Involvement of Bradykinin Receptor 2 in Nerve Growth Factor Neuroprotective Activity

Neurotrophin nerve growth factor (NGF) has been demonstrated to upregulate the gene expression of bradykinin receptor 2 (B2R) on sensory neurons, thus facilitating nociceptive signals. The aim of the present study is to investigate the involvement of B2R in the NGF mechanism of action in nonsensory neurons in vitro by using rat mixed cortical primary cultures (CNs) and mouse hippocampal slices, and in vivo in Alzheimer’s disease (AD) transgenic mice (5xFAD) chronically treated with NGF. A significant NGF-mediated upregulation of B2R was demonstrated by microarray, Western blot, and immunofluorescence analysis in CNs, indicating microglial cells as the target of this modulation. The B2R involvement in the NGF mechanism of action was also demonstrated by using a selective B2R antagonist which was able to reverse the neuroprotective effect of NGF in CNs, as revealed by viability assay, and the NGF-induced long-term potentiation (LTP) in hippocampal slices. To confirm in vitro observations, B2R upregulation was observed in 5xFAD mouse brain following chronic intranasal NGF treatment. This study demonstrates for the first time that B2R is a key element in the neuroprotective activity and synaptic plasticity mediated by NGF in brain cells.


Introduction
Neurotrophin nerve growth factor (NGF) is characterized by the ability to improve the growth and differentiation of sensory and sympathetic nerve cells [1]. Numerous papers have proposed NGF as a possible therapeutic option in the treatment of Alzheimer's disease (AD) due to its ability to sustain cholinergic activity [2][3][4] and its neuroprotective function [5,6], together with its ability to directly inhibit amyloidogenesis [7,8].
Following this period, CNs were washed twice with Neurobasal + 1% B27 medium and incubated for 6 h (−NGF 6 h), while other cultures were treated with NGF (100 ng/mL) for the same period (+NGF 6 h).
Total RNA was extracted with Trizol (Invitrogen, Milano, Italy) from four biological replicates (derived from the same litter) for each of the experimental conditions (CTR, +NGF, −NGF 6 h and +NGF 6 h).
RNA integrity was confirmed using a RNA chip and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with the protocol outlined by the manufacturer. Complementary RNAs (cRNAs) labeled with Cy3-CTP were synthesized from 1 µg of total RNA of each sample using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies), following the manufacturer's protocol. Aliquots (750 ng) of Cy3 labeled cRNA targets were hybridized on Whole Rat Genome Oligo Microarrays (Agilent Technologies). Microarray hybridization and washing were performed using reagents and instruments (hybridization chambers and rotating oven), as indicated by the manufacturer. Microarrays were scanned at 5-µm resolution using a GenePix Personal 4100A microarray scanner and the GenePix Pro 6.0 acquisition and data-extraction software (Molecular Devices, San Jose, CA, USA). Raw data were processed and analyzed by GeneSpring GX 11.5 software 13 (Agilent Technologies).

Immunocytochemistry
Cultured cells were washed in PBS and fixed in 4% (w/v in PBS) paraformaldehyde for 30 min at room temperature. Fixed cells were washed in PBS, pH 7.4, permeabilized using 0.1% Triton X100-Tris-HCl, pH 7.4, for 10 min, and then treated with the following primary antibody: rabbit antiB2R Nuclei were stained with Hoechst 33258 (0.25 µg/mL) for 5 min at room temperature. Controls to assess primary antibody specificity were performed by including the omission of the primary antibody.

ELISA
For the determination of bradykinin (BK) levels, cell media were loaded directly onto enzyme-linked immunosorbent assay (ELISA) BK plates, in accordance with the manufacturer's instructions (Enzo Life Sciences, Ann Arbor, MI, USA). BK concentration was corrected by referring to the volume of the collected sample (1 mL). The minimum detectable level for this assay was 10 pg/mL. This assay can recognize B2 receptor ligands such as BK and Lys-BK, whereas it does not recognize B1 receptor ligands (i.e., [desArg9]-BK and [desArg9]-Lys-BK).

