P2X1 and P2X5 subunits form the functional P2X receptor in mouse cortical astrocytes

ATP plays an important role in signal transduction between neuronal and glial circuits and within glial networks. Here we describe currents activated by ATP in astrocytes acutely isolated from cortical brain slices by non-enzymatic mechanical dissociation. Brain slices were prepared from transgenic mice that express enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp. Exogenous ATP evoked inward currents in 75 of 81 astrocytes. In the majority (~65%) of cells, ATP-induced responses comprising a fast and delayed component; in the remaining subpopulation of astrocytes, ATP triggered a smoother response with rapid peak and slowly decaying plateau phase. The fast component of the response was sensitive to low concentrations of ATP (with EC50 of ~40 nM). All ATP-induced currents were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS); they were insensitive to ivermectin. Quantitative real-time PCR demonstrated strong expression of P2X1 and P2X5 receptor subunits and some expression of P2X2 subunit mRNAs. The main properties of the ATP-induced response in cortical astrocytes (high sensitivity to ATP, biphasic kinetics, and sensitivity to PPADS) were very similar to those reported for P2X1/5 heteromeric receptors studied previously in heterologous expression systems

Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a beta-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells

Mice Lacking the Circadian Modulators SHARP1 and SHARP2 Display Altered Sleep and Mixed State Endophenotypes of Psychiatric Disorders

Increasing evidence suggests that clock genes may be implicated in a spectrum of psychiatric diseases, including sleep and mood related disorders as well as schizophrenia. The bHLH transcription factors SHARP1/DEC2/BHLHE41 and SHARP2/DEC1/ BHLHE40 are modulators of the circadian system and SHARP1/DEC2/BHLHE40 has been shown to regulate homeostatic sleep drive in humans. In this study, we characterized Sharp1 and Sharp2 double mutant mice (S1/2(-/-)) using online EEG recordings in living animals, behavioral assays and global gene expression profiling. EEG recordings revealed attenuated sleep/wake amplitudes and alterations of theta oscillations. Increased sleep in the dark phase is paralleled by reduced voluntary activity and cortical gene expression signatures reveal associations with psychiatric diseases. S1/2(-/-) mice display alterations in novelty induced activity, anxiety and curiosity. Moreover, mutant mice exhibit impaired working memory and deficits in prepulse inhibition resembling symptoms of psychiatric diseases. Network modeling indicates a connection between neural plasticity and clock genes, particularly for SHARP1 and PER1. Our findings support the hypothesis that abnormal sleep and certain (endo) phenotypes of psychiatric diseases may be caused by common mechanisms involving components of the molecular clock including SHARP1 and SHARP2

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

Molecular matched targeted therapies for primary brain tumors—a single center retrospective analysis

PURPOSE: Molecular diagnostics including next generation gene sequencing are increasingly used to determine options for individualized therapies in brain tumor patients. We aimed to evaluate the decision-making process of molecular targeted therapies and analyze data on tolerability as well as signals for efficacy. METHODS: Via retrospective analysis, we identified primary brain tumor patients who were treated off-label with a targeted therapy at the University Hospital Frankfurt, Goethe University. We analyzed which types of molecular alterations were utilized to guide molecular off-label therapies and the diagnostic procedures for their assessment during the period from 2008 to 2021. Data on tolerability and outcomes were collected. RESULTS: 413 off-label therapies were identified with an increasing annual number for the interval after 2016. 37 interventions (9%) were targeted therapies based on molecular markers. Glioma and meningioma were the most frequent entities treated with molecular matched targeted therapies. Rare entities comprised e.g. medulloblastoma and papillary craniopharyngeoma. Molecular targeted approaches included checkpoint inhibitors, inhibitors of mTOR, FGFR, ALK, MET, ROS1, PIK3CA, CDK4/6, BRAF/MEK and PARP. Responses in the first follow-up MRI were partial response (13.5%), stable disease (29.7%) and progressive disease (46.0%). There were no new safety signals. Adverse events with fatal outcome (CTCAE grade 5) were not observed. Only, two patients discontinued treatment due to side effects. Median progression-free and overall survival were 9.1/18 months in patients with at least stable disease, and 1.8/3.6 months in those with progressive disease at the first follow-up MRI. CONCLUSION: A broad range of actionable alterations was targeted with available molecular therapeutics. However, efficacy was largely observed in entities with paradigmatic oncogenic drivers, in particular with BRAF mutations. Further research on biomarker-informed molecular matched therapies is urgently necessary. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11060-022-04049-w

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

Disturbed Clockwork Resetting in Sharp-1 and Sharp-2 Single and Double Mutant Mice

BACKGROUND: The circadian system provides the basis to anticipate and cope with daily recurrent challenges to maintain the organisms' homeostasis. De-synchronization of circadian feedback oscillators in humans causes 'jet lag', likely contributes to sleep-, psychiatric-, metabolic disorders and even cancer. However, the molecular mechanisms leading to the disintegration of tissue-specific clocks are complex and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Based on their circadian expression and cell culture experiments, the basic Helix-Loop-Helix (bHLH) transcription factors SHARP-1(Dec2) and SHARP-2(Stra13/Dec1) were proposed as novel negative regulators of the molecular clock. To address their function in vivo, we generated Sharp-1 and Sharp-2 single and double mutant mice. Our experiments reveal critical roles for both factors in regulating period length, tissue-specific control of clock gene expression and entrainment to external cues. Light-pulse experiments and rapid delays of the light-dark cycle (experimental jet lag) unravel complementary functions for SHARP-1 and SHARP-2 in controlling activity phase resetting kinetics. Moreover, we show that SHARP-1 and 2 can serve dual functions as repressors and co-activators of mammalian clock gene expression in a context-specific manner. This correlates with increased amplitudes of Per2 expression in the cortex and liver and a decrease in the suprachiasmatic nucleus (SCN) of double mutant mice. CONCLUSIONS/SIGNIFICANCE: The existence of separate mechanisms regulating phase of entrainment, rhythm amplitude and period length has been postulated before. The differential effects of Sharp-deficiency on rhythmicity and behavioral re-entrainment, coupled to tissue-dependent regulatory functions, provide a new mechanistic basis to further understand the complex process of clock synchronizations

The multispecific thyroid hormone transporter OATP1C1 mediates cell-specific sulforhodamine 101-labeling of hippocampal astrocytes

Sulforhodamine 101 (SR101) is widely used for astrocyte identification, though the labeling mechanism remains unknown and the efficacy of labeling in different brain regions is heterogeneous. By combining region-specific isolation of astrocytes followed by transcriptome analysis, two-photon excitation microscopy, and mouse genetics, we identified the thyroid hormone transporter OATP1C1 as the SR101-uptake transporter in hippocampus and cortex