system and peripheral immune system imbalances. However, most studies have not yielded conclusive results due to limitations such as small sample size, dissimilarities in the clinical status of patients and the high variability of cytokine levels within the normal human population. Here, we have attempted to account for these limitations by carrying out standardised multiplex immunoassay analyses of 9 cytokines in serum from 180 antipsychotic-naïve first-episode schizophrenia patients and 350 matched controls across 5 clinical cohorts. All subjects were matched for potential confounding factors including age, gender, smoking and body mass index. We found that the levels of interleukin (IL)-1RA, IL-10 and IL-15 were increased significantly in patients across the cohorts. We also found that the levels of IL-1RA and IL-10 were decreased in 32 patients who had been followed up and treated for 6 weeks with atypical antipsychotics. Interestingly, we found that the changes in IL-10 levels were significantly correlated with the improvements in negative, general and total symptom scores. These results indicate that mixed pro- and anti-inflammatory responses may be altered in first onset patients, suggesting a role in the aetiology of schizophrenia. The finding that only the anti-inflammatory cytokine IL-10 responded to treatment in parallel with symptom improvement suggests that this could be used as a potential treatment response biomarker in future studies of schizophrenia.

Peripheral profiling analysis for bipolar disorder reveals markers associated with reduced cell survival

Little is known about the molecular factors that are altered in remitting bipolar disorder (BD) patients. We carried out proteome profiling of peripheral blood mononuclear cells (PBMCs) and serum from BD patients who were not experiencing mania or major depression (euthymia) compared to matched healthy controls using liquid chromatography–mass spectrometry (LC‐MSE) and Multi‐Analyte Profiling (Human Map®) platforms. This resulted in the identification of approximately 60 differentially expressed molecules involved predominantly in cell death/survival pathways. In PBMCs, this was manifested in cytoskeletal and stress response‐associated proteins, whereas most serum analytes were associated with the inflammatory response. The predicted effect of serum analytes on physiological systems was tested by treating PBMCs with serum obtained from the same patients, resulting in reduced cellular survival. These preliminary results suggest that BD patients carry a peripheral fingerprint that has detrimental effects on cell function and that could be used to distinguish BD patients from healthy controls despite being in a remission phase. It is hoped that additional studies of BD patients in the manic and depressed stages could lead to the identification of a molecular fingerprint that could be used for predicting episodic switching and for guiding treatment strategies.

The peripheral benzodiazepine receptor ligand PK11195 binds with high affinity to the acute phase reactant alpha1-acid glycoprotein: implications for the use of the ligand as a CNS inflammatory marker.

The peripheral benzodiazepine receptor ligand PK11195 has been used as an in vivo marker of neuroinflammation in positron emission tomography studies in man. One of the methodological issues surrounding the use of the ligand in these studies is the highly variable kinetic behavior of [(11)C]PK11195 in plasma. We therefore undertook a study to measure the binding of [(3)H]PK11195 to whole human blood and found a low level of binding to blood cells but extensive binding to plasma proteins. Binding assays using [(3)H]PK11195 and purified human plasma proteins demonstrated a strong binding to alpha1-acid glycoprotein (AGP) and a much weaker interaction with albumin. Immunodepletion of AGP from plasma resulted in the loss of plasma [(3)H]PK11195 binding demonstrating: (i) the specificity of the interaction and (ii) that AGP is the major plasma protein to which PK11195 binds with high affinity. PK11195 was able to displace fluorescein-dexamethasone from AGP with IC(50) of <1.2 microM, consistent with a high affinity interaction. These findings are important for understanding the behavior of the ligand in positron emission tomography studies for three reasons. Firstly, AGP is an acute phase protein and its levels will vary during infection and pathological inflammatory diseases such as multiple sclerosis. This could significantly alter the free plasma concentrations of the ligand and contribute to its variable kinetic behavior. Secondly, AGP and AGP-bound ligand may contribute to the access of [(11)C]PK11195 to the brain parenchyma in diseases with blood brain barrier breakdown. Finally, local synthesis of AGP at the site of brain injury may contribute the pattern of [(11)C]PK11195 binding observed in neuroinflammatory diseases.

Expression Profiling of Fibroblasts Identifies Cell Cycle Abnormalities in Schizophrenia

Many previous studies have attempted to gain insight into the underlying pathophysiology of schizophrenia by studying postmortem brain tissues of schizophrenia patients. However, such analyses can be confounded by artifactual features of this approach such as lengthy agonal state and postmortem interval times. As several aspects of schizophrenia are also manifested at the peripheral level in proliferating cell types, we have studied the disorder through systematic transcriptomic and proteomic analyses of skin fibroblasts biopsied from living patients. We performed comparative transcriptomic and proteomic profiling to characterize skin fibroblasts from schizophrenia patients compared to healthy controls. Transcriptomic profiling using cDNA array technology showed that pathways associated with cell cycle regulation and RNA processing were altered in the schizophrenia subjects (n = 12) relative to controls (n = 12). LC-MS(E) proteomic profiling led to identification of 16 proteins that showed significant differences in expression between schizophrenia (n = 11) and control (n = 11) subjects. Analysis in silico revealed that these proteins were also associated with proliferation and cell growth pathways. To validate these findings at the protein level, fibroblast protein extracts were analyzed by Western blotting which confirmed the differential expression of three key proteins associated with these pathways. At the functional level, we confirmed the decreased proliferation phenotype by showing that cultured fibroblasts from schizophrenia subjects (n = 5) incorporated less (3)H-thymidine into their nuclei compared to those from controls (n = 6) by day 4 over an 8 day time course study. Similar abnormalities in cell cycle and growth pathways have been reported to occur in the central nervous system in schizophrenia. These studies demonstrate that fibroblasts obtained from living schizophrenia subjects show alterations in cellular proliferation and growth pathways. Future studies aimed at characterizing such pathways in fibroblasts and other proliferating cell types from schizophrenia patients could elucidate the molecular mechanisms associated with the pathophysiology of schizophrenia and provide a useful model to support drug discovery efforts.