Electrophysiology
Mice (three-months old) were anesthetized with halothane and their brains were removed and placed in ice-cold artificial cerebrospinal fluid solution (ACSF) containing 124 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO 4 , 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 2.4 mM CaCl 2 , and 10 mM glucose. Hippocampal slices (350 µm) were cut with a Vibroslice (VT 1000S; Leica, Wetzlar, Germany) and kept for 1 h in oxygenated medium at room temperature (20-22 • C) before recordings. A single slice was then placed on a nylon mesh, completely submerged in a small chamber (0.5 mL), and superfused with oxygenated ACSF (30 • C) at 3 mL/min constant flow rate. Experiments were performed in the CA1 region and field excitatory synaptic potentials (fEPSPs) were recorded in stratum radiatum by stimulating Schaffer Cells 2020, 9, 2651 5 of 16 collaterals. Long-term potentiation (LTP) was induced by conventional HFS applied to the Schaffer collateral-CA1 synapses (1 train of 100 Hz). All data are presented as mean ± SEM and assessed for significance using the unpaired Student's t test.

Transgenic Mice
Transgenic mice (three months old) with five familial Alzheimer's disease mutations (5xFAD) and coexpressing FAD mutant forms of human APP and presenilin 1 were purchased from the Jackson Laboratory [37].
The 5xFAD mice used were hemizygotes with respect to the transgenes, while nontransgenic wild-type littermates were used as controls. Genotyping was performed by a PCR analysis of tail DNA. All analyses were done blind with respect to the genotype of the mice and treatment. All experiments with mice were performed according to the national and international laws for laboratory animal welfare and experimentation (EU directive no. 2010/63/EU and Italian DL no. 26 04/03/2014). Mice were kept under a 12 h dark to light cycle, with food and water ad libitum.

Intranasal Treatment with NGF and Tissue Processing
NGF diluted in 1M PBS was administered intranasally to mice, 3 µL at a time, alternating nostrils, with a lapse of 2 min between each administration, for a total of 14 times at a dose of µg/kg (equivalent to 0.51 pmol), as previously described by Capsoni et al. [38].
As control treatments, wild-type and 5xFAD mice were treated with PBS. The frequency of administration for intranasal delivery was three times per week (every two days). Administrations were repeated nine times over a 21-day period, followed by seven days of washout during which mice were not dosed with the protein. Following anesthesia with 2,2,2-tribromethanol, the caudal part of the brain was removed, stored at −80 • C, and used for Western blot analysis.

Data Analysis
A statistical analysis was performed using SPSS 11.0.0 for Windows (SPSS Inc., Chicago, IL, USA). All results are expressed as mean ± SEM, with n the number of independent experiments. The significance of the effect was performed by one-way analysis of variance (ANOVA) followed by Bonferroni's test for multiple comparison. The significance level was set at p < 0.05 (*) and p < 0.01 (**).

Expression of Bradykinin (BK) and BK Receptors in CNs Following NGF Treatment and Deprivation
Aiming to define the transcriptional changes regulating BK and its receptor proteins involved in NGF treatment, deprivation, and rescue, we measured the steady-state mRNA levels of gene encoding for BK (Kng1), B1R (Bdkrb1), and B2R (Bdkrb2). As shown in Figure 1, Bdkrb2 was the only upregulated gene following NGF treatment, while following NGF deprivation, we observed significantly increased levels of both Kng1 and Bdkrb1. monitored by normalization to β-actin. Blots were scanned, and a quantitative densitometric analysis was performed using ImageJ software (http://imagej.nih.gov/ij/).

Data Analysis
A statistical analysis was performed using SPSS 11.0.0 for Windows (SPSS Inc., Chicago, IL, USA). All results are expressed as mean ± SEM, with n the number of independent experiments. The significance of the effect was performed by one-way analysis of variance (ANOVA) followed by Bonferroni's test for multiple comparison. The significance level was set at p < 0.05 (*) and p < 0.01 (**).

