The diadenosine homodinucleotide P18 improves in vitro myelination in experimental Charcot-Marie-Tooth type 1A.

Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca(2+) concentration ([Ca(2+)]i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca(2+) levels through down-regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5',5'''-P1, P2-diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11-overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild-type (wt) cells. P18 increased [cAMP]i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non-phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]i-increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype.

Oxydative phosphorylation in sciatic nerve myelin and its impairment in a model of dysmyelinating peripheral neuropathy.

Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot-Marie-Tooth type 1A (CMT1A) rats. The latter used as a model of dys-demyelination. O2 consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O2 consumption by IMV from Wt and CMT1A 1-month-old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys-demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys-demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.

P2X7-mediated increased intracellular calcium causes functional derangement in Schwann cells from rats with CMT1A neuropathy.

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca(2+)](i)) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca(2+)](i) is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca(2+) into CMT1A SC. Correction of the altered [Ca(2+)](i) in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca(2+)](i) and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.

Impaired expression of ciliary neurotrophic factor in Charcot-Marie-Tooth type 1A neuropathy.

We investigated the contribution of Schwann cell-derived ciliary neurotrophic factor (CNTF) to the pathogenesis of Charcot-Marie-Tooth disease type 1A (CMT1A) and addressed the question as to whether it plays a role in the development of axonal damage observed in the disease, with aging. Ciliary neurotrophic factor was underexpressed in experimental CMT1A but not in other models of hereditary neuropathies. Sciatic nerve crush experiments and dosage of CNTF at different time points showed that expression of this trophic factor remained significantly lower in CMT1A rats than in normal controls; moreover, in uninjured CMT1A sciatic nerves CNTF levels further decreased with ageing, thus paralleling the molecular signs of axonal impairment, that is increased expression of non-phosphorylated neurofilaments and amyloid precursor protein. Administration of CNTF to dorsal root ganglia cultures reduced dephosphorylation of neurofilaments in CMT1A cultures, without improving demyelination. Taken together, these results provide further evidence that the production of CNTF by Schwann cells is markedly reduced in CMT1A. Moreover, the observations suggest that trophic support to the axon is impaired in CMT1A and that further studies on the therapeutic use of trophic factors or their derivatives in experimental and human CMT1A are warranted.