Main Project Areas
Mitochondria, oxidative stress and apoptosis
Mitochondria are good and bad for cells
Life is a balance between processes that lead to properly organised form and function in the various tissues of the body, and factors that initiate disease and death. This balance can be seen at the level of single cells and their internal components. Mitochondria are the components of cells in which ATP is made by oxidative phosphorylation. They have many other important roles required for the maintenance of cellular health, mostly relating to energy conservation, as well as participating in ion homeostasis in cells, particularly Ca++ ions. Recently it was discovered that mitochondria have an entirely opposite role that leads to the orderly destruction of cells in the process of apoptosis (Fig. 1).
Fig.1 Mitochondria in Cell Life and Death
Mitochondria have a regulated system, monitored and activated by members of the Bcl-2 family, for releasing apoptotic signalling proteins from the intermembrane space, through the outer membrane, into the cytosol. Such proteins act in various ways to drive the cell towards apoptosis. Three examples are shown in Fig. 2 below. Cytochrome c in the cytosol is the trigger for assembly of the apoptosome (a large molecular machine that activates caspase-9 on an aggregate of Apaf-1). Smac/DIABLO antagonises the action of endogenous inhibitors of caspases, thus enabling caspase-mediated progression towards apoptosis. AIF is thought to activate DNA degradation in the nucleus. A fascinating aspect of these proteins is that they themselves have both life sustaining roles and cell death signalling function. Thus, in the intermembrane space of mitochondria cytochrome c is an electron carrier in the respiratory chain while AIF acts as an antioxidant enzyme, protective against the prevailing oxidative stress brought about by the reactive oxygen species (ROS) in mitochondria. The role of Smac/DIABLO inside mitochondria is presently unknown.
Fig.2: Main modes of action of signalling molecules released from mitochodria.
The dual functions of mitochondria and some of its constituent proteins provide the backdrop to the research projects in the Nagley Laboratory outlined below. Other aspects of research on the energy-generating properties of mitochondria are described under the ATP synthase project (PDF document: Please refer to page 7) elsewhere in the description of departmental research projects. The main methods of investigation of apoptosis and other aspects of mammalian cell biology discussed below include: cell culture (both primary cells and immortalised cancer cells), confocal microscopy using live cell imaging techniques as well as immunocytochemistry, flow cytometry (FACS analysis), molecular biology and genetic manipulation of cells (expression of genes transfected into cells) and protein analysis by Western blotting and other techniques.
Neurones are highly dependent on oxidative metabolism, which is one of the reasons why maintaining a continuous oxygen supply to the brain is important for survival. When regions of the brain suffer a deprivation of oxygen, such as in stroke, damage to neurones occurs that is exacerbated by the subsequent reperfusion of the affected region by oxygenated blood. A high level of oxidative stress that is damaging to the neurones occurs under these conditions, inducing cell death, both necrosis and apoptosis. The neuronal injury is compounded by the uncontrolled release of neurotransmitters, such as glutamate, which induce a phenomenon called excitotoxicity whereby the affected neurones flood with Ca++ ions and mitochondria are activated to proceed into apoptotic signalling. Thus, understanding the detailed mechanism of neuronal cell death, particularly the involvement of mitochondria and oxidative stress, will lead to insight into stroke-related injury and will help identify potential drug targets for minimising cell death and promoting better recovery.
Moreover, many neurodegenerative diseases are characterised by oxidative stress-induced damage and consequent neuronal dysfunction and death. Mitochondria are directly or indirectly implicated in neuronal degeneration in such diseases. One example is Parkinson's disease, in which defects in Complex I (NADH-CoQ reductase) of the respiratory chain are known to occur. This disease is characterised by the degeneration and loss of dopaminergic neurones, a process accompanied by oxidative stress and excessive apoptosis. By elucidating the precise mechanisms of neuronal death and the role played by mitochondria, it may be possible to not only identify drug targets but also to develop strategies for replenishment of the missing neuronal populations with implantation of suitably prepared exogenous neuronal stem cells.