Expression of Bradykinin (BK) and BK Receptors in CNs Following NGF Treatment and Deprivation
Aiming to define the transcriptional changes regulating BK and its receptor proteins involved in NGF treatment, deprivation, and rescue, we measured the steady-state mRNA levels of gene encoding for BK (Kng1), B1R (Bdkrb1), and B2R (Bdkrb2). As shown in Figure 1, Bdkrb2 was the only upregulated gene following NGF treatment, while following NGF deprivation, we observed significantly increased levels of both Kng1 and Bdkrb1.
Together, these data suggest that NGF regulates BK and its receptor system, specifically acting through the constitutive B2R in neuroprotective conditions and through BK and the inducible B1R in apoptosis due to NGF deprivation.

Steady-State Levels of B2R Protein
Since Bdkrb2 was the only increased gene following NGF treatment, we analyzed the corresponding B2R expression level in CNs lysates by Western blot analysis. As shown in Figure 2, Figure 1. mRNA expression profiles of BK and its receptors genes after NGF treatment, deprivation, and rescue. Transcript levels of genes encoding BK (Kng1; NM_012696), Bradykinin receptor 2 (Bdkrb2; NM_001270713), and Bradykinin receptor 1 (Bdkrb1; NM_030851) in cortical neurons (CNs) following 48 h NGF treatment (+NGF), induction of apoptosis by 6 h NGF deprivation (−NGF 6 h), and rescue by 6 h NGF replacement (+NGF 6 h). Data represent means (±S.E.M.) from four replicates. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni's test for multiple comparison (* p < 0.001 vs CTR; § p < 0.05 vs CTR 6 h; # p < 0.05 vs. +NGF replacement (+NGF).
Together, these data suggest that NGF regulates BK and its receptor system, specifically acting through the constitutive B2R in neuroprotective conditions and through BK and the inducible B1R in apoptosis due to NGF deprivation.

Steady-State Levels of B2R Protein
Since Bdkrb2 was the only increased gene following NGF treatment, we analyzed the corresponding B2R expression level in CNs lysates by Western blot analysis. As shown in Figure 2, NGF treatment provoked a significant increase (about 1.5 fold upregulation), while NGF deprivation caused a Furthermore, an immunofluorescence analysis of CNs revealed the exact localization of B2R in the basal condition after NGF treatment. As shown in Figure 3, a merged analysis indicated that B2R immunoreactivity was present in microglial cells (IBA1) in control conditions (CTR) and overexpressed after NGF treatment (+NGF). Conversely, no colocalization was detected in neurons (MAP2 cells) or astrocytes (GFAP cells). Furthermore, an immunofluorescence analysis of CNs revealed the exact localization of B2R in the basal condition after NGF treatment. As shown in Figure 3, a merged analysis indicated that B2R immunoreactivity was present in microglial cells (IBA1) in control conditions (CTR) and overexpressed after NGF treatment (+NGF). Conversely, no colocalization was detected in neurons (MAP2 cells) or astrocytes (GFAP cells).

Expression of B2R in Cultured Microglial Cells
Since B2R seems to be selectively upregulated by NGF treatment in microglia, we performed Western blot and immunofluorescence analyses of this receptor in enriched microglial cultures. As shown in Figure 4a,b, B2R was significantly upregulated, confirming the results obtained in mixed cortical cultures.

Expression of B2R in Cultured Microglial Cells
Since B2R seems to be selectively upregulated by NGF treatment in microglia, we performed Western blot and immunofluorescence analyses of this receptor in enriched microglial cultures. As shown in Figure 4a,b, B2R was significantly upregulated, confirming the results obtained in mixed cortical cultures.

