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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...
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| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2014
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| Online Access: | PDF Full Text |
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| Summary: | 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. |
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| DOI: | 10.17192/z2014.0354 |