Ectopic expression of the TrkA receptor in adult dopaminergic mesencephalic neurons promotes retrograde axonal NGF transport and NGF-dependent neuroprotection.

摘要

A recombinant adeno-associated virus (rAAV) was used to investigate the impact of an ectopic expression of the NGF high-affinity receptor in adult neurons. The rat TrkA cDNA cloned in a pCMX vector was first tagged with a human c-Myc sequence. The resulting vector was shown to encode a functional receptor which promoted the expression of TrkA immunoreactivity upon transfection of 293 fibroblasts or nnr5 cells, a TrkA-defective variant of PC12 cells. These cells also accumulate TrkA transcripts upon transfection and extended neurites in the presence of NGF. Therefore, the TrkA(myc) cassette was inserted into the pSSV9 plasmid. The new vectors shared properties similar to pCMX TrkA(myc) in 293 and nnr5 cells and enabled the preparation of rAAV TrkA(myc) viruses. Unilateral injection of this rAAV into the substantia nigra (SN) resulted in a protracted expression of TrkA (or c-Myc) immunoreactivity in numerous cell bodies, including tyrosine-hydroxylase (TH)-positive dopaminergic neurons. The presence of TrkA receptors in corresponding striatal dopaminergic endings was demonstrated by the advent of a striato-nigral retrograde axonal transport of (125)I-NGF. Likewise, ectopic expression of TrkA in neurons of the parafascicular thalamic nucleus promoted a striatofuge transport of NGF toward this structure. To investigate whether ectopic expression of TrkA in SN neurons may confer neuroprotection, lesions were induced by 6-hydroxydopamine in striata located ipsilateral to the virus injection site. NGF or vehicle were next delivered dorsally to the virus-treated SN for 2 weeks, before sacrifice and processing of brains for TH-immunohistochemistry. NGF treatment, in contrast to treatment with vehicle, significantly enhanced the number of dopaminergic neurons counted in the lesioned SN. These data suggest that ectopic TrkA can mediate the trophic actions of NGF and influence neuronal plasticity in vivo.