Extensive Transcriptional Regulation of Chromatin Modifiers during Human Neurodevelopment

Epigenetic changes, including histone modifications or chromatin remodeling are regulated by a large number of human genes. We developed a strategy to study the coordinate regulation of such genes, and to compare different cell populations or tissues. A set of 150 genes, comprising different classes of epigenetic modifiers was compiled. This new tool was used initially to characterize changes during the differentiation of human embryonic stem cells (hESC) to central nervous system neuroectoderm progenitors (NEP). qPCR analysis showed that more than 60% of the examined transcripts were regulated, and >10% of them had a >5-fold increased expression. For comparison, we differentiated hESC to neural crest progenitors (NCP), a distinct peripheral nervous system progenitor population. Some epigenetic modifiers were regulated into the same direction in NEP and NCP, but also distinct differences were observed. For instance, the remodeling ATPase SMARCA2 was up-regulated >30-fold in NCP, while it remained unchanged in NEP; up-regulation of the ATP-dependent chromatin remodeler CHD7 was increased in NEP, while it was down-regulated in NCP. To compare the neural precursor profiles with those of mature neurons, we analyzed the epigenetic modifiers in human cortical tissue. This resulted in the identification of 30 regulations shared between all cell types, such as the histone methyltransferase SETD7. We also identified new markers for post-mitotic neurons, like the arginine methyl transferase PRMT8 and the methyl transferase EZH1. Our findings suggest a hitherto unexpected extent of regulation, and a cell type-dependent specificity of epigenetic modifiers in neurodifferentiation

Characterization of the putative outer membrane protein TTC0322 of Thermus thermophilus

The bacterium Thermus thermophilus lives in hot thermal springs and has potential importance for biotechnology, as it contains thermostable proteins.In this study the characterization of the protein TTC0322 was initiated. Bioinformatical research predicts either a localization in the periplasm or in the outer membrane. Considering the localization on the chromosome, upstream genes encode for an ABCtransporter for glucose and an energizing system like ExbB/ExbD or Tol/Pal. This led to the model of a function of TTC0322 in the active uptake of glucose across the outer membrane. In this model TTC0322 is an active outer membrane transporter for glucose, which is activated by the putative energizing proteins encoded by the three direct upstream genes. To test the model a TTC0322-knockout-mutant was constructed by inserting a bleomycin-resistence-cassette into the gene TTC0322. In a following phenotypically characterization it was found that TTC0322 is essential for growth on glucose, but not for other sugars. Nevertheless, our hypothesis was disapproved, because the knockout-mutant continued transporting glucose. Besides, by knocking out TTC0322, the glucose-ABC-transport-system was constitutively present, which normally is regulated through presence of glucose. That implicates a regulatory effect of TTC0322 on induction. Whether this effect is primarily related to this protein itself or carried out by some second protein influenced through it, was not determined. At this point, a conclusion about the exact role of TTC0322 in T. thermophilus is not possible. Furthermore I tried to express a his-tagged version of TTC0322 in E. coli and T. thermophilus in order to purify it and start some localization studies with it. Low expression only worked in E. coli, whereas T. thermophilus showed no expression, making it impossible to purify the protein or localize it in cellular extracts of T. thermophilus. Additionally there were efforts made to localize the protein by cell-fractionating T. thermophilus wild-type and TTC0322 mutant strains. However, this method was unsuggestive of the localization of TTC0322. By using some bioinformatic research tools extensively in order to find out other hints on this proteins function, it could be shown that the encoding gene isn t necessarily located in one operon with the glucose-ABC-transporter-genes, since anotherpromoter in front of these genes was found. It might form an own operon, including only additional the genes for the energizing system.Another interesting observation alongside these experiments is that MalK, whichis known as the ATP-hydrolyzing subunit of both ABC-transporters for glucose andmaltose, might also be part of a so far unknown fructose-ABC-transporter. Experiments showed that a knockout-mutant of malK lost its ability to grow on fructose

