Pluripotent stem cells for uncovering the role of mitochondria in human brain function and dysfunction

Mitochondrial dysfunctions are a known pathogenetic mechanism of a number of neurological and psychiatric disorders. At the same time, mutations in genes encoding for components of the mitochondrial respiratory chain cause mitochondrial diseases, which commonly exhibit neurological symptoms. Mitochondria are therefore critical for the functionality of the human nervous system. The importance of mitochondria stems from their key roles in cellular metabolism, calcium handling, redox and protein homeostasis, and overall cellular homeostasis through their dynamic network. Here, we describe how the use of pluripotent stem cells (PSCs) may help addressing the physiological and pathological relevance of mitochondria for the human nervous system. PSCs allow the generation of patient-derived neurons and glia and the identification of gene-specific and mutation-specific cellular phenotypes via genome engineering approaches. We discuss the recent advances in PSC-based modeling of brain diseases and the current challenges of the field. We anticipate that the careful use of PSCs will improve our understanding of the impact of mitochondria in neurological and psychiatric disorders and the search for effective therapeutic avenues

Cell-type-specific modulation of feedback inhibition by serotonin in the hippocampus

Midbrain raphe nuclei provide strong serotonergic projections to the hippocampus, in which serotonin (5-HT) exerts differential effects mediated by multiple 5-HT receptor subtypes. The functional relevance of this diversity of information processing is poorly understood. Here we show that serotonin via 5-HT(1B) heteroreceptors substantially reduces synaptic excitation of cholecystokinin-expressing interneurons in area CA1 of the rat hippocampus, in contrast to parvalbumin-expressing basket cells. The reduction is input specific, affecting only glutamatergic synaptic transmission originating from CA1 pyramidal cells. As a result, serotonin selectively decreases feedback inhibition via 5-HT(1B) receptor activation and subsequently increases the integration time window for spike generation in CA1 pyramidal cells. Our data imply an important role for serotonergic modulation of GABAergic action in subcortical control of hippocampal output

Forecast, observation and modelling of a deep stratospheric intrusion event over Europe

A wide range of measurements was carried out in central and southeastern Europe within the framework of the EU-project STACCATO (Influence of Stratosphere-Troposphere Exchange in a Changing Climate on Atmospheric Transport and Oxidation Capacity) with the principle goal to create a comprehensive data set on stratospheric air intrusions into the troposphere along a rather frequently observed pathway over central Europe from the North Sea to the Mediterranean Sea. The measurements were based on predictions by suitable quasi-operational trajectory calculations using ECMWF forecast data. A predicted deep Stratosphere to Troposphere Transport (STT) event, encountered during the STACCATO period on 20-21 June 2001, could be followed by the measurements network almost from its inception. Observations provide evidence that the intrusion affected large parts of central and southeastern Europe. Especially, the ozone lidar observations on 20-21 June 2001 at Garmisch-Partenkirchen, Germany captured the evolution of two marked tongues of high ozone with the first one reaching almost a height of 2 km, thus providing an excellent data set for model intercomparisons and validation. In addition, for the first time to our knowledge concurrent measurements of the cosmogenic radionuclides <sup>10</sup>Be and <sup>7</sup>Be and their ratio <sup>10</sup>Be/<sup>7</sup>Be are presented together as stratospheric tracers in a case study of a stratospheric intrusion. The ozone tracer columns calculated with the FLEXPART model were found to be in good agreement with water vapour satellite images, capturing the evolution of the observed dry streamers of stratospheric origin. Furthermore, the time-height cross section of ozone tracer simulated with FLEXPART over Garmisch-Partenkirchen captures with many details the evolution of the two observed high-ozone filaments measured with the IFU lidar, thus demonstrating the considerable progress in model simulations. Finally, the modelled ozone (operationally available since October 1999) from the ECMWF (European Centre for Medium-Range Weather Forecasts) atmospheric model is shown to be in very good agreement with the observations during this case study, which provides the first successful validation of a chemical tracer that is used operationally in a weather forecast model. This suggests that coupling chemistry and weather forecast models may significantly improve both weather and chemical forecasts in the future

