Epidermal Growth Factor Receptor Inhibition Reverses Cellular and Transcriptomic Alterations Induced by Hypoxia in the Neonatal Piglet Brain.

Acute hypoxia (HX) causes extensive cellular damage in the developing human cerebral cortex. We found increased expression of activated-EGFR in affected cortical areas of neonates with HX and investigated its functional role in the piglet, which displays a highly evolved, gyrencephalic brain, with a human-like maturation pattern. In the piglet, HX-induced activation of EGFR and Ca2+/calmodulin kinase IV (CaMKIV) caused cell death and pathological alterations in neurons and glia. EGFR blockade inhibited CaMKIV activation, attenuated neuronal loss, increased oligodendrocyte proliferation, and reversed HX-induced astrogliosis. We performed for the first time high-throughput transcriptomic analysis of the piglet cortex to define molecular responses to HX and to uncover genes specifically involved in EGFR signaling in piglet and human brain injury. Our results indicate that specific molecular responses modulated by EGFR may be targeted as a therapeutic strategy for HX injury in the neonatal brain.

Introduction

Hypoxic (HX) encephalopathy is the major cause of death and neurodevelopmental disability in newborns (Lawn et al., 2005; Kurinczuk et al., 2010). Decreased oxygen and energy failure in the brain led to neuronal cell death (Martin et al., 1997b; Mehmet et al., 1998; DiGiacomo et al., 1992), but the cellular and molecular mechanisms of HX-induced neuronal and glial cell damage are still largely undefined. Future cell-based therapeutic interventions depend on identifying specific signaling pathways and their selective role in the pathophysiology of HX, leading to long-term neurological sequelae.

Cell death signaling in the neonatal brain involves the apoptotic and the rat sarcoma/mitogen-activated protein kinase (Ras/MAPK) pathways (Chin et al., 1997; Hognason et al., 2001), and the initial HX insult linked to activation of these pathways causes oxygen free radical formation and lipid peroxidation of the neuronal membrane. We have previously shown that HX activates a set of apoptotic enzymes in the area of focal adhesions (FAs) and a variety of membrane receptors, including the epidermal growth factor receptor (EGFR) (Kratimenos et al., 2017b; Delivoria-Papadopoulou and Malaeb, 2014). Located at the cell membrane, the EGFR plays an essential role in cell growth and proliferation and has been shown to be neuroprotective following HX via nuclear factor (NF)-κB-dependent transcriptional upregulation of cyclin D1 (Chen et al., 2016). However, EGFR activation can also induce neural cell damage and apoptotic cell death (Armstrong et al., 1994; Jackson and Ceresa, 2017).

There is significant cross-interaction between different molecular elements of the FAs network, which ultimately result in either promoting cell proliferation and motility (mitogenic) or apoptotic cell death (Kratimenos et al., 2014, 2017c). It has been shown that EGFR overexpression induces apoptosis through molecular alterations of the glutamate ionotropic receptor N-methyl-D-aspartate (NMDA)-type subunit 2B (GluN2B) (Tang et al., 2015). Modification of the NMDA receptor by oxygen free radicals promotes calcium influx into the cytosol, deactivation of protein tyrosine phosphatases, and activation of EGFR kinase (Maulik et al., 2008; Vibert et al., 2008). This series of events causes downstream activation of nuclear CaMKIV, CREB transcription, and the formation of the apoptosome and caspase activation, ultimately leading to DNA fragmentation and cell death (Mishra et al., 2009, 2010).

The newborn piglet is a powerful model to study human brain development, as it displays a highly evolved, gyrencephalic brain (Ishibashi et al., 2012; Imai et al., 2006). Furthermore, cortical development and anatomical structure are similar in piglet and human, and—like in human—approximately 50% of the piglet brain volume is represented by white matter (Felix et al., 1999; Imai et al., 2006). The similar white/gray matter ratio and developmental age at term with human brain (Thoresen et al., 1996; Haaland et al., 1997; Bjorkman et al., 2006; Odden et al., 1989; Jain et al., 2017; Martin et al., 1997a, 1997b; Brambrink et al., 1999; Guerguerian et al., 2002; Ezzati et al., 2017; Groenendaal et al., 1999; Mehmet et al., 1994, 1998; Yue et al., 1997) make the piglet an ideal preclinical model to study the cellular and developmental consequences of neonatal brain injury, including HX, and potential therapeutic interventions. Finally as the piglet shares many metabolic and physiological similarities with humans, the effects of pharmacological treatment in pigs resemble those in humans more closely than other laboratory animals (Forster et al., 2010).