Degeneration mechanisms in human dopaminergic neurons

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. The extensive loss of dopaminergic neurons in the Substantia nigra pars compacta and the resulting lack of dopamine in the target regions lead to severe motor symptoms. The single most consistent risk factor is aging. With a steadily increasing age of the world population, the prevalence of the global burden of PD will further rise in the future.So far, only symptomatic treatments that reduce motor complications are available. Neuroprotective and –restorative therapies are still lacking. To make things worse, at the time point of diagnosis around 60% of nigral dopaminergic neurons are already lost. Thus, strategies to prevent or at least to slow disease progression remain the central aim of PD-related research. We think that a deeper understanding of molecular mechanisms of the selective neurodegeneration is needed.Also, new and optimized models for the study of protective strategies are essential for further achievements in PD research. Animal models are often used, but differences in signaling pathways to humans exist. Furthermore, PD is a disease affecting solely humans. In vitro models, based on human material, represent a relevant alternative to in vivo experiments.In this thesis, the human LUHMES cell line was introduced as model system for studies on neurodevelopment and neurodegeneration. We characterized the cell line with a focus on the suitability for PD research. We optimized the differentiation protocol and demonstrated a fast, homogeneous and irreversible differentiation to post-mitotic dopaminergic neurons. A distinct sensitivity to the parkinsonian model toxin 1-methyl-4-phenylpyridinium (MPP+) was shown. This allowed mechanistic studies on parkinsonian neurodegeneration processes and the investigation of possible intervention strategies. The LUHMES/MPP+ model reproduced many molecular pathways of the complex disease. A newly established differentiation protocol, allowing the generation of dopamine-free LUHMES cells, demonstrated that dopamine contributes to MPP+-toxicity. Furthermore, different strategies using diverse classes of pharmacological inhibitors were found to protect LUHMES from MPP+-induced neurodegeneration.Major findings were that (i) the LUHMES cell line represents an advanced in vitro model for PD research, and that (ii) dopaminergic neurons can be protected from degeneration, although a strong MPP+-mediated energy depletion via inhibition of complex I of the respiratory chain occured. We showed that ATP-depletion and cell death are not inevitably coupled. Transferred to a general view on PD, with mitochondrial dysfunctions as key contributor to disease progression, novel strategies for the protection from degeneration of dopaminergic neurons might be developed. The primary effect of MPP+ (in the cell model) and of mitochondrial dysfunction (in the disease), does not necessarily have to be prevented to be able to protect cells from dying. This approach might, in combination with an earlier diagnosis of the disease, make an important contribution to the development of neuroprotective therapies for PD

Targeting chelatable iron as a therapeutic modality in Parkinson's disease

Aims: The pathophysiological role of iron in Parkinson's disease (PD) was assessed by a chelation strategy aimed at reducing oxidative damage associated with regional iron deposition without affecting circulating metals. Translational cell and animal models provided concept proofs and a delayed-start (DS) treatment paradigm, the basis for preliminary clinical assessments. Results: For translational studies, we assessed the effect of oxidative insults in mice systemically prechelated with deferiprone (DFP) by following motor functions, striatal dopamine (HPLC and MRI-PET), and brain iron deposition (relaxation-R2*-MRI) aided by spectroscopic measurements of neuronal labile iron (with fluorescence-sensitive iron sensors) and oxidative damage by markers of protein, lipid, and DNA modification. DFP significantly reduced labile iron and biological damage in oxidation-stressed cells and animals, improving motor functions while raising striatal dopamine. For a pilot, double-blind, placebo-controlled randomized clinical trial, early-stage Parkinson's patients on stabilized dopamine regimens enrolled in a 12-month single-center study with DFP (30 mg/kg/day). Based on a 6-month DS paradigm, early-start patients (n=19) compared to DS patients (n=18) (37/40 completed) responded significantly earlier and sustainably to treatment in both substantia nigra iron deposits (R2* MRI) and Unified Parkinson's Disease Rating Scale motor indicators of disease progression (p<0.03 and p<0.04, respectively). Apart from three rapidly resolved neutropenia cases, safety was maintained throughout the trial. Innovation: A moderate iron chelation regimen that avoids changes in systemic iron levels may constitute a novel therapeutic modality for PD. Conclusions: The therapeutic features of a chelation modality established in translational models and in pilot clinical trials warrant comprehensive evaluation of symptomatic and/or disease-modifying potential of chelation in PD

