Blood biomarker discovery in drug-free schizophrenia: the contribution of proteomics and multiplex immunoassays

<p><b>Introduction:</b> Recent evidence supports an association between systemic abnormalities and the pathology of psychotic disorders which has led to the search for peripheral blood-based biomarkers.</p> <p><b>Areas covered:</b> Here, we summarize blood biomarker findings in schizophrenia from the literature identified by two methods currently driving biomarker discovery in the human proteome; mass spectrometry and multiplex immunoassay. From a total of 14 studies in the serum or plasma of drug-free schizophrenia patients; 47 proteins were found to be significantly altered twice or more, in the same direction. Pathway analysis was performed on these proteins, and the resulting pathways discussed in relation to schizophrenia pathology. Future directions are also discussed, with particular emphasis on the potential for high-throughput validation techniques such as data-independent analysis for confirmation of biomarker candidates.</p> <p><b>Expert commentary:</b> We present promising findings that point to a convergence of pathophysiological mechanisms in schizophrenia that involve the acute-phase response, glucocorticoid receptor signalling, coagulation, and lipid and glucose metabolism.</p

Epigenetic factors in schizophrenia: mechanisms and experimental approaches.

Schizophrenia is a chronic mental disorder that is still poorly understood despite decades of study. Many factors have been found to contribute to the pathogenesis, including neurodevelopmental disturbance, genetic risk, and environmental insult, but no single root cause has emerged. While evidence from twin studies suggests a strong heritable component, few individual loci have been identified in genomewide screens, suggesting a role for epigenetic effects. Rather, large numbers of weakly acting loci may cumulatively increase disease risk, including several mapping to epigenetic pathways. In this review, we discuss mechanisms of epigenetic regulation and evidence for an epigenetic contribution to disease phenotype. We further describe the range of experimental tools currently available to study epigenetic effects associated with the disease

Adolescent Risperidone treatment alters protein expression associated with protein trafficking and cellular metabolism in the adult rat prefrontal cortex.

The prefrontal cortex (PFC) is associated with mental health illnesses including schizophrenia, depression, bipolar disorder, and autism spectrum disorders. It richly expresses neuroreceptors which are the target for antipsychotics. However, as the precise mechanism of action of antipsychotic medications is not known, proteomic studies of the effects of antipsychotic drugs on the brain are warranted. In the current study, we aimed to characterize protein expression in the adult rodent PFC (n = 5 per group) following low-dose treatment with Risperidone or saline in adolescence (postnatal days 34-47). The PFC was examined by triplicate 1 h runs of label-free LC-MS/MS. The raw mass spectral data were analyzed with the MaxQuant(TM) software. Statistical analysis was carried out using SAS® Version 9.1. Pathway and functional analysis was performed with IngenuityPathway Analysis and in the Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively, the most implicated pathways were found to be related to mitochondrial function, protein trafficking, and the cytoskeleton. This report adds to the current repertoire of data available concerning the effects of antipsychotic drugs on the brain and sheds light on their biological mechanisms. The MS data have been deposited with the ProteomeXchange Consortium with dataset identifier PXD000480.</p

Butyrate limits human natural killer cell effector function

The gut microbiota regulates chronic inflammation and has been implicated in the pathogenesis of a broad spectrum of disease including autoimmunity and cancer. Microbial short-chain fatty acids (SCFAs) e.g., butyrate have demonstrated immunomodulatory effects and are thought to be key mediators of the host-microbiome interaction. Here, we investigated the effect of butyrate on effector functions of blood derived human NK cells stimulated for 18 h with a combination of IL-12/ IL-15, a potent mix of cytokines that drive NK cell activation. We show that butyrate has a strong anti-inflammatory effect on NK cells. NK cells cultured in the presence of butyrate expressed lower levels of activating receptors (TRAIL, NKp30, NKp44) and produced lower levels of cytokines (IFNγ, TNF-α, IL-22, granzyme B, granzyme A, perforin) in response to IL-12/IL-15. Butyrate restricted NK cell function by downregulation of mTORC1 activity, c-Myc mRNA expression and metabolism. Using a shotgun proteomic approach, we confirmed the effect of butyrate on NK cell cytokine signaling and metabolism and identified BRD2, MAT2A and EHD1 as downstream mediators of these effects. This insight into the immunomodulatory activity of butyrate on human NK cell function might help to develop new ways to limit NK cell function during chronic inflammation.</p

Maternal immune activation induces changes in myelin and metabolic proteins, some of which can be prevented with risperidone in adolescence.