In the present study, we first performed a postmortem assessment of the cerebral cortex of human neonates, and compared HX brains with appropriate controls. We found that expression of the activated (phosphorylated) EGFR was increased in the affected cortical areas in infants with HX. Therefore, we aimed to explore the functional role of EGFR-related signaling pathways in the cellular and molecular changes induced by HX in the cerebral cortex of newborn piglets. We established a regulatory role of EGFR kinase in the FAs network, by demonstrating that EGFR blockade before HX significantly reduced the cellular and anatomical damage induced by the injury. We also used RNA sequencing (RNA-seq) and transcriptomic bioinformatics to define for the first time gene regulatory networks induced by HX in the piglet cerebral cortex, and their regulation by EGFR signaling. Our transcriptomic analysis showed that a significant number of genes crucial for neuronal development and functional differentiation were differentially expressed during HX in piglet cortex, but were normalized following EGFR blockade. Importantly, a number of these genes are also known to be involved in human brain injury, further supporting the notion that phosphorylated EGFR plays a crucial role in HX-induced apoptotic cell death in the piglet and human perinatal brain (Dietrick et al., 2020; Korhonen et al., 1998; Maussion et al., 2019; Yu et al., 2016; Pardini et al., 2014; Yue et al., 2017).

Results

HX Induces EGFR Activation in the Cerebral Cortex of Human Newborns

In a term neonate, acute HX mainly affects the cerebral cortex and basal ganglia. Therefore, we initially performed a neuropathological assessment of the cerebral cortex of postmortem human neonates with HX (n = 5) (Figure 1A, red box). We included neonates with the diagnosis of hypoxic-ischemic encephalopathy (moderate or severe) that were not subjected to hypothermia protocol for neuroprotection. We utilized postmortem brain tissue from subjects who succumb to sudden infant death syndrome to serve as controls (neonates without or with minimal exposure to HX and inflammation, n = 5) from National Institutes of Health NeuroBioBank (Table S1). Using H&E, we noticed that in neonates with HX the deep cortical layers (III–V) were depopulated, with numerous apoptotic profiles and profound edema, when compared with controls (Figure 1B). It has been demonstrated that HX activates the EGFR and its downstream cell death pathways (Chen et al., 2016; Delivoria-Papadopoulos et al., 2011a; Mishra et al., 2010; Scafidi et al., 2014). Therefore, we assessed expression of the activated EGFR in deep cortical layers and found enhanced expression of activated EGFR (number of phosphorylated EGFR-expressing cells) in the affected cortical areas of neonates with HX, when compared with controls (Figure 1C).

Figure 1

HX Induces EGFR Activation in the Cerebral Cortex of Human Newborns with HX

(A) Tissue sections from the parietal cerebral cortex of neonates with HX and controls were analyzed.

(B) Representative H&E photomicrographs. In controls, well-demarcated pyramidal neurons with well-defined nucleolus and dense neuropil were observed (upper left panel). In contrast, in HX (upper right panel) pyknotic neurons (black arrowhead) were present, with damaged irregular nucleolus, diffuse edematous matrix, and hypodense neuropil. Phosphorylated EGFR immunostaining (brown) is depicted in the lower panels. Almost no EGFR staining was present in the controls (lower left panel), whereas multiple cortical neurons were positive for EGFR in the HX group (white arrowheads).

(C) Immunostaining intensity from (B) is quantified as the number of EGFR-immunopositive cells. HX, hypoxic (n = 5), controls (n = 5). Data are represented as mean ± SD, t test ∗p < 0.05. (B) Photographed at 40X. Scale bar, 10 μm. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com [smart.servier.com].

