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. 2018 Jul 20;7(7):1685-1693.
doi: 10.1021/acssynbio.8b00126. Epub 2018 Jul 12.

Optical Activation of TrkA Signaling

Liting Duan  1 , Jen M Hope  1 , Shunling Guo  1 , Qunxiang Ong  1 , Amaury François  2 , Luke Kaplan  1 , Grégory Scherrer  2   3 , Bianxiao Cui  1
Affiliations

Optical Activation of TrkA Signaling

Liting Duan et al. ACS Synth Biol. .

Abstract

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. As TrkA signaling is highly dynamic, mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Design scheme of the light-controlled TrkA systems. We have constructed four methods of optogenetic activation of TrkA signaling by fusion to CRY2. The molecular architecture of each construct is detailed. (A) The full length TrkA receptor consists of an extracellular domain (ECD), a transmembrane domain (TM) and an intracellular domain (iTrkA). (B) Opto-full-TrkA is a fusion of CRY2 to the C-terminus of full length TrkA. (C) Opto-Lyn-iTrkA appends CRY2 to the C-terminal end of the intracellular domain of TrkA (iTrkA), and features an N-terminal Lyn membrane-targeting sequence. (D) Opto-iTrkA fuses CRY2 to the N-terminus of iTrkA. (E) Opto-iTrkA + CAAX uses CIBN-CAAX, localized to the plasma membrane, in combination with opto-iTrkA to recruit iTrkA to the membrane in a light-dependent manner.
Figure 2.
Figure 2.
OptoTrkA systems activate PI3K/AKT and Raf/MEK/ERK signaling pathways. (A) Opto-Lyn-iTrkA, opto-iTrkA, and opto-iTrkA + CAAX exhibit activation of the PI3K/AKT signaling pathway, assayed by the membrane translocation of PHAKT-GFP in response to blue light stimulation. Opto-full-TrkA does not induce obvious PHAKT-GFP membrane translocation (200 ms, 9.7 W/cm2, blue light pulses delivered at 5 s intervals for 10 min). (B) Opto-Lyn-iTrkA, opto-iTrkA, and opto-iTrkA + CAAX systems induce ERK activation upon blue light stimulation, assayed by the nuclear translocation of ERK-GFP. Opto-full-TrkA does not induce obvious nuclear translocation of ERK-GFP (200 ms, 9.7 W/cm2, blue light pulses delivered at 10 s intervals for 20 min). (C) Average translocation of PHAKT-GFP and ERK-GFP upon blue light stimulation for the four optoTrkA systems (for PHAKT-GFP assay, n = 10, 11, 11, 10 cells and for ERK-GFP assay, n = 11, 10, 11, 10 cells, respectively). (D) Western blot analysis of optoTrkA systems for phosphorylated ERK (Thr202 and Tyr204) and phosphorylated AKT (Ser473). Opto-iTrkA and opto-iTrkA + CAAX exhibit AKT and ERK phosphorylation upon blue light stimulation (200 μW/cm2 continuous blue light illumination for 20 min). Scale bars, 5 μm.
Figure 3.
Figure 3.
Light-induced activation of TrkA promotes neurite growth in PC12 cells in the absence of NGF. PC12 cells were subjected to continuous blue light illumination at 200 μW/cm2 for 24 h. (A) PC12 cells expressing opto-full-TrkA and GFP displayed no obvious neurite growth. (B) Some PC12 cells expressing opto-Lyn-iTrkA and GFP extend short neurites. (C) Some PC12 cells expressing opto-iTrkA and GFP exhibit neurite growth. (D) Most PC12 cells expressing opto-iTrkA + CAAX grow long neurites. (E) PC12 expressing only CIBN-GFP-CAAX did not show noticeable neurite growth. (F) Quantification of percentage of cells with neurite growth for different systems. (G) Quantification of the length of the longest neurite in cells bearing neurites. (H) Quantification of the average number of neurite branches in cells bearing neurites. Results are averaged from three independent sets of experiments (detailed in Supplementary Table S1) and presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. Student’s t test was used between dark or light cells under the same transfection conditions (*P < 0.05, **P < 0.005). Scale bars, 10 μm.
Figure 4.
Figure 4.
Light-induced TrkA activation supports DRG neuron survival in the absence of NGF. After transfection, primary rat DRG neurons were allowed to recover in NGF-supplemented medium for 24 h. Then, NGF was withdrawn and replaced with anti-NGF for 48 h while the cultures were subjected to continuous blue light stimulation at 200 μW/cm2 or kept in dark for 2 days. (A) DRG neurons transfected with opto-full-TrkA and GFP did not survive under light or dark. (B) Some DRG neurons expressing opto-Lyn-iTrkA and GFP survived upon blue light stimulation, but few survived in the dark control. (C) Many DRG cells expressing opto-iTrkA survived and had extended long axons upon blue light stimulation, but not in the dark controls. (D) Most DRG neurons expressing opto-iTrkA and CIB1-GFP-CAAX were alive with long axons with blue light, but not in the dark controls. (E) Quantification of survival rate of DRG neurons for optoTrkA systems, in light and dark conditions. Results are averaged from three independent sets of experiments (see numbers of cells in each set of experiment in Table S2) and presented as means ± SEM. One-way ANOVA with Dunnett’s post hoc test was performed between cells with different transfection. t test was performed between cells in dark or in light with the same transfection (*P < 0.05, **P < 0.005). Scale bars, 50 μm.
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