BACKGROUND: Maternal infection is a risk factor for schizophrenia but the molecular and cellular mechanisms are not fully known. Myelin abnormalities are amongst the most robust neuropathological changes observed in schizophrenia, and preliminary evidence suggests that prenatal inflammation may play a role. METHODS: Label-free liquid chromatography-mass spectrometry was performed on the prefrontal cortex (PFC) of adult rat offspring born to dams that were exposed on gestational day 15 to the viral mimic polyinosinic:polycytidylic acid [poly(I:C), 4 mg/kg] or saline and treated with the atypical antipsychotic drug risperidone (0.045 mg/kg) or saline in adolescence. Western blotting was employed to validate protein changes. RESULTS: Over 1,000 proteins were quantified in the PFC with pathway analyses implicating changes in core metabolic pathways, following prenatal poly(I:C) exposure. Some of these protein changes were absent in the PFC of poly(I:C)-treated offspring that subsequently received risperidone treatment in adolescence. Particularly interesting reductions in the expression of the myelin-related proteins myelin basic protein isoform 3 (MBP1) and rhombex 29 were observed, which were reversed by risperidone treatment. Validation by Western blotting confirmed changes in MBP1 and mitogen-activated kinase 1 (MAPK1). Western blotting was extended to assess the MAPK signalling proteins due to their roles in inflammation, namely phosphorylated MAPK1 and phosphorylated MAPK-activated protein kinase 2. Both were upregulated by poly(I:C) treatment and reversed by risperidone treatment. CONCLUSIONS: Overall, our data suggest that maternal inflammation may contribute to an increased risk for schizophrenia through mechanisms involving metabolic function and myelin formation and that risperidone in adolescence may prevent or reverse such changes.</p

Adolescent-onset and adult-onset schizophrenia: reduced ribosomal protein expression via mTOR signalling in patient-derived olfactory cells.

Schizophrenia is a heterogeneous disorder associated with many genetic and environmental risk factors that could affect brain development. It is unknown whether adolescent-onset and adult-onset schizophrenia have similar aetiology. To address this we used discovery-based proteomics to find proteins differentially expressed in olfactory neurosphere-derived cells from adolescents with schizophrenia compared to age- and gender-matched healthy controls. Of 1638 proteins identified, 241 were differentially expressed in patient cells, with significant down-regulation of ribosomal and cytoskeletal proteins, and dysregulation of protein synthesis pathways. We then re-analysed our previous adult-onset proteomic data to compare directly with adolescent-onset protein expression. Schizophrenia-associated protein expression in adult-onset patients was remarkably similar to adolescent-onset patients. To increase sample size and power we combined the two datasets for a bioinformatic meta-analysis. Schizophrenia-associated protein expression indicated significant downregulation of the mTOR signalling pathway, which regulates protein synthesis, indicated by the reduced expression of all ribosomal proteins and other mTOR-dependent proteins: RPS6, VIM, LDHB and PPP2R1A. A protein-protein interaction network built from differentially expressed proteins in the combined dataset was significantly associated with schizophrenia-associated risk genes and with proteins regulating neural stem cell differentiation, cell adhesion and growth cones in the developing brain. This study demonstrates that despite the divergent age of onset, the proteomes of olfactory neural stem cells of adolescent- and adult-onset patients are remarkably similar. The dysregulated proteins in patient cells form a tightly interconnected protein-protein interaction network associated with mTOR signalling, protein translation, neurogenesis and axon growth - all key components of brain development

Pcl3 promotes ESC self-renewal.

<p>(A) <i>Pcl3</i> expression levels measured by qRT-PCR in ESC clones transduced with scramble or multiple <i>Pcl3</i> shRNAs. Graph represents average expression from 3–6 different clones. (B) A portion of cells transduced with <i>Pcl3</i> shRNA, but not scramble shRNA, are larger, flatter, and less dense, signifying a decrease in ESC cell morphology. Scale bar 25 µm. These pictures are representative of 2–3 different clones of scramble and <i>Pcl3</i> shRNA cells taken at three different time points. (C) Expression levels of <i>Oct4</i> and <i>Nanog</i> in scramble, <i>Pcl3</i> shRNA, and <i>Pcl3</i> overexpressing cells. (D) Quantification of Oct4 and Nanog staining in scramble and <i>Pcl3</i> shRNA treated ESCs. +++indicates bright staining, ++indicates less bright staining, and+indicates little or no staining as assessed by eye. Graphs are representative of two clones and between 5–10 fields of view at 10× magnification. (E) Alkaline phosphatase activity in scramble and <i>Pcl3</i> shRNA cells. Graph represents average activity from 3–6 different clones in three experiments assayed in duplicate. (F) Quantification of the number of colonies formed per well from scramble and <i>Pcl3</i> shRNA cells plated at 100 cells/well in a 6-well plate. Experiment was performed four times in duplicate with two clones each of scramble and <i>Pcl3</i> shRNA ESCs. (G) Quantification of colonies formed by plating 100 cells/well of wild type and Pcl3 overexpressing cells in a 6-well plate. LIF was reduced to 5% and was performed four times in duplicate. (H) Images of teratomas derived from scramble or <i>Pcl3</i> shRNA ESCs containing all three germ layers stained with hematoxylin and eosin. Abbreviations: EN-endoderm, NE-neuroectoderm, B-bone, C-cartilage, M-muscle, N-neural tissue. Scale bar 25 µm. Error bars indicate standard deviation. Expression analysis experiments represent 3–4 experiments assayed in quadruplet. For all experiments, asterisk denotes statistical significance of <i>p</i><0.05. Staining was performed 2–3 times in two or more clones.</p

Pcl2 and Pcl3 localize to CpG islands.

