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. 2010 Mar 26;285(13):9908-9918.
doi: 10.1074/jbc.M109.080689. Epub 2010 Feb 4.

Neuropilin 1 directly interacts with Fer kinase to mediate semaphorin 3A-induced death of cortical neurons

Susan X Jiang  1 , Shawn Whitehead  1 , Amy Aylsworth  2 , Jacqueline Slinn  1 , Bogdan Zurakowski  1 , Kenneth Chan  3 , Jianjun Li  3 , Sheng T Hou  4
Affiliations

Neuropilin 1 directly interacts with Fer kinase to mediate semaphorin 3A-induced death of cortical neurons

Susan X Jiang et al. J Biol Chem. .

Abstract

Neuropilins (NRPs) are receptors for the major chemorepulsive axonal guidance cue semaphorins (Sema). The interaction of Sema3A/NRP1 during development leads to the collapse of growth cones. Here we show that Sema3A also induces death of cultured cortical neurons through NRP1. A specific NRP1 inhibitory peptide ameliorated Sema3A-evoked cortical axonal retraction and neuronal death. Moreover, Sema3A was also involved in cerebral ischemia-induced neuronal death. Expression levels of Sema3A and NRP1, but not NRP2, were significantly increased early during brain reperfusion following transient focal cerebral ischemia. NRP1 inhibitory peptide delivered to the ischemic brain was potently neuroprotective and prevented the loss of motor functions in mice. The integrity of the injected NRP1 inhibitory peptide into the brain remained unchanged, and the intact peptide permeated the ischemic hemisphere of the brain as determined using MALDI-MS-based imaging. Mechanistically, NRP1-mediated axonal collapse and neuronal death is through direct and selective interaction with the cytoplasmic tyrosine kinase Fer. Fer RNA interference effectively attenuated Sema3A-induced neurite retraction and neuronal death in cortical neurons. More importantly, down-regulation of Fer expression using Fer-specific RNA interference attenuated cerebral ischemia-induced brain damage. Together, these studies revealed a previously unknown function of NRP1 in signaling Sema3A-evoked neuronal death through Fer in cortical neurons.

