Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Matsuda W, Furuta T, Nakamura KC, Hioki H, Fujiyama F, Arai R et al. Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum. J Neurosci 2009; 29: 444–453.
Lin AC, Holt CE . Function and regulation of local axonal translation. Curr Opin Neurobiol 2008; 18: 60–68.
Campbell DS, Holt CE . Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 2001; 32: 1013–1026.
Wang W, van Niekerk E, Willis DE, Twiss JL . RNA transport and localized protein synthesis in neurological disorders and neural repair. Dev Neurobiol 2007; 67: 1166–1182.
Kim SR, Chen X, Oo TF, Kareva T, Yarygina O, Wang C et al. Dopaminergic pathway reconstruction by Akt/Rheb-induced axon regeneration. Ann Neurol 2011; 70: 110–120.
Kim SR, Kareva T, Yarygina O, Kholodilov N, Burke RE . AAV transduction of dopamine neurons with constitutively active Rheb protects from neurodegeneration and mediates axon regrowth. Mol Ther 2012; 20: 275–286.
Kislauskis EH, Zhu X, Singer RH . Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. J Cell Biol 1994; 127: 441–451.
Zhang HL, Eom T, Oleynikov Y, Shenoy SM, Liebelt DA, Dictenberg JB et al. Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility. Neuron 2001; 31: 261–275.
Aronov S, Aranda G, Behar L, Ginzburg I . Axonal tau mRNA localization coincides with tau protein in living neuronal cells and depends on axonal targeting signal. J Neurosci 2001; 21: 6577–6587.
El-Husseini Ael D, Craven SE, Brock SC, Bredt DS . Polarized targeting of peripheral membrane proteins in neurons. J Biol Chem 2001; 276: 44984–44992.
Satpute-Krishnan P, DeGiorgis JA, Conley MP, Jang M, Bearer EL . A peptide zipcode sufficient for anterograde transport within amyloid precursor protein. Proc Natl Acad Sci USA 2006; 103: 16532–16537.
Babetto E, Beirowski B, Janeckova L, Brown R, Gilley J, Thomson D et al. Targeting NMNAT1 to axons and synapses transforms its neuroprotective potency in vivo. J Neurosci 2010; 30: 13291–13304.
Biermann B, Ivankova-Susankova K, Bradaia A, Abdel Aziz S, Besseyrias V, Kapfhammer JP et al. The Sushi domains of GABAB receptors function as axonal targeting signals. J Neurosci 2010; 30: 1385–1394.
Aranda-Abreu GE, Behar L, Chung S, Furneaux H, Ginzburg I . Embryonic lethal abnormal vision-like RNA-binding proteins regulate neurite outgrowth and tau expression in PC12 cells. J Neurosci 1999; 19: 6907–6917.
Farina KL, Huttelmaier S, Musunuru K, Darnell R, Singer RH . Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment. J Cell Biol 2003; 160: 77–87.
Ross AF, Oleynikov Y, Kislauskis EH, Taneja KL, Singer RH . Characterization of a beta-actin mRNA zipcode-binding protein. Mol Cell Biol 1997; 17: 2158–2165.
Schon EA, Przedborski S . Mitochondria: the next (neurode)generation. Neuron 2011; 70: 1033–1053.
Blichenberg A, Rehbein M, Muller R, Garner CC, Richter D, Kindler S . Identification of a cis-acting dendritic targeting element in the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. Eur J Neurosci 2001; 13: 1881–1888.
Sawamoto K, Nakao N, Kobayashi K, Matsushita N, Takahashi H, Kakishita K et al. Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons. Proc Natl Acad Sci USA 2001; 98: 6423–6428.
Acknowledgements
This work was supported by NIH NS38370, DOD W81XVVH-12-1-0051, the Parkinson’s Disease Foundation and the Parkinson Alliance (R.E.B).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on Gene Therapy website
Rights and permissions
About this article
Cite this article
Padmanabhan, S., Kareva, T., Kholodilov, N. et al. Quantitative morphological comparison of axon-targeting strategies for gene therapies directed to the nigro-striatal projection. Gene Ther 21, 115–122 (2014). https://doi.org/10.1038/gt.2013.74
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/gt.2013.74
Keywords
This article is cited by
-
In vivo measurement of afferent activity with axon-specific calcium imaging
Nature Neuroscience (2018)