Mechanismen DNMT1-abhängiger Regulation des neuronalen Überlebens

• DNMT1-dependent mechanisms of neuronal survival regulation

Bayer, Cathrin; Zimmer-Bensch, Geraldine Marion (Thesis advisor); Lüscher, Bernhard (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

The cerebral cortex as the seat of higher cognitive functions consists of different, highly interconnected cellular components that form neural circuits in a network of excitation and inhibition. The establishment of these neuronal networks during development ensures their proper functionality upon maturation, which needs to be maintained over lifetime due to the restricted capacity of neurons to regenerate. Apart from excitatory principal neurons, the cortex' inhibitory interneurons are a significant part of these structures and modulate incoming as well as outgoing signals, and therefore the activity of participating neurons. Hence, cortical inhibitory interneurons are key for cortical information processing. From the cells’ birth and over their entire lifespan a tight orchestration but also flexible regulation of gene expression is essential, crucially impacting developmental programs, functionality and cell survival. Transcriptional control is accomplished by various epigenetic mechanisms including DNA methylation, histone modifications and regulatory RNAs. These mechanisms are involved in regulating the accessibility of genomic sequences, gene activity and posttranscriptional events. DNA methylation as one major epigenetic mechanism is catalyzed by the enzymes of the protein family of DNA methyltransferases and is often associated with gene silencing. The largest protein of the DNA methyltransferases with several protein binding domains is DNMT1, being strongly expressed in cortical inhibitory interneurons. DNMT1 is critical for both orchestrating developmental processes of cortical interneurons and their proper function in the adult cortex. During embryonic development, DNMT1 promotes the migration of interneurons from their site of origin in the basal telencephalon to the cortex and supports their survival. In adult interneurons, DNMT1 regulates intracellular processes such as endocytosis and through this vesicle recycling in a DNA methylation dependent manner, which affects the GABAergic neurotransmission. In contrast, in the aging brain, DNMT1 regulates the survival of interneurons independent of its DNA methylation function through indirect impact on components of the proteostasis network. Intracellular processes such as protein transport and protein degradation among others are essential for this network and ensure neuronal long-term survival. A defect in one of these processes may have fatal consequences and can lead to neurodegeneration. Previous data showed that DNMT1 conducts stage- and cell-type specific functions even independent of its DNA methylating activity. Hence, this work aims to investigate DNA methylation-independent modes of action of DNMT1, and how DNMT1 regulates proteostasis and cell survival.In this thesis, it was highlighted that apart from its DNA methylating function, DNMT1 triggers changes in histone modifications. In POA derived interneurons as well as in the neuroblastoma cell line N2a, DNMT1 affects the trimethylation of histone 3 lysine 27 (H3K27me3). Experiments in which Dnmt1 was depleted by target-specific siRNA led to a decrease of global H3K27me3 levels, indicating that DNMT1 is required for proper establishment of H3K27me3 marks. In support of this, an interaction of DNMT1 with EZH2, which mediates the trimethylation of H3K27, was identified in N2a cells as well as in murine tissue of interneuron-generating regions. Analyses on further interaction partners of DNMT1 by mass spectrometry, revealed putative interactions with proteins of different cell compartments including the nucleus but also the cytoplasm. In line with the identification of potential cytosolic binding partners, a cytosolic localization of the DNMT1 protein was verified, which additionally points to possible cytosolic functions of DNMT1. The identified DNMT1-interacting proteins, TPD54, DOCK7, DPYSL2, CRMP1 and Gephyrin, are strongly related to intracellular transport processes and especially retrograde trafficking was already shown to be negatively influenced by DNMT1. Besides this microtubule-based transport mechanism, in this study DNMT1 was found to adversely impact also autophagy. Both processes are crucial for proteostasis and the elimination of aggregation-prone proteins. In this context, knockdown experiments provided evidence that DNMT1 delays the aggregation of mutant Huntingtin in perinuclear regions. The formation of aggresomes that contained mutant Huntingtin was found to be associated with an improved survival of cells expressing the mutant protein. In this regard, a functional implication of DNMT1 in mutant Huntingtin-induced cytotoxicity was revealed, as cell survival of mutant Huntingtin-expressing cells was reduced in the presence of DNMT1. This is in line with previous observations of an impaired long-term survival of parvalbuminergic interneurons in the aging brain when Dnmt1 is expressed.The here mentioned effects of DNMT1 on the mutant Huntingtin-induced cytotoxicity propose DNA methylation-independent mechanisms as an inhibition of the DNA methylase activity did not impact cell survival of mutant Huntingtin-expressing cells. Instead, these effects seem to rely at least in part on DNMT1-dependent regulation of protein degradative processes presumably through DNMT1’s interaction with cytosolic proteins.Collectively, results of this thesis have verified that DNMT1 influences a histone methylation through the protein interaction with the histone-modifying enzyme EZH2 in neurons. Besides its well-known nuclear location, DNMT1 also acts in the cytoplasm, where it putatively interacts with cytosolic proteins that are associated with the proteostasis-relevant microtubule-based transport. Through this process, DNMT1 negatively influences aggresome formation of mutant Huntingtin and decreases cell survival of mutant Huntingtin-expressing cells. Overall, DNMT1 seems to have important functions apart from its gene regulation activity, being proposed to negatively affect proteostasis-relevant processes and cell survival, which could rely on the possible binding to cytoplasmic interaction partners.