Uncoupling of ATP-depletion and cell death in human dopaminergic neurons

The mitochondrial inhibitor 1-methyl-4-phenylpyridinium (MPP(+)) is the toxicologically relevant metabolite of 1-methyl-4-phenyltetrahydropyridine (MPTP), which causes relatively selective degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic LUHMES cells were used to investigate whether ATP-depletion can be uncoupled from cell death as a downstream event in these fully post-mitotic human neurons. Biochemical assays indicated that in the homogeneously differentiated cell cultures, MPP(+) was taken up by the dopamine transporter (DAT). MPP(+) then triggered oxidative stress and caspase activation, as well as ATP-depletion followed by cell death. Enhanced survival of the neurons in the presence of agents interfering with mitochondrial pathology, such as the fission inhibitor Mdivi-1 or a Bax channel blocker suggested a pivotal role of mitochondria in this model. However, these compounds did not prevent cellular ATP-depletion. To further investigate whether cells could be rescued despite respiratory chain inhibition by MPP(+), we have chosen a diverse set of pharmacological inhibitors well-known to interfere with MPP(+) toxicity. The antioxidant ascorbate, the iron chelator desferoxamine, the stress kinase inhibitor CEP1347, and different caspase inhibitors reduced cell death, but allowed ATP-depletion in protected cells. None of these compounds interfered with MPP(+) accumulation in the cells. These findings suggest that ATP-depletion, as the initial mitochondrial effect of MPP(+), requires further downstream processes to result in neuronal death. These processes may form self-enhancing signaling loops, that aggravate an initial energetic impairment and eventually determine cell fate

Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line

We characterized phenotype and function of a fetal human mesencephalic cell line (LUHMES, Lund human mesencephalic) as neuronal model system. Neurodevelopmental profiling of the proliferation stage (d0, day 0) of these conditionally-immortalized cells revealed neuronal features, expressed simultaneously with some early neuroblast and stem cell markers. An optimized 2-step differentiation procedure, triggered by shut-down of the myc transgene, resulted in uniformly post-mitotic neurons within 5 days (d5). This was associated with down-regulation of some precursor markers and further up-regulation of neuronal genes. Neurite network formation involved the outgrowth of 1–2, often > 500 μm long projections. They showed dynamic growth cone behavior, as evidenced by time-lapse imaging of stably GFP-over-expressing cells. Voltage-dependent sodium channels and spontaneous electrical activity of LUHMES continuously increased from d0 to d11, while levels of synaptic markers reached their maximum on d5. The developmental expression patterns of most genes and of the dopamine uptake- and release-machinery appeared to be intrinsically predetermined, as the differentiation proceeded similarly when external factors such as dibutyryl-cAMP and glial cell derived neurotrophic factor were omitted. Only tyrosine hydroxylase required the continuous presence of cAMP. In conclusion, LUHMES are a robust neuronal model with adaptable phenotype and high value for neurodevelopmental studies, disease modeling and neuropharmacology

Generation of Genetically-Modified Human Differentiated Cells for Toxicological Tests and the Study of Neurodegenerative Diseases

Human differentiated cell types, such as neurons or hepatocytes, are of limited availability, and their use for experiments requiring ectopic gene expression is challenging. Using the human conditionally-immortalized neuronal precursor line LUHMES, we explored whether genetic modification in the proliferating state could be used for experiments in the differentiated post-mitotic neurons. First, alpha-synuclein (ASYN), a gene associated with the pathology of Parkinson’s disease, was overexpressed. Increased amounts of the protein were tolerated without change of phenotype, and this approach now allows further studies on protein variants. Knockdown of ASYN attenuated the toxicity of the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP+). Different lentiviral constructs then were tested: cells labeled ubiquitously with green (GFP) or red fluorescent protein (RFP) allowed the quantification of neurite growth and of its disturbance by toxicants; expression of proteins of interest could be targeted to different organelles; production of two different proteins from a single read-through construct was achieved successfully by an expression strategy using a linker peptide between the two proteins, which is cleaved by deubiquitinases; LUHMES, labeled with GFP in the cytosol and RFP in the mitochondria, were used to quantify mitochondrial mobility along the neurites. MPP+ reduced such organelle movement before any other detectable cellular change, and this toxicity was prevented by simultaneous treatment with the antioxidant ascorbic acid. Thus, a strategy has been outlined here to study new functional endpoints, and subtle changes of structure and proteostasis relevant in toxicology and biomedicine in post-mitotic human cells

