Drug discovery for psychiatric disorders using high-content single-cell screening of signaling network responses ex vivo.

There is a paucity of efficacious new compounds to treat neuropsychiatric disorders. We present a novel approach to neuropsychiatric drug discovery based on high-content characterization of druggable signaling network responses at the single-cell level in patient-derived lymphocytes ex vivo. Primary T lymphocytes showed functional responses encompassing neuropsychiatric medications and central nervous system ligands at established (e.g., GSK-3ß) and emerging (e.g., CrkL) drug targets. Clinical application of the platform to schizophrenia patients over the course of antipsychotic treatment revealed therapeutic targets within the phospholipase Cγ1-calcium signaling pathway. Compound library screening against the target phenotype identified subsets of L-type calcium channel blockers and corticosteroids as novel therapeutically relevant drug classes with corresponding activity in neuronal cells. The screening results were validated by predicting in vivo efficacy in an independent schizophrenia cohort. The approach has the potential to discern new drug targets and accelerate drug discovery and personalized medicine for neuropsychiatric conditions.

Pathway sensor-based functional genomics screening identifies modulators of neuronal activity.

Neuronal signal transduction shapes brain function and malfunction may cause mental disorders. Despite the existence of functional genomics screens for proliferation and toxicity, neuronal signalling has been difficult to address so far. To overcome this limitation, we developed a pooled screening assay which combines barcoded activity reporters with pooled genetic perturbation in a dual-expression adeno-associated virus (AAV) library. With this approach, termed pathScreener, we comprehensively dissect signalling pathways in postmitotic neurons. This overcomes several limitations of lentiviral-based screens. By applying first a barcoded and multiplexed reporter assay, termed cisProfiler, we identified the synaptic-activity responsive element (SARE) as top performance sensor of neuronal activity. Next, we targeted more than 4,400 genes and screened for modulatory effects on SARE activity in primary cortical neurons. We identified with high replicability many known genes involved in glutamatergic synapse-to-nucleus signalling of which a subset was validated in orthogonal assays. Several others have not yet been associated with the regulation of neuronal activity such as the hedgehog signalling members Ptch2 and Ift57. This assay thus enhances the toolbox for analysing regulatory processes during neuronal signalling and may help identifying novel targets for brain disorders.

Spironolactone is an antagonist of NRG1-ERBB4 signaling and schizophrenia-relevant endophenotypes in mice.

Enhanced NRG1-ERBB4 signaling is a risk pathway in schizophrenia, and corresponding mouse models display several endophenotypes of the disease. Nonetheless, pathway-directed treatment strategies with clinically applicable compounds have not been identified. Here, we applied a cell-based assay using the split TEV technology to screen a library of clinically applicable compounds to identify modulators of NRG1-ERBB4 signaling for repurposing. We recovered spironolactone, known as antagonist of corticosteroids, as an inhibitor of the ERBB4 receptor and tested it in pharmacological and biochemical assays to assess secondary compound actions. Transgenic mice overexpressing Nrg1 type III display cortical Erbb4 hyperphosphorylation, a condition observed in postmortem brains from schizophrenia patients. Spironolactone treatment reverted hyperphosphorylation of activated Erbb4 in these mice. In behavioral tests, spironolactone treatment of Nrg1 type III transgenic mice ameliorated schizophrenia-relevant behavioral endophenotypes, such as reduced sensorimotor gating, hyperactivity, and impaired working memory. Moreover, spironolactone increases spontaneous inhibitory postsynaptic currents in cortical slices supporting an ERBB4-mediated mode-of-action. Our findings suggest that spironolactone, a clinically safe drug, provides an opportunity for new treatment options for schizophrenia.

A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated with Borrelia Seropositivity.

In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl(-) channels and phospholipid scramblases. ANO10 augments volume-regulated Cl(-) currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca(2+) signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.

A myelin gene causative of a catatonia-depression syndrome upon aging.

