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. 2008 Jun 25;582(15):2303-2308.
doi: 10.1016/j.febslet.2008.05.034. Epub 2008 Jun 2.

Deregulation of PKN1 activity disrupts neurofilament organisation and axonal transport

Catherine Manser #  1 , Alison Stevenson #  1 , Steven Banner  1 , Jennifer Davies  1 , Elizabeth L Tudor  1 , Yoshitaka Ono  2 , P Nigel Leigh  1 , Declan M McLoughlin  1 , Christopher E Shaw  1 , Christopher C J Miller  1
Affiliations

Deregulation of PKN1 activity disrupts neurofilament organisation and axonal transport

Catherine Manser et al. FEBS Lett. .

Abstract

Neurofilaments are synthesised in neuronal cell bodies and then transported through axons. Damage to neurofilament transport is seen in amyotrophic lateral sclerosis (ALS). Here, we show that PKN1, a neurofilament head-rod domain kinase is cleaved and activated in SOD1G93A transgenic mice that are a model of ALS. Moreover, we demonstrate that glutamate, a proposed toxic mechanism in ALS leads to caspase cleavage and disruption of PKN1 in neurons. Finally, we demonstrate that a cleaved form of PKN1 but not wild-type PKN1 disrupts neurofilament organisation and axonal transport. Thus, deregulation of PKN1 may contribute to the pathogenic process in ALS.

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Figures

Fig. 1
Fig. 1
Transfection of PKN1 elevates cellular PKN1 activity and PKN1561–942 displays greater activity than PKN1wt. (A) shows in vitro PKN1 assays of cells transfected with PKN1wt or CAT as a control. The higher level of activity seen with the PKN1 compared to the FLAG antibody may be because the PKN1 antibody pulls down endogenous + transfected whereas the FLAG antibody pulls down only transfected kinase. Also, the different antibodies may differentially immunoprecipitate active kinase. (B) shows assays from cells transfected with PKN1wt or PKN1561–942; cells transfected with CAT are again shown as a control. PKN1 was immunoprecipitated using either PKN antibody to isolate total (transfected plus endogenous PKN1) or an antibody to the FLAG tag on transfected PKN1 as indicated; − indicates omission of immunoprecipitating antibody as a control. Both the Coomassie stained gels showing substrate and corresponding autoradiographs are displayed.
Fig. 2
Fig. 2
Expression of PKN1561–942 but not PKN1wt disrupts neurofilament organisation in rat cortical neurons. (A) shows normal distribution of neurofilaments in a non-transfected neuron. (B–D) show neurofilaments in neurons co-transfected with PKN1wt (B) or with PKN1561–942 (C, D). In (C) note the disruption to neurofilaments in the PKN1561–942 transfected neuron (arrowhead) but not in its non-transfected near neighbour. Neurofilaments were detected using antibody NA1216 and PKN1wt/PKN1561–942 with FLAG antibody. Scale bars = 20 μm.
Fig. 3
Fig. 3
PKN1561–942 but not PKN1wt disrupts axonal transport of neurofilaments. (A) shows distances moved by GFP-NFM through axons of rat cortical neurons co-transfected with CAT (control), PKN1wt or PKN1561–942 at different time-points post-transfection as indicated. No significant differences were detected in GFP-NFM transport between CAT and PKN1wt co-transfected neurons at any time point. GFP-NFM in PKN1561–942 co-transfected neurons moved significantly less than both CAT and PKN1wt co-transfected neurons (P ≤ 0.05 at 220 min; P ≤ 0.001 at 240 and 260 min; n = 35–60, Mann–Whitney). (B) shows representative images of GFP-NFM labelling in neurons co-transfected with PKN1 or PKN1561–942 at 260 min post-transfection; cells were stained with tubulin to show presence of axon. Scale bar = 50 μm.
Fig. 4
Fig. 4
Glutamate induces caspase-mediated cleavage and activation of PKN1 in cultured cortical neurons. (A) shows immunoblot of PKN1 in untreated neurons (Un) or neurons treated with 100 μm glutamate for 15–960 min as indicated. Also shown is cleavage of PARP, a known caspase substrate, but not NFL, which is not a substrate, following glutamate treatment. (B) shows inhibition of glutamate-induced PKN1 cleavage (480 min 100 μm glutamate treatment) by pre-treatment with caspase inhibitors. Z-VAD-FMK and Z-DEVD-FMK were used at 100 μm and applied 2 h prior to glutamate stimulation. (C) In vitro kinase assays for PKN1 activity in untreated neurons and neurons treated with 100 μm glutamate for 480 min. Both the Coomassie stained gel showing substrate and corresponding autoradiograph are shown along with reaction mix controls with no substrate (RM). + and − refer to presence or absence of the PKN1 antibody in the immunoprecipitations to isolate PKN1. Glutamate induces a 2.07-fold increase in activity (t-test; n = 7).
Fig. 5
Fig. 5
PKN1 is cleaved and activated in SOD1G93A transgenic mice. (A) Immunoblots to show PKN1 in spinal cord (upper) and brain (lower) from SOD1G93A and non-transgenic littermate (NT) mice at ages 40, 80 and 120 days. Full-length PKN1 (120 kDa) and an approx. 65 kDa species are seen in all samples. An additional approx. 55 kDa migrating species (arrowhead) is seen in spinal cord but not brains of SOD1G93A mice at 80 and 120 days; this species is not detected in non-transgenic littermates. The samples were also probed with an antibody to SOD1 to confirm the genotype (bottom panel). Two mice at each age are shown but an additional two mice were analysed with highly similar results. (B) shows in vitro kinase assays for PKN1 activity in spinal cords of SOD1G93A and non-transgenic littermate (NT) mice. Both the Coomassie stained gel showing substrate and corresponding autoradiograph are shown along with reaction mix controls with no substrate (RM). + and − refer to presence or absence of the PKN1 antibody in the immunoprecipitations to isolate PKN1. PKN1 activity is elevated approximately 2.25-fold in SOD1G93A mice (n = 8, t-test).
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