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

Aims

This research aims to develop and characterise an in vitro model of neuronal cell death in response to accumulation of the tau protein, reducing and replacing the use of live animal models in the study of the molecular basis of neurodegeneration and dementia.

Background

In Alzheimer's disease the neuronal protein, tau, becomes misfolded, forming neurofibrillary tangles (NFT), thought to be critical for neurodegeneration in the disease, making tau of interest as a therapeutic target. However, how tau causes neurons to die is still an open question, and dying cells in the brain are not easily accessible for study. Current cellular models of ‘tauopathy’ fail to exhibit NFT formation and subsequent neuronal death, meaning animal models are the only currently viable model for study.

This research aims to develop and characterise a cell culture system where neurons develop pathological forms of tau and go on to die, allowing the molecular mechanisms underlying this and potential therapies to be investigated in vitro, minimising the use of animals and associated suffering with advanced disease pathology.

Preliminary data suggests that dorsal root ganglia (DRGs) neurons from adult transgenic P301S human mutant tau mice can be cultured for at least eight weeks during which they develop pathological forms of tau and go on to die. Up to 12 replicate cultures can be obtained from a single mouse prior to the stage when the animals develop severe pathology.

Research details and methods

Long lived DRG neuronal cultures will be prepared from a small number of adult transgenic mice at different stages of tau pathology. These cultures will be characterised to elucidate the mechanisms by which tau positive neurons die and to probe the causes of this cell death by studying the function of cellular organelles, such as mitochondria, and structural proteins called microtubules.

Whether inflammatory cytokines or immune cells, such as macrophages and microglia, exacerbate the pathology of the disease will be investigated. Finally, neuronal cell cultures will be developed as a platform for testing drugs that potentially decrease various stages of tau pathology.

This research could offer a new option, with distinct scientific advantages such as allowing neuronal death related to tau pathology to be visualised, probed and monitored. Furthermore, it would allow for much more rapid screening of potential therapies which impact tau and NFT formation, preventing neuronal death, hastening the drug development process. Alongside the scientific advantages, it is estimate that the in vitro model could reduce animal use around eight-fold. 

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.

Brelstaff J, Ossola B, Neher JJ, Klingstedt T, Nilsson KP, Goedert M, Spillantini MG, Tolkovsky AM (2015) The fluorescent pentameric oligothiophene pFTAA identifies filamentous tau in live neurons cultured from adult P301S tau mice. Frontiers in Neuroscience 9: 184 doi:10.3389/fnins.2015.00184

Brelstaff J, Spillantini MG, Tolkovsky AM (2015) pFTAA - a high affinity oligothiophene probe that detects filamentous tau in vivo and in cultured neurons. Neural Regeneration Research 10(11): 1746-7 doi:10.4103/1673-5374.165298

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Project grant

Status:

Closed

Principal investigator

Professor Maria Grazia Spillantini

Institution

University of Cambridge

Co-Investigator

Dr Aviva Tolkovsky

Grant reference number

NC/L000741/1

Award date:

Jan 2014 - Dec 2016

Grant amount

£301,421