Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv

Aneuploidy is a change in number or structure of one or more chromosomes that are not a multiple of the whole chromosome set. One of the best known pathological aneuploidies is trisomy 21 (Down syndrome), with chromosome 21 present in three instead of two copies. Patients with Down syndrome display severe mental retardation and growth defects. In fact, most abnormal aneuploid karyotypes lead to spontaneous abortions during embryogenesis, indicating that aneuploidy is not well tolerated in humans. Aneuploidy was also shown to be a common hallmark of cancer tissues; however, the debate is ongoing whether aneuploidy is rather a by-product or a trigger of tumorigenesis. Even though aneuploid karyotypes were already identified more than 100 years ago little is understood about cellular physiology of aneuploidy cells, especially in humans. To uncover the consequences of numerical aneuploidy in human cells, I generated aneuploid cell lines derived from the human cell lines HCT116 and RPE-1 hTERT. First, we showed that aneuploid cells proliferate slower compared to their disomic counterparts. A detailed cell cycle analysis revealed that this delay was due to a prolonged G1 and S phase, whereas G2 and M phase remained unperturbed. Furthermore, we conducted an in depth genome wide comparison of DNA, mRNA and protein levels in aneuploid cells. Using CGH, mRNA array and SILAC technology, we quantified the changes in DNA, mRNA and protein abundance. We revealed that extra chromosomes are actively transcribed and translated. However, the abundance of some proteins, particularly subunits of protein complexes and protein kinases, are adjusted towards disomic levels. Additionally, we asked how the cellular physiology is affected by the addition of a specific chromosome. Two scenarios are possible: either the cellular response depends on the additional chromosomes or all aneuploid cells show the same changes of cellular physiology. Indeed, we found that all aneuploid cell lines show similar physiological responses, irrespective of the type of additional chromosome. All aneuploid cell lines down-regulate DNA and RNA metabolism and up-regulate among others energy metabolism, lysosome function and membrane biosynthesis pathways. Lysosomes which are involved in autophagy are besides the ubiquitin-proteasome system important for cellular protein turn over. We found p62-dependent selective autophagy increased in all analyzed cell lines with extra chromosomes suggesting a role of p62-dependent selective autophagy in maintenance of protein homeostasis upon expression of extra protein in these cell lines.

Generation of the brain depends on proper regulation of progenitor proliferation and differentiation during development. Many such factors known to affect proliferation and differentiation are transcription factors. In particular, the transcription factor Pax6 has received much attention because of its potency to control various aspects of brain development. During development of the telencephalon Pax6 regulates patterning, cell proliferation and neurogenesis, but how Pax6 mediates and coordinates these diverse functions at the molecular level is not well understood. It has previously been demonstrated that the homeodomain of Pax6 plays a role in establishing the pallial-subpallial boundary. However it is not involved in other processes during telencephalic development as shown by the analysis of Pax64NEU mice, which are characterized by a point-mutation in the DNA-binding helix of the homeodomain. In order to gain more insights into the molecular network underlying the mild homeodomain function in the developing telencephalon, transcriptome analysis with Pax64NEU mice was performed. Almost no transcriptional changes were detected, suggesting that transcriptional regulation by the homeodomain of Pax6 has no major impact on forebrain development. Additionally, these results implied that the majority of effects exerted through Pax6 during telencephalic development are mediated by the bipartite paired-domain (PD). Therefore the main focus of this thesis was to examine the specific roles of the Pax6 paired-domain and its individual DNA-binding subdomains (PAI and RED) during forebrain development. The role of these DNA-binding domains was examined using mice with point-mutations in the PAI (Pax6Leca4, N50K) and RED (Pax6Leca2 R128C) subdomains and showed that the mutations in these subdomains exert opposing roles regulating proliferation in the developing cortex. While the mutated PAI domain resulted in reduced proliferation of both apical and basal progenitors, the mutated RED domain provoked increased proliferation. However, the PAI domain largely mediates the neurogenic function of Pax6. Additionally, genome-wide transcriptome analysis was able to unravel the key signatures mediated by the distinct domains. In summary, Pax6 exerts its key roles during forebrain development by use of distinct subdomains to regulate proliferation and differentiation. Thus Pax6 is able to coordinate and fine tune patterning, neurogenesis and proliferation in a simultaneous manner in different radial glial subpopulations. The transcriptional regulation through Pax6 may not only be restricted to protein coding genes, but may also include control of microRNA (miRNA) expression. Such small RNA molecules have recently been implicated in proliferation and differentiation during development, however expression and the role of single microRNAs is still poorly understood. Towards this end, miRNA expression profiling was performed using an embryonic stem cell differentiation system at different stages of neuronal differentiation in order to identify new miRNAs involved in radial glia specification and differentiation. This analysis revealed a number of microRNAs induced during differentiation from neural progenitors to neurons. Most strikingly only four miRNA candidates were found with exclusively high expression in progenitor cells. These data suggest that also Pax6 may play a role in transcriptional regulation beyond mRNAs.

