Figure 9.

Diagram illustrating a putative mechanism by which intra-axonal disulfide reduction with PB1 and the Wld^S mutation promote axonal protection. A, In the intact axon, the distribution of phosphatidylserine (red circles) is actively restricted to the cytosolic (inner) leaflet of the plasma membrane. Scramblases (ovals) randomly shuffle phosphatidylserine between the inner and outer leaflet of membrane. Floppases (blue triangles) move the phosphatidylserine from the inner leaflet to the outer leaflet of the membrane, whereas flippases (beige triangles) actively remove phosphatidylserine from the outer leaflet and insert them in the inner leaflet. The net effect is to maintain phosphatidylserine asymmetry in the inner leaflet. B, After axotomy, the generation of intracytoplasmic ROS affects these transporters and the balance shifts, with floppases moving more phosphatidylserine to the outer leaflet while the rate of inward transport of phosphatidylserine by flippases slows. Scramblases possibly increase their rate of shuffling. The result is a net accumulation of phosphatidylserine in the outer leaflet of membrane. The exposure of phosphatidylserine to the outer leaflet and its interaction with annexin B12-IANBD (gray circles) switches “on” the fluorescence signal (green circles). C, Either the Wld^S mutation or intra-axonal disulfide reduction with membrane-permeable PB1 (which is inactive outside the axon) can inhibit the effects of ROS on flippases, floppases, and scramblases and are thereby able to maintain the asymmetry of phosphatidylserine across the membrane. D, Poorly permeable disulfide-reducing agents 1,4-dithiothreitol (DTT) and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) reduce extra-axonal disulfides and inhibit transporter activity, with the effect on flippases maintaining phosphatidylserine at the outer leaflet, thereby reversing the Wld^S phenotype.