Molecular Analysis of Neurofibromin Deficient Muscle

Abstract

The genetic disease neurofibromatosis type 1 (NF1) is classically associated with central nervous system and tumour manifestations. However, recent clinical and basic studies have confirmed anecdotal reports of muscle weakness associated with NF1, which can have substantial impact on quality of life. This thesis investigates underlying mechanisms of muscle weakness in NF1, utilising several genetic mouse models as well as patient muscle biopsies. In Chapter 2, CreERT2 inducible Cre/loxP technology was used to generate an inducible muscle-specific model of Nf1 deletion. It was hypothesised that the conditional deletion of Nf1 in adult muscle stem cells using the Pax7 promotor would better model NF1-muscle weakness in adult mice. However, Nf1Pax7i-/- mice treated with tamoxifen showed limited recombination efficiencies in adult muscle resulting in no observable phenotype. Subsequent fluorescent reporter studies assessed the efficiency of myofiber targeting by the muscle- specific Pax7-CreERT2 allele, and confirmed substantially lower than predicted rates of muscle stem cell integration into adult muscle. These studies highlight fundamental limitations of the Pax7-CreERT2 system. In Chapter 3, the ability of a small molecular mitogen-activated protein kinase (MEK) inhibitor targeting canonical NF1-RAS/MAPK signalling to modulate the NF1-muscle phenotype was tested in two mouse models of Nf1 deficiency. These were the muscle-specific -/- knockout mouse and the limb-specific Nf1 knockout model. Developmental Nf1MyoD -/- Prx1 dosing with a MEK inhibitor rescued the reduced bodyweight and increased lipid storage seen in neonatal Nf1Myo -/- pups. However, treatment failed to affect adult Nf1Prx1 -/- mice, which maintained a myopathic phenotype. These results indicate that MEK/ERK signalling- dependant pathways are responsible for the development of NF1-associated muscle weakness, but persistence of the myopathy appears independent of MEK/ERK signalling. This suggests MEK inhibitor would have a limited clinical efficacy for management of NF1 muscle weakness. In Chapter 4, we conduct the first analyses of human NF1 muscle biopsy specimens, and confirm several disease features in patient muscle that align with previous mouse data, including lipid storage and muscle fibrosis. For subsequent mechanistic studies, molecular technologies were employed, including RNAseq transcriptomic and lipidomic analyses of -/- muscle tissues from the Nf1MyoD mouse. This identified specific changes in muscle metabolism that are hypothesized to underlie muscle weakness. These include shifts in gene expression associated with metabolic glucose regulation, and the accumulation of neutral lipids, particularly triglycerides and cholesterol esters containing long-chain fatty acids (LCFAs). The Nf1Prx1-/- mouse was histologically characterized and shown to recapitulate the prominent features of human NF1-myopathy, including muscle fibrosis, lipid storage, and muscle weakness. Unlike the Nf1Myo -/- line, the Nf1Prx1 -/- mouse survived postnatally and was therefore suitable for dietary interventions aimed at lowering neutral lipid accumulations and improving muscle function. A dietary intervention was thus designed to be enriched for medium chain fatty acid (MCFA) containing lipids as well as L-carnitine, a cofactor used in the mitochondrial metabolism of LCFAs. Nf1Prx1-/- mice placed on the modified diet showed a significant reduction in muscle lipid staining as well as a 45% increase in grip strength following 8-weeks of intervention. In summary, this body of work makes substantial progress in our understanding of the developmental and biochemical nature of NF1 associated muscle weakness and proposes a novel treatment option. The dietary therapy trialled in the mouse model is promising for human clinical trials, and it is speculated that translation could yield dramatic improvements in quality of life for individuals with NF1.