Alternative early neural differentiation to neural crest progenitors.

<p>(A) hESC were differentiated towards NCP and stained with antibodies specific for <i>OCT4</i> (no stain observed), <i>PAX6</i> (no stain observed), <i>NESTIN</i> and <i>HNK-1</i>. Cell nuclei were labeled with the DNA dye Hoechst H-33342 (blue). Scale bars: 100 µm. (B) Pairwise comparisons of hESC, NEP or NCP yielded 4277 differentially expressed transcripts. The heat map displays the genes after clustering according to the Pearson's correlation of their expression values across samples. The colors represent Z-scores of the row-wise normalized expression values for each gene. The dendrogram indicates the pattern similarities indicated by Spearman correlation distances (1- Spearman correlation coefficient) and shows a large separation of NCP from NEP and hESC. (C) The expression of early neuronal marker genes was measured in three preparations each of hESC, NEP and NCP by qPCR. The transcript levels of NEP and NCP were calculated relative to hESC. The relative gene expression levels were color coded (significant down-regulation vs. hESC in blue; significant up-regulation in red; non-significant changes marked by “N”. The genes showing different behavior in NEP vs NCP are displayed. All measures of variance and p-values are indicated in the supplemental material.</p

Transcriptional regulation of epigenetic modifiers during neuroepithelial differentiation.

<p>(A) The levels of epigenetic modifier transcripts of NEP and hESC were analyzed by qPCR in three independent cell preparations, and relative abundances were calculated. The data were color-coded, with up-regulated genes displayed in red and down-regulated genes in blue. Measures of variance and p-values are indicated in supplemental material, and only significantly regulated genes are displayed. Genes up-regulated >5-fold are displayed in bold. (B) The transcript levels of HDACs were determined for hESC, NEP, NCP and CTX. All expression levels of differentiated cells were normalized to those of hESC, and relative abundances are displayed. For instance, seven different HDACs were up-regulated in NEP compared to hESC. The dotted lines indicate 2-fold regulation levels. Data are means ± SEM of three independent differentiations. *: p<0.05</p

Effect of neuroectodermal differentiation on localization of histone marks.

<p>(A) hESC were differentiated towards NEP and stained with antibodies specific for Oct4, HNK-1 (neural crest marker), Pax6 (NEP marker) and nestin (neural stem cell marker). Nuclei were stained with the DNA dye H-33342 (blue). Scale bars: 100 µm. (B) GO analysis of the up-regulated genes in NEP compared to hESC (C) Whole cell extracts from hESC and NEP were analyzed by Western blot with antibodies specific for the indicated histone H3 modifications. Total histone H3 (Pan-H3) was used as loading control. (D) hESC and differentiated NEP were grown on glass cover slips and immunostained with antibodies specific for H3K9me3 or H4K20me3. The upper panels show grey-scale signal intensities of the stain, the lower panels show a superimposition of the same histone stain as above (red) with a DNA counter-stain (DAPI, blue). Arrows mark two cells with a diffuse H4K20me3 stain, which differs from the spot-like pattern always observed in hESC. Scale bars: 10 µm. (E) Chromatin immunoprecipitiation was perfomed from nuclei of hESC or NEP with antibodies specific for H3K4me3 and H3K27me3. The abundance of promoter regions of <i>OCT4, NANOG, PAX6</i> and <i>SOX2</i> was measured by qPCR with specific primers for the indicated genes. Data were compared to control samples prepared without specific antibody and are indicated as relative enrichment. Data are means ± SD from 2 experiments.</p