Severe mental illnesses have been linked to white matter abnormalities, documented by postmortem studies. However, cause and effect have remained difficult to distinguish. CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase) is among the oligodendrocyte/myelin-associated genes most robustly reduced on mRNA and protein level in brains of schizophrenic, bipolar or major depressive patients. This suggests that CNP reduction might be critical for a more general disease process and not restricted to a single diagnostic category. We show here that reduced expression of CNP is the primary cause of a distinct behavioural phenotype, seen only upon aging as an additional 'pro-inflammatory hit'. This phenotype is strikingly similar in Cnp heterozygous mice and patients with mental disease carrying the AA genotype at CNP SNP rs2070106. The characteristic features in both species with their partial CNP 'loss-of-function' genotype are best described as 'catatonia-depression' syndrome. As a consequence of perturbed CNP expression, mice show secondary low-grade inflammation/neurodegeneration. Analogously, in man, diffusion tensor imaging points to axonal loss in the frontal corpus callosum. To conclude, subtle white matter abnormalities inducing neurodegenerative changes can cause/amplify psychiatric diseases.

Convergent evidence of the contribution of TP53 genetic variation (Pro72Arg) to metabolic activity and white matter volume in the frontal lobe in schizophrenia patients.

Abnormalities in white matter (WM) volumes and integrity in schizophrenia, together with post-mortem studies showing reduced expression of oligodendrocyte/myelination genes and apoptotic processes taking place in oligodendrocytes, suggest the interest of major regulators of apoptosis as candidate genes for some features related to myelin integrity in schizophrenia. Protein p53, encoded by TP53 gene, has a central role in the control of apoptosis and is involved in oligodendrocyte development. TP53 gene polymorphisms may account for variability in WM features, metabolic activity and biochemical markers of neuronal integrity and membrane turnover. Pro72Arg and Ins16bp polymorphisms at TP53 gene were analyzed in 20 DSM-IV schizophrenia patients. T1/T2-weighted sequences of these patients were acquired using a 1.5T Philips Gyroscan system. Scans were transformed into Talairach space and segmented into gray matter (GM), WM and cerebrospinal fluid (CSF) using Statistical Parametric Mapping under a ROI approach. Likewise dorsolateral prefrontal cortex (DLPFC) metabolic activity was measured using a procedure based on MRI/PET image fusion. In 13 of these patients proton magnetic resonance spectroscopy was used to examine N-acetylaspartate (NAA), creatine (Cr) and choline (Cho) levels in dorsolateral-medial prefrontal cortex (DLMPFC). MRI data were adjusted for age and brain volume using regression parameters from a healthy control group (n=45). Patients Pro/Arg heterozygous (Pro72Arg polymorphism) showed a generalized deficit in whole-brain WM that was especially prominent in frontal lobe and a lower metabolic activity in the DLPFC as compared to Pro/Pro homozygous. Pro/Arg subjects also showed decreased NAA/Cho and increased Cho/Cr ratios in right DLMPFC. TP53 genetic variability influences WM volumes in frontal lobes and it seems to modulate the metabolic activity in this region. Our results suggest that TP53 might influence aspects of myelin and white matter integrity which may account for some of the frontal dysfunction features commonly described in these patients.

Analysis of polymorphisms at the tumor suppressor gene p53 (TP53) in contributing to the risk for schizophrenia and its associated neurocognitive deficits.

Owing to the role of the nuclear phosphoprotein p53 in the regulation of neurodegeneration and neurodevelopmental processes, some authors have suggested TP53 as a candidate gene for schizophrenia and/or the neurocognitive deficits commonly observed in these patients. In the present study we have analyzed two polymorphisms (Pro72Arg and 16 bp insertion) located on the TP53 gene in order to investigate their role in the risk of developing schizophrenia and their effect on the neurocognitive profile of these patients in the context of an association study. The distribution of genotypes, alleles and haplotypes did not differ between cases and controls. Additionally, we did not detect any influence of this genetic variability in the neurocognitive functions of schizophrenic patients. Our findings suggest that the analyzed variability of the TP53 gene does not influence (i) the risk of suffering from schizophrenia and (ii) the deficits in the neurocognitive profile of these patients.