CNs Viability
Considering the B2R upregulation by NGF, we tested the possible involvement of this receptor in NGF neuroprotective activity by analyzing CN survival ( Figure 5). A quantitative analysis revealed that incubation of CNs with NGF (100 ng/mL) for 48 h (+NGF) did not alter cell viability, while 24 h of NGF deprivation (−NGF) caused a ~55% reduction in the number of surviving cells compared to control conditions (CTR). After adding back NGF (100 ng/mL) for 24 h, we observed a complete rescue of cell viability.
Since it has been suggested that B2R is neuroprotective against different toxic insults both in vitro and in vivo [39][40][41][42], we treated CNs for 24 h with RPM-7 (Labradimil, Cereport), a specific B2R agonist (RPM-7 100 nM). As shown in Figure 5b, RPM-7 significantly rescued the cell death induced by NGF deprivation (−NGF), and this effect was antagonized by HOE140 co-administration (HOE140+RPM-7), confirming the neuroprotective role exerted by B2R in this experimental model. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) followed by Bonferroni's test for multiple comparisons (* p < 0.05). (b) Representative immunofluorescence images of microglial cells stained with antibodies for B2R (red) or microglia (Iba1, green) and nuclei (Hoechts, blue) in control conditions (CTR) and after NGF treatment (+NGF). Scale bar: 15 µm.

CNs Viability
Considering the B2R upregulation by NGF, we tested the possible involvement of this receptor in NGF neuroprotective activity by analyzing CN survival ( Figure 5). A quantitative analysis revealed that incubation of CNs with NGF (100 ng/mL) for 48 h (+NGF) did not alter cell viability, while 24 h of NGF deprivation (−NGF) caused a~55% reduction in the number of surviving cells compared to control conditions (CTR). After adding back NGF (100 ng/mL) for 24 h, we observed a complete rescue of cell viability. Since we demonstrated an increase in the BK precursor gene Kng1 (Figure 1) in apoptotic conditions (−NGF), and the B2R antagonist was able to significantly reverse apoptosis due to NGF deprivation ( Figure 5), we can hypothesize that in such conditions, endogenous BK could be produced by the cleavage of kininogen derived from Kng1.
To this end, we performed an ELISA analysis of cell media from CNs in control conditions (CTR), treated with NGF (+NGF), and deprived of NGF (−NGF). The results from the ELISA test indicated no differences among the groups (Supplementary Figure S1).

Electrophysiology
Electrophysiology recordings showed that pretreatment of hippocampal slices with NGF (100 ng/mL for 1 h) was able to significantly enhance LTP at CA1 hippocampal synapses (1.77 ± 0.18) compared to the control (1.45 ± 0.06) ( Figure 6). Notably, the application of BK (1 μM for 1h) mimicked the NGF-mediated facilitatory action on CA1-LTP (1.64 ± 0.06). The effect of NGF was prevented by the pretreatment of slices with the B2R antagonist HOE140 (100 nM) (1.4 ± 0.05), further suggesting that NGF modulates synaptic plasticity via interaction with B2R. These results indicate that B2R, endowed with neuroprotective activity, plays a role in the NGF mechanism of action. Since it has been suggested that B2R is neuroprotective against different toxic insults both in vitro and in vivo [39][40][41][42], we treated CNs for 24 h with RPM-7 (Labradimil, Cereport), a specific B2R agonist (RPM-7 100 nM). As shown in Figure 5b, RPM-7 significantly rescued the cell death induced by NGF deprivation (−NGF), and this effect was antagonized by HOE140 co-administration (HOE140+RPM-7), confirming the neuroprotective role exerted by B2R in this experimental model.
Since we demonstrated an increase in the BK precursor gene Kng1 (Figure 1) in apoptotic conditions (−NGF), and the B2R antagonist was able to significantly reverse apoptosis due to NGF deprivation ( Figure 5), we can hypothesize that in such conditions, endogenous BK could be produced by the cleavage of kininogen derived from Kng1.
To this end, we performed an ELISA analysis of cell media from CNs in control conditions (CTR), treated with NGF (+NGF), and deprived of NGF (−NGF). The results from the ELISA test indicated no differences among the groups (Supplementary Figure S1).

Electrophysiology
Electrophysiology recordings showed that pretreatment of hippocampal slices with NGF (100 ng/mL for 1 h) was able to significantly enhance LTP at CA1 hippocampal synapses (1.77 ± 0.18) compared to the control (1.45 ± 0.06) ( Figure 6). Notably, the application of BK (1 µM for 1 h) mimicked the NGF-mediated facilitatory action on CA1-LTP (1.64 ± 0.06). The effect of NGF was prevented by the pretreatment of slices with the B2R antagonist HOE140 (100 nM) (1.4 ± 0.05), further suggesting that NGF modulates synaptic plasticity via interaction with B2R. These results indicate that B2R, endowed with neuroprotective activity, plays a role in the NGF mechanism of action.