HX Induces EGFR Activation and Changes in Biochemical Energetics in Piglet Cortex

Based on our findings in the cortex of human neonates with HX, we assessed the effect of HX on EGFR function in the piglet cortex (Figure S1A). We first measured the enzymatic activity of EGFR. In line with our findings in human neonates, phosphorylated EGFR activity was significantly increased following HX (Figure S1B). To test the functional role of EGFR signaling in HX, we used a pharmacological inhibitor (EGFRi; PD-168393; 1 mg/kg) and established its potential to cross the blood-brain barrier by measuring enzymatic activity of the receptor in the brain. PD-168393 is a selective, cell-permeable pharmacologic compound with anti-tumor action that was initially used in cancer-related research for the treatment of EGFR-expressing tumors (Pu et al., 2006). Pretreatment with EGFRi significantly reduced the activity of EGFR kinase in the piglet cortex (Figure S1B), demonstrating its potential to cross the blood-brain barrier.

To optimize our study design, and ensure that all piglets exposed to HX (with and without EGFRi) achieved similar and comparable degree of injury, we performed direct biochemical measurements of two oxidative biometabolites—ATP and phosphocreatine (PCr)—in cortical tissue immediately after HX. The extent of HX in the piglet cortex was reflected in changes in biochemical energetics. Both ATP and PCr levels were significantly reduced in all HX groups, when compared with the normoxic (NX) groups (Figures S1C and S1D). No significant difference in cerebral tissue high-energy compounds was found in the HX groups with and without EGFRi, indicating that similar levels of HX were achieved in both groups (Figures S1C and S1D). Pretreatment with EGFRi did not reverse the energy failure due to HX. Finally, a number of physiological parameters examined were also similar in the HX and EGFRi-HX groups, and both groups displayed similar and significant differences from physiologic data obtained in NX piglets (Table S2), confirming that a similar degree of HX was achieved in HX and EGFRi-HX groups.

EGFR Blockade Attenuates the Neuropathological Alterations Induced by HX in Piglet Cerebral Cortex

Based on the biochemical results, we aimed to define the cellular effects of HX in the piglet cerebral cortex, and determined whether EGFRi pretreatment also affected HX-induced changes in cellular dynamics. First, we evaluated the overall cortical neuropathology following HX using H&E and a previously established and validated newborn piglet neuropathology score (Hoque et al., 2014; Kratimenos et al., 2017a, 2017b). The neuropathology score [median (interquartile range [IQR])] was 0 (0–1) in NX (n = 5) (p < 0.05 versus HX), 4 (3–4) in HX (n = 6) (p < 0.05 versus NX), and 2 (1–3) in EGFRi-HX (n = 7) (p < 0.05 versus HX) (Figures 2A–2C). In the superficial layers of the cortex (layers I and II), HX resulted in diffuse edema, very prominent laminar necrosis, disrupted neuropil, vacuolization, and a significant increase in necrotic neurons (Figures 2A, 2B, and 2D). Although we observed a positive trend, EGFRi did not significantly reverse the HX-induced edema of layers I and II in the cortex (p = 0.06, NS). In deep cortical layers III–V, HX resulted in neuronal cell death in the form of apoptosis, necrosis, or a continuum of hybrid forms, identified by hyperchromatic, pyknotic nuclei with ruptured and irregularly bordered nuclear membranes, and hypodense neuropil (Figure 2A). In contrast, EGFRi treatment resulted in fewer injured neurons, decreased number of apoptotic profiles, and the presence of well-defined round nuclei, i.e., it improved the overall neuropathology associated with HX (Figures 2A–2C).

Figure 2

EGFR Blockade Attenuates Neuropathological Alterations Induced by HX in Piglet Cerebral Cortex

(A–D) (A–C) Representative photomicrographs of H&E staining from the cingulate and parietal cortex of NX, HX, and EGFRi HX piglets. In the superficial layers of the cerebral cortex (layers I–II), HX resulted in diffuse edema, very prominent laminar necrosis (depicted as pale zone, red arrowheads), disrupted neuropil (hatched box, layers I–II), vacuolization (black arrowheads, layers III–IV), and necrotic neurons (blue arrowheads, layer V). (A and D) The thickness of the molecular layer (ML) was increased in HX compared with NX. In the deep cortical layers (III–V), HX resulted in neuronal cell death hyperchromatic, pyknotic nuclei with ruptured and irregularly bordered nuclear membranes, and hypodense neuropil (A–C). In contrast, EGFRi blockade normalized or improved the neuropathological alterations observed in the HX group (C). The cortical neuropathology was assessed using a validated newborn piglet neuropathology score (Hoque et al., 2014) and the level of section was at level 5 (L5) based on the stereotaxic atlas of pig brain (Felix et al., 1999). NX, normoxic (n = 5), HX, hypoxic (n = 6); EGFRi HX, epidermal growth factor receptor blockade and HX (n = 7); ML, molecular layer; data are represented as mean ± SD, one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (A) Photographed at 2.5X (upper panel) and at 40X (lower panels). Scale bar, 100 μm in the upper and 20 μm in the lower panels (layers I –V). This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com [smart.servier.com].

