Applying the 3Rs to elucidate the mechanisms of tau pathology using DRG neurons in culture

Nerve cells are complicated highly branched cells that are essential for our brains and our bodies to function. Without nerve cells we cannot think, feel, walk, pump blood or digest our food. The protein tau is a nerve cell protein that enables neurons to distribute essential proteins to distant parts of the cell in the brain, spinal cord, and in neurons that mediate sensation and control the actions of our internal organs. In Alzheimer's disease, tau ceases to function normally; it becomes misfolded and eventually forms neurofibrillary tangles (NFT) that are thought to be critical for neurodegeneration in the disease. Currently there are over 20 different human neurodegenerative diseases that show NFT, some of which are caused by a single mutation in the gene that codes for tau. This observation demonstrates that mutant forms of tau are enough to cause neurodegnerative disease. However, in many cases tau is not mutated, yet it still forms tangles. Tau is therefore an important therapeutic target. Quite a lot is known about pathological changes that occur in tau but one fundamental problem is still unanswered: what form of tau is truly pathological and how tau causes the nerve cells to die? These problems are difficult to solve by looking at the nervous system in post mortem human brains because it is difficult to catch a dying nerve cell as dead nerve cells are rapidly cleared by neighbouring cells. For this reason mouse models of tauopathy have been created where several features of the disease are replicated. However, even in mice, the dying cells in the brain are not easily accessible. To reduce animal use and still be able to investigate how tau kills cells, we have turned to a cell culture system of nerve cells from the sensory nervous system. What is intriguing is that the neurons that are cultured develop aspects of tau pathology as time goes by. Moreover, those nerve cells that express pathological forms of tau go on to die. This allows us to gain access to individual nerve cells to study the causes and mechanisms of tau pathology. and at the same time, reduce, refine, and replace a large number of mice that would have been otherwise necessary for this study. We now know that cell death is not necessarily a random process. It can involve the orderly demolition of the cell. Here we propose to develop this model to determine the pathways by which tau causes nerve cell pathology and death. We also wish to explore whether we can keep the sick nerve cells alive using drugs that are being developed for eventual use in humans. And, importantly, this model will reduce use of mice in research aimed at solving the mystery of why tau disables nerve cells and causes them to die.

Good cellular models of tauopathy would be hugely beneficial as a platform for understanding the causes of tau-related dysfunction and neuron degeneration, and for testing potential drugs that ameliorate tauopathies. Several cellular models of tauopathy have been described but none appears to sustain spontaneous tau aggregation and filament formation, and few, if any, of these die as a consequence of tau expression. In preliminary studies, we have found that we can culture DRG neurons from adult transgenic P301S human mutant tau for several weeks during which they develop pathological forms of tau and they go on to die. Given that we can obtain several replicate cultures form a single mouse prior to the stage when the mice develop severe pathology, this means that we can meet the aims of the 3R programme as well as test fundamental questions concerning the mechanisms of tau-induced neurodegeneration. In this proposal we aim to further characterise these cultures and uncover how the neurons die, which intracellular processes are disturbed such that they contribute to the ultimate pathology, whether inflammatory cytokines or microglia/macrophages exacerbate the pathology, and finally test drugs we have been given to see if the platform can be used to uncover potential anti-tau therapies. Our research will significantly reduce and replace the number of animals needed for tau-related research, and refine husbandry because DRG can be cultured from the mice prior to overt pathology. At the same time, we will contribute to the basic understanding of mechanisms of tau pathology and hopefully to the identification of therapies for these devastating diseases.

In this project, neuronal cultures will be established from adult transgenic mice. This amplification clearly reduces the number of animals required to obtain a significant result. Indeed, the reason we need to use more than one animal per experiment is to obtain a measure of biological variation between cohorts of cells. This is absolutely necessary for a proper statistical evaluation. Because we can age the neurons in culture, this model also replaces and refines animal use: it allows us to humanely kill the mice prior to the development of severe pathology, thus minimising the severity of animal suffering, and provides a platform for testing drugs that may have deleterious effects on the mice (or have no effect). The cultures also allow us to screen for possible toxicity at the cellular level, and test for possible effects on neuron growth, recovery from damage, and physiological functions, such as axonal transport. Altogether, a conservative estimate of the scale of reduction given that occasional problems can arise with the culture, such as infections, is about 8-fold. We already practice economies of scale by sharing the mice with others in the lab and at the Centre who are researching other organs and processes involving tau. Conversely, we can take DRG neurons from those using the mice for their research. We believe that use of DRG cultures in studies of neurodegeneration will gain wider use and indeed propel more clinicians to test for sensory function deficits in dementias, a neglected area of research due to the difficulty of collecting DRG from post mortem patients. Our study should also encourage those who are interested in diabetic and other sensory neuropathies to use DRG cultures instead of mice for studying cellular aspects of the condition; little use has been made of long term cultures of DRG to observe how these pathologies may develop and be treated.