A Novel Netrin-1-Derived Peptide Enhances Protection Against Neuronal Death And Mitigates Of Intracerebral Hemorrhage in Mice Part 1

Abstract: 

It has been reported that Netrin-1 is involved in neuroprotection following injury to the central nervous system. However, the minimal functional domain of Netrin-1 which can preserve the neuroprotection but avoid the major side effects of Netrin remains elusive. 

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Here, we investigated the neuroprotective effect of a peptide E1 derived from Netrin-10 s EGF3 domain (residues 407–422). We found that it interacts with deleted colorectal carcinoma (DCC) to activate focal adhesion kinase phosphorylation exhibiting neuroprotection. 

The administration of the peptide E1 was able to improve functional recovery through reduced apoptosis in an experimental murine model of intracerebral hemorrhage (ICH). 

In summary, we reveal a functional sequence of Netrin-1 that is involved in the recovery process after ICH and identify a candidate peptide for the treatment of ICH.

Keywords: intracerebral hemorrhage; hemin; neuroprotection; Netrin-1; peptide.

1. Introduction

Intracerebral hemorrhage (ICH) is a devastating stroke subtype with a high mortality and morbidity rate [1–3] that involves abnormal rupture of cerebral blood vessels within the brain [4,5]. 

Surgical removal of hematomas is preferred for achieving hemostasis and relieving intracranial pressure [6]. However, remnant hematic residues and blood breakdown products can still promote secondary brain injury following ICH, inducing neuronal damage and delaying functional recovery [7–9]. 

Thus, the development of effective strategies targeting secondary brain injury-induced neuronal damage would be beneficial for stroke treatment. Netrin-1 is one of the most well-studied proteins that regulate axonal guidance and synaptogenesis [10–12] via interaction with its receptors deleted in colorectal cancer (DCC) and UNC5H (unco-ordinated-5 homolog) to activate cell survival, differentiation, and proliferation [13–15]. 

The receptors induce a death signal to mediate apoptosis when the Netrin-1 is absent [16]. It is known that the binding of Netrin-1 with DCC results in dimerization of DCC and initiates tyrosine 861 phosphorylation of focal adhesion kinase (FAK) to induce neurite outgrowth and axonal guidance [17,18]. 

The binding of Netrin-1 with DCC involves the EGF3 domain of Netrin-1 and the FN5 domain of DCC in the activation of the Netrin-1 signaling pathway [19,20]. Recently, Netrin-1 was shown to exert neuroprotective effects in ischemic regions [21]. 

The serum's expression level of Netrin-1, which inhibits cell apoptosis and promotes neuronal regeneration, is positively correlated with patient recovery from ischemic stroke [22,23]. 

Interestingly, transplantation of bone marrow mesenchymal stem cells that secrete Netrin-1 into the damaged rat sciatic nerve initiates axonal regeneration [24] and reduces motor neuron death [25]. Stereotaxic injection of the Netrin-1 protein into the ventricles reduces nerve cell death after subarachnoid hemorrhage through activation of DCC/APPL1/AKT signaling [26]. 

A polypeptide derived from the EGF3 domain of Netrin-1 (amino acids 423–433) markedly activates ERK phosphorylation and promotes the production of NO, which has cardioprotective effects [27,28]. However, Netrin-1 has been identified as a potential biomarker for tumorigenesis [29–31] because regulation of the Netrin-1 status is found in multiple tissue-derived cancers [32]. 

When Netrin-1 is bound to its receptor DCC or Unc5H, the receptors transduce a positive signal leading to survival, inflammation, angiogenesis, and anti-apoptosis, which in turn regulates tumorigenesis [31]. 

Therefore, it is very important to identify the minimal functional domain of Netrin-1 that can preserve the neuroprotection and avoid the major side effects of Netrin-1. Here, we found that peptide E1 (residues 407–422), which is derived from the EGF3 domain of Netrin-1, interacts with DCC to protect neurons from hemin-induced toxicity. 

In an experimental model of ICH mice, the application of peptide E1 improved functional recovery, as determined by behavioral assays. Furthermore, peptide E1 can prevent neurons from degenerating after ICH. 

Based on these results, we identified a functional sequence of Netrin-1 that is involved in recovery after ICH and determined that this peptide may be used for the treatment of ICH.

2. Results

2.1. EGF3 Domain Is Critical for the Netrin-DCC Interaction

Netrin-1 consists of a VI domain, three EGF repeats, and a C-terminal domain [33]. The crystal structure of the human Netrin-1/DCC complex reveals that the Netrin-1 EGF3 domain is crucial for the Netrin-1/DCC interaction, but not the EGF1 and EGF2 domains [34]. 

