FROM CELL SIGNALLING TO CELL DEATH: GLUTAMATE NEUROTOXICITY

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Apoptosis, or programmed cell death: this is the orderly destruction of a cell from within, the systematic execution of a sequence of active measures that result in the quiet removal of a cell from active service. It is an active process that requires internal organisation and the activation of enzyme pathways. It involves a disruption of cytoskeleton by enzymatic disruption of actin, the dismantling of the nuclear envelope, the splicing a DNA, and the transfer of the phospholipid phosphatidyl serine to the outer leaflet of the cell membrane, which targets the cell for removal  by phagocytes. No cell components are released, it is tidy, and no immune response results.

The major players in the apoptotic process are a family of enzymes - proteases - called caspases. Most of these exist in an inactive precursor form and they are activated as a cascade often catalysing the activation of each other. Of this, more below.

Apoptotic cell death plays a central role in development in shaping organ systems, and it is an essential mechanism to get rid of cells which have been damaged and which pose a potential threat of malignant growth. Disorders of the apoptotic pathway cause disease as indicated above - in 50% of human cancers, genes involved in the apoptotic pathway are suppressed, whiel premature or inappropriate activation of cell death pathways by subacute injury is implicated in the neurodegenerative disorders.

Necrosis is a passive process. It results from energy depletion, the dissipation of ionic gradients, cell swelling, disruption and permeabilisation of the plasma membrane and loss of intracellular contents, resulting in an inflammatory response.

Mitochondria: we all know of mitochondria as the seat of oxidative phosphorylation. However,  mitochondria may play a central determining role in pathways to cell death.

To remind you of basic mechanisms: the delivery of substrates to the citric acid cycle provies NADH and FADH₂ to the mitochondrial respiratory chain. This consists of a series of 4 enzyme complexes which transfer electrons from one to the next, eventually reducing molecular oxygen to generate water. In the process, protons are transferred from the matrix across the inner mitchondrial membrane into theintermembrane space by each complex. This proton gradient is expressed largely as  a transmembrane poential, often referred to as Dy_m ,which is about -150mV to the cytosol. This  proton gradient provides the force that drives proton influx into the mitochondria, through the enzyme called the F1F0 ATP synthase. This consists of a proton channel and an ATPase and proton influx drives the motor that phosphorlyates ADP to generate ATP (you can see images of the motor working on a web site at  //www.bmb.leeds.ac.uk/illingworth/oxphos/atpase.htm.

Collapse of the mitochondrial membrane potential stops ATP synthesis by oxidatiev  phosphorylaton, but also may allow the ATPase to run 'backwards' driven by the ATP now that the proton gradient has gone, and so mitochondrial ATP consumption may serve to hasten ATP depletion.

ALSO: it turns out that one of the major triggers for apoptosis is mitochondrial cytochrome c. Cyt c is a major  component of the final enzyme complex of the respiratory chain. It sits in the intermembrane space, and shuttles electrons between complex  2 and 3. Cyt c appears in the cytosol in response to initiation of apoptosis by a number of pathways, and release of cyt c into the cytosol may trigger apoptosis through the activation of a caspase.

Mitochondria and calcium: mitochondria will take up Ca²⁺  via a 'uniporter' - the uptake is not balanced by counter movement of any other ion. For years there has been controversy about whether mitochondria take up calcium under conditions of normal cell signalling, but it is now clear that they do.

Mitochondrial calcium regulates mitochondrial metabolism but may also trigger mitochondrial pathology, especially when combined with an oxidative stress imposed by excess free radical generation.

Glutamate toxicity: Glutamate is a major excitatory amino acid neurotransmitter in the CNS. It is released by exocytosis (Ca²⁺ dependent) at synaptic terminals and it acts at postsynaptic membranes at a variety of different classes of receptors. We are concerned with the ionotropic receptors, receptors coupled to ion channels that permit the influx of cations into the cell causing depolarisation of the postsynaptic cell. The 'AMPA' receptor allows flux mostly of Na⁺, but the 'NMDA' receptor has a high permeability to Ca²⁺ and activation allows significant Ca²⁺ influx. There are also metabotropic glutamate  receptors that involve activation of second messenger pathways, but we will not consider these now.

