Cyclophilin A and serine proteases : Targets of neuronal cell death upstream of mitochondrial demise

Many neurological disorders and neurodegenerative diseases are associated with mitochondrial abnormalities. Mitochondria are essential organelles regulating the energy metabolism of the cell, thereby, determining essential cellular functions and viability. This accounts particularly for neurons w...

Ausführliche Beschreibung

Gespeichert in:
1. Verfasser: Reuther, Christina
Beteiligte: Culmsee, Carsten (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2014
Pharmakologie und Toxikologie
Ausgabe:http://dx.doi.org/10.17192/z2014.0354
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Zusammenfassung:Many neurological disorders and neurodegenerative diseases are associated with mitochondrial abnormalities. Mitochondria are essential organelles regulating the energy metabolism of the cell, thereby, determining essential cellular functions and viability. This accounts particularly for neurons which show a pronounced energy demand and where mitochondria play a pivotal role in balancing the Ca2+ homeostasis, controlling ROS formation and providing most of the energy for neurotransmitter metabolism and maintenance of the membrane potential. Further, mitochondria are essential organelles controlling the ‘point of no return’ in intrinsic pathways of programmed cell death where the decision between cellular life and death involves post-translational protein modifications determining mitochondrial dysfunction and release of detrimental proteins such as AIF. Therefore, major aims of the present thesis included the characterization of the key regulators of protein modifications that occur upstream of mitochondrial demise and AIF release. In this context, the role of two PPIases, CypA and Pin1, and the involvement of serine proteases in paradigms of PCD were investigated. Most experiments were performed in an immortalized neuronal cell line (HT22 cells) which depicts a well-established model to study caspase-independent cell death induced by glutamate. The most prominent feature in this model of cell death is the mitochondrial AIF release and the subsequent translocation to the nucleus where it induces chromatinolysis. To substantiate the importance of these results further experiments were performed in a model of glutamate-induced excitotoxicity in primary cortical neurons. The findings of the first part of this study revealed a prominent role for CypA in glutamate-induced mitochondrial AIF release and cell death. Glutamate toxicity resulted in the translocation of CypA to the nucleus where it built a pro-apoptotic complex with AIF, thereby inducing chromatinolysis. Silencing of CypA protected HT22 cells against glutamate toxicity. Moreover, the depletion of CypA preserved mitochondrial fission and the loss of MMP and also prevented the release of AIF from the mitochondria. Furthermore, lipid peroxidation arising from the mitochondria was attenuated which supported increased cell viability. Further experiments addressed the involvement of a second family member of PPIases, Pin1, in neuronal cell death pathways. The inhibition of Pin1 with Br57 resulted in decreased susceptibility of HT22 cells to glutamate toxicity. This increase in cell viability was attended by changes at the level of mitochondria. Pin1 inhibition led to enhanced mitochondrial fission, but further, complete mitochondrial fragmentation induced by glutamate was attenuated. This elevated mitochondrial fission rate was accompanied by a slight decrease in ATP levels in Br57-treated controls, however, the strong ATP depletion that occurred after the glutamate challenge was prevented and the impairment of MMP was abolished. In summary, these findings depict a pivotal role for CypA and Pin1 in glutamate-induced cell death in HT22 cells upstream of mitochondrial demise. Since this model exhibits common features of neurological disorders the results obtained here may give a platform to investigate new neuroprotective strategies. The second part of this thesis dealt with the impact of activated trypsin-like serine proteases on cell viability and mitochondrial function. Inhibition of serine proteases with TLCK resulted in increased cell viability in HT22 cells and in primary cortical neurons. Furthermore, this work revealed that activation of trypsin-like serine proteases occurred upstream of Bid activation and moreover, in a very initial phase of this PCD pathway in HT22 cells. Further, lipid peroxidation was blocked and mitochondrial morphology alterations were prevented. In addition, ATP depletion and the impairment of the MMP were preserved and the decrease in mitochondrial respiration after glutamate toxicity was abolished. Interestingly, the interaction of Drp1 and CypA was strengthened by TLCK. However, the release of Drp1 from the actin cytoskeleton and the following redistribution of Drp1 from the cytoplasm to the mitochondria were not prevented. In conclusion, the second part of this thesis highlighted trypsin-like serine proteases as mediators of glutamate-induced cell death in HT22 cells. The initial activation in this PCD pathway is a promising target for therapeutic intervention strategies in neurological diseases.
DOI:http://dx.doi.org/10.17192/z2014.0354