Replacing animals in motor neuron disease research
Motor neuron disease is a devastating and fatal adult-onset disorder, with very limited treatments available. Mice and rats are commonly used as ‘models’ in basic research and drug efficacy testing for motor neuron disease (MND). These experiments can cause substantial pain and suffering to the animals involved and do not effectively represent the human disease.
Here we describe some limitations of the rodent models and briefly discuss examples where results have failed to translate into clinical benefit. We then summarise some of the non-animal replacement methods that can be used to study MND and argue that these should be replacing the animal research.
Rodents as models
A common model of motor neuron disease in basic research and drug development uses mice and rats genetically modified to carry the abnormal human gene SOD1, which codes for the enzyme superoxide dismutase. When allowed to run its course, this results in substantial suffering for the animals, including a tremor from the age of 90 days, locomotor problems especially in the hind legs, and eventually paralysis. These symptoms last until around 140 days which represents the end stage of the disease in rodents [e.g. 1].
However MND is a label used for a range of illnesses, with highly variable clinical courses [2]. Much of the time the human disorder does not have an identifiable genetic component and SOD1 is only involved in a fraction of human MND cases. Manipulation of SOD1 as the sole component of the rodent model is clearly not representative of the human disorder. Even the small number of patients who do have an SOD1 abnormality are not effectively represented, as the human gene is expressed against a mouse or rat genetic background, not a human background, with significant implications for gene/gene interactions.
This approach therefore has severe limitations and unsurprisingly the translational steps from genes to transgenic animals and finally to the patient have proven difficult [3]. Therapies found effective in the SOD1 model have not translated to clinical trials, seriously calling into question the utility of animal models as the basis for identifying which therapeutic agents to progress to human trials [4]. Early high expectations of mouse models among some researchers have not been fulfilled, hence there are many, inside and outside the field, who are seriously questioning the value of these models in MND research [3].
Mouse ‘model’ has been misleading in drug trials
Studies on animal models of MND found that Copaxone, licensed for the treatment of multiple sclerosis, extended survival and motor activity. SOD1 mice received an injection of Copaxone at the time of symptom onset, followed by daily oral administration [5]. However, a subsequent human trial designed to test the drug’s effectiveness found that Copaxone did not slow disease progression or extend survival [6].
Another drug, minocycline, has previously been shown to delay onset and prolong survival in animal models of MND [7]. In 2002, Nature published further research on the effects of minocycline in animals, in which scientists reported that they had discovered the exact site of action for the drug [8]. However a phase III clinical trial of minocycline showed a 25% increase in symptom severity compared to controls [9].
Human genetic studies
The most fundamental step towards finding a treatment for MND is finding the cause or causes. Genetic studies of DNA samples from large numbers of patients and healthy controls enable researchers to identify the genes that cause the onset of MND or modify features of the disease, such as age of onset or site of onset. They can also provide clues about the mechanisms underlying motor neuron degeneration; can increase understanding of the physiological and biochemical processes underlying the disease progression, and may indicate potential mechanisms for pharmacological intervention to delay or prevent disease.
A £1 million project to set up a MND DNA bank and national database in the UK is underway. This collects donated blood from patients, family members and other healthy controls, and cell lines will be available to researchers worldwide. The DNA bank will enable research scientists to study the genetics of the human MND, not a surrogate in non-human animals [10].
In February 2008 Professor Christopher Shaw and other researchers involved in the DNA bank announced the most significant breakthrough in MND research since 1993. Using donated blood samples, they have discovered that a gene associated with the disease in patients, which codes for the protein TDP-43, in fact causes toxicity to motor neurons; whereas before, expression of this protein was thought to be a harmless by-product of the disease [11]. Whilst it is likely that this genetic abnormality will be transferred into mouse models, this could prove misleading. The important breakthrough could be better and more ethically progressed using other non-animal methods, outlined below.
Using cells from the DNA bank, as part of a UK-US collaboration, Dr Al-Chalabi and colleagues at Kings College London analysed over 5,000 samples, performing genome-wide scans. The heritability of age of onset in MND is estimated to be about 50% and preliminary studies have identified several regions of the human genome that are likely to harbour such genes [12,13].
Human imaging
Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) are used to explore degeneration in parts of the brain and spinal cord affected in patients. These scans can enable doctors to differentiate between different types of the disease which may have similar symptoms in patients. For example, one study by Turner and colleagues [14] used PET to examine two patients with similar symptoms. They found that while one had abnormalities in the cerebral cortex, the other had no damage in this area. This demonstrates the specificity of disease in individual patients and shows that a surrogate animal model can never effectively represent patients with MND.
Imaging in drug development
Imaging can also be used to monitor the effects of drugs on volunteers during clinical trials. Imaging is being applied to assess any changes in the brain in a clinical trial in the USA, to investigate the effects of the antioxidant co-enzyme Q10 in MND [15]. This is the second study, following encouraging results in a very small, preliminary trial in the same institute. The previous trial found that Co-Q10 was safe and well tolerated by patients when taken for a nine-month period [16].
Post-mortem tissue and living tissue samples
Post-mortem and living tissue samples donated by patients and healthy controls can provide first-hand knowledge of the effects of MND.
For example, one study used post-mortem brain and spinal cord samples to study genetic control of the manufacture of glutamate transporters. It is thought that these transporters cause motor neuron degeneration if not working effectively, as they lead to the presence of glutamate in excessive amounts [17]. Results indicated that in tissue from MND patients there is more RNA editing, causing amino acid changes in central nervous system proteins, than in controls. This may prevent the glutamate transporters from ‘mopping up’ excess glutamate.
This discovery is specific to human patients and could not have been made by studying animal ‘models’ of the disease. Further studies using mini-gene constructs in cell culture and a study of spinal cord RNA are now underway to investigate the genetics behind this finding, which could lead to potential treatments [17].
Cell culture studies
Professor Pamela Shaw, at the University of Sheffield, is undertaking cellular studies of MND, exploring the specific features of motor neurons that make them vulnerable to this disease. The research group is using cellular imaging techniques to study the physiological processes in motor neurons, including handling of calcium and free radicals, mitochondrial function and axonal transport [18].
Pamela Shaw also uses cellular models to study the effects of mutations in SOD1 by transfecting a motor neuron cell line with normal and mutant forms of the human SOD1 gene. This work has already produced fascinating results, including that mutant SOD1 causes apoptosis under oxidative stress, and that there is altered intracellular handling of superoxide and nitric oxide free radicals [19].
Conclusions
There are no perfect research methods, but using ethical, human-relevant means to study a human disease provides confidence that the models are relevant to the species and illness of interest. Combining human-based studies at the genetic, molecular, cellular, volunteer and epidemiological levels could open the door to faster progress in understanding and treating motor neuron disease.
References
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10. MND Association DNA Bank
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16. MND Association enzyme Q10
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