Genomic characterization of murine monocytes reveals C/EBPβ transcription factor dependence of Ly6C(-) cells

Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C(+) monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C(-) monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C(+) into Ly6C(-) monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C(-) monocytes involved induction of the transcription factor C/EBP{beta} and C/EBP{beta}-deficient mice lacked Ly6C(-) monocytes. Mechanistically, C/EBP{beta} bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor

1,2-(13)C(2)-glucose tracing approach to assess metabolic alterations of human monocytes under neuroinflammatory conditions

Neuroinflammation is one of the common features in most neurological diseases including multiple sclerosis (MScl) and neurodegenerative diseases such as Alzheimer's disease (AD). It is associated with local brain inflammation, microglial activation, and infiltration of peripheral immune cells into cerebrospinal fluid (CSF) and the central nervous system (CNS). It has been shown that the diversity of phenotypic changes in monocytes in CSF relates to neuroinflammation. It remains to be investigated whether these phenotypic changes are associated with functional or metabolic alteration, which may give a hint to their function or changes in cell states, e.g., cell activation. In this article, we investigate whether major metabolic pathways of blood monocytes alter after exposure to CSF of healthy individuals or patients with AD or MScl. Our findings show a significant alteration of the metabolism of monocytes treated with CSF from patients and healthy donors, including higher production of citric acid and glutamine, suggesting a more active glycolysis and tricarboxylic acid (TCA) cycle and reduced production of glycine and serine. These alterations suggest metabolic reprogramming of monocytes, possibly related to the change of compartment (from blood to CSF) and/or disease-related. Moreover, the levels of serine differ between AD and MScl, suggesting different phenotypic alterations between diseases

Assessment of ethanol-induced toxicity on iPSC-derived human dopaminergic neurons using a novel high-throughput mitochondrial neuronal health (MNH) assay

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human neurons, and particularly dopaminergic neurons that are crucial for the development of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human dopaminergic neuron-containing cultures (DNs) from induced pluripotent stem cells (iPSCs). The assay, dubbed mitochondrial neuronal health (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DNs decreases MMP and neuronal outgrowth in a dose-dependent manner. The toxic effect of ethanol on DNs was already detectable after 1 h of exposure, and occurred similarly in DNs derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate compounds modulating acute ethanol toxicity in human DNs. We found that disulfiram and baclofen, which are used for AUD treatment, and lithium caused neurotoxicity also in the absence of ethanol, while the spasmolytic drug flavoxate positively influenced MNH. Altogether, we developed an HT assay to probe human MNH and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of MNH and toxicity in live human neurons

Early and rapid engraftment of bone marrow-derived microglia in scrapie

Prion neuroinvasion is accompanied by maximal activation of microglia, the significance of which for pathogenesis is unknown. Here, we used bone marrow (BM) cells expressing GFP (green fluorescent protein) to study the turnover of microglia in mouse scrapie. We found that >or=50% of all brain microglia were replaced by BM-derived cells before clinical disease onset. In terminally sick mice, microglia density increased threefold to fourfold. Hence BM-derived microglia rapidly and efficaciously colonize the brain in scrapie. Whereas reconstitution of wild-type mice with prion protein-deficient (Prnp(o/o)) BM did not alter scrapie pathogenesis, Prnp(o/o) mice transplanted with wild-type BM cells were resistant to peripherally administered prions despite high levels of infectivity in the spleen. Cerebellar homogenates from prion-inoculated Prnp(o/o) mice reconstituted with >10% of wild-type microglia failed to infect transgenic mice overexpressing the cellular prion protein. Hence, in contrast to previous reports, microglia are not competent for efficient prion transport and replication in vivo

Alternative Splicing and Extensive RNA Editing of Human TPH2 Transcripts

Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre- mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour