Peptidyl-prolyl isomerase FKBP52 controls chemotropic guidance of neuronal growth cones via regulation of TRPC1 channel opening

Abstract

Immunophilins, including FK506-binding proteins (FKBPs), are protein chaperones with peptidyl-prolyl isomerase (PPIase) activity. Initially identified as pharmacological receptors for immunosuppressants to regulate immune responses via isomerase-independent mechanisms, FKBPs are most highly expressed in the nervous system, where their physiological function as isomerases remains unknown. We demonstrate that FKBP12 and FKBP52 catalyze cis/trans isomerization of regions of TRPC1 implicated in controlling channel opening. FKBP52 mediates stimulus-dependent TRPC1 gating through isomerization, which is required for chemotropic turning of neuronal growth cones to netrin-1 and myelin-associated glycoprotein and for netrin-1/DCC-dependent midline axon guidance of commissural interneurons in the developing spinal cord. By contrast, FKBP12 mediates spontaneous opening of TRPC1 through isomerization and is not required for growth cone responses to netrin-1. Our study demonstrates a novel physiological function of proline isomerases in chemotropic nerve guidance through TRPC1 gating and may have significant implication in clinical applications of immunophilin-related therapeutic drugs.

Figure 1. Binding and isomerization of TRPC1 by FKBP52 and FKBP12

(A) A schematic diagram of human TRPC1 indicating regions critical for FKBP52 (¹⁹LPSSP²³ and ⁶⁴⁴LPPPF⁶⁴⁸) and C-terminal Homer binding (⁶⁴⁵PPPF⁶⁴⁸). (B) FKBP52 and Homer require similar, but not identical sequences of TRPC1 C-terminus for binding. Lysates of HEK293 cells expressing HA-TRPC1 constructs (C1) were assayed for co-IP with native FKBP52 or Homer 3 (H3). (C) FKBP52 binds TRPC1 N-terminus (NT) and requires canonical Leu-Pro residues for binding. Lysates of HEK293 cells expressing HA-TRPC1 NT constructs were assayed for binding to GST-FKBP52. (D) Catalysis of L19-P20 and L644-P645 peptidyl-prolyl bonds in TRPC1 mimic peptides by substoichiometric amounts of FKBP12 and FKBP52. Expansion of ¹H-¹⁵N heteronuclear exchange spectra at 25°C showing amide signals of cis and trans isomers of L19 and L644 residues within TRPC1(14–30) and TRPC1(535–656) peptides, respectively. Conformational exchange between cis and trans isomers was slow on the NMR timescale (k_ex < 0.1s⁻¹) in absence of FKBPs resulting in distinct resonance signals for the two conformations (left). Upon addition of catalytic amounts of either FKBP12 or FKBP52 [30:1 TRPC1(635–656)/FKBP and 133:1 TRPC1(14–30)/FKBP] exchange peaks were observed connecting the cis and trans isomer resonances, denoted by dashed lines, indicating efficient acceleration of the isomerization rate by the enzymes.

Figure 2. Differential roles of FKBP12 and FKBP52 in TRPC1 gating

(A–C) Sample traces of electrophysiology recordings of TRPC1 currents. HEK293 cells transfected with TRPC1 and empty vector (A), TRPC1 and FKBP12-WT (○) or FKBP12-D37L)(□)(B), TRPC1 and FKBP52-WT(△), or FKBP52-FD67DV(■)(C) were bathed in media containing 140 mM Na⁺, then stimulated with 100 μM carbachol and finally incubated in Na⁺-free media to determine the zero current. (D) Summary of the spontaneous, receptor-activated and total current. Values represent mean ± SEM. (*: p < 0.01, Student t-test). (E) Agonist-stimulated currents of select TRPC1 mutants were restored by co-expression of FKBP52. HEK293 cells were transfected with TRPC1-WT and the indicated TRPC1 mutants alone or together with FKBP52. Values represent mean ± SEM (n = 4–5) of the total, spontaneous and agonist-stimulated current (*: p < 0.01, Student t-test).

Figure 3. PPIase-dependent regulation of netrin-1-induced Ca²⁺ influx by FKBP52

(A–C) Bright-field and fluorescence images of growth cones of Xenopus spinal neurons loaded with Fluo-4 at different time points after stimulation with netrin-1 (10 ng/ml). Sample images show neurons from an un-injected embryo without (control, A) or with GPI-1046 (500 nM, B) in the bath, or from an FKBP52-FD67DV expressing neuron (C). Pseudocolor indicates Ca²⁺ levels with red as the highest and blue as the lowest. Scale bar: 10 μm. (D) Time course of netrin-1-induced Ca²⁺ rise in growth cones under different conditions. The fluorescence intensity of each time point was normalized to the average fluorescence intensity of 5 mins baseline period before netrin-1 application for each neuron. Values represent mean ± SEM. (n = 8–11; *: p < 0.01, Bootstrap test). (E) Summary of netrin-1-induced Ca²⁺ changes in growth cones. The fluorescence intensity was normalized to the average fluorescence intensity of 5 mins baseline levels prior to the netrin-1 application (5 ng/ml). Values represent mean ± SEM. (*: p < 0.01, Bootstrap test).

