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PirB is a Functional Receptor for Myelin Inhibitors of Axonal Regeneration

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Science  07 Nov 2008:
Vol. 322, Issue 5903, pp. 967-970
DOI: 10.1126/science.1161151

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  1. Fig. 1.

    LILRB2 and PirB can bind Nogo66 and MAG. (A) Schematic diagram of LILRB, PirB, and NgR receptors. ITIM, immunoreceptor tyrosine-based inhibitory motif; LRR, leucine-rich repeat. (B) COS cells were transfected with empty vector (control) or PirB or LILRB2 cDNA. After 48 hours, cells were incubated with AP-Nogo66 or MAG-Fc, and bound ligand was detected. NgR and NgR2 were used as positive controls for AP-Nogo66 and MAG-Fc binding, respectively. Scale bars, 200 μm. (C) The affinity of the MAG-Fc-PirB interaction is shown by one representative enzyme-linked immunosorbent assay binding curve. The experiment was repeated twice with similar results.

  2. Fig. 2.

    Blocking PirB reverses inhibition of CGN outgrowth on AP-Nogo66 or myelin. Dissociated mouse P7 CGNs were plated on PDL/laminin (control), AP-Nogo66, or myelin to test inhibition by these substrates after various manipulations. In each panel, representative photomicrographs are shown on the left, and a graph measuring average neurite length (±SE, error bars) from one representative experiment is shown on the right. (A) Neurons were grown on PDL/laminin or AP-Nogo66, either alone or mixed with a fivefold excess of PirB extracellular domain (PirB-His). Neuronal inhibition by AP-Nogo66 was largely rescued by the presence of PirB-His. (B) Neurons grown on PDL/laminin, AP-Nogo66, or myelin were cultured in the presence or absence of anti-PirB.1 (50 μg/ml), which significantly reduced inhibition by either substrate. (C) Neurons cultured from either WT control mice or PirBTM mice were grown on PDL/laminin, AP-Nogo66, or myelin. PirBTM neurons were significantly less inhibited on either substrate (Student's t test,*P <0.01; n = 6 wells per condition). Scale bars, 50 μm.

  3. Fig. 3.

    PirB and NgR together contribute to myelin inhibition. Neurons were cultured on PDL/laminin, AP-Nogo66, or myelin substrates, and the PirB and NgR receptors were functionally blocked, either independently or in combination, to assess the contribution of these two pathways to inhibition. Representative photomicrographs are shown (top), and a graph measuring average neurite length (±SE, error bars) from one representative experiment is shown (bottom). NgR functional blockade was achieved by culturing neurons from NgR-null mice. PirB blockade was achieved by culturing neurons in the presence of anti-PirB.1 (50 μg/ml) (Student's t test,*P <0.01; n = 6 per condition). Scale bars, 50 μm.

  4. Fig. 4.

    Both PirB and NgR are required to mediate growth-cone collapse by myelin inhibitors. Growth cones of postnatal DRG axons were treated with medium alone (control), myelin (3 μg/ml), or AP-Nogo66 (100 nM) for 30 min to stimulate collapse and were stained with rhodamine-phalloidin to visualize growth cones. Representative photomicrographs are shown on the left, and a graph measuring percent of growth-cone collapse (±SE, error bars) from cumulative experiments is shown on the right (n ≥ 4 per condition). Scale bars, 50 μm.

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