NGF Treated AD Mice
Considering the B2R upregulation by NGF treatment in vitro, we examined by Western blot analysis the effect of NGF chronically administered to 5xFAD mice. As shown in Figure 7, the B2R expression level in brain extracts was slightly detectable in the brain of wild type and 5xFAD mice treated with PBS, while its amount was significantly increased following NGF administration.
Both the 45kDa band, corresponding to the nonglycosylated B2R, and the 75kDa band, consistent to the mature fully glycosylated B2R, showed about a four-fold increase, confirming the crucial role of this receptor also in vivo.

NGF Treated AD Mice
Considering the B2R upregulation by NGF treatment in vitro, we examined by Western blot analysis the effect of NGF chronically administered to 5xFAD mice. As shown in Figure 7, the B2R expression level in brain extracts was slightly detectable in the brain of wild type and 5xFAD mice treated with PBS, while its amount was significantly increased following NGF administration.

NGF Treated AD Mice
Considering the B2R upregulation by NGF treatment in vitro, we examined by Western blot analysis the effect of NGF chronically administered to 5xFAD mice. As shown in Figure 7, the B2R expression level in brain extracts was slightly detectable in the brain of wild type and 5xFAD mice treated with PBS, while its amount was significantly increased following NGF administration.
Both the 45kDa band, corresponding to the nonglycosylated B2R, and the 75kDa band, consistent to the mature fully glycosylated B2R, showed about a four-fold increase, confirming the crucial role of this receptor also in vivo. Both the 45 kDa band, corresponding to the nonglycosylated B2R, and the 75kDa band, consistent to the mature fully glycosylated B2R, showed about a four-fold increase, confirming the crucial role of this receptor also in vivo.

