Perturbed Interactions of Mutant Proteolipid Protein/DM20 with Cholesterol and Lipid Rafts in Oligodendroglia: Implications for Dysmyelination in Spastic Paraplegia

Abstract

Missense mutations in the human PLP1 gene lead to dysmyelinating diseases with a broad range of clinical severity, ranging from severe Pelizaeus-Merzbacher disease (PMD) to milder spastic paraplegia type 2 (SPG-2). The molecular pathology has been generally attributed to endoplasmic reticulum (ER) retention of misfolded proteolipid protein (PLP) (and its splice isoform DM20) and induction of the unfolded protein response. As opposed to previous studies of heterologous expression systems, we have analyzed PLP/DM20 trafficking in oligodendroglial cells, thereby revealing differences between PMD and SPG-2-associated PLP/DM20 isoforms. PLP A242V and DM20 A242V ( jimpy-msd in mice), associated with severe PMD-like phenotype in vivo, were not only retained in the ER but also interfered with oligodendroglial process formation. In contrast, glial cells expressing SPG-2-associated PLP I186T or DM20 I186T (rumpshaker in mice) developed processes, and mutant PLP/DM20 reached a late endosomal/lysosomal compartment. Unexpectedly, PLP/DM20 with either substitution exhibited impaired cholesterol binding, and the association with lipid raft microdomains was strongly reduced. Turnover analysis demonstrated that mutant PLP was rapidly degraded in oligodendroglial cells, with half-lives for PLP Ͼ PLP I186T Ͼ PLP A242V . Protein degradation was specifically sensitive to proteasome inhibition, although PLP/DM20I186T degradation was also affected by inhibition of lysosomal enzymes. We conclude that, in addition to ER retention and unfolded protein response (UPR) induction, impaired cholesterol binding and lipid raft association are characteristic cellular defects of PLP1-missense mutations. Mutant protein is rapidly cleared and does not accumulate in oligodendroglial cells. Whereas UPR-induced cell death governs the PMD phenotype of the msd mutation, we propose that impaired cholesterol and lipid raft interaction of the rsh protein may contribute to the dysmyelination observed in SPG-2.

Transport of the Major Myelin Proteolipid Protein Is Directed by VAMP3 and VAMP7

“…Expression vectors used were as follows: pCMV-PLP (Krämer-Albers et al, 2006); pEYFP-N1-MOG has been subcloned from pcDNA3.1-MOG using BglII and HindIII restriction sites; pEGFP-C3-TI-VAMP, pEGFP-C3-Nter-TI-VAMP, and pEGFP-C3-cellubrevin (Martinez-Arca et al, 2000, 2003; pCMV5-TeNT and pCMV5-TeNT-E234Q (McMahon et al, 1993) kindly provided by T. Binz (Institute for Physiological Chemistry, Medizinische Hochschule Hannover, Hannover, Germany); pcDNA4/TO/ myc-HisA-Syntaxin-2, pcDNA4/TO/myc-HisA-Syntaxin-3, and pcDNA4/ TO/myc-HisA-Syntaxin-4 , kindly provided by T. Weimbs (University of California, Santa Barbara, Santa Barbara, CA).…”

“…Oli-neu cells were transfected by electroporation using a GenePulser Xcell (Bio-Rad) as described previously (Krämer-Albers et al, 2006). Small interfering RNA (siRNA) transfection of primary oligodendrocytes (performed immediately after shake-off from astrocyte monolayer) and Oli-neu cells was performed using Amaxa Biosystems technology according to the manufacturer's instructions (AmaxaNucleofector kit, Primary Neurons; program O-005).…”

ERdj4 and ERdj5 Are Required for Endoplasmic Reticulum-associated Protein Degradation of Misfolded Surfactant Protein C

“…In contrast to mutant SP-C, ERdj4 and ERdj5 formed complexes with both WT and mutant insulin 2, a nonglycosylated, secreted protein ( Figure 5A and Table 2). Because WT connexin 32 and proteolipid protein were reported previously to be degraded in a proteasome-dependent manner (Vanslyke et al, 2000;Kramer-Albers et al, 2006), we determined whether WT insulin2 was part of a complex destined for degradation. Both WT and mutant insulin 2 coprecipitated with p97, suggesting that the WT protein was unstable and degraded when expressed transiently in HEK293 cells ( Figure 4B).…”

Transcriptional Expression of Myelin Basic Protein in Oligodendrocytes Depends on Functional Syntaxin 4: a Potential Correlation with Autocrine Signaling

“…Yet, we cannot exclude that in this particular case, sheet-directed transport, mediated by the SNARE machinery, might have exploited alternatives for syntaxin 4, e.g., syntaxin 2, which is present in mature OLGs (18,19), in a nonpolarized manner (our unpublished observations) or for the VAMP3 v-SNARE, e.g., VAMP2 (18,19) or VAMP7, which has been implicated in PLP trafficking (32). If so, it should also be emphasized that "t-SNARE substitution" does not apply to the apical machinery, driven by syntaxin 3, as in this case its downregulation did effectively preclude PLP trafficking to the plasma membrane of the cell body, which precedes subsequent transport to the myelin sheet (1,34,36,(63)(64)(65). Thus, the specificity of the effect of syntaxin 4 is emphasized by similar observations of a reduction of MBP expression upon downregulation of its v-SNARE, VAMP3, the absence of an effect of syntaxin 3, and the apparent inability to potentially maintain MBP mRNA transcription via a syntaxin 2-mediated pathway.…”