Suzanne Kadereit

Sensitivity of dopaminergic neuron differentiation from stem cells to chronic low-dose methylmercury exposure

Perinatal exposure to low doses of methylmercury (MeHg) can cause adult neurological symptoms. Rather than leading to a net cell loss, the toxicant is assumed to alter the differentiation and neuronal functions such as catecholaminergic transmission. We used neuronally differentiating murine embryonic stem cells (mESC) to explore such subtle toxicity. The mixed neuronal cultures that formed within 20 days contained a small subpopulation of tyrosine hydroxylase (TH)-positive neurons with specific dopaminergic functions such as dopamine transport (DAT) activity. The last 6 days of differentiation were associated with the functional maturation of already preformed neuronal precursors. Exposure to MeHg during this period downregulated several neuronal transcripts, without affecting housekeeping genes or causing measurable cell loss. Profiling of mRNAs relevant for neurotransmitter systems showed that dopamine receptors were coordinately downregulated, whereas known counterregulatory systems such as galanin receptor 2 were upregulated. The chronic (6 days) exposure to MeHg, but not shorter incubation periods, attenuated the expression levels of endogenous neurotrophic factors required for the maturation of TH cells. Accordingly, the size of this cell population was diminished, and DAT activity as its signature function was lost. When mixed lineage kinase activity was blocked during MeHg exposure, DAT activity was restored, and the reduction of TH levels was prevented. Thus, transcriptional profiling in differentiating mESC identified a subpopulation of neurons affected by MeHg, and a pharmacological intervention was identified that specifically protected these cells.

microRNA 184 regulates expression of NFAT1 in umbilical cord blood CD4+ T cells

The reduced expression of nuclear factor of activated T cells-1 (NFAT1) protein in umbilical cord blood (UCB)-derived CD4+ T cells and the corresponding reduction in inflammatory cytokine secretion after stimulation in part underlies their phenotypic differences from adult blood (AB) CD4+ T cells. This muted response may contribute to the lower incidence and severity of high-grade acute graft-versus-host disease (aGVHD) exhibited by UCB grafts. Here we provide evidence that a specific microRNA, miR-184, inhibits NFAT1 protein expression elicited by UCB CD4+ T cells. Endogenous expression of miR-184 in UCB is 58.4-fold higher compared with AB CD4+ T cells, and miR-184 blocks production of NFAT1 protein through its complementary target sequence on the NFATc2 mRNA without transcript degradation. Furthermore, its negative effects on NFAT1 protein and downstream interleukin-2 (IL-2) transcription are reversed through antisense blocking in UCB and can be replicated via exogenous transfection of precursor miR-184 into AB CD4+ T cells. Our findings reveal a previously uncharacterized role for miR-184 in UCB CD4+ T cells and a novel function for microRNA in the early adaptive immune response.

A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles

Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood–brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6+ precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.

GFAP-independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells

The directed generation of pure astrocyte cultures from pluripotent stem cells has proven difficult. Generation of defined pluripotent‐stem‐cell derived astrocytes would allow new approaches to the investigation of plasticity and heterogeneity of astrocytes. We here describe a two‐step differentiation scheme resulting in the generation of murine embryonic stem cell (mESC) derived astrocytes (MEDA), as characterized by the upregulation of 19 astrocyte‐associated mRNAs, and positive staining of most cells for GFAP (glial fibrillary acidic protein), aquaporin‐4 or glutamine synthetase. The MEDA cultures could be cryopreserved, and they neither contained neuronal, nor microglial cells. They also did not react to the microglial stimulus lipopolysaccharide, while inflammatory activation by a complete cytokine mix (CCM) or its individual components (TNF‐α, IL1‐β, IFN‐γ) was readily observed. MEDA, stimulated by CCM, became susceptible to CD95 ligand‐induced apoptosis and produced NO and IL‐6. This was preceded by NF‐kB activation, and up‐regulation of relevant mRNAs. Also GFAP‐negative astrocytes were fully inflammation‐competent. Neurotrophic support by MEDA was found to be independent of GFAP expression. In summary, we described here the generation and functional characterization of microglia‐free murine astrocytes, displaying phenotypic heterogeneity as is commonly observed in brain astrocytes.

Metabolite profiling in posttraumatic stress disorder

BackgroundTraumatic stress does not only increase the risk for posttraumatic stress disorder (PTSD), but is also associated with adverse secondary physical health outcomes. Despite increasing efforts, we only begin to understand the underlying biomolecular processes. The hypothesis-free assessment of a wide range of metabolites (termed metabolite profiling) might contribute to the discovery of biological pathways underlying PTSD.MethodsHere, we present the results of the first metabolite profiling study in PTSD, which investigated peripheral blood serum samples of 20 PTSD patients and 18 controls. We performed liquid chromatography (LC) coupled to Quadrupole/Time-Of-Flight (QTOF) mass spectrometry. Two complementary statistical approaches were used to identify metabolites associated with PTSD status including univariate analyses and Partial Least Squares Discriminant Analysis (PLS-DA).ResultsThirteen metabolites displayed significant changes in PTSD, including four glycerophospholipids, and one metabolite involved in endocannabinoid signaling. A biomarker panel of 19 metabolites classifies PTSD with 85% accuracy, while classification accuracy from the glycerophospholipid with the highest differentiating ability already reached 82%.ConclusionsThis study illustrates the feasibility and utility of metabolite profiling for PTSD and suggests lipid-derived and endocannabinoid signaling as potential biological pathways involved in trauma-associated pathophysiology.

Metabolite fingerprinting reveals new serum metabolites and associated pathways in posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) is associated with an increased risk for adverse physical health outcomes. However, the underlying bio-molecular processes and associated pathways remain poorly elucidated. Using time-of-flight mass spectrometry (TOF-MS), the untargeted and holistic investigation of the metabolome – the total of hydrophilic and amphiphilic metabolites - holds the potential to provide novel insights into PTSD pathophysiology. To address metabolome changes associated with PTSD, serum from 20 individuals with a PTSD diagnosis and 18 healthy controls was analyzed with TOF-MS. Symptom severity of PTSD was assessed using the Clinician-administered PTSD scale (CAPS). Univariate and multivariate approaches, namely Partial Least Square Discriminant Analysis, were applied for statistical analyses. The group comparison revealed 13 metabolites significantly altered in PTSD, including four glycerophospholipids and one metabolite involved in endocannabinoid signaling. Out of the 13, eleven metabolites showed a correlation between the serum level and the CAPS score. In the multivariate approach, a metabolite profile of 19 biomolecules predicted PTSD with an accuracy of 85%. Here, we initially illustrate the potential of metabolite fingerprinting to identify novel pathophysiological underpinnings of PTSD. It further provides the possibility to highlight associated pathways, such as lipid-derived and endocannabinoid signaling. More research will help to gain not only a deeper understanding of the molecular mechanisms and associated pathways in PTSD, but also of the biological processes stimulated by (psycho) therapeutical treatment.