The vast majority of mitochondrial proteins are encoded in the nucleus and synthesized as precursor proteins on cytosolic ribosomes. After translation, these precursor proteins are imported in a largely, if not completely, unfolded state into one of the four mitochondrial subcompartments, the outer membrane, the intermembrane space, the inner membrane or the matrix. Once the precursor proteins reach their compartment of destination, they can fold into the functionally active three-dimensional native structure. Therefore, internal mitochondrial folding systems are needed in each subcompartment to assist folding of these precursor proteins upon import. Members of several “classical” chaperone families are present in the mitochondrial matrix and have been shown to support import and folding of newly imported polypeptides. However, folding of proteins in the mitochondrial intermembrane space is only poorly understood. Recently, a disulfide relay system in the intermembrane space that mediates import and folding was described, but this system is limited to proteins that form disulfide bonds. For the majority of intermembrane proteins, folding helpers that promote folding have not yet been discovered. In order to identify general folding helpers of the intermembrane space, the well studied model substrate mouse dihydrofolate reductase (DHFR) was targeted to the mitochondrial intermembrane space of S. cerevisiae and its folding analyzed. DHFR assumes its mature fold in the intermembrane space and heat shock induces DHFR aggregation. Interestingly, aggregation is counteracted by an ATP-dependent process. The i-AAA protease Yme1 that is anchored in the inner mitochondrial membrane and exposes its functional domains to the intermembrane space was able to prevent the aggregation of DHFR. A number of proteins of diverse structural and functional classes were found in the aggregate fractions of mitochondria lacking Yme1. Amongst them were factors that are involved in the establishment and maintenance of the mitochondrial ultrastructure, lipid metabolism, protein translocation and respiratory growth. Considering the diversity of the proteins affected in the absence of Yme1 and their function in mitochondria, the pleiotropic effects of the deletion of Yme1 can be readily explained. The findings of the present in vivo study confirm previous hints to a chaperone-like function of Yme1 resulting from in vitro experiments. Yme1 thus has a dual role as protease and as chaperone and occupies a key position in the protein quality control system of the mitochondrial intermembrane space.

Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. Conversion of postnatal astroglia from the cerebral cortex of mice into functional neurons in vitro can be achieved by forced expression of a single transcription factor. Also skin fibroblasts have been successfully reprogrammed into functional neurons yet through the synergistic action of several transcription factors. A major challenge for the translation of neuronal reprogramming into therapy concerns the feasibility of this approach in adult human tissues. This work demonstrates the potential of perivascular cells isolated from the adult human brain to serve as a substrate prompted to neuronal reprogramming by forced co-expression of neurogenic transcription factors, namely the SRY-related HMG box protein Sox2 and the basic helix loop helix (bHLH) mammalian homologue of achaete-schute-1 Mash1 (also known as Ascl1). The cells used in this study display characteristics of pericytes assessed by immunocytochemistry, fluorescence-activated cell sorting (FACS) and real time RT-PCR. The presence of neural progenitor cells was excluded by real time RT-PCR analysis of mRNAs typically expressed by these cell lineages. Upon expression of Sox2 and Mash1, these cells adopt a neuronal phenotype characterized by the expression of neuronal markers such us ßIII-Tubulin, MAP2, NeuN, GABA and calretinin. Electrophysiological recordings reveal the ability of these cells to fire repetitive action potentials and to integrate into neuronal networks when co-cultured with mouse embryonic neurons. The pericytic nature of the reprogrammed cells was further demonstrated by isolation of PDGFRß-positive cells from adult human brain cultures by FACS and monitoring the Mash1/Sox2-induced neuronal conversion by time-lapse video microscopy. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue non-specific alkaline phosphatase promoter corroborated that pericytes from the adult cerebral cortex can be expanded and reprogrammed in vitro into neurons by co-expression of Sox2 and Mash1. These results demonstrate the feasibility of an in vitro neuronal reprogramming approach on somatic cells isolated from the adult human cerebral cortex which could have important implications in the development of in vivo direct repair strategies in neurodegenerative diseases and brain injury.

