Axonal regeneration in the mature CNS differs from other forms of axon elongation. In tissue culture, peripheral nerve and developing CNS, growth cones migrate rapidly via a pulling action by filopodia and lamellipodia, which contain microfilaments but no neurofilaments (NFs). By contrast, growth cones in regenerating CNS migrate slowly, have no filopodia or lamellipodia, and are packed with NFs. It is therefore possible that NF transport into the regenerating CNS growth cone helps to push the axon tip forward. Spinal cord transection in the lemprey is an excellent model for this type of regeneration. Preliminary data show that, following an initial down-regulation of NF, neurons that regenerate well exhibit an increase in NF expression, while those that regenerate poorly exhibit permanent down regulation. It is now proposed to use molecular manipulations of NF expression in microinjected reticulospinal neurons of the lamprey, in order to determine the role of NFs in functional regeneration of CNS axons.
AIM 1 will be to determine whether the increase in NF expression that distinguishes regenerating from non-regenerating neurons is potentially part of the mechanism of regeneration or merely a consequence of it. Semiquantitative in situ hybridization and immunohistochemistry will be used both in animals permitted to regenerate and in those mechanically prevented from regenerating. If the secondary increase in NF occurs even when regeneration is blocked, then NF upregulation cannot be a consequence of regeneration and may be part of an intrinsic regeneration program.
AIM 2 will be to determine whether blocking NF production blocks regeneration. Fabs to lamprey NF-180) will be microinjected (together with a long-term fluorescent tracer) into the giant reticulospinal neurons (GRNs) in order to block NF assembly. After 3-6 weeks of recovery, the probability and distance of regeneration will be compared in cells previously injected with anti-NF and those injected with a non-specific Fab. Antisense oligonucleotide probes will be constructed from the NF-180 cDNA sequence and microinjected into axotomized GRNs. The effect on regeneration will be compared with that seen in cells injected with non-specific oligonucleotides.
AIM 3 will be to determine whether inhibiting NF phosphorylation can enhance axonal regeneration. Since NFs in the growth cones are highly phosphorylated, and since phosphorylation is thought to slow transport of NFs, it may be possible to enhance regeneration by the use of drugs that block NF phosphorylation. An inhibitor of serine/threonine kinase, K-252a, will be microinjected into GRNs and at the effect on regeneration will be compared with that of the serine/threonine phosphatase inhibitor, okadaic acid. In order to test the specificity of these results, NF phosphorylation will also be blocked by microinjection of Fabs specific for the unphosphorylated form of the NF phosphorylation will also be blocked by microinjection of Fabs specific for the unphosphorylated form of the NF-180 sidearm, and the effect on axonal regeneration determined. The elucidation of mechanisms of regeneration in CNS axons may lead to improved therapeutic approaches to inducing regeneration and functional recovery in human patients following spinal cord injury, head trauma or stroke.
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