A NOVEL ANIMAL MODEL FOR AMYOTROPHIC LATERAL SCLEROSIS: THE SOD1G93A TRANSGENIC SWINE.
Creators: name: CHIEPPA, MARIA NOVELLA
Publisher: name: Universit� degli Studi di Milano
Year: 2013

description: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that may occur in two clinically indistinguishable forms: sporadic (sALS) and familial (fALS), the latter linked to several gene mutations, mostly inheritable in a dominant fashion [Rosen et al., 1993]. The disease is characterized by selective and progressive degeneration of upper and lower motor neurons, leading to muscle weakness, atrophy and evolving to complete paralysis that results in patient death in 2 to 5 years after symptoms onset. Research on ALS has mainly relied so far on experimental rodent models carrying a variety of Cu/Zn Superoxide Dismutase 1 (SOD1) mutations. Currently, the most widely employed model is a transgenic mouse with a glycine to alanine conversion at the 93rd codon (G93A) of the SOD1 gene. These mice reliably reproduce the ALS patients phenotype progression, developing a rapidly progressive motor neuron degeneration. Death occurs about four months after symptoms onset [Turner &amp; Talbot, 2008], not reflecting the disease course in human patients. Although the use of these murine models is currently widespread both in clinical trials and in basic research, aimed at a resolution of the pathogenic mechanisms underlying the disease, doubts have been recently raised, from numerous reliable sources [Schnabel, 2008; Benatar, 2007; Van Den Bosch, 2011; Gordon et al., 2007] about rodents suitability to faithfully reproduce the human disease. Since human and rodent species differ in life-span, physiology, anatomy and biochemical aspects, data extrapolation has proved to be difficult. As a matter of fact, encouraging results of drug tests in rodents have never been so far successfully translated to humans, and, in some cases, molecules delaying disease progression in transgenic mice, such as minocycline, have resulted even detrimental in ALS patients [Scott et al., 2008] also because of the heterogeneity of mouse genetic background [Schnabel, 2008]. The scientific community has already accepted swine as an attractive model, alternative to non-human primates, for pharmacological and surgical testing as well as for biomedical research on the basis of its anatomical, physiological and biochemical features that are more closely related to human species than the rodent ones. Furthermore, the prospect of obtaining genetically modified pigs further extended their biomedical potential especially to mimic inherited human diseases [Bendixen et al., 2010]. In particular, regarding Central Nervous System (CNS) anatomy, pig brain cortical surface resembles human gyrencephalic neocortex and similarities with the human brain have also been demonstrated for the hippocampus, subcortical and diencephalic nuclei and brainstem structures. Furthermore, pig brain size permits an easy identification of cortical and subcortical structures by conventional imaging techniques and offers invaluable opportunities for microsurgical techniques and intrathecal drugs administration. Consistently, the swine large size and long lifespan allow to perform numerous and repetitive samplings from the same animal, thus enabling to get a higher amount of data to characterize in detail preclinical and clinical phases. The longer lifespan makes swine also a suitable animal model for long-term evaluation of safety and efficacy of innovative therapies. Moreover cloning techniques are well established in this species, allowing thus to solve problems related to the variability of genetic background. On this basis, our group has produced by in vitro transfection of cultured somatic cells combined with Somatic Cell Nuclear Transfer (SCNT) the first swine ALS model. To achieve this goal, an ubiquitous SOD1G93A expression vector has been used, which is characterized by the ability to maintain high expression levels through the next generation of pigs [Brunetti et al., 2008], wherein the pCAGGS promoter is inserted between two insulators (5? MAR of chicken lysozyme gene) to prevent silencing effects. This vector was used to transfect primary porcine adult male fibroblasts (PAFs), thus obtaining transgenic cell colonies to use as nuclei donor in SCNT procedures. After SCNT, SOD1G93A embryos were transferred in recipient sows, and four pregnancies developed to term. Five piglets survived artificial hand raising, weaning, developed normally and reached adulthood. The remaining piglets died within 48?96 h after farrowing due to events commonly reported in commercial herds (i.e. neonatal diarrhoea or pneumonia, etc). Fibroblasts obtained from ear biopsy of living piglets were analyzed by immunocitochemistry (ICC) and revealed the transgenic protein expression. Furthermore Western Blot (WB) and immunohistochemistry (IHC) analysis were performed on dead piglets tissues, that proved to be all positive for the transgenic protein presence. Immunohistochemistry revealed granular mutant protein aggregates in the CNS. Unlike rodent models that show an extremely high expression transgene level and a rapid disease course [Turner &amp; Talbot, 2008], our swine model presents an expression level comparable to that of human patients, where a single allele mutation results in a toxic gain of function. In rodents, the mutant SOD1 expression level for a given mutation determines disease severity, higher levels yielding a more aggressive phenotype [Bento-Abreu et al., 2010]. However, since a SOD1G93A swine model has never been produced before, no data are available about the correlation between transgene expression level and disease onset timing and we can only make rough estimates as to when the first neurological symptoms may occur. Piglets are expected to show ALS symptoms in two or three years, while we cannot exclude a longer period. On one hand this could result in a longer pre-clinical phase and in an increase of animal maintaining costs, on the other hand our SOD1G93A swine could represent an invaluable opportunity to find early biomarkers and a closer and more faithful model to reproduce human pathology since ALS is typically an adult-onset disease. Currently, an animal model recapitulating all the ALS crucial aspects has not yet been produced, although some transgenic mouse lines modulate a relatively faithful subset of disease features. However, since increasing difficulties are emerging in translating information gleaned from rodent models into therapeutic options for ALS patients, there is an urgent need for an intermediate research system. I do believe that a swine model could provide this essential bridge between insights gained from rodent models and the reality of treating a human disease.