Mutagenesis of key amino acid residues (His407, Gln442, or Gln443) at the EGF3 domain completely disrupted Netrin-1/DCC binding [19]. EGF1/2 domains are required for switching attractive signaling into repulsive signaling when Unc5 coexists with DCC [20]. 

Thus, we first generated full-length and EGF3-deleted (Netrin-1 ∆407–443) Netrin-1 constructs (Figure 1A) and transiently expressed these constructs in cultured HEK293T cells for GST pulldown. 

Binding was observed for the constructs that expressed Netrin-1 but not Netrin-1 (∆407–443) (Figure 1C). These results suggest that the domain of EGF3 containing amino acids 407–443 is responsible for the interaction of Netrin-1 with DCC. To investigate the role of the EGF3 domain in Netrin-DCC, we first purified myc-tagged Netrin-1 or truncated Netrin-1 (∆407–443) (Figure 1B). 

Next, HEK293T cells were transfected with a plasmid carrying DCC cDNA and incubated with Netrin-1 protein, and then Netrin-1 binding was assessed by immunostaining. 

We found that full-length Netrin-1, but not truncated Netrin-1, was able to bind to DCC at the plasma membrane (Figure 1D). To determine whether the EGF3 domain can interact with DCC, we generated a GST-EGF3 construct to pull down lysed cells overexpressing DCC. We observed specific binding of DCC with GST-EGF3 (Figure 1E). 

Furthermore, we synthesized the FITC-labelled peptide EGF3 and confirmed that, like full-length Netrin-1, it was able to bind to DCC at the plasma membrane (Figure 1F). Taken together, our data indicate that the EGF3 domain is responsible for the interaction of Netrin-1 with DCC.

2.2. The EGF3 Domain Induces Phosphorylation of Downstream Pathways of Netrin-1

The interaction of Netrin-1 and DCC activates multiple signaling pathways, including FAK, SFK, and ERK [16,35]. Focal adhesion kinase (FAK) is one of the major tyrosine phosphorylation activities linked to Netrin-1/DCC signaling. 

Netrin-1 activates FAK phosphorylation and Src family protein tyrosine kinases (SFKs) phosphorylation [17]. Both FAK and SFK signaling pathways play a pivotal role in the developmental stage of the nervous system and injury repair [36,37]. Moreover, Netrin-1 can activate the extracellular signal-regulated kinase-1/2 (ERK1/2), which mediates the action of the Mitogen-activated protein kinase (MAPK) pathway [38–40]. 

To investigate whether the EGF3 domain is critical for the activation of downstream signaling pathways via the binding of Netrin-1 to DCC, we first examined the effect of the EGF3 domain on the tyrosine 861 phosphorylation of FAK (Figure 2A). We found that deletion of EGF3 inhibited FAK phosphorylation in cultured cortical neurons (Figure 2B, C), thus confirming an essential role for EGF3 in the Netrin-1 signaling pathway. 

Next, cultured cortical neurons were stimulated with the GST-EGF3 fusion protein for 20 min, and western blotting further confirmed that treatment with the GST-EGF3 fusion protein induced FAK, ERK, and SFK phosphorylation (Figure 2D–G). 

These results suggest that the EGF3 domain of Netrin-1 functions as a potent stimulator to activate downstream signaling pathways.

Figure 1. The EGF3 domain of Netrin-1 is the critical region for Netrin-1/DCC interaction. (A) Illustrations of Netrin-1 constructs. The Netrin-1 constructs are Netrin-1 full-length, Netrin-1-(∆407–443) and GST-EGF3. (B) Purified Netrin-1- (∆407–443)-Myc/His were quantified through a comparison with the BSA standard curve. (C) Immunoblotting of the pull-down fraction by the GST-DCC FN5 domain fusion protein and GST alone. (D) Immunofluorescence images of Flag-DCC expressing 293T cells incubated with control medium, Netrin-1-myc/his and Netrin-1-(∆407–443) -Myc/his for 30 min, respectively, and the quantitative analysis. Scale bar, 10 µm. (E) Immunoblotting of the pull-down fraction by the GST-EGF3 fusion protein and GST along. (F) Immunofluorescence images of Flag-DCC expressing 293T cells incubated with control medium and FITC labeled Pep EGF3 for 30 min, respectively, and the quantitative analysis. Scale bar, 10 µm.

