Activity-dependent NG2 cleavage and release of the ectodomain into the ECM.

(A) Primary OPC were isolated from total mouse brain of postnatal day 9 (P9) by magnetic cell sorting (MACS). After 1 day of culture (90%–95% OPC), the cells were incubated with PFR or 4AP + BCC for 20 min or with Glut for 10 min. To test the involvement of NMDARs, in some experiments cells were pre-incubated for 15 min with the NMDAR inhibitor MK801 (Glut+MK801) and to confirm shedding by ADAM10 with its inhibitor GI (PFR + GI, Glut + GI). Cells were harvested and PN cell-lysates were obtained by differential centrifugation prior to subjecting to Western blot. Western blot quantification (bar graphs) revealed a reduction in the NG2 FL protein for 4AP + BCC, Glut, and Glut+MK801 stimulation, no change was observed if the cells were stimulated with PFR or if the cells were pre-incubated with the ADAM10 inhibitor GI (unpaired Student's t test). (B) Soluble (saline) and membrane fractions (triton) were obtained from forebrains of adult WT and NG2^−/− mice. Only a small amount of the NG2 290 kD ectodomain is soluble in the saline-fraction in the WT (see Figure S1) as reported before. (C) Western blot of ECM extracts of acute hippocampal slices from P70 rats by digestion of the glycosaminoglycan chains by chondroitinase ABC without detergent. An increase of the NG2 ectodomain was detected after chemical LTP (PFR) stimulation for 15 min (15′). The NG2 ectodomain levels within the ECM return to starting levels after 180 min of recovery time. NG2 ectodomain levels were normalized against total protein from a matching coomassie gel (Figure S5). (D) NG2 ectodomain levels increase 1.5-fold 15 min after stimulation with PFR and 40 min recovery time detected by Western blot from ECM extracts of acute hippocampal slices. Inhibition of metalloproteases (GM6001), or ADAM10 (GI), prevented the increase of ECM-associated NG2 ectodomain. Incubation with GI reduced levels of ECM-associated NG2 ectodomain to below the control value. Treatment of the slices with 4AP + BCC for 10 min with 20 min recovery time showed the same increase in levels of ECM-associated NG2 ectodomain as seen with the PFR treatment (one way ANNOVA with Dunnett's multiple comparison test).