Dokument

Summary:
Neuronal cell death causes progressive loss of brain tissue and function after acute brain injury and in chronic neurodegenerative diseases. Although the pathological features of stroke and brain trauma or Alzheimer’s and Parkinson’s disease differ greatly, the underlying neuronal damage shares common molecular and cellular mechanisms. Despite extensive research and increasing knowledge on the molecular pathology, no efficient therapy has been born from these efforts until today. As a promising concept to overcome this plight, it has been suggested to enhance endogenous survival signaling pathways like the transcription factor NF-κB and thus obtain neuroprotection. Increasing neuroprotective NF-κB signaling can be achieved by blocking repressors of NF-κB transcriptional activity such as p53 and CYLD. Both factors may mediate cell death by mechanisms dependent on and independent of NF-ΚB signaling. Therefore, the major aim of this study was to explore the roles of p53 and CYLD in neuronal cell death and to connect their detrimental effects with NF-κB activity. This issue was addressed in immortalized mouse hippocampal HT-22 neurons and in primary neuronal cultures exposed to glutamate toxicity. Furthermore, an in vivo model system of traumatic brain injury was employed to compare infarct development after controlled cortical impact in wild-type and CYLD-/- mice. The present study revealed that both approaches, inhibiting p53 and CYLD successfully preserved mitochondrial integrity and function, and significantly attenuated neuronal cell death. Surprisingly, however, the pronounced neuroprotective effect of the p53-inhibitor pifithrin-α occurred independently of enhanced NF-κB activity in HT-22 cells. In addition, neuroprotection induced by silencing of CYLD was completely independent of NF-κB, despite of the previously established role of CYLD as a negative regulator of NF-κB in keratinocytes. In line with that notion, the NF-κB subunit expression and NF-κB transcriptional activity were not significantly altered in HT-22 neurons undergoing glutamate dependent cell death. In conclusion, these data suggested that the NF-κB pathway was neither significantly affected by glutamate dependent cell death, nor did it mediate the neuroprotective response of CYLD and p53 inhibition in this model system of glutamate toxicity. Interestingly, inhibiting p53 with pifithrin-α maintained mitochondrial morphology and mitochondrial membrane potential in HT-22 cells. This effect occurred independently of p53 dependent transcription. Investigating the underlying cause of neuroprotection associated with CYLD depletion, it was unveiled that glutamate-induced oxytosis in HT-22 cells occurred through mechanisms of necroptosis. This conclusion is based on the detection of RIP1/RIP3 complexes as a hallmark of necroptotic cell death in HT-22 cells exposed to glutamate. Further, silencing either RIP-kinase provided strong protection of the cells. Repressing CYLD, in turn, prevented the formation of the RIP1/RIP3 necrosome, suggesting that inhibition of necroptosis was the underlying mechanism of neuroprotection after CYLD depletion. In contrast, CYLD depletion had no effect on cell death in a model of glutamate excitotoxicity in primary cultured neurons, while inhibition of RIP1 kinase by necrostatin-1 significantly enhanced neuronal survival. These data suggest a CYLD independent but RIP1 dependent mechanism of glutamate toxicity in primary neurons, which requires further investigation. In vivo, however, using a model of traumatic brain injury, CYLD knockout mice showed a significantly reduced infarct size compared to wild-type littermates suggesting a potent neuroprotective effect inherent with CYLD repression. In summary the data from this thesis highlight a yet unknown role of CYLD in neuronal cell death and unravel CYLD and p53-dependent mechanisms of cell death as a putative therapeutic approach for the treatment of acute and chronic neurodegenerative diseases. Future research, however, is warranted to further elucidate the exact mechanisms leading to CYLD and RIP-kinase activation in neurons and to determine the exact molecular link to mitochondria.

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