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. 2011 Nov 2;60(2):203–217. doi: 10.1002/glia.21255

Fig. 1.

Fig. 1

Proteases serve as positive and negative regulators of myelination. (A) Pharmacological BACE1 inhibition blocks myelination in DRG dissociated explant cultures, whereas inhibition of ADAMs promotes myelination. Fluorescent imunostaining for MBP (green). Numbers of MBP-positive segments (mean ± SEM) after 14 days in vitro (DMSO, ADAMs inh n = 3 cultures; BACE1 inh n = 2 cultures; 2 coverslips/treatment; *P < 0.05, **P < 0.01). Scale bar, 200 μm. (B) Axonal association of Schwann cells is not impaired upon BACE1 or ADAMs inhibition in DRG neuron dissociated explant cultures. Immunostaining for axons (Tuj1; red) and counterstaining for nuclei (DAPI; blue) after 9 d of protease inhibitor treatment. Representative axon bundles used for quantification are delineated. Numbers of axon-associated Schwann cells (per bundle length in μm) are displayed as percentage ± SEM relative to DMSO treated controls (n = 2 cultures; 3 coverslips/condition; 3 bundles/coverslip; P = 0.38, **P < 0.01). Scale bar, 20 μm. (C) Cleavage of full-length (HA-NRG1FL), but not “processed” NRG1 type III (HA-NRG1GIEF) by BACE1. Western blot of transfected HEK293T cell lysates probed with an anti-HA antibody. Expression of BACE1 (+B) strongly reduced the level of full-length HA-NRG1FL (black arrowheads) and resulted in the accumulation of a ∼75 kD N-terminal processing product (white arrowhead), which corresponds in size to HA-NRG1GIEF. HA-NRG1FL processing by BACE1 was blocked by BACE1 inhibitor treatment (+B/Bi). Expression of BACE1 had no visible impact on HA-NRG1GIEF levels. Note that “base line” processing of HA-NRG1FL (+Bi) was not affected by BACE1 treatment. (D) BACE1 cleaves HA-NRG1FL in the juxtamembrane “stalk” region. Western blot of transfected HEK293T cell lysates probed with an antibody against the C-terminal peptide sequence “GIEF”. Note that the C-terminal in ‘GIEF’ epitope only accumulates in HA-NRG1FL lysates when BACE1 was coexpressed (+B). HA-NRG1GIEF lysates served as a positive control. The membrane was reprobed for tubulin as a loading control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]