Altered sensory neuron development in CMT2D mice is site-specific and linked to increased GlyRS levels

Abstract

Dominant, missense mutations in the widely and constitutively expressed GARS1 gene cause a peripheral neuropathy that usually begins in adolescence and principally impacts the upper limbs. Caused by a toxic gain-of-function in the encoded glycyl-tRNA synthetase (GlyRS) enzyme, the neuropathology appears to be independent of the canonical role of GlyRS in aminoacylation. Patients display progressive, life-long weakness and wasting of muscles in hands followed by feet, with frequently associated deficits in sensation. When dysfunction is observed in motor and sensory nerves, there is a diagnosis of Charcot-Marie-Tooth disease type 2D (CMT2D), or distal hereditary motor neuropathy type V if the symptoms are purely motor. The cause of this varied sensory involvement remains unresolved, as are the pathomechanisms underlying the selective neurodegeneration characteristic of the disease. We have previously identified in CMT2D mice that neuropathy-causing Gars mutations perturb sensory neuron fate and permit mutant GlyRS to aberrantly interact with neurotrophin receptors (Trks). Here, we extend this work by interrogating further the anatomy and function of the CMT2D sensory nervous system in mutant Gars mice, obtaining several key results: 1) sensory pathology is restricted to neurons innervating the hindlimbs; 2) perturbation of sensory development is not common in mouse models of neuromuscular disease; 3) in vitro axonal transport of signalling endosomes is not impaired in afferent neurons of all CMT2D mouse models; and 4) Gars expression is selectively elevated in a subset of sensory neurons and linked to sensory developmental defects. These findings highlight the importance of comparative neurological assessment in mouse models of disease and shed light on key proposed neuropathogenic mechanisms in GARS1-linked neuropathy.

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…This protocol and associated tools can be used to isolate ganglia from mice aged P5 onwards. The resulting DRG can be subsequently used for analyses of RNA and protein, immunohistochemically investigated as sections [15] and when cleared [14], or cultured as explants and dissociated primary neurons [19,25] for the in vitro study of live cellular processes [22]. Moreover, assessment of sensory neuron biology can be simultaneously compared and contrasted with that of the motor nervous system [23,[26][27][28] for detailed peripheral nerve characterisation.…”

“…Mice have 30 or 31 DRG pairs, depending on genetic background [17][18][19]: eight cervical (C1 to C8), 13 thoracic (T1 to T13), ve or six lumbar (L1 to L5/L6) and four sacral (S1 to S4). Lumbar DRG are most often studied; however, ganglia at different spinal levels show clear distinctions not only in development, size, cell number and proportion of functional subtypes, but also in their disease involvement [20][21][22]. It is therefore important to examine DRG from several distinct and de ned spinal levels.…”

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…By following this step-by-step video-assisted protocol, you will be able to swiftly learn how to dissect mouse sensory ganglia from defined cervical, thoracic, and lumbar levels of the spinal column (> 50 DRG/animal). The resulting DRG can be subsequently used for analyses of RNA and protein, immunohistochemically investigated as sections [15] and when cleared [14], or cultured as explants and dissociated primary neurons [19,25] for the in vitro study of live cellular processes [22].…”

“…Mice have 30 or 31 DRG pairs, depending on genetic background [17][18][19]: eight cervical (C1 to C8), 13 thoracic (T1 to T13), five or six lumbar (L1 to L5/6) and four sacral (S1 to S4). Lumbar DRG are most often studied; however, ganglia at different spinal levels show clear distinctions not only in development, size, cell number and proportion of functional subtypes, but also in their disease involvement [20][21][22]. It is therefore important to examine DRG from several distinct and defined spinal levels.…”

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…Lumbar DRG are most often studied; however, ganglia at different spinal levels show clear distinctions not only in development, size, cell number and proportion of functional subtypes, but also in their disease involvement [20][21][22]. It is therefore important to examine DRG from several distinct and defined spinal levels.…”