Discussion
NGF, discovered in the 1950s by Levi Montalcini and Hamburger, has been identified as a trophic factor for sympathetic and sensory neurons, inducing the survival, development, and neurite growth of these neurons [43]. It has also been reported that NGF deprivation of sympathetic neurons leads to their degeneration and massive death due to the activation of programmed cell death [44]. The NGF-promoting action found in sensory neurons was subsequently extended to a significantly and pronounced "trophic" effect in other nonsensory neurons [45].
NGF, synthesized in brain structures innervated by the basal forebrain cholinergic neurons and then retrogradely transported via axons to the bodies of cholinergic cortical neurons, is extremely important for their survival [46]. Indeed, the trophic support by NGF of developing and mature basal forebrain cholinergic neurons is required for normal plastic rearrangements during the development and functioning of adult cholinergic neurons, affecting the extent of connection between these and innervated targets [47,48].
NGF deprivation of these neurons leads to their degeneration, as demonstrated by evidence that the levels of NGF in brain structures are modified in neurodegenerative diseases, including AD [49]. The well-documented nociceptive activity of NGF, representing a substantial side effect for the development of an NGF-based prospective therapy for human diseases, is characterized by the upregulation of B2R expression in sensitive neurons [15,50].
Here, we demonstrated that also in nonsensitive cells (mixed cortical cultures), NGF was able to modify B2R expression. From microarray data, it emerged that following NGF exposure, B2R mRNA was significantly upregulated and its expression levels decreased in NGF-deprived conditions. In the same apoptotic conditions, B2R reduction was accompanied by an increase in BK and in inducible B1R mRNA levels, thus emphasizing that BK and B1R could be related to neurodegeneration, while B2R seems to have a neuroprotective role.
We established B2R upregulation by NGF also at a protein level; the results showed a selective expression in microglial cells. Since it is well known that B2R is widely distributed in rat brain within the neuronal compartment, including the cerebral cortex, our results could appear quite surprising. However, it is worth mentioning that while it was showed a specific B2R immunoreactivity in neurons in the whole rat brain, the expression of this receptor in glial cells could not be excluded [51]. In fact, the presence of B2R has been identified in rat primary cultures of cortical astrocytes [52][53][54] and microglia [55,56].
In the present study, we demonstrated that in mixed cortical cultures, B2R is expressed only in microglial cells, while no labeling was seen in neurons or astrocytes. On the other hand, by the use of the same mixed primary cortical cultures it was showed a weak expression of B2R immunoreactivity only in neurons [57]. Therefore, the observed discrepancies could be explained by different culture conditions, since in our experiments, we extended DIV and reduced B27 supplement concentration to amplify the effect of NGF.
Our results, indicating microglia as the main target of NGF activity, are in agreement with a previous work showing that following NGF treatment, microglial cells change into a neuroprotective and anti-inflammatory phenotype both in vitro and ex vivo [58]. Indeed, these authors demonstrated that NGF exerts its neuroprotective and anti-inflammatory effects on neurons, reducing cytokines levels, rescuing spine density and LTP deficit, in a microglia-dependent way, through the NGF receptors present in these cells both in vivo and in vitro [58].
The neuroprotective role of B2R was confirmed in this in vitro experimental model by examining the cell viability in apoptotic conditions obtained by NGF deprivation. We demonstrated that not only NGF, but also a specific B2R agonist (RPM-7), significantly rescued cell death. Moreover, HOE140 (Icatibant), a selective B2R antagonist, reversed the rescue induced by NGF replacement, indicating a crucial role of B2R in the neuroprotective activity of NGF.
Interestingly, HOE140 was able to significantly reverse NGF deprivation-induced apoptosis, suggesting the potential production of endogenous BK from kininogen cleavage, whose gene (Kng1) was demonstrated to be upregulated in this apoptotic condition. In contrast, ELISA analysis indicated no differences in the BK amount after NGF deprivation. However, we cannot exclude the possibility that BK could be effectively secreted, since it is well known that BK is rapidly metabolized by kininases including aminopeptidase P and carboxypeptidase N [59].
Moreover, Kng1 enzymatic processing can lead to the production not only of BK, but also of other metabolites such as des-Arg9-BK and Lys-des-Arg9-BK. These peptides are highly pro-inflammatory since they have greater affinity for the inducible B1R than the homolog pair BK, and could contribute to the neurodegenerative development seen in NGF deprived conditions [19].
Corroborating the crucial role of B2R in the NGF neuroprotective activity, electrophysiological experiments also indicated that the enhancement of LTP by NGF was significantly mediated by B2R, since BK mirrored the NGF action and the selective B2R antagonist HOE140 prevented the NGF effect. Since B2R is present in microglial cells, it reasonable to suppose that NGF exerts its effect on synaptic plasticity via microglia, in line with previous data showing that the ability of NGF to rescue chemical LTP is completely dependent on the presence of microglia in the cultures [58].
The involvement of B2R in the NGF neuroprotective activity was further demonstrated in vivo in four-month-old 5xFAD mice chronically treated with a NGF variant [38]. As recently proved by our research group, the administration of the variant neurotrophin by intranasal delivery, a noninvasive method to transport neurotrophic factors to the brain [60], was able to reduce neurodegeneration, Aβ deposition, and memory deficits, and to promote the rescue of synaptic plasticity in the 5xFAD AD model. Interestingly, the authors established that these NGF activities were mediated by glial cells, modulating inflammatory proteins such as the soluble TNFa receptor II and the chemokine CXCL12 [38].
In our experiments, we evidenced that in brain extract of 5xFAD mice, the mature, fully glycosylated B2R form was significantly upregulated by NGF treatment, indicating a key role of this constitutive receptor in the mechanism of action of NGF.
Considering the selective overexpression of B2R in in vitro microglia, the results obtained in brain of 5xFAD treated mice indicated the same localization also in vivo. Of note, as already demonstrated [38], both NGF receptors p75NTR and TrKA are upregulated on 5xFAD microglia, as well as on human AD microglia, supporting the role of these cells as a target for the NGF neuroprotective activity.
Additional experiments should be performed to characterize the molecular mechanism through which the increased B2R is involved in NGF-mediated neuroprotection in order to identify new potential therapeutic strategies for AD.