Figure 2. EGF3 domain activates downstream pathways of Netrin-1. (A) Experimental procedure. (B) Cortical neurons (E17 and DIV3) were stimulated with Netrin-1, Netrin-1-(∆407–443) or control medium for 20 min. Cell lysates were incubated with an anti-p-FAK-861 antibody to confirm Netrin-1 activity. (C) Quantitative analysis of the western blot results is shown in (B). n = 3. (D) Cortical neurons (DIV3) were treated with 1.5 µM GST (+), 1.5 µM GST-EGF3 fusion protein (+), or 1.5 µM Netrin-1 (+) medium for 20 min. Cell lysates were incubated with anti-p-FAK-861, anti-p-SFK-418 and anti-p-ERK-202/204. (E–G) Quantitative analysis of the western blot results shown in (D). n = 3. Data are presented as the means ± SEM. One-way ANOVA was used for all statistical analyses shown in this figure (* p < 0.05, ** p < 0.01; ns, not significant). Details of data analysis are seen in Supplementary Table S1.

2.3peptide E1 and E2 Interact with DCC to Activate the Downstream Signaling Pathway of Netrin-1

As previously described, a small peptide derived from the EGF3 domain of Netrin-1 (amino acids 423–433) induced ERK phosphorylation [27]. We synthesized peptides E1 (residues 407–422) and E2 (residues 423–433) that were derived from the EGF3 domain of Netrin-1 (Figure 3A). 

We found that incubation of peptide E1 increased the levels of phosphorylated FAK and SFK (Figure 3B, D, E) in the culture cells in a dose-dependent manner but did not alter the levels of phosphorylated ERK (Figure 3B, F). 

Unlike peptide E1, peptide E2 not only induced the tyrosine phosphorylation of FAK (Figure 3C, G) and the tyrosine phosphorylation of SFK (Figure 3C, H) but also induced the tyrosine phosphorylation of ERK (Figure 3C, I) in cultured cortical neurons. 

The control peptide (Pep Ctrl) had no detectable effect on the tyrosine phosphorylation of FAK, SFK, and ERK (Figure S1). Furthermore, we observed that the phosphorylation of components of the FAK signaling pathway induced by peptide E1 was time-dependent (Figure S2). These results indicate that E1 and E2 are the minimal functional peptides of Netrin-1.

Figure 3. The minimal sequences of the EGF3 domain activate downstream pathways of Netrin-1. (A) Illustrations of the peptides. (B) Cortical neurons (DIV3) were stimulated with gradient concentrations of peptide E1. Cell lysates were incubated with anti-p-FAK-861, anti-p-SFK-418 and anti-p-ERK-202/204. (C) Cultured cortical neurons were treated with gradient concentrations of peptide E2. 

Lysates were collected and incubated with the indicated antibodies to measure the phosphorylation of FAK, SFK, and ERK. (D–F) Quantification of the extent of FAK, SFK, and ERK phosphorylation induced by peptide E1 in neurons is shown in (B). n = 3. (G–I) Quantification of the extent of FAK, SFK, and ERK phosphorylation induced by peptide E2 is shown in (C). n = 3. (J) Cortical neurons derived from DCC wild-type and homozygote mutant embryos were stimulated with Netrin-1 or Netrin-1-derived peptides and were lysed. 

The resulting lysates were subjected to immunoblotting. (K) Quantification of FAK PY861 and DCC levels in DCC mutant neurons is shown in (J). n = 3. The data are presented as the means ± SEMs. One-way ANOVA was used for all statistical analyses shown in this figure (* p < 0.05; ns, not significant). Details of data analysis are seen in Supplementary Table S1.

To determine whether the activation of the FAK signaling pathway is DCC-dependent, we measured the extent of tyrosine phosphorylation of FAK induced by these peptides in DCC+/+ and DCC−/− neurons. We found that both peptides E1 and E2 failed to induce the phosphorylation of FAK in DCC−/− neurons (Figure 3J, K). 

We also found that synthesized N-terminally Flag-tagged peptides E1 and E2 (Flag-E1 and Flag-E2) directly bind with the DCCFN5 domain (Figure S3).

2.4. Peptide E1 Protects Neurons from Hemin-Induced Cell Death In Vitro

To exhibit the protection against neuronal injury of peptide E1, we employed a Hemin inducible toxicity reaction to mimic secondary brain injury after intracerebral hemorrhage [41,42]. 

Cultured GnRH-expressing neuronal cells (NLT) [43–45] were incubated with hemin at different concentrations and durations. Incubation of NLT cells with hemin at the medial lethal dose (60 µM) resulted in a time-dependent decrease in the cell survival rate, which suggests that hemin was taken up by neurons and metabolized (Figure S4). 

