Spatio-temporal imaging of calcium in degenerating nerves

Axons are the long processes that link neurons together within our brain, spinal cord and peripheral nervous system, allowing neurons to communicate rapidly with one another. They are huge structures, up to 1 metre long in man, making human motor neurons, for example, the largest single cell within the human body. Naturally with such huge structures, there are significant logistical problems in maintaining axons. Failure to do so is the cause of a number of human neurological disorders, not only those where there is direct axonal injury such as spinal injury, but also many where the disorder is inherited such as motor neuron disease, or acquired such as exposure to neurotoxins. We are beginning to understand that the way in which axons die is similar in each of these seemingly unrelated circumstances. However, we do not yet fully understand what that mechanism is. Inorganic ions such as sodium and potassium play important roles in the transmission of the action potential, the means by which information passes rapidly along an axon from one end to another. However, another inorganic ion, calcium, plays a rather different but equally critical role in axons. Calcium is normally pumped out of all cells, including neurons, because high levels of calcium inside the cell are extremely dangerous. It is also pumped into specific stores within cells, including inside the axon, from where it may be released and used to signal certain events and processes so that the cell or axon can respond accordingly. We have some preliminary data suggesting that release of calcium from such stores occurs after an axon is injured, but long before the axon actually degenerates. When we repeated these experiments in the presence of a genetic mutation that protects injured axons from degenerating, the redistribution of calcium was also blocked. The possible causative link between these two events needs to be established by appropriate experiments. For example, if calcium release from the internal stores is part of the signalling mechanism that causes the axon to degenerate, then one would expect that causing calcium to be released from intracellular stores by another method (e.g., by adding an appropriate drug) would have a similar effect. We will carry out a series of experiments to test for such a causative link, and if we find it, to determine how it might be exploited. In the short term our aim is to understand better fundamental mechanisms of axon death. This is a very important issue because axons make up by far the largest part of most neurons and because they are essential for the function of that neuron and for the most part cannot be replaced if they are lost. In the longer term, understanding this process should lead to new ways to treat, or even prevent, a wide spectrum of neurodegenerative conditions.

Very little is known about how axons degenerate. As axons constitute up to 99.9 per cent of the cytoplasmic volume of neurons, are essential for neuronal activity, and in many cases are irreplaceable once lost, efforts to understand this aspect of neurodegeneration should be a high priority. The mechanism of axon degeneration differs from that of the cell body, so our understanding of neurodegeneration will never be complete without studying axons directly. Calcium entry, and mobilisation of intracellular calcium stores, are important events in axon degeneration. Calcium influx has long been implicated in the execution phase of injury-induced Wallerian degeneration, but recent data show that release of calcium from intra-axonal stores also occurs in ischaemic nerves. Our preliminary data now suggest that this occurs also during Wallerian degeneration. We have confirmed reports that calcium redistributes within 4 h of axon injury and find that the WldS mutation, which delays Wallerian degeneration, also blocks such calcium redistribution. Calcium redistributes long before axons actually degenerate, before the rise in total intra-axonal calcium and at sites distant from the injury, so it is unlikely to reflect calcium influx. We will investigate the mechanism of the calcium redistribution and test the hypotheses (a) that calcium redistribution is necessary and sufficient to trigger the later stages of Wallerian degeneration and (b) that the WldS mutation blocks either calcium release or its downstream effects. Progress in this field has been held back by a lack of suitable methodology for imaging calcium in intact myelinated nerves. We will overcome this in four ways. First, we will study primary neuronal cultures, where calcium indicator dyes targeted to various subcellular compartments can easily be introduced via the culture medium. Second, we will use an established method based on rapid calcium precipitation with an electron-dense material to study calcium in degenerating nerves in vivo with ultrastructural resolution. We have extensive experience of each of the above methods, having used them to study calcium release during excitotoxicity (Fig. 2) and calcium in the axons of transgenic WldS rats (Fig. 1) respectively. Third, we will study calcium dynamics in an integrated system, myelinated nerve, by introducing calcium indicators to small groups of axons teased apart from peripheral nerve. Sequestration of indicators by myelin has complicated earlier attempts to introduce them into myelinated nerve, but we have overcome this technical problem by teasing apart a few axons to improve indicator access (Fig. 4). Finally, we will use newly generated transgenic mice expressing proteinaceous calcium indicators to overcome altogether the need to load exogenous indicators into intact neuronal tissue. We will test whether axon injury induces calcium release from internal stores, using the above methods, together with drugs that block this process and confocal microscopy. We will then determine whether such drugs delay injury-induced axon degeneration and whether drugs that stimulate internal calcium release can cause axon degeneration in unlesioned wild-type nerves or in lesioned WldS nerves. In this way, we will build up a pathway of molecular events and their regulation during the early stages of axon degeneration. These studies may also identify pharmacological agents that, at least in ex vivo and in vitro models, can block axon degeneration after injury. We have already shown that axon degeneration after injury shares mechanism with axon degeneration in neurological disorders, so our results may enable future studies to test agents that preserve axons in such disorders. However, at this stage our primary aim is to determine the mechanism and its relationship to other forms of neuronal death.