Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

<div><p>Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.</p></div

Addition of Lidocaine Injection Immediately before Physiotherapy for Frozen Shoulder: A Randomized Controlled Trial

<div><p>The intraarticular injection of lidocaine immediately before a physiotherapy session may relieve pain during the stretching and mobilization of the affected joint in patients with a frozen shoulder, thus enhancing the treatment effect. To compare the effects of intraarticular injection of lidocaine plus physiotherapy to that of physiotherapy alone in the treatment of a frozen shoulder, a prospective randomized controlled trial was conducted in the rehabilitation department of a private teaching hospital. Patients with a frozen shoulder were randomized into the physiotherapy group or the lidocaine injection plus physiotherapy (INJPT) group. The subjects in the INJPT group underwent injection of 3 ml of 1% lidocaine into the affected shoulder 10 to 20 minutes before each physiotherapy session. In each group, the treatment lasted 3 months. The primary outcome measures were the active and passive range of motion of the affected shoulder. The secondary outcome measures were the results of the Shoulder Disability Questionnaire, the Shoulder Pain and Disability Index, and the 36-item Short-Form Health Survey (SF-36). The outcome measures were evaluated before treatment and 1, 2, 3, 4, and 6 months after the start of treatment. The group comparisons showed significantly greater improvement in the INJPT group, mainly in active and passive shoulder range of motion in flexion and external rotation and improvements in pain and disability (<i>P</i> < 0.05); however, no significant group difference was seen in the SF-36 results. The intraarticular injection of lidocaine immediately before a physiotherapy session might be superior to physiotherapy alone in the treatment of a frozen shoulder.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/results?term=LIN+FEN+HSIEH&Search=Search" target="_blank">NCT01817348</a></p></div

Dose-response studies of identified partial agonists of FGF14:Nav1.6 regulatory pathways.

<p>Fitting, pIC50/EC50 calculation, axes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117246#pone.0117246.g003" target="_blank">Fig. 3</a>. Partial agonists were defined as compounds that act as agonists (promote FGF14:Nav1.6 complementation with increasing dose).</p

Demographic and clinical characteristics of the subjects.

<p>Demographic and clinical characteristics of the subjects.</p

The consolidated standards for reporting trials: a flow diagram of the study.

<p>Abbreviations: PT, physiotherapy group; INJPT, lidocaine injection plus physiotherapy group.</p

Dose-response studies of identified partial inverse agonists of FGF14:Nav1.6 regulatory pathways.

<p>Fitting, pIC50/EC50 calculation, axes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117246#pone.0117246.g003" target="_blank">Fig. 3</a>. Partial inverse agonists were defined as compounds that act as inverse agonists (inhibit FGF14:Nav1.6 complementation with increasing dose) and have an efficacy value of less than 1.</p

Using LCA to measure real-time interaction between FGF14 and Nav1.6 C-tail in live cells.

<p>(<b>A</b>) Using the bioluminescence-based luciferase complementation assay (LCA) to measure protein:protein interactions. Two proteins of interest (FGF14, and CD4-Nav1.6-Ctail in this example) are fused to Cluc and Nluc fragments of <i>Photinus</i> luciferase. Upon interaction of the protein components, Nluc and Cluc fragments reconstitute into functional luciferase enzyme, which produces luminescence in the presence of luciferin substrate. The intensity of luminescence is linear to the strength of the protein:protein interaction identified. <b>(B)</b> Schematic of constructs used for LCA experiments, to scale. <b>Top</b>: Cluc (AAs 398–550), linker (GGGSSGGGQISYASRG), FGF14-b (AAs 1–252). <b>Bottom:</b> CD4-ΔCtail (AAs 1–395), Nav1.6-Ctail (AAs 1763–1976), linker (QISYASRGGGSSGGG), Nluc (AAs 2–416). <b>(C)</b> Schematic of protein inhibition by kinase inhibitors. ATP-competitive kinase inhibitors block the ATP-binding site of the target kinase, preventing the transfer of phosphate groups to the substrate. Non-ATP competitive kinase inhibitors work through other mechanisms, such as changing the conformation of the ATP-binding site to prevent docking of ATP.</p

Bioinformatics analysis of a GSK-3 centered kinase regulatory network.

<p>For protein interaction networks, Ingenuity Pathway Analysis (IPA) was applied to the list of identified major kinase targets for all of the inhibitors tested, in addition to GSK-3, and an unbiased network was generated using the “Connect” algorithm and subsequently submitted to the pathway analysis engine. Both direct (solid) and indirect (dashed) interactions, as classified by Ingenuity, are shown; the sub-network with edge length of 1 to GSK-3 is additionally highlighted (purple). <b>A)</b> Interaction network, showing direct interactions only. <b>B)</b> Interaction network, showing both direct (solid) and indirect (dashed) interactions. <b>C)</b> Western blot analysis of co-immunoprecipitation (<i>IP</i>:<i>myc</i>) and cell lysate from HEK293-Nav1.6 cells transfected with FGF14–6×myc. GSK3 inhibitor XIII (25 μM) treatment reduces the co-immunoprecipitated fraction of Nav1.6 without affecting FGF14–6×myc. <b>D)</b> and <b>E)</b> representative traces showing effect of 12 hour treatment with ether DMSO 0.25% (<b>D</b>) or CHIR99021 5μM (<b>E</b>) on neuronal excitability in cultured hippocampal neurons DIV 12–15. Single action potentials were evoked by brief (2.5 ms) depolarizing current injections. Grey squares indicate action potential threshold. <b>F)</b> CHIR99021 increases action potential threshold in cultured hippocampal neurons. Results represent mean ± SEM. n = 7 (DMSO), n = 6 (CHIR990221). *p<0.05, Student <i>t</i>-test. <b>G)</b> and <b>H)</b> CHIR99021 has no effect on input resistance (<b>G</b>) and action potential half width (<b>H</b>) threshold in cultured hippocampal neurons. Results represent mean ± SEM. n = 7 (DMSO), n = 6 (CHIR990221), NS—non significant, p>0.05, Student <i>t</i>-test.</p

Comparisons of the active and passive ROMs between the groups.

<p>Shown as bar charts for (a) flexion, (b) abduction, (c) external rotation, and (d) internal rotation for active ROM and for (e) flexion, (f) abduction, (g) external rotation, and (h) internal rotation for passive ROM with the corresponding standard deviations represented as error bars. An asterisk indicates significant differences between groups (<i>P</i><0.008). For the evaluation times (evaluation times: before and 1, 2, 3, 4, and 6 months after the start of treatment), a right arrow above the graph indicates a significant, linearly increasing trend, whereas a left arrow indicates a significant, linearly decreasing trend (<i>P</i><0.025). (Black bar: the PT group; gray bar: the INJPT group). Group differences were analyzed using Mann-Whitney <i>U</i> test. Treatment time effects were analyzed using Friedman's test for two groups respectively. Abbreviations: PT, physical therapy; INJPT, injection plus physical therapy.</p

Hierarchical clustering of inhibitors for FGF14:Nav1.6 regulatory pathways.

<p><b>A)</b> Heatmap and hierarchical clustering for individual inhibitors. Red, increased intensity relative to DMSO control. Green, decreased intensity. <b>Left</b>, Hierarchical clustering, based on differences in normalized interaction strength between Nav1.6 and FGF14 for each inhibitor, with equal weighing of all categories. <b>B)</b> Heatmap and hierarchical clustering for primary kinase targets of each inhibitor, derived from geometric averaging of all inhibitors of each primary kinase.</p