Oligodendrocytes expressing exclusively the DM20 isoform of the proteolipid protein gene: Myelination and development

Abstract

Oligodendroglia and Schwann cells synthesize myelin-specific proteins and lipids for the assembly of the highly organized myelin membrane of the motor-sensory axons in the central (CNS) and peripheral nervous system (PNS), respectively, allowing rapid saltatory conduction. The isoforms of the main myelin proteins, the peripheral myelin basic isoproteins (MBP) and the integral proteolipid proteins, PLP and DM20, arise from alternative splicing. Activation of a cryptic splice site in exon III of plp leads to the deletion of 105 bp encoding the PLP-specific 35 amino acid residues within the cytosolic loop 3 of the four-transmembrane domain (TMD) integral membrane protein. To study the different proposed functions of DM20 during the development of oligodendrocytes and in myelination, we targeted the plp locus in embryonic stem cells by homologous recombination by a construct, which allows solely the expression of the DM20 specific exon III sequence. The resulting dm20(only) mouse line expresses exclusively DM20 isoprotein, which is functionally assembled into the membrane, forming a highly ordered and tightly compacted myelin sheath. The truncated cytosolic loop devoid of the PLP-specific 35 amino acid residues, including two thioester groups, had no impact on the periodicity of CNS myelin. In contrast to the PLP/DM20-deficient mouse, mutant CNS of dm20(only) mice showed no axonal swellings and neurodegeneration but a slow punctuated disintegration of the compact layers of the myelin sheath and a rare oligodendrocyte death developing with aging.

PLP1 alternative splicing in differentiating oligodendrocytes: Characterization of an exonic splicing enhancer

“…We validated our oligodendrocyte cultures for developmentally regulated PLP alternative splicing and, as previously reported [Sporkel et al, 2002] we detected PLP and DM20 transcripts with ratios of 2:1 and 3:1 in oligodendrocytes differentiated in T3 for 24 and 72 h, respectively (data not shown). Similar ratios were found in oligodendrocytes cultured in N1 medium, although the transcript levels were significantly lower than in T3 medium (data not shown).…”

Splice-site contribution in alternative splicing ofPLP1 andDM20: molecular studies in oligodendrocytes

“…The DM20 splice site is more broadly utilized, and in the CNS it is used early during embryonic development prior to oligodendrocyte differentiation and myelination, suggesting that DM20 serves a more general function compared to PLP1 [Campagnoni and Skoff, 2001]. In contrast, PLP1-specific splicing occurs at high levels only in oligodendrocytes and in the postnatal period, supporting a specific function in glia and glial interactions [LeVine et al, 1990;Stecca et al, 2000;Sporkel et al, 2002]. Therefore, mutations that interfere with PLP1-specific splicing would be compatible with life and might generate a uniquely neurological phenotype.…”

“…In addition to cell-specific regulation, PLP1 splicing is subject to developmental regulation [Timsit et al, 1992;Stecca et al, 2000;Sporkel et al, 2002]. DM20 message is expressed in the embryonic CNS by the oligodendrocyte progenitor cells, whereas the PLP1 transcript is made in the postnatal period by differentiated oligodendrocytes.…”

“…[ LeVine et al, 1990;Sporkel et al, 2002]. The preferential use of the PLP1 splice site by differentiated oligodendrocytes suggests the existence of a specialized recognition system that favors the PLP1 splice-site selection over the DM20 site.…”

Evidence for possible interactions between PLP and DM20 within the myelin sheath

“…Thus, when DM20 alone was expressed at levels similar to those in wild-type, the myelin and axon abnormalities remained unchanged (Stecca et al, 2000). In contrast, when the level of DM20 alone equalled the total PLP/DM20 found in wild-type the myelin was compacted and axonal changes were absent; however, some degenerative changes occurred in the optic nerve with age (Spörkel et al, 2002). Therefore, it may be that if present at a sufficient level, one isoform can largely, but not entirely, compensate for the other.…”