To examine whether the expression of Netrin-1 and DCC responded to the hemin-induced neuronal cell death, cultured NLT cells were incubated with hemin for different durations. The western blot results demonstrated that the expression levels of Netrin-1 and DCC were increased significantly at 6 h after hemin incubation and then declined gradually (Figure 4A, B). 

To investigate whether peptides E1 and E2 were able to attenuate hemin-induced neuronal death, we performed a CCK-8 assay with Netrin-1, Pep Ctrl, peptide E1, and peptide E2. 

We found that these peptides had no detectable effect on cell viability (Figure 4C and Figure S5). Compared to incubation with Pep Ctrl and peptide E2, peptide E1 and Netrin-1 ameliorated neuronal death induced by hemin (Figure 4D). We also stained with calcein-AM and PI to distinguish live and dead cells. 

The results showed that both Netrin-1 and peptide E1 protected against hemin-induced death in vitro (Figure 4E–G). Furthermore, 1.5 µM peptide E1 alleviated hemin-induced death in primary cultured neurons (Figure 4H, I). Altogether, these results showed that peptide E1 protects neurons from hemin-induced cell death.

2.5. Netrin-1-Derived Peptide E1 Promotes Functional Recovery after ICH

To investigate whether peptide E1 derived from Netrin-1 plays an important role in functional recovery after hemorrhage stroke, we established an experimental ICH model in mice by injecting collagenase mixed with ink into the striatum (Figure S6A). 

After three days, images of the brain slice were shown to track the inject position and collagenase volume with ink (Figure S6B, C). Mice with striatal hemorrhage exhibited weakness on the ipsilateral side because of brain injury (Figure S7A–C). 

Hematoma in the striatum following ICH were visible compared with injection of saline (Figure S7D). The relative protein levels of Netrin-1 and DCC in the perihematomal cortex were increased in ICH mice compared to control mice on day 2 and the Netrin-1 was still higher significantly on day 3, indicating that Netrin-1 is important for recovery of brain injury (Figure S7E, F). 

Next, FITC-labelled TAT-peptide E1 (FITC-Pep TE1), which added a TAT sequence of the N-terminus of peptide E1, was injected into the ICH mice and the FITC-Pep TE1 was shown to cross the blood-brain barrier (Figure S8). 

The ICH and control mice were intraperitoneally injected with the peptide (12 mg/kg) daily or an equal volume of saline (Figure 5A). We found that administration with peptide TE1 improved the survival rate and rescued defects in body weight after ICH (Figure 5B, C). 

We also found collagenase volume traced with an equal ratio of ink in each group was similar, confirming this standard ICH model of collagenase resulted in reproducible intrastriatal hematomas (Figure 5D). Peptide TE1 improved the behavioral scores of the ICH mice in the tape removal test (Figure 5E) and cylinder test (Figure 5F). 

As expected, peptide TE1 reduced the infarct volume after ICH (Figure 5G, H). Taken together, these results suggest that peptide E1 was able to reduce the hematoma volume and improve functional recovery in a mouse model of ICH.

Figure 4. Peptide E1 ameliorates hemin-induced cell death in vitro. (A) Western blot of Netrin-1 and DCC in cultured NLT cells exposed to Hemin (60 µM). (B) Quantitative analysis of the western blot results is shown in (A). n = 3. (C) CCK-8 assays showed that series peptides did not change cell viability. n = 4. (D) 1.5 µM Pep E1 preserved NLT cell viability when added well after exposure to hemin (60 µM). n = 4. (E) Live (green)/Dead (red) assay of NLT cells exposed to hemin (60 µM, 6 h). Scale bar, 50 µm. (F, G) Quantitative analysis of the live/dead assay in (E). n = 9. (H) CCK-8 assays showed that Pep E1 could reduce hemin-induced cell death in primary culture neurons. n = 8. (I) Live/Dead assay imaging of primary culture neurons (50 µM, 6 h). Scale bar, 50 µm. Data are presented as the means ± SEM. One-way ANOVA or Two-way ANOVA was used for all statistical analyses shown in this figure (* p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant). Details of data analysis are seen in Supplementary Table S1.

Figure 5. Netrin-1-derived peptide E1 promotes the recovery process after ICH. (A) Schematic of the experimental paradigm of intraperitoneal (IP) injection of peptide TE1 in ICH modeling of mice. (B) The survival ratio post-ICH of each group was recorded, and the Kaplan-Meier method was employed. n = 10. (C) Body weight changes at 5 days after ICH. n = 6. (D) Collage/Saline mix with an equal ratio of ink (total 0.6 µL) injected in the striatum of C57 mice, representative images showing ink area was similar in mice treated with/without peptide TE1 5 days after ICH. (E, F) IP injection of peptide TE1 (12 mg/kg) but not TAT (12 mg/kg) control, improved behavior as monitored by a tape removal task (E) or a corner task (F). 

