AAV1.NT-3 gene therapy in a CMT2D model: phenotypic improvements in GarsP278KY/+ mice

Abstract

Glycyl tRNA synthetase (GARS) mutations are associated to the Charcot-Marie-Tooth type-2D. The GarsP278KY/+ model for Charcot-Marie-Tooth type-2D is known best for its early onset severe neuropathic phenotype with findings including reduced axon size, slow conduction velocities and abnormal neuromuscular junction. Muscle involvement remains largely unexamined. We tested the efficacy of neurotrophin 3 (NT-3) gene transfer therapy in two Gars mutants with severe (GarsP278KY/+) and milder (GarsΔETAQ/+) phenotypes via intramuscular injection of adeno-associated virus setoype-1, triple tandem muscle creatine kinase promoter, NT-3 (AAV1.tMCK.NT-3) at 1x1011 vg dose. In the GarsP278KY/+ mice, the treatment efficacy was assessed at 12 weeks post-injection using rotarod test, electrophysiology, and detailed quantitative histopathological studies of the peripheral nervous system including neuromuscular junction, and muscle. NT-3 gene transfer therapy in GarsP278KY/+ mice resulted in significant functional and electrophysiological improvements, supported with increases in myelin thickness and improvements in the denervated status of neuromuscular junctions as well as increases in muscle fiber size along with attenuation of myopathic changes. Improvements in the milder phenotype GarsΔETAQ/+ was less pronounced. Furthermore, oxidative enzyme histochemistry in muscles from Gars mutants revealed alterations in the content and distribution of oxidative enzymes with increased expression levels of Pgc1a. Cox1, Cox3 and Atp5d transcripts were significantly decreased suggesting that the muscle phenotype might be related to mitochondrial dysfunction. NT-3 gene therapy attenuated these abnormalities in muscle. This study shows that NT-3 gene transfer therapy has disease modifying effect in a mouse model for Charcot-Marie-Tooth type-2D, leading to meaningful improvements in peripheral nerve myelination and neuromuscular junction integrity as well as in a unique myopathic process, associated with mitochondria dysfunction, all in combination contributing to functional outcome. Based on the multiple biological effects of this versatile molecule, we predict NT-3 has the potential to be beneficial in other aminoacyl-tRNA synthetase (ARS)-linked CMT subtypes.

AAV1.NT-3 gene therapy prevents age-related sarcopenia

“…We also assessed the status of NMJ in response to NT-3 gene therapy in this model as there is evidence that changes in endplate morphology and NMJ remodeling occur with aging and precede loss of fast motor units [ 20 ]. Using immunohistochemistry-based parameters [ 11 ], we analyzed a total of 330 NMJs derived from intrinsic foot muscles of NT-3 treated and untreated aged C57BL/6 mice. This analysis showed that AAV1.NT-3 gene therapy, at 6 months of treatment gave rise to a 29.7% increase of innervated NMJs ( p = 0.0123).…”

“…Lumbrical muscles collected from treated and untreated mice ( n = 4 for both cohorts with equal sex distribution) were processed as described previously [ 11 ]. Muscles were fixed and stained with primary antibodies (Acetylcholine receptor (AChR) antibody, α Bungarotoxin, T1175, 1:500; Anti Neurofilament 200 antibody, N4142, 1:500; SV2 antibody, AB_2315387, 1:50) [ 49 , 50 ], followed by incubation with secondary antibodies (Alexa Fluor 488 conjugated anti-rabbit and anti-mouse IgG, 1:500).…”

“…Samples were imaged at 60× magnification using a Zeiss LSM 800 confocal microscope. NMJs were considered to be innervated when nerve completely overlapped the AChRs, as partially innervated when some parts of the AChRs were not overlapping with nerve, and as denervated when there was not any nerve co localizing with AChRs [ 11 , 51 ]. An average of 41.3 NMJs per mouse were evaluated from NT-3-treated and UT-mice ( n = 4 mice per group with equal sex distribution).…”

The emerging role of furin in neurodegenerative and neuropsychiatric diseases

“…The mature neurotrophins then bind to their corresponding receptors, the Trk family of receptor tyrosine kinases, and regulate neuronal survival and synaptic plasticity [100,101]. Aberrant expressions of NT-3 and NT-4/5 participate in pathophysiological conditions including motor dysfunction, cognitive decline, stroke, and SCZ [102][103][104][105][106][107].…”

Hereditary motor neuropathies

“…It also increased both myelin thickness and muscle fiber size and improved the denervated state of NMJ. On the basis of the multiple effects of this molecule, a potential benefit could be predicted in patients with mutations in GARS1 and other aminoacyl tRNA synthetases [55 ▪▪ ]. Zuko et al found that mutant glycyl-tRNA synthetases cause an impairment in mRNA translation elongation and that transgenic tRNA Gly overexpression rescues protein synthesis and peripheral neuropathy in mice and fly models of GARS1-CMT/CMT2D.…”