It is becoming increasingly clear by current research that the continuum of physiological anxiety up to psychopathology is not merely dependent on genes, but is orchestrated by the interplay of genetic predisposition, gene x environment and epigenetic interactions. To consider this interplay, we here took advantage of the rigid genetic predisposition of a selectively bred mouse model exhibiting high anxiety-related behavior (HAB) and tested whether and how enriched environment, a manipulation of housing conditions, is capable of rescuing the genetically driven high anxiety phenotype via gene x environment and/or epigenetic interactions. Indeed, enriched environment exerts a significant anxiolytic effect on HABs of both sexes indicating for the first time that even a rigid genetic predisposition of high anxiety can be rescued by beneficial environmental stimuli. Thereby, a reduced neophobia and a bigger behavioral repertoire of HABs (e.g. social interactions) have been observed with a stronger anxiolysis in males than in females. The behavioral shift is accompanied by an attenuated release of corticosterone after application of a mild stressor. A hyperreactive hypothalamic-pituitary-adrenal (HPA) axis and amygdala constitute the most common symptoms of anxiety disorders, and decreased corticosterone release seems to entail a reduced release of noradrenaline from locus caeruleus (LC) to the medial prefrontal cortex (mPFC), thereby increasing the top-down control of mPFC on amygdala. This would entail less activation of amygdala and thus HPA axis, a consequence we indeed can observe as decreased neuronal activity flow through the amygdala of enriched housed (EE) compared to standard housed (SE) HABs. We suggest that corticotropin-releasing hormone receptor 1 (Crhr1) is critically involved in this phenomenon since (i) HABs compared to low anxiety-related behavior (LAB) mice exhibit higher Crhr1 mRNA in the basolateral amygdala (BLA), (ii) this overexpression can be significantly reduced when HABs are housed in enriched environment and (iii) a bilateral application of a CRHR1 antagonist in the BLA of SE HABs induced a significant anxiolytic effect. Subsequent pyrosequencing identified that enriched environment increased methylation at a CpG site in the promoter of Crhr1, which is located next to a transcription factor binding site (TFB) of the epigenetic transcription factor Yin Yang 1 (YY1), whose mRNA levels are indeed decreased in EE HABs. In silico analysis identified Nr4a1 and D3Ertd300e as critical co-transcription factors, whereas Nr4a1 seems to be regulated by the quantity of available glucocorticoid receptor (GR) and D3Ertd300e positively regulates YY1. Thus, we hypothesize that reduced corticosterone release decreases the availability and thus binding of corticosterone to GR in the BLA. This, in turn decreases the binding affinity of Nr4a1 to D3Ertd300e, which then cannot positively regulate YY1 to decrease or even prevent methylation at the identified CpG site of Crhr1. This would finally result in a differentially methylated region (DMR) with higher methylation levels in EE HABs, which underlies the observed gene expression differences. The identified DMR might therefore be used as a biomarker for high or pathological anxiety. This hypothesized mechanism highlights the possibility that even a rigid genetic predisposition modeling pathological anxiety might be rescued by an epigenetic process that seems to be triggered by beneficial environmental stimuli, thereby raising the exciting possibility for new treatment strategies, which can be utilized complementary to already existing ones.