6 General discussion
6.2 Epigenetics in normal and diseased postmitotic neurons
As the knowledge about the importance of epigenetic regulation on cellular function grows it becomes evident that epigenetic mechanisms either cause or influence many human disorders. First links to pathology were made, not surprisingly, in cancer studies (215). But other so-called “complex disorders” like neurodegenerative diseases are also in the focus of research. The idea of a connection between dysfunctional epigenetic control and neurodegeneration is not too farfetched (227). In fact, the treatment of neurodegeneration has been advancing towards epigenetic drug targeting recently
(261). One good example is the use of sodium butyrate and other HDAC-inhibitors as neuroprotective agents in Alzheimer`s and Parkinson`s disease models (282-284).
HDACs, in general, have been recognized as important targets for therapeutic approaches in neurological disorders. This includes not only neurodegeneration but also depression, anxiety and cognitive deficits caused by neurodevelopmental disorders (283). Cases of neurological disease types where epigenetic processes are the direct cause for malfunction are also known (256). Some of them are associated with mutations in important epigenetic regulators, like MECP2. MECP2 is a conserved binding protein for methylated DNA and is associated with repressor recruitment. A mutation in this gene causes the intellectual disability and delayed psychomotor development that is seen in the Rett-Syndrome phenotype (285). Even if epigenetic modifiers are not the direct cause of the disorder, many neurological diseases are characterized by altered DNA methylation or histone modification patterns (286). That is why some researchers try to find disease specific epigenetic signatures in order to discover patterns of disease formation (287) (Fig. 29).
Figure 29: Possible context between risk factors, epigenome and neurodegenerative disease
The combination of genetic and environmental factors could be relevant to the pathogenesis of complex neurodegenerative disorders (adapted from Fischer, 2013)
In this respect, the concept of epigenetic transcriptional regulation is both intriguing and concerning at the same time. On the one hand targeting those dynamic processes can help to repair intrinsic malfunctions of the transcriptional apparatus that cause disease and disorders; on the other hand those processes are also vulnerable to extrinsic disturbances especially because they are so dynamically regulated. Those disturbances can be environmental pollutants like pesticides to which we are exposed on a daily basis. Two prominent examples are the herbicide paraquat (288) and the insecticide dieldrin. Both are cytotoxic for dopaminergic neurons; partly because they induce histone hyperacetylation in those cells. This shows that understanding the underlying
principles of epigenetic regulation is important to expose, protect against and ultimately eliminate the causes of neurological disorders.
In the work presented in chapter 5, we focused on one particular epigenetic modification that is also associated with neurodegenerative phenotypes (289); the methylation of H3K27. This histone modification is mediated by EZH 1 and 2. Both are members of the polycomb group (PcG). This protein family is associated with transcriptional repression and the establishment and maintenance of cell fate decisions during development (290-292). PcG proteins also play a role in neuronal development and tumorigenesis (293,294). While this seems contradictory a tight regulation of cell cycle is crucial for both processes; the maturation of neurons and tumorigenesis. Cell cycle exit and the prevention of re-entry is a major prerequisite for the proper differentiation into mature neurons, whereas an uncontrolled regulation of the cell cycle leads to cancer. Therefore PcG family members connect two seemingly contrary processes; postmitotic cell cycle exit in neurons and uncontrolled cell proliferation in cancer. Interestingly, the methyltransferase EZH2 is reported to be overexpressed in aggressive and progressive tumours (295), it is downregulated in postmitotic neurons.
Instead the second H3K27 methyltransferase EZH1 is expressed in those cells (see chapter 3). This indicates a major role of PcG proteins in the delicate balance between cell cycle regulation and differentiation.
With our work we wanted to answer the question if H3K27me has a functional role even after cell cycle exit. We observed a striking enrichment of H3K27me at the nuclear periphery of several neuronal lineages once the cells entered a postmitotic state. This was true even for non-neuronal postmitotic macrophages indicating that this peripheral localization is more a postmitotic than a neuronal feature. Since a tight regulation of gene expression is especially important for long lived cells like neurons, H3K27me could mark a terminally restricted type of chromatin which prevents cell cycle re-entry. This seems to contradict with the aforementioned overexpression of EZH2 in cancer, but the genomic targets of EZH2 and the postmitotic EZH1 are only partially overlapping (86). It was also shown, at least in vitro, that chromatin methylated by EZH1 is more condensed than chromatin methylated by EZH2 (86).
That means H3K27me might mark two separate chromatin environments in proliferating and postmitotic cells. The connection between expression, chromatin modification and higher-order chromatin structure we established in our experiments also indicated major H3K27me rearrangement; both functional and spatial redistribution. Our view is supported by recent works on nuclear architecture (266). It has been suggested that different nuclear territories are responsible for transcriptional stabilization and that the nuclear envelope plays an important part in the establishment of such territories (268). In addition, the nuclear periphery has also been associated with transcriptional repression (296) which is in line with our findings. In an attempt to link the transcriptional repressive and H3K27me-enriched nuclear periphery to the postmitotic phenotype of neurons we stimulated the cell cycle re-entry in our model system. The stimulated cells showed re-expression of cell cycle markers like Cyclin A1 and we could observe EdU incorporation which suggested S-phase entry. But contrary to this observation we could not find any significant upregulation of mitotic markers.
This indicated activation but not progression of cell cycle in stimulated cells; a condition also found in neurodegenerative disease models (244,247). Further, the upregulation of cell cycle markers was reported in the early stages of many
neurodegenerative diseases (144). The observation that stimulated cells still showed peripheral H3K27me but that there was no overlap with the S-phase marker EdU lead us to a new hypothesis. In this model the restrictive H3K27me-marked chromatin represents a transcriptional barrier in neurons which prohibits cell cycle progression and sequentially leads to replicative stress. Indeed, we found markers of replicative stress in stimulated neurons compared to untreated cells. Upregulation of cell cycle markers is a common feature of diseased brains in PD and AD patients. The “cell cycle switch” which is part of the LUHMES system and that we have extensively characterized (chapter 5), can therefore be utilized to evoke another kind of neurodegenerative phenotype. We could show that these events do resemble physiological conditions. As it was shown before there was no mitotic activity in stimulated cells and markers for replicative stress were upregulated in those cells.
Those observations fit well with previous studies on the subject (144). However, a clear link to cell death which is the most prominent feature of neurodegenerative diseases remains to be investigated. This could also help to understand some of the phenotypes seen in other models of neurodegeneration (297).