<p>(A) The 500 bp central regions of Pcl3 ChIP-seq peaks were scanned for enriched motifs by using a 9th order Markov background dependence model <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002576#pgen.1002576-Carroll1" target="_blank">[86]</a>. Two examples of 10- and 14-mer enriched motifs are shown. (B–C) Smoothed scatter plots of maximum position specific-scoring matrix (PSSM) scores for the two motifs and CpG density are shown for (B) Suz12 binding sites depleted upon <i>Pcl3</i> knockdown overlapping with Pcl2 and Pcl3 and (C) Suz12 binding sites unaffected upon <i>Pcl3</i> knockdown and that do not overlap with Pcl2 and Pcl3. (D) Shown are the decision boundaries of a support vector machine classifier using these three features, where the purple regions correspond to Suz12 co-localizing with Pcl2 and Pcl3. The predictor had a cross validation accuracy of 75%. (E) A model of Pcl3 and Pcl2 regulation of PRC2 binding and activity. In wild type ESCs, Pcl3 promotes PRC2 binding and H3K27me3. Pcl2 antagonizes Pcl3-mediated Suz12 binding at sites bound by both but promotes PRC2 function at sites solely regulated by Pcl2. Knockdown of <i>Pcl3</i> causes decreased PRC2 binding and H3K27me3. Pcl2 does not compensate at Pcl2 and Pcl3 targets and continues to inhibit or promote PRC2 function depending on the gene.</p

Pcl3 is a component of PRC2.

<p>(A) Protein levels of Suz12 and Suz12-TAP were measured in wild type, <i>Suz12^Gt/+</i>, <i>Suz12^Rev/+</i>, and <i>Suz12^Suz12TAP/+</i> cell lines by immunoblot. (B) Proteins detected by mass spectrometry that specifically co-purified with Suz12-TAP, their symbol, unique hits, and percent coverage. (C) Pcl3-V5 binds to Suz12-TAP. <i>Suz12^Suz12TAP/+</i> ESCs were transfected with empty vector, Pcl3-V5, or Mks1-V5 (control), and lysates were immunoprecipitated with FlagM2 and probed with anti-V5. (D) Pcl3-V5 binds Suz12, Ezh2, and Eed. Lysates from ESCs transfected with empty vector, Pcl3-V5, and Mks1-V5 (control) vectors were subjected to immunoprecipitation with anti-V5. Samples were then probed with anti-Suz12, anti-Ezh3, and anti-Eed. All westerns and co-immunoprecipitations were performed three times.</p

Pcl3 promotes PRC2 function.

<p>(A) Immunoblot showing levels of H3K27me3 in multiple clones of scramble and <i>Pcl3</i> shRNA ESCs and EBs. (B) H3K27me3 levels in <i>Suz12</i> and <i>Pcl3</i> siRNA treated cells. (C) Increased levels of H3K27me3 as measured by immunoblot in cells overexpressing Pcl3. (D) Immunoblot of H3K27me3, H2AK119Ub, H3K9me3, H3K4me3, and H3K27ac levels in histones from scramble and <i>Pcl3</i> shRNA-expressing cells. (E) Pcl3-TAP resistant to <i>Pcl3</i> shRNA was reintroduced into <i>Pcl3</i> shRNA cells, immunoprecipitated, and detected with anti-FlagM2. <i>Suz12^Suz12TAP/+</i> cells were used as a positive control. (F) Pcl3-TAP binds Suz12, Eed, and Ezh2. Lysates from scramble and <i>Pcl3</i> shRNA cells containing Pcl3-TAP were immunoprecipitated with FlagM2 and immunoblotted for Suz12, Eed, and Ezh2. (G) qRT-PCR shows partial rescue of <i>Pcl3</i> expression in <i>Pcl3</i> shRNA clones expressing <i>Pcl3-TAP</i>. Error bars indicate standard deviation. Graph represents average expression from 3–6 different clones in three experiments assayed in quadruplet. (H) Immunoblot showing restoration of H3K27me3 levels in <i>Pcl3</i> shRNA cells transduced with Pcl3-TAP. Histone H3 and α-tubulin were used as loading controls. All westerns and immunoprecipitations were performed three or more times with 2–6 clones.</p