Dynamic composition of myelin basic protein mRNA-containing ribonucleoprotein complexes

Myelin Basic Protein (MBP) is a major component of the myelin sheath orchestrating the assembly of compact myelin in the central nervous system and thus ensuring saltatory signal propagation and maintenance of the neuronal network. MBP expression is precisely regulated at the posttranscriptional level and depends on the assembly of MBP mRNA-containing granules, which mediate transport and localized translation at the axoglial contact site. This study focused on the identification of the dynamic composition of these RNP complexes and a detailed functional analysis of the previously identified potential MBP mRNA-associating proteins DDX5 and FUS. DDX5 was shown to associate with MBP mRNA in subpopulations of cytoplasmic RNP complexes. In oligodendroglial cells DDX5 functioned as an inhibitor of MBP protein synthesis, acting at the posttranscriptional level and depending on the DDX5 helicase activity. Consequently, knockdown of DDX5 in primary OL correlates with the elevation of MBP protein levels and an imbalance of the known MBP-associating RNA-binding proteins hnRNP A2 and hnRNP F. In addition, DDX5-knockdown selectively increased the expression of the exon 2-containing MBP isoforms 17.22-kDa and 21.5-kDa, possibly by affecting alternative splicing of the pre-mRNA. Alteration of FUS levels did not show a major impact on the myelin protein expression, although MBP mRNA levels were slightly reduced in line with changes in the expression of MBP mRNA-associated proteins hnRNP A2 and DDX5. During oxidative stress, FUS localized to oligodendroglial stress granules and FUS levels inversely correlated with MBP RNA stability upon increasing concentrations of sodium arsenite. To further examine the dynamic composition of MBP mRNA complexes in a more RNA-centric approach, the MS2-RNA-labeling system was adapted to MBP mRNA. The introduction of MS2 hairpin loops into the MBP transcript allowed its visualization and the purification of associated MBP mRNP complexes. Oligodendroglial cell lines stably expressing moderate levels of MS2-labeled MBP14-MS2 mRNA were generated and single molecule FISH confirmed localization and the physiological behavior of the transcript, which reacted to cellular cues such as oxidative stress. The following affinity purification of MBP14-MS2 mRNP complexes under oxidative stress conditions resulted in the identification of numerous candidates potentially playing a role in stress-dependent RNA granule formation and the regulation of MBP. This list includes several proteins connected to neurodegenerative or psychiatric diseases and may thus aid in shedding light on mechanisms regulating MBP expression during OL maturation and myelination in health and disease

Oligodendroglial p130Cas is a target of Fyn kinase involved in process formation, cell migration and survival

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation

p130Cas is involved in oligodendroglial apoptosis.

<p>(A) Primary OPCs were treated with control or p130Cas siRNA. 48 hours later, cells were lysed and tested for levels of p130Cas and cleaved Caspase 3 (indicative of apoptosis) by Western analysis. GAPDH serves as loading control. (B) Primary OPCs were treated with siRNA as in A. Additionally, some cells were treated with the apoptosis inducing agent staurosporine as positive control. After 42 hours, the cells were subjected to a TUNEL assay to test for apoptotic cells. Data show the mean ± SEM from 11 independent experiments; ** p<0.01 (Student’s <i>t</i> test). (C) Relative cell numbers from B were counted as additional indication of cell death. Data show the mean ± SEM from 11 independent experiments; ** p<0.01, *** p<0.001 (Student’s <i>t</i> test).</p

The effect of p130Cas knockdown on spreading and migration in Oli-<i>neu</i> does not result from changes in cellular viability.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 6 h after re-plating a TUNEL assay was performed to test for apoptosis. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (B) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control. (C) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 h later, the cells were detached from their culture vessel and 0.5 h and 6 h after re-plating MTT and LDH assays were carried out to test for cell viability and membrane integrity, respectively. Data show the mean ± SEM from 3 independent experiments; ns, not significant (Student’s <i>t</i> test). (D) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in C were lysed and p130Cas levels were assessed by Western blotting. α-tubulin serves as loading control.</p

The effect of p130Cas on spreading and migration of oligodendroglial cells.

<p>(A) Oli-<i>neu</i> cells were treated with p130Cas or control siRNA. 24 hours later, the cells were detached from their culture vessel and 30 min after re-plating they were subjected to immunofluorescence analysis of cell spreading. Cells were classified as either not spread (arrowhead), showing lamellipodia (asterisk) or lamella (arrow). (B) Statistical evaluation of A. Data represents the mean ± SEM from 3 independent experiments; ** p<0.01 (Student’s <i>t</i> test). (C) Oli-<i>neu</i> cells were treated as in A. Here, immunofluorescence analysis of process thickness was carried out 4 hours after re-plating. Three examples per condition are shown. Areas of measurement are outlined by white dashed rectangles. (D) Statistical evaluation of C. The thickest process for each individual cell was measured and plotted on a chart (from high to low diameters) and the average ratio ± SEM from 3 independent experiments is presented; ** p<0.01 (Student’s <i>t</i> test). (E) Oli-<i>neu</i> cells were treated with siRNA as in A. After 24 hours, they were re-plated in Boyden chambers and allowed to migrate for 6 hours in the presence of 5 ng/ml bFGF before fixation and migration analysis were carried out. Data are expressed as a percentage of basal migration, i.e. the migration of Oli-<i>neu</i> without chemoattractant. Data represent the mean ± SEM from 3 independent experiments; * p<0.05 (Student’s <i>t</i> test). (F) Knockdown of p130Cas in Oli-<i>neu</i> cells. The siRNA-treated cells as analyzed in A-E were lysed and p130Cas levels were assessed by Western blotting. GAPDH serves as loading control.</p

Identification of p130Cas as an oligodendroglial tyrosine-phosphorylated protein in the presence of active Fyn.

<p>(A) Tyrosine-phosphorylated proteins were immunoprecipitated from Oli-<i>neu</i> cells overexpressing active Fyn. A Coomassie-stained SDS-Polyacrylamide gel with first and second elution (E1 and E2), antibody control (control) and MW marker (M) is shown. The indicated band (p130Cas) was excised and analyzed by mass spectrometry. Molecular weights in kilodalton are indicated on the right. (B) Primary structure of p130Cas. The identified peptides are shown in bold red and cover 20% of the protein sequence. (C) p130Cas expression at different developmental stages in cultured oligodendrocytes (DIV, days in vitro, 1, 2, 4, 6, 8) was analyzed by Western blots with antibodies indicated on the left. CNP and MOG represent early and late maturation markers, respectively, and GAPDH serves as a loading control.</p