EGFR Blockade Reverses HX-Induced Reduction in Neurons and Cell Death in Piglet Cerebral Cortex

Next, we determined the specific role of EGFR in neuronal survival in the piglet cortex. Positive immunostaining for NeuN was used as a mature neuronal marker. HX caused a decrease in the number of NeuN+ neurons in deep cortical layers III–V (Figures 3A–3C), indicating that deep cortical neurons were significantly affected by injury. EGFRi blockade significantly reversed the effects of HX in neurons of these layers (Figures 3A–3C). In addition to neuropathology, DNA fragmentation was also assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and immunofluorescence microscopy. NX piglets did not display any significant TUNEL labeling (Figures 3A, 3B, and 3D). HX increased the total number of TUNEL-labeled cells, but fewer TUNEL-positive cells were found in HX piglets treated with EGFRi (Figures 3A, 3B, and 3D). Finally, we evaluated overall cell survival by cleaved Caspase-3 immunostaining. HX caused a 4/5-fold increase in the number of cleaved Caspase-3+ cells (Figures 3A, 3B, and 3E), which was almost completely reversed by EGFRi treatment (Figures 3A, 3B, and 3E).

Figure 3

EGFR Blockade Reverses HX-Induced Reduction in Neurons and Cell Death in Piglet Cerebral Cortex

(A–E) (A and B) Representative photomicrographs of NeuN, TUNEL, and cleaved Caspase-3 immunostaining in layers III–V of the cingulate and parietal cortex (A) of NX, HX, and EGFRi HX piglets. HX resulted in a decrease in the number of NeuN+ neurons in the cortical layers III–V (B and C) and increased cell death, as shown by TUNEL+ and cleaved Caspase-3+ cells (B, D, and E). Pretreatment with EGFR blockade reversed the effects of HX on neurons as well as overall on cell death after injury (B–E). Note the characteristic apoptotic profiles in TUNEL+ cells (white arrowheads) and the cleaved Caspase-3+ cells (black arrowheads). TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′, 6-diamidino-2-phenylindole; NX, normoxic (n = 5), HX, hypoxic (n = 5); EGFRi HX, epidermal growth factor receptor blockade and HX (n = 5). Data are represented as mean ± SD, one-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (B) Photographed at 60X (NeuN and TUNEL) and 100X (cleaved Caspase-3). Scale bar, 10 μm (NeuN and TUNEL) and 5 μm (cleaved Caspase-3).

EGFR Blockade Reverses HX-Induced Gliosis in Piglet Cerebral Cortex

In the next set of experiments, we determined the specific role of the EGFR in the response of different types of glial cells to HX in the piglet cortex. HX induced a 3-fold increase in the number of GFAP+ reactive astrocytes and in Olig2+ GFAP+ cells (Figures 4A–4C), indicating that distinct glial cell types were affected by injury. Conversely, HX caused a significant decrease in the number of Olig2+ GFAP− oligodendrocyte lineage cells (Figures 4A and 4D), partially due to a reduction in cell proliferation, as demonstrated by anti-Ki67 immunostaining in GFAP− cells (Olig2+Ki67+ cells that do not express GFAP) (Figures 4A and 4E). Increased astrogliosis was due to enhanced cell proliferation, as demonstrated by anti-Ki67 immunostaining in GFAP+ astrocytes (Figures 4A and 4F). EGFRi treatment reversed the effects of HX on the number of Olig2+ oligodendrocytes, and normalized reactive gliosis after injury (Figures 4A–4C, 4E, and 4F). Finally, EGFRi treatment also reversed the overall inhibitory effects of HX on oligodendrocyte proliferation (Figures 4A and 4E). Altogether, these findings indicate that HX induces differential effects on distinct types of glia in the piglet cortex, i.e., causes reactive gliosis in astrocytes and inhibits oligodendrocyte lineage cell proliferation. Importantly, both effects are reversed by EGFR inhibition before HX, indicating that EGFR activation induces the reactive astrocytosis and the oligodendrocyte lineage injury following HX.

