The Growth Arrest-Specific Gene Product Gas6 Promotes the Survival of Human Oligodendrocytes via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Axl/Mer/Rse are expressed in human fetal spinal cord oligodendrocytes. cDNA was prepared from O4⁺ oligodendrocyte RNA at 22 gw and the human oligodendroglioma TC620 cells. The Axl and Rse primers are near the start site and stop within the 3′-UTR. A, Axl (lane 1), Rse (lane3), and Mer (lane5) are expressed in O4⁺ oligodendrocytes. Axl and Rse are expressed in TC620 cells also (lanes 2, 4, respectively); Mer was not examined in TC620 cells. B, C, Western blot analysis confirmed the presence of Axl and Rse in human fetal spinal cord at 22 gw. Total protein homogenates (20–50μg) from two human oligodendrogliomas, TC620 cells (lanes 1, 4) and HOG cells (lanes 2, 5), and human fetal spinal cord (22 gw; lanes 3, 6) were analyzed for the expression of Axl (B) and Rse (C). Preincubation of the Axl antibody (1:250) with 20μg/ml Axl-Fc (B, lanes 4–6) or the preincubation of the Rse antibody (1:100) with its blocking peptide reduced immunoreactivity (C, lanes 4–6). M, Marker lane (in B). To verify protein loading in each lane, we stripped and incubated the blots with a β-tubulin monoclonal antibody (1:1000). Visualization is by ECL. D, Analysis of Axl receptor expression during human fetal spinal cord development. Lanes 1–3 contain 50 μg of total protein isolated from human fetal spinal cord at 18, 20, and 22 gw, respectively. First the blot was incubated with the affinity-purified Axl polyclonal antibody (1:250; Amgen) overnight at 4°C; so that equal loading could be confirmed, the blot was stripped and incubated with a β-tubulin monoclonal antibody (1:1000). Blots were scanned in a linear range; the data are presented as a ratio of Axl over β-tubulin. The relative quantities of the 140 kDa Axl were 18 gw (lane 1), 0.528; 20 gw (lane 2), 0.287; 22 gw (lane 3), 0.160.

Axl is expressed on oligodendrocytes in fetal spinal cord at 21 gw. Maximum pixel projection of z-series images was obtained by confocal microscopy of the white matter from the lumbar region. A–C, Single staining of the polyclonal antibody to Axl (A) and a monoclonal antibody to MAP-2 + 13 (B) and to O4 (C). D–F, Double-label immunofluorescence of Axl (red fluor Alexa 568) and O4 (blue fluor Alexa 647). G–I, Double-label immunofluorescence of Axl (red fluor Alexa 568) and MAP-2 + 13 (Alexa 647 fluor is shown as green in the images). Arrowheads in F and I denote a higher magnification of the double-labeled oligodendrocyte in the inset boxes. Axl staining was always performed first. J, Preincubation of the Axl antibody with Axl-Fc before incubation eliminated Axl staining. Double-label immunofluorescence of adsorbed Axl antibody with O4 (K) or MAP-2 + 13 (L) detected staining with only the respective monoclonal antibody. No cross-reactivity was observed when the O4 or MAP-2 + 13 monoclonal antibody was eliminated and the respective secondary antibody and fluor were included (data not shown). Scale bar, 20μm.

The number of CNP⁺ and MBP⁺ human oligodendrocytes is increased when plated on irradiated NIH3T3 cells secreting Gas6. NIH3T3 cells with and without Gas6 were plated at a density of 5 × 10⁴ cells in 24-well plates. After 24 hr the NIH3T3 cells were γ-irradiated at 12,500 rads to prevent cell division but permitted the secretion of Gas6 into the medium from the NIH3T3–Gas6 cells. Enriched oligodendrocytes isolated from 20–23 gw human fetal spinal cord were plated on the irradiated 3T3 cells. N2 basal medium and 10 ng/ml PDGF, 5 ng/ml NT-3, or PDGF and NT-3 were used to identify a reasonable basal medium to support the oligodendrocytes in the presence and the absence of Gas6. After 6 d in culture the oligodendrocyte–3T3 cocultures with or without Gas6 were fixed and immunostained with antibodies to CNP and MBP. Data represent the number of CNP⁺ and MBP⁺ oligodendrocytes counted from duplicate wells. Each error bar represents the mean ± SEM (n = 6) obtained from three independent experiments (**p < 0.01; *p < 0.05; unpaired Student's t test).

