Unbiased Metabolite Profiling of Schizophrenia Fibroblasts under Stressful Perturbations Reveals Dysregulation of Plasmalogens and Phosphatidylcholines.

We undertook an unbiased metabolite profiling of fibroblasts from schizophrenia patients and healthy controls to identify metabolites and pathways that are dysregulated in disease, seeking to gain new insights into the disease biology of schizophrenia and to discover potential disease-related biomarkers. We measured polar and nonpolar metabolites in the fibroblasts under normal conditions and under two stressful physiological perturbations: growth in low-glucose media and exposure to the steroid hormone dexamethasone. We found that metabolites that were significantly different between schizophrenia and control subjects showed separation of the two groups by partial least-squares discriminant analysis methods. This separation between schizophrenia and healthy controls was more robust with metabolites identified under the perturbation conditions. The most significant individual metabolite differences were also found in the perturbation experiments. Metabolites that were significantly different between schizophrenia and healthy controls included a number of plasmalogens and phosphatidylcholines. We present these results in the context of previous reports of metabolic profiling of brain tissue and plasma in schizophrenia. These results show the applicability of metabolite profiling under stressful perturbations to reveal cellular pathways that may be involved in disease biology.

Disease signatures for schizophrenia and bipolar disorder using patient-derived induced pluripotent stem cells.

Schizophrenia and bipolar disorder are complex psychiatric disorders that present unique challenges in the study of disease biology. There are no objective biological phenotypes for these disorders, which are characterized by complex genetics and prominent roles for gene-environment interactions. The study of the neurobiology underlying these severe psychiatric disorders has been hindered by the lack of access to the tissue of interest - neurons from patients. The advent of reprogramming methods that enable generation of induced pluripotent stem cells (iPSCs) from patient fibroblasts and peripheral blood mononuclear cells has opened possibilities for new approaches to study relevant disease biology using iPSC-derived neurons. While early studies with patient iPSCs have led to promising and intriguing leads, significant hurdles remain in our attempts to capture the complexity of these disorders in vitro. We present here an overview of studies to date of schizophrenia and bipolar disorder using iPSC-derived neuronal cells and discuss potential future directions that can result in the identification of robust and valid cellular phenotypes that in turn can lay the groundwork for meaningful clinical advances.

Stitched α-helical peptides via bis ring-closing metathesis.

Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides.

Mapping targetable sites on human telomerase RNA pseudoknot/template domain using 2'-OMe RNA-interacting polynucleotide (RIPtide) microarrays.

Most cellular RNAs engage in intrastrand base-pairing that gives rise to complex three-dimensional folds. This self-pairing presents an impediment toward binding of the RNA by nucleic acid-based ligands. An important step in the discovery of RNA-targeting ligands is therefore to identify those regions in a folded RNA that are accessible toward the nucleic acid-based ligand. Because the folding of RNA targets can involve interactions between nonadjacent regions and employ both Watson-Crick and non-Watson-Crick base-pairing, screening of candidate binder ensembles is typically necessary. Microarray-based screening approaches have shown great promise in this regard and have suggested that achieving complete sequence coverage would be a valuable attribute of a next generation system. Here, we report a custom microarray displaying a library of RNA-interacting polynucleotides comprising all possible 2'-OMe RNA sequences from 4- to 8-nucleotides in length. We demonstrate the utility of this array in identifying RNA-interacting polynucleotides that bind tightly and specifically to the highly conserved, functionally essential template/pseudoknot domain of human telomerase RNA and that inhibit telomerase function in vitro.

Perturbational Profiling of Metabolites in Patient Fibroblasts Implicates α-Aminoadipate as a Potential Biomarker for Bipolar Disorder.

Many studies suggest the presence of aberrations in cellular metabolism in bipolar disorder. We studied the metabolome in bipolar disorder to gain insight into cellular pathways that may be dysregulated in bipolar disorder and to discover evidence of novel biomarkers. We measured polar and nonpolar metabolites in fibroblasts from subjects with bipolar I disorder and matched healthy control subjects, under normal conditions and with two physiologic perturbations: low-glucose media and exposure to the stress-mediating hormone dexamethasone. Metabolites that were significantly different between bipolar and control subjects showed distinct separation by principal components analysis methods. The most statistically significant findings were observed in the perturbation experiments. The metabolite with the lowest p value in both the low-glucose and dexamethasone experiments was α-aminoadipate, whose intracellular level was consistently lower in bipolar subjects. Our study implicates α-aminoadipate as a possible biomarker in bipolar disorder that manifests under cellular stress. This is an intriguing finding given the known role of α-aminoadipate in the modulation of kynurenic acid in the brain, especially as abnormal kynurenic acid levels have been implicated in bipolar disorder.

HDAC6 inhibitors modulate Lys49 acetylation and membrane localization of ß-catenin in human iPSC-derived neuronal cells.

We examined the effects of isoform-specific histone deacetylase (HDAC) inhibitors on ß-catenin posttranslational modifications in neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs). ß-catenin is a multifunctional protein with important roles in the developing and adult central nervous system. Activation of the Wnt pathway results in stabilization and nuclear translocation of ß-catenin, resulting in activation of multiple target genes. In addition, ß-catenin forms a complex with cadherins at the plasma membrane as part of the adherens junctions. The N-terminus of ß-catenin has phosphorylation, ubiquitination, and acetylation sites that regulate its stability and signaling. In the absence of a Wnt signal, Ser33, Ser37, and Thr41 are constitutively phosphorylated by glycogen synthase kinase 3ß (GSK3ß). ß-Catenin phosphorylated at these sites is recognized by ß-transducin repeat-containing protein (ßTrCP), which results in ubiquitination and degradation by the ubiquitin-proteasome pathway. The N-terminal regulatory domain of ß-catenin also includes Ser45, a phosphorylation site for Casein Kinase 1α (CK1α) and Lys49, which is acetylated by the acetyltransferase p300/CBP-associated factor (PCAF). The relevance of Lys49 acetylation and Ser45 phosphorylation to the function of ß-catenin is an active area of investigation. We find that HDAC6 inhibitors increase Lys49 acetylation and Ser45 phosphorylation but do not affect Ser33, Ser37, and Thr41 phosphorylation. Lys49 acetylation results in decreased ubiquitination of ß-catenin in the presence of proteasome inhibition. While increased Lys49 acetylation does not affect total levels of ß-catenin, it results in increased membrane localization of ß-catenin.