Figure 5.

RNA processing in paracrine is stress inducible and restricted to a protein secreted by neurons acting specifically on astroglia. A, RNA gels showing cleavage of astrocyte RNA collected under serum withdrawal (−Ser, left) or in full medium (FM; right) after treatment with NSC34-conditioned medium (NCM; for 3 h). B, NSC34 RNA collected under serum withdrawal treated (3 h) with NSC34 (left)- or astrocyte (right)-conditioned medium (NCM/ACM; 24 h). C, Intracellular RNA cleavage in NSC34 cells overexpressing wt hAng, Ang K40I, or GFP. D, Human MZ-294 astrocytoma RNA collected under serum withdrawal treated (3 h) with conditioned medium (cMed) collected from NSC-34 cells overexpressing wt hAng, Ang K40I, or GFP (24 h). E, Comparison of endogenous (NSC-34) and paracrine (MZ-294) RNA cleavage fragments and demonstration of lack of dominant-negative effect of Ang K40I on Ang-induced RNA cleavage. NSC-34 cells were expressed with equal amounts of wt hAng, Ang K40I, or GFP as indicated. Experiments were performed as described in C and D. E′, Represents the same gel overexposed to highlight the endogenous cleavage products in NSC34 cells. F, NSC34 RNA collected under serum withdrawal treated (3 h) with conditioned medium collected from primary astrocytes overexpressing wt hAng, Ang K40I, or GFP (24 h). All experiments were performed in duplicate with similar results. G, RNA cleavage in primary mixed motoneuron cultures under normal culture conditions (Ctrl) or treated with heparin (1 μg/ml; 1 or 8 h; left; performed in triplicate with similar results) and treated with heparin (Hep) or an Ang-inactivating antibody (αAng; 2 μg/ml; 24 h; right).