Saline, n = 10 individual animals; ICH, n = 6 individual animals; ICH + TAT, n = 6 individual animals; ICH + TE1, n = 8 individual animals. (G) Representative hematoxylin and eosin-stained sections in mice treated with/without peptide TE1 5 days after ICH. Scale bar, 1 mm. (H) Quantitation of hemorrhage volume in brain sections shown in (G). 

Saline, n = 10 individual animals; ICH, n = 6 individual animals; ICH + TAT, n = 6 individual animals; ICH + TE1, n = 8 individual animals. Data are presented as the means ± SEM. One-way ANOVA or Two-way ANOVA was used for all statistical analyses shown in this figure (* p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant). Details of data analysis are seen in Supplementary Table S1.

2.6. Netrin-1-Derived Peptide E1 Reduces Neuronal Apoptosis after ICH

To explore the underlying mechanisms of peptide E1 in the process of functional recovery of ICH. We detected neuronal degeneration in the perihematomal region by fluoro-jade C (FJC) staining. 

We found that the administration of peptide TE1 significantly reduced the number of FJC-positive cells in perihematoma regions, indicating that pep TE1 inhibited neuronal degeneration after ICH (Figure 6A, B). We performed TUNEL staining and found that peptide TE1 significantly decreased the number of TUNEL-positive neurons (Figure 6C, D). 

The number of TUNEL-positive astrocytes was not detectable change after peptide TE1 treatment (Figure 6E, F). In addition, by using FITC-labelled 4 kDa dextran as a tracer to indicate the permeability of the blood-brain barrier (BBB), we found that peptide TE1 did not affect the recovery of BBB permeability (Figure S9A, B). 

The serum raw fluorescence units (RFUs) levels were similar, confirming the presence of injected tracer in all the animals analyzed (Figure S9C). We also used fluorescein-labeled lectin to label microvessels but failed to observe any improvement after TE1 treatment (Figure S9D, E). Above all, these findings suggest that the peptide E1 promotes neuronal recovery after ICH by inhibiting neuronal apoptosis.

Figure 6. Netrin-1-derived peptide E1 can protect against neuronal death and degeneration, but not astrocytes after ICH. (A) Representative Fluoro-jade C-stained sections in mice treated with/without TE1 at 5 days after ICH. Scale bar, 50 µm, and 10 µm, respectively. (B) Quantitation of Fluoro-jade C positive cells in the perihematomal region (n = 4). (C) TUNEL staining showing effects of TE1 on secondary brain injury (SBI) at 5 days after ICH onsets. Representative images from Saline, ICH, ICH + TAT, and ICH + TE1 groups. 

Each group was subjected to ICH except for the Saline group. Scale bar, 50 µm, and 10 µm respectively. (D) Quantitative analyses for the percentage of TUNEL-positive neurons are shown in (C). n = 4. (E) Colocalization of TUNEL with GFAP at 5 days after ICH. Representative images from Saline, ICH, ICH + TAT, and ICH + TE1 groups. Scale bar, 50 µm, and 10 µm, respectively. (F) Quantitative analyses for the percentage of TUNEL-positive astrocytes are shown in (E). n = 4. Data are presented as the means ± SEM. 

One-way ANOVA was used for all statistical analyses shown in this figure (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant). Details of data analysis are seen in Supplementary Table S1.

3. Discussion

Our study identified a core peptide sequence of Netrin-1 that is crucial to functional recovery after ICH. By interacting with DCC, this Netrin-1-derived peptide protects against neuronal death due to secondary damage after ICH and is thus a promising treatment for ICH. 

The therapeutic application of Netrin-1, a high-molecular-weight protein, requires the identification of the minimal functional sequence of Netrin-1 that promotes downstream signaling. In our study, peptides E1 (residues 407–422) and E2 (residues 423–433), which are derived from the EGF3 domain of Netrin-1, respectively, were generated. We found that peptide E1 (residues 407–422) protected against neuronal death after ICH. 

We found that Netrin-1 and DCC expression levels were increased in NLT cells after hemin treatment. Hemin is released from hemoglobin after ICH and induces secondary injury to both glia and neuronal cells [46–48]. 

Additionally, we also observed increased protein levels of Netrin-1 and DCC following experimental ICH. Additionally, we also observed increased protein levels of netrin-1 and DCC following experimental ICH. 

These results are consistent with previous results showing that netrin-1 plays an important role in recovery after ICH [49].

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