Cdk5 inhibits anterograde axonal transport of neurofilaments but not that of tau by inhibition of mitogen-activated protein kinase activity.

Cyclin-dependent kinase 5 (cdk5) inhibits neurofilament (NF) anterograde axonal transport while p42/44 mitogen-activated protein kinase (MAPk) promotes it. Since cdk5 is known to inhibit MAP kinase activity, we examined whether or not cdk5 inhibits anterograde NF transport via inhibition of MAPk activity. To accomplish this, we manipulated the activity of these kinases in differentiated NB2a/d1 cells, and monitored anterograde axonal transport of green fluorescent protein-conjugated-NF-M (GFP-M) and cyan fluorescent protein-conjugated (CFP)-tau. The cdk5 inhibitor roscovitine increased anterograde axonal transport of GFP-M and CFP-tau; transfection with cdk5/p25 inhibited transport of both. Inhibition of MAPk activity by PD98059 or expression of dominant-negative MAPk inhibited anterograde GFP-M transport, while expression of constitutively active MAPk enhanced it; these treatments did not affect CFP-tau transport. PD98059 prevented roscovitine-mediated enhancement of GFP-M transport, but did not prevent enhancement of CFP-tau transport. Co-transfection with constitutively activated MAPk prevented the inhibition of GFP-M transport that normally accompanied transfection with cdk5/p25, but did not prevent inhibition of tau transport by cdk5/p25. Finally, the extent of inhibition of GFP-M axonal transport by PD98059 was not additive to that derived from transfection with cdk5/p35, and the increase in NF transport that accompanies roscovitine treatment was not additive to that derived from transfection with constitutively activated MAPk, suggesting that the influence of these kinases on NF transport was within the same, rather than distinct, pathways. These findings suggest that axonal transport of tau and NFs is under the control of distinct kinase cascades, and that cdk5 inhibits NF transport at least in part by inhibiting MAPk.

Mitogen-activated protein kinase regulates neurofilament axonal transport.

Mitogen-activated protein kinase (MAP) kinase plays a pivotal role in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. Here we examined whether p42/44 MAP kinase plays a role in axonal transport and the organization of neurofilaments (NFs) in axonal neurites. Dominant-negative p42/44 MAP kinase, anti-MAP kinase antisense oligonucleotides and the MAP kinase inhibitor PD98059 all reduced NF phospho-epitopes and inhibited anterograde NF axonal transport of GFP-tagged NF subunits in differentiated NB2a/d1 neuroblastoma cells. Expression of constitutively active MAP kinase and intracellular delivery of active enzyme increased NF phospho-epitopes and increased NF axonal transport. Longer treatment with PD98059 shifted NF transport from anterograde to retrograde. PD98059 did not inhibit overall axonal transport nor compromise overall axonal architecture or composition. The p38 MAP kinase inhibitor SB202190 did not inhibit NF transport whereas the kinase inhibitor olomoucine inhibited both NF and mitochondrial transport. Axonal transport of NFs containing NF-H whose C-terminal region was mutated to mimic extensive phosphorylation was substantially less affected by PD98059 compared to a wild-type construct. These data suggest that p42/44 MAP kinase regulates NF anterograde transport by NF C-terminal phosphorylation. MAP kinase may therefore stabilize developing axons by promoting the accumulation of NFs within growing axonal neurites.