A porcine model of neurofibromatosis type 1 that mimics the human disease

Abstract

Loss of the NF1 tumor suppressor gene causes the autosomal dominant condition, neurofibromatosis type 1 (NF1). Children and adults with NF1 suffer from pathologies including benign and malignant tumors to cognitive deficits, seizures, growth abnormalities, and peripheral neuropathies. NF1 encodes neurofibromin, a Ras-GTPase activating protein, and NF1 mutations result in hyperactivated Ras signaling in patients. Existing NF1 mutant mice mimic individual aspects of NF1, but none comprehensively models the disease. We describe a potentially novel Yucatan miniswine model bearing a heterozygotic mutation in NF1 (exon 42 deletion) orthologous to a mutation found in NF1 patients. NF1+/ex42del miniswine phenocopy the wide range of manifestations seen in NF1 patients, including café au lait spots, neurofibromas, axillary freckling, and neurological defects in learning and memory. Molecular analyses verified reduced neurofibromin expression in swine NF1+/ex42del fibroblasts, as well as hyperactivation of Ras, as measured by increased expression of its downstream effectors, phosphorylated ERK1/2, SIAH, and the checkpoint regulators p53 and p21. Consistent with altered pain signaling in NF1, dysregulation of calcium and sodium channels was observed in dorsal root ganglia expressing mutant NF1. Thus, these NF1+/ex42del miniswine recapitulate the disease and provide a unique, much-needed tool to advance the study and treatment of NF1.

Sex-dependent differences in pain and sleep in a porcine model of Neurofibromatosis type 1

“…DRG neurons were isolated from 7-9-month-old Yucatan miniswine using methods described previously [55]. At necropsy, a bilateral laminectomy was made and DRGs were collected then digested in 3 ml neurobasal media (cat# 21103049, Thermofisher Scientific) containing collagenase Type II (Cat# LS004205, Thermofisher, 12 mg/ml) and dispase II (Cat# 17-105-041, Thermofisher, 20 mg/ml) and incubated for 45-75 minutes at 37°C under gentile agitation.…”

“…To design a better NF1 animal model, a miniswine harboring a disruptive NF1 mutation (exon 42 deletion) was engineered [55]. This miniswine spontaneously exhibited café-au-lait macules, learning impairment and neurofibromas, thus recapitulating classic features of NF1, many of which are not displayed in conventional heterozygotic rodent models [55]. However, pain perception and signaling in NF1 have not been investigated.…”

Validating indicators of CNS disorders in a swine model of neurological disease

“…This has led to development of large animal 67 models of disease, such as genetically modified swine, which are more similar to humans 68 anatomically, genetically, physiologically, and metabolically. [1][2][3][4] This increased similarity is 69 especially important when studying neurological disorders, where the anatomical and 70 physiological differences found in the rodent systems, cannot recapitulate human disease. 71…”

“…Successful genetically modified miniswine models have been established to study a number of 72 human diseases including atherosclerosis, cancer, ataxia telangiectasia, cystic fibrosis, and 73 neurofibromatosis type 1. 1,[4][5][6][7] However, while these new larger animal systems can better 74 recapitulate many of the hallmarks of human disease, there are limited tools and reagents that 75 have been well described and validated in these models. Herein, we test a number of 76 antibodies relevant to the study of the brain and neurological disorders in porcine brain tissue.…”

“…As some antibodies are predominantly reactive in a disease state, here we use a 110 recently developed miniswine model of NF1 to validate a number of CNS cell-specific 111 antibodies. 1 As patients with NF1 experience a host of CNS-specific impairments, this porcine 112 model is ideal for validation of neurologically relevant antibodies. We validate and explore the 113 expression of antibodies implicated in glial inflammation, oligodendrocyte differentiation, 114 neuronal signaling, and nociceptive function.…”