Figure 4

EGFR Blockade Reverses HX-Induced Gliosis in Piglet Cerebral Cortex

(A–F) (A) Representative photomicrographs of Olig2+, GFAP+, and Ki67+ cells from the cingulate and parietal cortex of NX, HX, and EGFRi HX piglets. (A, B, and C) HX induced a significant increase in the number of GFAP+ reactive astrocytes and of Olig2+ GFAP+ cells (yellow arrowheads). (A, D, and E) HX also caused a significant decrease in the number of Olig2+ GFAP− oligodendrocyte lineage cells, due to a reduction in cell proliferation, as demonstrated by the number of Olig2+Ki67+ that did not express GFAP (white arrowheads). (A and F) Anti-Ki67 immunostaining in GFAP+ cells (A, green arrowheads) indicates that increased astrogliosis was due to an increase in cell proliferation. (A, B, C, and F) EGFRi treatment reversed all the effects of HX on astrogliosis. (A and E) Finally, EGFRi treatment also reversed the overall inhibitory effects of HX on oligodendrocyte proliferation. GFAP, glial fibrillary acidic protein; Olig2, oligodendrocyte transcription factor; DAPI, 4′,6-diamidino-2-phenylindole; NX, normoxic (n = 5); HX, hypoxic (n = 5); EGFRi HX, epidermal growth factor receptor blockade and HX (n = 5). Data are represented as mean ± SD, one-way ANOVA, ∗p < 0.05, ∗∗p < 0.001, ∗∗∗∗p < 0.0001; (A) Photographed at 40X (GFAP and Olig2/ki67) and at 60X (Olig2/GFAP and Ki67/GFAP). Scale bar, 10 μm in GFAP, Olig2/GFAP/DAPI, and Olig2/ki67/DAPI and 5 μm in ki67/GFAP/DAPI.

EGFR Blockade Reverses the Effects of HX on CaMKIV Activation in Piglet Cortex

We aimed to explore whether EGFR interferes with specific signaling pathways activated by HX in the piglet brain (Chakraborty et al., 2014; Chin et al., 1997; Ferriero, 2004; Jackson and Ceresa, 2017; Kratimenos et al., 2017a; Soderling, 1999; Treda et al., 2016). As calcium plays a central role in cell signaling, we focused on the relationship between EGFR activation and intracellular Ca2+. Expression of tyrosine-phosphorylated CaMKIV and its enzymatic activity were significantly enhanced in the piglet cortex after HX (Figures S2A and S2B). EGFRi treatment prevented HX-induced increase in CaMKIV expression and activity (Figures S2A and S2B), indicating that EGFR inhibition directly interfered with the calcium signaling pathway activated by HX in the piglet cortex.

Transcriptomic Changes Induced by HX in Piglet Cortex Are Reversed by EGFR Blockade

To further investigate molecular mechanisms underlying the effects of HX in the piglet cortex and to establish the functional role of EGFR, we defined the transcriptome of early postnatal piglet cerebral cortex using RNA-seq. All reported differentially expressed genes (DEGs) had normalized read counts above 5 in all samples and reached statistical significance (adjusted p < 0.05). Distinct expression patterns were evident between NX, HX, and EGFRi HX (Figure 5A). Differential gene expression analysis between the groups revealed 522 DEGs between HX and NX cerebral cortex tissue (318 up-regulated, 204 down-regulated), 786 DEGs between EGFRi-HX and HX (331 up-regulated, 456 down-regulated), and 158 DEGs between EGFRi-HX and NX (118 up-regulated and 40 down-regulated) (Figure 5A and Data S1, S2, and S3).

Figure 5

Transcriptomic Changes Induced by HX in Piglet Cortex Are Reversed by EGFR Blockade

(A) Heatmap of average expression of the total DEGs across the three study groups (NX, HX, and EGFRi HX). The color bar to the right indicates expression levels, where cool/blue represents lowest levels and warm/red indicates highest levels. Source data are provided as a Source Data file.

