Abstract
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.
Highlights
The biochemical mechanisms correlating PMP22 dysfunction with demyelination are still unclear
By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca2؉]i is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca2؉ into CMT1A Schwann cells (SC)
Characterization of SC Cultures—The phenotypic characterization of 5-day-old SC cultures from CMT1A and wild type rats revealed expression of PMP22, myelin protein zero (MPZ), and myelin basic protein (MBP), which are up-regulated during terminal SC differentiation and myelination [18], together with L1 and glial fibrillary acidic protein (GFAP), which should only be expressed by nonmyelinating SC and immature SC precursors [18, 19] (Fig. 1)
Summary
Antibodies—Commercially available monoclonal antibodies against myelin basic protein (MBP) (MAB386, Millipore, Milano, Italy), myelin protein zero (MPZ) All experiments were performed on 5-day-old cultures from adult CMT1A or from wild type rats. Schwann Cells Characterization—Characterization of CMT1A and wild type SC by real time PCR analyses for PMP22, MPZ, MBP, L1, Krox-20, and GFAP was performed as described below. RNA was extracted, and real time PCR analyses were performed as described above; myelination was tested on transfected cells co-cultured with normal sensory neurons (see below). Myelination Studies—Primary SC from newborn rats (wild type and CMT1A) transfected with the shRNA-P2X7 plasmid or with the negative control shRNA plasmid were seeded (3 ϫ 105 cells/dish) onto DRG neurons in neurobasal medium, supplemented with 15% newborn calf serum, 2 mM glutamine, 5 ng/ml NGF, and 100 g/ml ascorbic acid. Statistical Analyses—All parameters were tested by paired t test or by a one-way analysis of variance. p values Ͻ 0.05 were considered significant
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