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Figures

FIGURE 1.
FIGURE 1.
NRP1 inhibitory peptide ameliorates Sema3A-mediated inhibition of neurite outgrowth, growth cone collapse, axonal damage and neuronal death. Cortical brain tissue explants were cultured on a collagen matrix gel for 2–5 DIV, followed by treatment with brain-derived neurotrophic factor (50 ng/ml), or Sema3A (5 μg/ml) with or without pre-treatment with the NRP1 inhibitory peptide1 or the control peptide (20 μm) (a–d). Neurite outgrowth, as indicated by solid arrows in a, was measured using ImageJ on digitized images and plotted in b. Growth cone collapse induced by Sema3A treatment was visualized using phalloidin labeled with Alexa 488, and the representative images are shown in c. Increased number of collapsed growth cone and amelioration of growth cone collapse by NRP1 inhibitory peptide1 were quantified and plotted in d. To determine Sema3A-induced neuronal death, cortical neurons were cultured in the B27 and N2 supplemented neural basal medium for 7 DIV. These cells were untreated (e), treated with Sema3A (f), pretreated using the control peptide (g), NRP1 inhibitory peptide1 (h), or NRP1 inhibitory peptide2 (i). After 8-h incubation, PI (1 μg/ml) was added to the culture wells and incubated with cells for 30 min at 37 °C. Cells were examined under a florescence microscope to detect PI (e–i). The percentage of PI-positive cells were counted per 40× microscopic objective field and plotted in j. Changes in neurite morphology and length were shown in k–n. Solid arrows in l indicate dead cells, whereas open arrows in k–n indicate live cells under the phase-contrast microscope. Error bars = ±S.E. **, statistical significance by one-way analysis of variance and further post hoc test for significant groups using Tukey's test with p < 0.01. Data were from at least three independent repeats. Scale bars = 20 μm.
FIGURE 2.
FIGURE 2.
Early induction in the expression of Sema3A and NRP1 in ischemic mouse brain. Brains were removed from mice subjected to 1-h MCAO followed by 2-, 4-, or 8-h reperfusion. Proteins were isolated from both the contralateral (C) and ipsilateral (I) side of the brain. Western blotting was performed on 25 μg of brain protein to detect the expression of NRP1 (a), NRP2 (c), and Sema3A (e). The expression levels of NRP1, NRP2, and Sema3A were quantified against GAPDH (b, d, and f, respectively). A representative gel of at least three repeats is presented. **, statistical significance compared with the sham-operated group by one-way analysis of variance and further post hoc test for significant groups using Tukey's test with p < 0.01.
FIGURE 3.
FIGURE 3.
Inhibition of NRP1/Sema3A interaction provides neuroprotection during cerebral ischemia. Mice were injected with the control peptide or the NRP1 inhibitory peptide stereotactically into the lateral ventricles 1 h prior to a 1-h MCAO, followed by various reperfusion time. After 1-, 6-, and 24-h reperfusion, mice were killed and the brains were removed. Fresh frozen coronal brain sections (10 μm in thickness) were sectioned for MALDI-MSI (a and b; 1- and 6-h reperfusion, respectively) to demonstrate the increased dissemination of the injected peptide in the brain parenchyma. Arrows in a indicate the injection site and the presence of high level of the peptide along the needle track at 1 h after reperfusion. At 6 h after reperfusion, as shown in b, both the injected NRP1 inhibitory peptide1 and the control peptide were widely distributed in the parenchyma on the ischemic side of the brain (ipsi = ipsilateral side; contra = contralateral side). Total ion chromatograph of brain tissue from the boxes indicated in b (the top panel) depicts the spectrum of the NRP1 inhibitory peptide1 (m/z 1952), as well as the Na+ (m/z 1975) and K+ (m/z 1991) ionized forms of the injected peptide (c). Brain infarction was measured using both a spectrophotometric-based method detecting soluble TTC color intensities extracted from the ischemic brains after 24-h reperfusion (d), and by staining the 2-mm thick coronal brain sections using TTC (e). Lane 1 in e shows a representative image of serial coronal brain sections treated with the control peptide; lanes 2 and 3 show representative images of brain infarctions from the NRP1 inhibitory peptide1- and 2-treated mice. During reperfusion, two motor behaviors were measured, including the forepaw grip strength test (f) and the six-point neurological deficit scores in turning behavior (g). Symbols in panels f and g are ● is for the control peptide, ■ for NRP1 inhibitory peptide1, and ▴ for NRP1 inhibitory peptide2. The total number of mice used were n = 11 for the control peptide-treated and n = 9 for both NRP1 inhibitory peptide1- and 2-treated mice. Error bars = ±S.E. Statistical analysis was performed to compare the control peptide and the NRP1 inhibitory peptide-treated brains using the non-parametric Mann-Whitney U test with ** indicating statistically significant at p < 0.01.
FIGURE 4.
FIGURE 4.
Neuronal protection conferred by NRP1 inhibitory peptide. MCAO mouse brains injected with the control peptide or the NRP1 inhibitory peptide1 were frozen-fixed and serially sectioned. Immunostainings for NeuN, βIII-tubulin, and Iba1 (a) and double immunostaining with TUNEL and MAP2 were performed to show changes in NeuN positive neurons, microtubules in axons, the activated microglial cells and neuronal death in the ischemic brain, respectively (b). The numbers of NeuN-positive cells were counted from high resolution digital images (1300 × 1030 pixels) taken from in the ischemic cortex under a 40 × objective and plotted as shown in c. The intensity of βIII-tubulin immunostaining was measured on digitized images taken from ischemic brains using the ImageJ software. An arbitrary intensity unit was used to show the relative changes in βIII-tubulin intensity in the core and peri-infarct area of the brain (d). The intensity of Iba1 staining was also measured using the same method and shown in e. Brain sections adjacent to those used for NeuN staining were subjected to TUNEL and MAP2 double immunostaining (b). The number of TUNEL-positive cells per 40× objective field were counted and plotted in f. Error bars = ±S.E. Statistical significance was determined by the Mann-Whitney U test using data from at least three independent repeats. **, p < 0.01. Scale bars = 50 μm.