(B and C) Bar diagrams depicting the distribution of GO terms in HX versus NX and EGFRi HX versus HX. Criteria for inclusion as a highly enriched function required a –log adjusted p value of ≥5, shown by the horizontal p threshold line. DEGs based on statistical analysis using the Wald test with Benjamini-Hochberg correction (adjusted p < 0.05). GO terms in red color highlight nervous system-specific functions.

(D) Venn diagram depicting DEG overlap in HX versus NX, EGFRi HX versus HX, and EGFRi HX versus NX. GO, Gene Ontology; NX, normoxic (n = 2); HX, hypoxic (n = 3); EGFRi HX (n = 3), epidermal growth factor receptor blockade and HX. GO term lists, specific transcripts and names of genes associated with them are shown for (C) and (D) in Data S5 and S6.

Interestingly, specific genes that are known to be involved in human brain injury, such as BDNF, CAMKK2A, CAMK2A, DRD1, DRD2, FEZF2, and CREB5 (Dietrick et al., 2020; Korhonen et al., 1998; Maussion et al., 2019; Yu et al., 2016; Pardini et al., 2014; Yue et al., 2017), were also altered in the piglet cortex, further emphasizing the significance and clinical relevance of the piglet model of neonatal HX brain injury. The above transcripts identified through RNA-seq as altered by HX and EGFRi HX were confirmed by RT-PCR (Figures S3A–S3G).

Next, using g:Profiler (Raudvere et al., 2019), we sorted DEGs via enriched functional Gene Ontology (GO) terms, to reveal HX-induced alterations in biological functions and to define the effects of EGFRi treatment (Figures 5B and 5C). Of the top 25 predicted biological functions to be altered in HX, 12 specifically represented functions of synaptic signaling, synaptic transmission, and regulation of nervous system development, neuronal differentiation, and neurogenesis. After EGFRi treatment, 13 of the top 25 biological functions were specifically associated with nervous system development (top enriched function), including neurogenesis, neuronal differentiation, and development of neuronal projections, and 6 GO terms were associated with synaptic signaling/synaptic transmission (HX versus NX: 521 DEG annotated genes, 142 human homologs; EGFRi-HX versus HX: 656 DEG annotated genes, 773 human homologs; EGFRi-HX versus NX: 128 DEG annotated genes, 37 human homologs) (Figures 5C and 5D and Data S4). A Venn diagram of overlap between the 3-group comparisons showed that HX resulted in 521 DEGs versus NX, and that 183 of these DEGs were also significantly altered by EGFRi (Figure 5D).

As neuronal development and synaptic transmission were predicted to be altered based on the GO analysis, heatmaps of DEGs from both groups were created. This analysis revealed distinct groups of DEGs related to these functions that were affected by HX, and were either responsive to EGFRi (Figures 6A and 6B) or unresponsive to EGFRi treatment (Figures 6C and 6D). Descriptive names for all genes are provided in Supplemental Data (Data S5 and S6). Of note, one of the neurotrophins, the brain-derived neurotrophic factor (BDNF), was significantly altered by HX and fully reversed by EGFRi treatment (Figures 6A, 6B, and S3G and Table S3).

Figure 6

EGFR Blockade Normalizes HX-Induced Alterations in Specific DEGs Associated with Nervous System Development and Synaptic Transmission

(A–D) Heatmaps derived from genes identified in the GO term analysis that are associated with either nervous system development (A and C) or synaptic transmission (B and D). Expression levels of DEGs that were altered by HX and by HX EGFRi are shown in the top maps (A and B). On the bottom (C and D) DEGs that were altered by HX, but not by HX EGFRi are shown. Transcripts in (A) and (B) are distinct from those in (C) and (D), indicating that although EGFRi affects the expression of a broad group of genes altered by HX, other genes involved in neuronal development and synaptic transmission that are altered by HX are not affected by EGFRi. DEG, differentially expressed genes based on statistical analysis using the Wald test with Benjamini-Hochberg correction (adjusted p < 0.05). GO, Gene Ontology; NX, normoxic (n = 2); HX, hypoxic (n = 3); EGFRi HX (n = 3), epidermal growth factor receptor blockade and HX. Gene symbols and individual descriptions for the transcripts in the maps are shown in Data S5.