During a stroke or period of anoxia/ischaemia in the CNS, cells may die because they are starved of substrates for oxidative phosphorylation - glucose and oxygen, ATP is depleted and the cells die by necrosis. However, it is a common clinical observation that clinical state may deteriorate rather slowly after an initial event. This is often seen in a stroke - described as the 'evolution' of a stroke - or after perinatal asphyxia, during which a baby may seem OK for a day or so after delivery but then slowly deteriorates. Much of this delayed cell death is attributed to activation of death pathways by the accumulation of glutamate in the extracellular space during the period of anoxia, the excessive activation of NMDA receptors and cellular calcium overload. Glutamate may accumulate to concentrations >100mM largely because the glutamate transporter in glial cells reverses during ischaemia and pumps glutamate out of the cell into the narrow extracellular spaces. (Normally, the pump allows glial cells to remove glutamate from the synaptic cleft, but the pump is driven by ionic gradients for Na⁺, K⁺, H⁺, and the reversal of gradients especially for K⁺ and H⁺ during ischaemia can drive the pump 'backwards' ).

Application of glutamate to neurons in culture may cause a delayed cell death 24 hours later. In many instances in animal models and to some extent in patients, cell injury may be limited by the delivery of NMDA receptor antagonists. It is also clear that the cell death induced by NMDA receptor activation is dependent on Ca²⁺ influx. But why should Ca²⁺ cause the death of the cell?  After all, [Ca²⁺]_i is changing all the time in relation to cell signalling, so what changes here?

It turns out that glutamate induced cell death, while dependent on changes in [Ca²⁺]_i , does not show a simple direct relationship to changes in [Ca²⁺]_i . We can measure [Ca²⁺]_i in cells using intracellular fluorescent indicators. We can also follow changes in mitochondrial membrane potential.

Depolarisation of  the plasmamembrane with high K⁺ concentrations (depolarises, activates voltage-gated Ca²⁺ influx) raises [Ca²⁺]_i , as does application of glutamate. If we measure [Ca²⁺]_i and Dy_m systematically, we find that mitochondria depolarise in response to glutamate in cells in which glutamate is toxic, but not in response to a similar [Ca²⁺]_i change induced by K⁺. On a cell to cell basis, there is no correlation between the [Ca²⁺]_i signal and the loss of mitochondrial potential, even though the loss of potential is Ca²⁺ dependent and will lead to cell death. How can this be?

Perhaps some other variable is involved in addition to Ca²⁺?

It turns out that inhibition of NO production (L-NAME) will protect cells from glutamate toxicity and suppresses the mitochondrial response. Further, addition of exogenous NO under conditions in which Ca²⁺ is not toxic (i.e. to high K⁺) will cause mitochondrial depolarisation.

The interesting problem here, is that neuronal nitric oxide synthase (nNOS) is a Ca²⁺ dependent enzyme. So, if the K⁺ and glutamate both cause a similar rise in [Ca²⁺]_i why do they both not cause  a similar production of NO???.

The answer was apparently provided very recently in a paper in Science (Sattler et al, below) which showed that nNOS is held in place closely localised with the NMDA receptor by a protein (called psd - post-synaptic density-) 95, a so-called scaffolding protein. This means that the nNOS will be exposed to microdomains of very high calcium concentration as it comes into the cell through the NMDA gated channel, while the enzyme nNOS will only experience the rather more diluted Ca²⁺ signal  when the [Ca²⁺]_i is raised by depolarisation with K⁺, even though the average [Ca²⁺]_i through the cell is the same in the two cases.

It is also clear that preventing mitochondrial calcium uptake in response to glutamate is protective, and so mitochondrial Ca²⁺ uptake seems to trigger the downstream processes that lead to cell death. One possibility is that Ca²⁺ uptake triggers a mitochondrial patjology called the permeability transition pore (PTP). There is great interest in this now, as the PTP has been implicated in apoptosis, will cause collapse of Dy_m and so failure of oxidative phosphorylation, and also has pharmacological modulators, so that it becomes accessible as a therapeutic target.

There is still much that we don't fully understand. What is the basis for the loss of mitochondrial membrane potential? What determines whether the cells will go on to die by apoptosis or by necrosis? Are there other participants in this cell death cascade - free radicals, for example?

It seems likely in some of the neurodegenerative disorders, this kind of process is going on in the brain to cause the gradual attrition of neurons which are for some reason sensitised to glutamate. Cells die like this in response to beta-amyloid (found in Alzheimer's) in response to the AIDS virus coat protein gp120 (implicated in AIDS dementia). Similar mechanisms underlie cell death in the CNS following perinatal asphyxia or the brain damage that follows a cardiac arrest. Almost certainly similar processes take place in the heart or kidney following infarction or ischaemic injury. So, being able to understand the fundamental events that lead to cell death raises the possibility that we may be able to manipulate these processes systematically one day in people to help avoid disease and considerable human suffering.