Abstract
Many cell surface signaling receptors, such as the neurotrophin receptor, TrkB, have emerged as potential therapeutic targets for diverse diseases. Reduced activation of TrkB in particular is thought to contribute to neurodegenerative diseases. Unfortunately, development of therapeutic reagents that selectively activate particular cell surface receptors such as TrkB has proven challenging. Like many cell surface signaling receptors, TrkB is internalized upon activation; in this proof-of-concept study, we exploited this fact to isolate a pool of nuclease-stabilized RNA aptamers enriched for TrkB agonists. One of the selected aptamers, C4-3, was characterized with recombinant protein-binding assays, cell-based signaling and functional assays, and, in vivo in a seizure model in mice. C4-3 binds the extracellular domain of TrkB with high affinity (KD ∼2 nM) and exhibits potent TrkB partial agonistic activity and neuroprotective effects in cultured cortical neurons. In mice, C4-3 activates TrkB upon infusion into the hippocampus; systemic administration of C4-3 potentiates kainic acid-induced seizure development. We conclude that C4-3 is a potentially useful therapeutic agent for neurodegenerative diseases in which reduced TrkB activation has been implicated. We anticipate that the cell-based aptamer selection approach used here will be broadly applicable to the identification of aptamer-based agonists for a variety of cell-surface signaling receptors.
Footnotes
This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant NS056217]; and by a postdoctoral fellowship from the American Heart Association (to F.J.H.).
Duke University (B.G. and Y.Z.H.) and the University of Iowa (J.O.M., F.J.H., and P.H.G.) have applied for a patent on this technology. M.A.B. is employed by Integrated DNA Technologies, Inc. (IDT), which offers oligonucleotides for sale that are similar to some of the compounds described in the article. IDT, however, is not a publicly traded company, and M.A.B. does not personally own any shares/equity in IDT.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- CNS
- central nervous system
- BDNF
- brain-derived neurotrophic factor
- ECD
- extracellular domain
- SELEX
- systematic evolution of ligands by exponential enrichment
- E18
- embryonic day 18
- HEK
- human embryonic kidney
- PCR
- polymerase chain reaction
- DPBS
- Dulbecco's phosphate-buffered saline
- SPR
- surface plasmon resonance
- BSA
- bovine serum albumin
- PAGE
- polyacrylamide gel electrophoresis
- p
- phospho
- FAM
- fluorescein amidite
- FITC
- fluorescein isothiocyanate
- LDH
- lactate dehydrogenase
- KA
- kainic acid
- AP
- anteroposterior
- ML
- mediolateral
- DV
- dorsoventral
- PBS
- phosphate-buffered saline
- EEG
- electroencephalogram
- shRNA
- short hairpin RNA
- Scr
- scrambled
- ANOVA
- analysis of variance
- MK-801
- 5H-dibenzo[a,d]cyclohepten-5,10-imine
- RTK
- receptor tyrosine kinase.
- Received February 9, 2012.
- Accepted June 29, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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