Figure 4. PPIase activity-dependent regulation of netrin-1 induced attractive growth cone turning responses of Xenopus spinal neurons by FKBP52

(A–D) Growth cone turning responses in a gradient of netrin-1 by neurons in the control medium (A), in the presence of bath application of FK506 (10 nM, B) or GPI-1046 (10 nM, C), and neurons derived from embryos injected with the mRNA encoding FKBP52-FD67DV (D), respectively. The left two columns of images show growth cones at the start (0′) and the end of exposure (30′) in a netrin-1 gradient (10 μg/ml in the pipette). Scale bar: 20 μm. The right column shows superimposed trajectories of neurite extension during the 30′ period for a sample population of 12 neurons under each condition. The origin is the center of the growth cone and the original direction of growth is vertical. Arrows indicate the direction of the gradient. Scale bar: 10 μm. (E) The PPIase activity is required for netrin-1-induced growth cone attraction. Shown is the summary of turning angles under different conditions. Values represent mean ± SEM. (*: p < 0.01, Bootstrap test). (F) FKBP52 regulates netrin-1-induced growth cone turning through its association with TRPC1. Values represent mean ± SEM. (*: p < 0.01, Bootstrap test). (G–H) Netrin-1-induced PPIase activation is sufficient for attractive growth cone turning. Shown in (G) is a schematic illustration of experimental paradigms and models. Netrin-1 (orange); PPIase activity (gray); GPI-1046 (blue). Similar as in (A–D), shown in (H) are growth cone turning responses in a gradient of GPI-1046 (100 μM in the petite) in the normal medium, or with netrin-1 (10 ng/ml) in the bath.

Figure 5. Requirement of PPIase activity of FKBP52 for midline axon guidance of commissural interneurons in the developing Xenopus spinal cord

(A) A schematic diagram of commissural interneuron projections in the developing embryonic Xenopus spinal cord. (B–G) Sample images of the sagittal view of commissural interneurons and their axonal projections in the Xenopus spinal cord from stage 25 embryos. Shown are projections of Z-stack confocal images of 3A10 immunostaining of commissural interneuron axons from un-injected embryos (control, B), with treatment of GPI-1046 (0.5 μM, D) or FK506 (0.5 μM, E), or embryos injected with XDCC-MO (C), FKBP52-WT mRNA (F), FKBP52-FD67DV mRNA (G), respectively. Arrows point to axons with defect in projection to the ventral middle line. Also note axons with premature fasciculation (highlighted with “*”). Scale bar: 40 μm. (H) Quantification of the percentage of 3A10⁺ commissural interneurons with normal midline crossing under different experimental conditions. Values represent mean ± SEM. (n = 10–20 embryos; *: p < 0.01, Bootstrap test).

Figure 6. Requirement of association between TRPC1 and FKBP52, but not FKBP12, for midline axon guidance of commissural interneurons in the developing Xenopus spinal cord

(A–F) Sample images of the sagittal view of commissural interneurons and their axonal projections in the Xenopus spinal cord from stage 25 embryos. Shown are projections of Z-stack confocal images of 3A10 immunostaining of commissural interneuron axons from embryos injected with mRNAs encoding TRPC1-WT (A), TRPC1-P645L (B), TRPC1-P645A (C), TRPC1-P645A and FKBP12-WT (D), TRPC1-P645A and FKBP52-WT (E), and FKBP12-WT (F), respectively. Arrows point to axons with defect in projection to the ventral middle line. Also note axons with premature fasciculation (highlighted with “*”). Scale bar: 40 μm. (F) Quantification of the percentage of 3A10⁺ commissural interneurons with normal midline crossing under different experimental conditions. Values represent mean ± SEM. (n = 10–20 embryos; *: p < 0.01, Bootstrap test).

Figure 7. Requirement of PPIase activity of FKBP52 for MAG-, but not Sema3A-induced growth cone repulsion

(A) Axonal growth cone responses in a gradient of MAG (150 μg/ml in the pipette) of primary hippocampal neurons expressing GFP alone, or co-expressing GFP and FKBP52-FD67DV, TRPC1-P645A, TRPC1-P645A and FKBP52-WT, respectively. Images show growth cones at the start (0′; middle panel) and the end of exposure (30′; right panel) in a MAG gradient (150 μg/ml in the pipette). Also shown are images of the whole neuron at the end of the assay (left panel). (B) Summary of growth cone turning angles of mammalian hippocampal neurons in response to a MAG gradient under different conditions. Values represent mean ± SEM (n = 13 to 32. *: p < 0.01, Bootstrap test). (C) Lack of effects of PPIase inhibitors on Sema 3A-induced growth cone turning. Shown is a summary of growth cone turning angles of Xenopus spinal neurons in a gradient of Sema3A (50 μg/ml in the pipette) in the absence or presence of GPI-1046 (10 nM) or FK506 (10 nM) in the bath. Values represent mean ± SEM (n = 12 to 14). Note that no significant differences were found (p < 0.05, Bootstrap test).