FIGURE 5.
FIGURE 5.
NRP1 inhibitory peptide1 and Sema3A protein has no effect on [Ca2+]i in cortical neurons. The effect of 5 μg/ml Sema3A (a) and 20 μm NRP1 inhibitory peptide 1 (b) on [Ca2+]i was determined by a ratiometric measurement of [Ca2+]i using Fura-2 following procedures as described under “Experimental Procedures.” [Ca2+]i concentration was represented by the ratio of fluorescent intensity between the two wavelengths of R340/380 of Fura-2 florescence. KCl (45 mm) and NMDA (100 μm) were used as controls to induce neuronal responses. Data were obtained from at least three independent experiments and the average of at least three experiments was plotted in a and b. Lines in a and b represent the averaged concentrations of [Ca2+]i. NRP1 inhibitory peptide1-treated and control peptide-treated ischemic brains were also collected for Western blotting to detect [Ca2+]i-evoked changes in pCaMKII (c). GAPDH protein was used as an internal loading control.
FIGURE 6.
FIGURE 6.
NRP1 directly and selectively interacts with Fer in ischemic brains. a, 200 μg of proteins from normal mouse brains were subjected to IP against NRP1 or NRP2. Equal amounts of IP products were subjected to Western blotting to detect NRP1 or NRP2 association with neuropilin-interacting protein, CDK5, Plexin A1, TCGAP, and Fer (a). In the IP reaction, a reaction without primary antibody was used as a negative control (a). To determine changes in Fer association with NRP1 during MCAO, ischemic brains were separated into contralateral (C) and ipsilateral (I) sides (b). Proteins from brain tissue were subjected to IP against NRP1 or NRP2. Western blotting was performed to detect Fer and IgG (as an internal control) as shown in b. The ischemic brains (200 μg) from both the NRP1 inhibitory peptide1-treated and the control peptide-treated mice were also subjected to IP against NRP1 (c). Western blotting was performed on these IP products to detect changes in Fer association with NRP1. IgG was used as a loading control. The intensities of the Fer band from two independent repeats were measured and quantified against those of the control peptide-treated brains shown in d. Total brain lysates were also subjected to Western blotting to detect the level of total Fer with GAPDH as an internal loading control (e). Densitometry measurements of band intensities were normalized against that of the sham-operated brain (f).
FIGURE 7.
FIGURE 7.
Knocking down Fer using RNAi provides neuroprotection against Sema3A-induced toxicity in cortical neurons. Cultured 7 DIV cortical neurons were transfected with Fer RNAi or its control negative RNAi at 1 μg/well in a 24-well plate. After 36- and 48-h transfection, cells were collected for Western blotting to demonstrate down-regulation of the expression of Fer as shown in a. GAPDH was detected on the Western blot and used as a loading control. Band intensity was measured using ImageJ, and the level of Fer was normalized against the level of GAPDH and plotted in b. After 36 h of Fer small interference RNA transfection, neurons were challenged with 5 μg/ml Sema3A. PI-positive cells, indicated by long arrows, are shown in c–e. The number of PI-positive cells was counted and plotted after 24 h of treatment (f). Neurite length, as indicated by short arrows in c–e, was also measured using ImageJ and plotted (g). At least 300 cells and their neurites were measured from at least three independent experiments. Scale bar in c = 50 μm. Error bars = ±S.E. Statistical significance was determined by one-way analysis of variance and further post hoc test for significant groups using Tukey's test with p < 0.01.
FIGURE 8.
FIGURE 8.
Knocking down Fer is neuroprotective against MCAO-induced neuronal death and improves neurological functions. a, schema of Fer RNAi delivery to mouse brain. Fer RNAi or the control negative RNAi was injected into mouse brains using stereotaxic injection. After 24, 48, 72, and 90 h of injection, mice were killed and brain was collected for protein extraction and Western blotting to detect changes in Fer expression level (b). Each brain was separated into the control side (c) and the injected side (I). Control mice were only subjected to skull surgery (labeled as Sham in b). The band intensities of Fer and GAPDH in Fer RNAi-injected mice brains were measured using ImageJ software, and the average -fold changes of the ratio of Fer/GAPDH of two repeat experiments were plotted (c). Fer protein level was reduced by more than 1-fold in brains after 48- and 72-h injection with Fer RNAi. Therefore, mice injected with Fer RNAi for 48 h were subjected to 1-h MCAO and followed by 24-h reperfusion. The experimental scheme is shown in d. Brain infarction size as indicated by the percentage of decrease in TTC staining between the ipsilateral and contralateral side of the ischemic brain was determined using a spectrophotometric-based method to detect soluble TTC color intensity extracted from the brain as described under “Experimental Procedures” (n = 7 for each treatment) and the result is shown in e. Forepaw grip strength test (f) and neurological deficit scores (g) were also performed before sacrificing the mice. Error bars = ±S.E. Statistical significance between the Fer RNAi-treated and control negative RNAi-treated groups was determined by a non-parametric Mann-Whitney U test with ** indicating p < 0.01.
FIGURE 9.
FIGURE 9.
Knocking down Fer expression reduces the number of TUNEL-positive neurons in ischemic mouse brain. Mice injected with Fer RNAi for 48 h were subjected to 1-h MCAO, and followed by 24-h reperfusion. Mouse brains were subjected to frozen serial sectioning and immunostaining for TUNEL (a, b, e, and f; solid arrows in a indicate TUNEL-positive nuclei) and NeuN (c, d, g, and h; open arrows in c indicate NeuN-positive neurons). High resolution digital images of brain sections were taken, and the numbers of TUNEL- and NeuN-positive cells were counted on images covering the area of a 40× objective lens from the microscope (i and j). Scale bar = 80 μm. Error bars = ±S.E. Statistical significance was determined by a non-parametric Mann-Whitney U test with ** indicating p < 0.01 between the treated and control-treated groups using data from at least three independent repeats.
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