Figure 4
From: Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration
Effects of CRMP2 overexpression on axonal degeneration after axotomy of cortical neurons in vitro.
(a) The microfluidic chamber system consists of 4 wells and two channels. The two channels are connected by microgrooves. Rat cortical neurons are plated in the channel named ‘soma compartment’. Only axons can grow across the microgrooves to enter the other channel called ‘axonal compartment’. (b) Quantification of newly formed bulbs in the axons in the given areas proximal to the lesion site at the indicated time points after axotomy after transfection of p.CMV-EGFP. Error bars represent the standard error of the mean (SEM). N ≥ 19 axons. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Dunnett’s test. (c) Representative Western blot with anti-flag antibody in cortical neurons after transfection with p.CMV-EGFP or p.CMV-CRMP2-flag confirming the overexpression of the CRMP2-flag transcript from the plasmid. (d) Immunostaining with anti-flag antibody confirming co-transfection of p.CMV-CRMP2-flag and p.CMV-EGFP in the cortical neurons. (e) Representative axons proximal to the lesion site at 480 min after axotomy and previous transfection with p.CMV-EGFP as control or co-transfection with p.CMV-CRMP2-flag and p.CMV-EGFP. The number of bulbs was decreased in the axon overexpressing CRMP2 compared to control. (f) Quantification of the number of newly formed axonal bulbs within 400 μm proximal to the lesion site at the indicated time points after axotomy and previous transfection with p.CMV-EGFP or both p.CMV-CRMP2-flag and p.CMV-EGFP. N ≥ 19 axons per group. Error bars represent the standard error of the mean (SEM). *P < 0.05, **P < 0.01 by independent samples t-test.
