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Charcot–Marie–Tooth disease type 1A (CMT1A) is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin–proteasome system (UPS), autophagosomes and heat-shock proteins (HSPs). Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins., In peripheral nerves of neuropathic C22 mice the frequency of cytosolic PMP22 aggregates increases with age, which elicits a response from protein quality control mechanisms. The combined effects of aging and neuropathic genotype exacerbate disease progression leading to nerve defects.

A large fraction of hereditary demyelinating neuropathies, classified as Charcot-Marie-Tooth disease type 1A, is associated with misexpression of peripheral myelin protein 22. In this study, we characterized morphologic and biochemical changes that occur with diseaseprogression in neuromuscular tissue of Trembler-J mice, a spontaneous rodent model of Charcot-Marie-Tooth disease type 1A. Using age-matched, 2- and 10-month-old, wild-type and Trembler-J mice, we observed neuromuscular deficits that progress from distal to proximal regions. The impairments in motor performance are underlined by degenerative events at distal nerve segments and structural alterations at nerve-muscle synapses. Furthermore, skeletal muscle of affected mice showed reduced myofiber diameter, increased expression of the muscle atrophy marker muscle ring-finger protein 1, and fiber type switching. A dietary intervention of intermittent fasting attenuated these progressive changes and supported distal nerve myelination and neuromuscular junction integrity. In addition to the well-characterized demyelination aspects of this model, our investigations identified distinct degenerative events in distal nerves and muscle of affected neuropathic mice. Therefore, therapeutic studies aimed at slowing or reversing the neuropathic features of these disorders should include the examination of muscle tissue, as well as neuromuscular contact sites.

Misexpression and cytosolic retention of peripheral myelin protein 22 (PMP22) within Schwann cells (SCs) is associated with a genetically heterogeneous group of demyelinating peripheral neuropathies. PMP22 overproducer C22 and spontaneous mutant Trembler J (TrJ) mice display neuropathic phenotypes and affected nerves contain abnormally localized PMP22. Nutrient deprivation-induced autophagy is able to suppress the formation of PMP22 aggregates in a toxin-induced cellular model, and improve locomotor performance and myelination in TrJ mice. As a step toward therapies, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could facilitate the processing of PMP22 within neuropathic SCs and enhance their capacity to myelinate peripheral axons. Exposure of mouse SCs to RM induced autophagy in a dose- and time-dependent manner and decreased the accumulation of poly-ubiquitinated substrates. The treatment of myelinating dorsal root ganglion (DRG) explant cultures from neuropathic mice with RM (25 nm) improved the processing of PMP22 and increased the abundance and length of myelin internodes, as well as the expression of myelin proteins. Notably, RM is similarly effective in both the C22 and TrJ model, signifying that the benefit overlaps among distinct genetic models of PMP22 neuropathies. Furthermore, lentivirus-mediated shRNA knockdown of the autophagy-related gene 12 (Atg12) abolished the activation of autophagy and the increase in myelin proteins, demonstrating that autophagy is critical for the observed improvement. Together, these results support the potential use of RM and other autophagy-enhancing compounds as therapeutic agents for PMP22-associated demyelinating neuropathies.