Increased primary and secondary CNP⁺ oligodendrocyte processes when plated on irradiated NIH3T3 cells secreting Gas6. Shown are photomicrographs of 6 d oligodendrocyte cell cultures plated on NIH3T3 cells (A1, A2) or plated on irradiated NIH3T3 cells secreting Gas6 (B1, B2) in N2 basal medium containing PDGF. Immunocytochemistry of oligodendrocytes was performed with antibodies against CNP. Visualization was by DAB. Oligodendrocytes plated on Gas6-secreting NIH3T3 cells demonstrated a twofold increase in the number of CNP⁺ oligodendrocytes (B1, B2) relative to oligodendrocytes plated on irradiated NIH3T3 cells (A1, A2). Morphologic examination of the CNP⁺ oligodendrocytes plated on the Gas6-secreting NIH3T3 cells in N2 basal medium alone or supplemented with PDGF, NT-3, or PDGF and NT-3 media indicated that the oligodendrocytes showed increased numbers of primary and secondary processes (B2,Table 2). A1, B1, Magnification, 20×; A2, B2, magnification, 60×.

The Axl receptor is expressed on cultured oligodendrocytes. Cultures were grown in the absence (Minus Gas6) or the presence (Plus Gas6) of 400 ng/ml recombinant human Gas6 (rhGas6) in N2 basal medium containing PDGF and NT-3 and were fixed 6 d after plating. Oligodendrocytes were double-immunostained sequentially with Axl polyclonal antibody (green; B, E, H), followed by O4 monoclonal antibody (red; A, D, G). Red/green merged images are shown in C, F, I. A representative low-magnification field of oligodendrocytes with extensive process outgrowth and arborizations in the presence of rhGas6 is shown in A–C. G–I show a double-labeled oligodendrocyte with punctate Axl staining at high magnification. The O4⁺/Axl ⁺ cells in C, F, I confirm that Axl is expressed on oligodendrocytes. J, M, Single staining of the Axl receptor on oligodendrocytes and the corresponding phase-contrast image in the presence of rhGas6. Preincubation of the Axl antibody with an Axl-Fc (K, N) or omission of the primary antibody (L, O) eliminated the immunoreactivity and confirmed the specificity of Axl staining. Scale bars, 10 μm.

rhGas6/Axl signaling induces a dose-dependent reduction in human oligodendrocyte apoptosis. Oligodendrocytes were plated in N2 basal medium supplemented with PDGF and NT-3 on poly-l-lysine-coated plastic tissue culture wells in triplicate. Oligodendrocyte cultures were treated with varying concentrations of rhGas6 (25–400 ng/ml) for 6 d. Blocking experiments were performed with the said doses of rhGas6 and 10 μg/ml Axl-Fc. Apoptotic oligodendrocytes were detected by immunofluorescence staining with the O4 antibody, followed by TUNEL staining. The numbers of O4⁺/TUNEL⁺ double-labeled oligodendrocytes were counted from triplicate wells in 25 independent 20× microscopic fields and expressed as a percentage of the O4⁺ cells per field. Data are shown as the mean ± SEM of a representative experiment.

rhGas6 increases CNP⁺ oligodendrocyte cell number when plated on poly-l-lysine-coated wells. Oligodendrocyte cultures plated on poly-l-lysine-coated wells in N2 basal medium containing PDGF and NT-3 were treated with rhGas6 (400 ng/ml) in the absence or presence of 10 μg/ml Axl-Fc or TrkA-Fc for 6 d. Oligodendrocytes were immunostained with a polyclonal CNP antibody. The total number of oligodendrocytes was counted from triplicate wells. Each error bar represents the mean ± SEM (n = 6) obtained from two independent experiments (***p = 0.0002; *p = 0.0124; unpaired Student's t test).

PI-3-kinase/Akt pathway inhibitors LY 294006 and wortmannin block rhGas6 signaling and increase the number of TUNEL⁺ oligodendrocytes. For each experimental condition the oligodendrocytes were plated in N2 basal medium containing PDGF and NT-3 on poly-l-lysine-coated plastic tissue culture wells in duplicate. rhGas6 (400 ng/ml) was administered to oligodendrocyte cultures for 6 d in the presence and absence of 10 nm LY 294006 or 100 nm wortmannin. The O4⁺/TUNEL⁺ and CNP⁺/TUNEL⁺ double-labeled oligodendrocyte cell numbers were counted from duplicate wells in 25 independent 20× microscopic fields and expressed as a percentage of the O4⁺ or CNP⁺ cells per field. Data are shown as the mean ± SEM of a representative experiment (***p < 0.0001; **p < 0.01; unpaired Student's t test; ns, not significant).