Quantitative morphological comparison of axon-targeting strategies for gene therapies directed to the nigro-striatal projection.

Cellular targeting of mRNAs and proteins to axons is essential for axon growth during development and is likely to be important for adult maintenance as well. Given the importance and potency of these axon-targeting motifs to the biology of axons, it seems possible that they can be used in the design of transgenes that are intended to enhance axon growth or maintenance, so as to improve potency and minimize off-target effects. To investigate this possibility, it is first essential to assess known motifs for their efficacy. We have therefore evaluated four axon-targeting motifs, using adeno-associated viral vector-mediated gene delivery in the nigro-striatal dopaminergic system, a projection that is predominantly affected in Parkinson's disease. We have tested two mRNA axonal zipcodes, the 3' untranslated region (UTR) of β-actin and 3' UTR of tau, and two axonal-targeting protein motifs, the palmitoylation signal sequence in GAP-43 and the last 15 amino acids in the amyloid precursor protein, to direct the expression of the fluorescent protein Tomato in axons. These sequences, fused to Tomato, were able to target its expression to dopaminergic axons. Based on quantification of Tomato-positive axons, and the density of striatal innervation, we conclude that the C-terminal of the amyloid precursor protein is the most effective axon-targeting motif.