The culture of primary neurones of various types from normal and genetically modified mice is carried out at Monash in a collaborative project with Prof Philip Beart (Howard Florey Institute). This aspect of the Nagley Laboratory's research focusses on studies of the molecular events in neuronal apoptosis (see Fig. 3 below), coupled with application of a range of chemical antioxidants to understand better the role of oxidative stress.
[The work on neuroscience in the Nagley Laboratory at Monash University is carried out as part of an NHMRC Program Grant on Brain Injury and Recovery, in which Prof Nagley is a Chief Investigator, together with colleagues now based at the Howard Florey Institute, University of Melbourne.]
Fig 3. Apoptogenic signalling
Cancer represents a wide range of diseases characterised not only by uncontrolled proliferation of cells but also by failure of the cancer cells to undergo apoptosis when they should. Since mitochondria play a role in cellular apoptosis, it is important to understand how the response of cancer cells to internal and external apoptotic signals (e.g. through application of radiation or chemotherapeutic agents) involves mitochondria. The active role of mitochondria in cell death signalling may be complicated by the reduced importance of mitochondria in cellular energy production in many cancers, whereby the cells become much more glycolytic in their metabolic profile and less reliant on mitochondrial oxidative phosphorylation.
To address this interesting situation, we are systematically analysing groups of cancer cells of common origin but of different degrees of malignancy, in which mitochondrial contribution to cellular ATP production is reduced as the malignancy increases. This research has been initiated with a set of human neuroblastoma cells but other sets of cancer cells will be introduced into the research in the future. The aim is to precipitate apoptosis induced by a "pure" mitochondrial signal (by use of mitochondrially targeted photosensitisers or expression of pro-apoptotic Bcl-2 family members in cells using regulated expression vectors) to understand the role of mitochondria in the cell death execution pathways of the various cancer cells. This work is carried out in collaboration with Dr Nigel Waterhouse (Peter MacCallum Cancer Institute).
One of the most challenging aspects of the mitochondrial role in apoptotic signalling is to understand how the outer membrane (OM) of mitochondria is permeabilised to release the signalling proteins illustrated in the Figure above. A number of theories have been proposed. The different ideas invoke protein channels in the OM, lipidic pores in the OM (caused by local reorganisation of membrane phospholipids resulting from insertion of pro-apoptotic regulatory proteins into the OM), or gross rupture of the OM. Our approach is to study carefully the kinetics and extent of release of particular intermembrane space (IMS) proteins, such as those in the Fig. 2 above, during apoptosis induced by various means in diverse mammalian cell types. This work is enabled by use of antibodies and GFP tags on relevant proteins to monitor their release from mitochondria in individual cells and cell populations as a whole. Further constructs include specific homodimers or higher order aggregates of particular IMS proteins, as well as heterodimers between different IMS proteins. This involves use of genetic engineering of proteins and use of small molecular reagents for affinity-based dimerisation of proteins. By following the ability of such manipulated IMS proteins to exit through the OM under various apoptotic induction conditions we aim to understand the constraints on release of IMS proteins from mitochondria and thus to gain insight into the nature of the release apparatus. [This work is supported by the Australian Research Council.]
A recently initiated avenue of collaborative research in the Centre for Structural and Functional Microbial Genomics at Monash concerns host-pathogen interactions. In collaboration with Prof Ross Coppel (Department of Microbiology) we study bacteria in the mycobacterial group that cause disease in humans (such as tuberculosis) and animals of agricultural importance (such as Johne’s disease in cattle). In the Nagley Lab we focus on how mammalian macrophages respond to infectious episodes by mycobacteria, especially considering the cellular and molecular changes that invoke mitochondrial participation.
A challenge is to understand how macrophage apoptosis is initially blocked by the infecting mycobacteria after phagocytosis into these host cells (enabling the proliferation of mycobacteria shielded from the host’s immune system). This is later followed by enhanced macrophage apoptosis that leads to spread of the bacteria to other host cells. Mycobacterial strains in this research are derived from different species (pathogenic and non-pathogenic) as well as specific mutants affected in virulence properties. Techniques will include the cellular and molecular biological tools outlined in the sections above, as well as use of DNA microarray analysis to study the gene expression profile of the host cells and the bacteria subsequent to an infection episode.