30 Nov 2018

In the November 21 Neurobiology of Disease, researchers led by Cristiano Corona, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy, characterize the ALS-like signs and symptoms seen in pigs that express the mutant human SOD1 G93A transgene. After more than two years of living without symptoms, the pigs develop muscle problems, lose control of their limbs, and have trouble swallowing and breathing. Motor neuron degeneration, gliosis, and hSOD1 protein aggregates turn up in their brainstems and spinal cords, and they develop necrosis and inflammation in skeletal muscle. Disease in these transgenic pigs has a long presymptomatic phase marked by gradually increasing amounts of TDP-43 in peripheral blood mononuclear cells.

“The phenotype looks robust,” said Ida Holm, Aarhus University, Denmark. “I think it faithfully recapitulates human disease.”

“This is a great paper, which will contribute to the field in a major way,” said Martin Marsala, University of California, San Diego.

As Holm has reported, pigs are increasingly being used to model neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s (see Holm et al., 2016; Dolezalova et al., 2014). Pigs offer an advantage over rodents in that they are closer to humans in size, biochemistry, and physiology, and they have bigger brains that are more amenable to intervention.

Two transgenic pig models of ALS have been reported to date. One expresses mutant human SOD1 with the same G93A substitution, which causes ALS in humans (Yang et al., 2014). This model has motor defects in its hind limbs as well as motor neuron degeneration. It’s unclear what has become of these pigs, said Holm, and the authors did not respond to a request for comment.

The other model, by Corona and colleagues, also expresses hSOD1 G93A. It was created by implanting the nucleus of a pig fibroblast expressing hSOD1 G93A into a healthy egg cell from a pig. Five transgenic founder piglets were reported to develop normally, but no phenotypic characterization had been published thus far (Chieppa et al., 2013). 

In the current study, first author Paola Crociara followed up by studying disease in four of the founder pigs that reached adulthood. In three of them, transgene expression was too low to cause clinical signs within five years, so they weren’t examined further. In the remaining pig, transgene expression was high enough to cause progressive muscle weakness, motor neuron degeneration, and gliosis—symptoms seen in ALS patients. Breeding this pig produced first-, second-, and third-generation progeny that inherited disease in an autosomal-dominant pattern.

Five affected pigs, the founder and four descendants, are described in this paper. During a long preclinical phase of 27 months, TDP-43 slowly built up in their peripheral blood mononuclear cells. When the first symptoms arose, the pigs developed gait abnormalities, where one hind limb became lame and they walked less. A month later, they had even less control over hind limb movements, and as the phenotype gradually worsened, similar symptoms affected the front legs. The pigs spent more time lying down and coughed while eating. At 31 months of age, trembling when standing, severe ataxia, spastic tetraparesis, and labored breathing marked a humane endpoint. The animals were euthanized and studied postmortem.

Histological examination of their spinal cords revealed that motor neurons had shrunk and died away, accompanied by microgliosis and astrogliosis. SOD1-positive inclusions had settled into both spinal cord and brainstem. At this end stage of disease, neuromuscular junctions in leg muscles had become denervated.

Curiously, necrosis and inflammation had taken hold in skeletal muscles toward the end of disease, two signs not typically seen in people with ALS. This could be due to mutant hSOD1 expression in the muscle cells, wrote the authors.

“In these large animal models, the anatomical dimensions of the spinal cord are very similar to humans,” said Marsala. He sees this as especially promising for studies of gene therapy, where researchers can study methods of delivery and vector volume that resemble what humans require. A long preclinical phase followed by progressive disease is also exactly what researchers observe in humans, and will allow scientists to monitor treatment effects, Marsala said. He would have liked to see more animals available for characterization, to be sure they had a consistent disease onset at a specific time. He also cautioned about the added expense of keeping large animals for years in what will likely be specialized facilities.

Corona wrote to Alzforum that his group currently has seven transgenic pigs, all presymptomatic at 14 months of age, plus several wild-type pigs. They will use them to study the role of skeletal muscle and oxidative stress in ALS, as well as to try out new therapeutic strategies and identify biomarkers.

That this pig model has a phenotype that recapitulates human disease makes it valuable, Holm said. She added that future models may be fine-tuned with new genome-editing technologies, such as CRISPR-Cas9. For instance, more precise placement of the mutant gene would enable better control of protein levels and time of disease onset. In the current model, the transgene inserted randomly into the genome.—Gwyneth Dickey Zakaib

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References

Paper Citations

1. Holm IE, Alstrup AK, Luo Y. Genetically modified pig models for neurodegenerative disorders. J Pathol. 2016 Jan;238(2):267-87. Epub 2015 Nov 28 PubMed.
2. Dolezalova D, Hruska-Plochan M, Bjarkam CR, Sørensen JC, Cunningham M, Weingarten D, Ciacci JD, Juhas S, Juhasova J, Motlik J, Hefferan MP, Hazel T, Johe K, Carromeu C, Muotri A, Bui J, Strnadel J, Marsala M. Pig models of neurodegenerative disorders: Utilization in cell replacement-based preclinical safety and efficacy studies. J Comp Neurol. 2014 Aug 15;522(12):2784-801. Epub 2014 Apr 12 PubMed.
3. Yang H, Wang G, Sun H, Shu R, Liu T, Wang CE, Liu Z, Zhao Y, Zhao B, Ouyang Z, Yang D, Huang J, Zhou Y, Li S, Jiang X, Xiao Z, Li XJ, Lai L. Species-dependent neuropathology in transgenic SOD1 pigs. Cell Res. 2014 Apr;24(4):464-81. Epub 2014 Feb 28 PubMed.
4. Chieppa MN, Perota A, Corona C, Grindatto A, Lagutina I, Vallino Costassa E, Lazzari G, Colleoni S, Duchi R, Lucchini F, Caramelli M, Bendotti C, Galli C, Casalone C. Modeling Amyotrophic Lateral Sclerosis in hSOD1 Transgenic Swine. Neurodegener Dis. 2013 Oct 23; PubMed.

Further Reading

Papers

1. Tan RH, Ke YD, Ittner LM, Halliday GM. ALS/FTLD: experimental models and reality. Acta Neuropathol. 2017 Feb;133(2):177-196. Epub 2017 Jan 5 PubMed.

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