HOW DO REACTIVE OXYGEN SPECIES
AFFECT CELL FUNCTION AND SURVIVAL?
The term reactive oxygen species
(ROS) is a generalized description for a collection of reactive
oxygen molecules of biological significance. There is intensive
interest in ROS because they are thought to contribute to the
complications of many chronic diseases including blindness,
Alzheimerfs, emphysema, Parkinsonfs disease, diabetes, renal
failure and aging. ROS are often thought of as being directly
detrimental to cell survival because at elevated concentrations
they can damage key macromolecules such as nucleic acids,
proteins and lipids and cause rapid cell death.
An exciting development has been
the realization that low levels of ROS can also act as signaling
molecules influencing key physiological processes. One example
serves to illustrate the value of understanding how ROS affect
cell function. Recently, overexpression of catalase (an
antioxidant enzyme) in the mitochondria was found to extend
maximum lifespans of mice by 20%. This provides very strong
support for the free radical theory of aging and reinforces the
importance of mitochondria as a source of these radicals. ROS, at
low levels, likely affect many aspects of cellular function (e.g.
growth, metabolic rate, cell division) by reversibly modifying a
variety of intracellular proteins. One of the challenges is to
identify proteins targeted by low levels of ROS.
Current projects focus on
identifying the signaling pathways by which ROS affect cell
function or cause cell death. If any of these projects interest
you or the research area in general, then please do not hesitate
to discuss them with me. I am also happy to discuss and develop
specific projects to suit your interests.
1. Techniques to sensitively
monitor the effects of ROS on protein function (collaborative
with Proteomics International)
There is convincing evidence that
ROS can have profound impacts on cell functions by reversibly
modifying a variety of intracellular proteins. Yet in spite of a
very great scientific interest, only in a few cases has the
biological significance of these modifications been identified.
In part, this poor understanding reflects the complexity of the
reactions, the scarce knowledge of relationships with other
antioxidant systems, and the difficulty in identifying specific
biological effects. A particular problem is that efforts to
demonstrate the biological relevance of protein modifications
have been hampered by the lack of sensitive and specific
techniques for the detection of these protein modifications.
The objective of this project is
to develop proteomic methods which will be useful in detecting
modifications of proteins in cell exposed to oxidising
environments.
2. ROS as signaling molecules in
heart cells (collaborative with Dr L. Hool, physiology)
ROS are a feature of a variety
disease states including ischemic heart disease, hypertension,
and congestive heart failure. One likely source of ROS are
mitochondria, and abnormalities in mitochondrial structure and
function have been observed during heart failure and following
myocardial ischemia-reperfusion injury (ie heart attack or
stroke). ROS generation by mitochondria has the potential to
acutely affect cell function (eg contraction in heart cells) as
well as cause longer term changes by altering the cellular
proteome. However, little is understood about the nature of ROS
generation by damaged mitochondria, particularly in intact
cells.
The objective of this project is
to use proteomic techniques to identify the protein targets of
ROS in mammalian hearts subjected to an oxidising
environment.
 |
Figure 1. Electron spin resonance
(ESR) signal of a hydroxyl radical. Hydroxyl radicals are a
particularly reactive ROS and are potent oxidants thought
to be involved causing cell death following a heart
attack. |
Retinal diseases with a vascular
component account for the majority of new blindness in our
community. The retina is particularly vulnerable to vascular
disease because of the very delicate balance between oxygen
supply and demand. The oxygen consumption rate of the retina as a
whole is one of the highest in the body and on a per gram basis
is higher than that of the brain. It is not understood how
changes in the availability of oxygen affects retinal function
and survival but there is good evidence that ROS are mediating
the cellular responses. Possible responses to a lack of oxygen
include rapid death, loss of function without death or adaptation
to a chronic lack of oxygen (figure 3).
Research on the retina has been
hindered by the lack of techniques to work on such small amounts
of tissue (less than 1 mg). However, we have been developing
methods to work with small amounts of retinal tissue.
The objective of this project will
be to examine how retinal cells respond to a lack of oxygen by
testing the hypothesis that ROS are acting as signalling
molecules to affect retinal cell function.
 |
Figure 2. The is similar to that of
human in that it has a structured and layered vascular
supply |
4. How does c-Jun NH2-terminal
kinase mediate cell death induced by ROS in neurons?
Injury to the brain or spinal cord
triggers, for the survivors of the initial ordeal, delayed death
of nerve cells over the following hours and days. There is
evidence that ROS are involved in delayed cell death. ROS
activate a variety of cell signalling pathways including those
involved in apoptotic cell death. We have focussed on c-Jun
NH2-terminal kinases (JNKs) which are one subgroup of
mitogen-activated pathways activated by ROS and are proposed to
have a central role in apoptotic cell death. Recently we
discovered JNKs are also involved in necrotic cell death. This is
particularly significant because delayed cell death is caused by
both necrosis and apoptosis. Therefore by inhibiting JNKs or some
downstream component, it may be possible to prevent delayed cell
death. This could mean the difference between retaining the
ability to walk, talk and control bodily functions or be confined
to a wheelchair or require 24 hour nursing care. If this delayed
cell death could be prevented, then the quality of life for the
survivors could be improved.
The objective of this project is
to identify how JNKs are causing cell death by necrosis.
 |
Figure 3. Evidence of apoptosis and
necrosis in neuronal cultures following a simulated stroke
in the neuronal model. Magnified example of neurons stained
with DAPI 24 h after simulated stroke (B). Neurons were
characterised by brightly stained fragmented nuclei
characteristic of PCD (->) or by bright even staining
characteristic of necrosis (ª). |
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