HX Induces Transcriptional Alterations in Ca2+ and EGFR-Mediated Signaling Pathways that Are Prevented by EGFR Blockade

Next, we utilized Ingenuity Pathway Analysis (IPA; Qiagen) to identify specific canonical pathways and upstream regulators altered by HX either in the presence or absence of EGFR blockade. Importantly, EGFR was identified as upstream regulator in HX-induced expression of many genes (SCUBE3, NELL1, EDIL3, ERBB3, Neuregulin 3, GAB1, DRD1, DRD2, MAPK10, MAPK11, MAPK8IP2, MAP3K10, MAP3K5, MAP4K4, ABL, FEFZ2, and MAPK1) (Figure 7A; see also Figures 5 and 6), and EGFR blockade normalized many of these alterations (Figure 7B). Interestingly, HX also altered the expression of numerous genes directly related to calcium signaling (Figure 7A). In line with our results on CaMKIV signaling, EGFR blockade reversed HX-induced transcriptomic alterations related to calcium signaling, indicating that EGFR mediates HX-induced neuronal injury through calcium signaling pathways (Figure 7B).

Figure 7

HX Induces Molecular Alterations in EGFR-Mediated Signaling Pathways That Are Prevented by EGFR Blockade

(A and B) Circos plots depicting the main EGFR-associated signaling pathways altered by HX (A) and reversed by EGFR blockade (B) in piglet cerebral cortex. Transcript symbols outside the circle depict individual DEGs. The log fold change (fc) for each DEG is shown in the outer circle bars where fc for HX versus NX is shown in (A) and fc for HX EGFRi versus HX is shown in (B). Interactions between EGFR-associated pathways are shown by the colored-coded connections; G-protein-coupled receptor signaling pathway (red), calcium-mediated signaling (light green), cAMP-mediated signaling (fluorescent green); ErbB signaling (blue), neuregulin signaling (purple). DEG, statistically differential expression of genes, using the Wald test with Benjamini-Hochberg correction (adjusted p < 0.05). NX, normoxic (n = 2); HX, hypoxic (n = 3); EGFRi HX (n = 3), epidermal growth factor receptor blockade and HX.

Of note, EGFR blockade also prevented HX-induced differential expression of genes related to G-protein, ErbB, cyclic AMP (cAMP), and Neuregulin signaling pathways (Figure 7B). G-proteins, cAMP, and ErbB signaling pathways play a significant role in cell proliferation and death, and their differential expression in the cerebral cortex might contribute to altered neuropathology and developmental disabilities. The Neuregulin pathway (ERBB3, NRG3, AKT3) plays a significant role in the plasticity of the developing brain and is known to have a protective role following HX injury (Corfas et al., 2004; Yoo et al., 2019).

Altogether, our RNA-seq analysis offers a wealth of new information regarding complex changes in specific gene networks that govern neuronal functions in the piglet brain under normal physiological conditions and after HX. This analysis also defines the impact of EGFR inhibition on a variety of dynamic molecular changes underlying cellular recovery from perinatal HX in the developing piglet cortex.

Discussion

In the present study, we define a regulatory function of EGFR in acute HX of the cerebral cortex of the newborn piglet. We also establish a role of EGFR-associated signaling pathways in the molecular, cellular, and neuropathological outcome of the injury. Finally, we present for the first time a high-throughput analysis of the newborn piglet brain, which provides novel and crucial insights into HX-induced molecular alterations in neurotransmitters and neurotrophins, as well as abnormalities in neuronal development and synaptogenesis. Importantly, we identified genes that are directly associated with HX-induced EGFR signaling that are also altered in human brain injury (Dietrick et al., 2020; Korhonen et al., 1998; Maussion et al., 2019; Yu et al., 2016; Pardini et al., 2014; Yue et al., 2017). Moreover, we identify transcripts regulated by HX that could not be mapped to the annotated pig genome, but were mapped to the more complete human genome (human homologs) (Data S4). Overall, these data not only support the significance of our molecular findings, but—more broadly—point to the translational impact of the piglet injury model used in this study.

EGFR kinase is a well-established promoter of cell proliferation, neuronal growth, and regeneration. However, EGFR activation may induce cell proliferation and growth in some cell populations, and apoptosis in others, depending on type of injury, brain region, and developmental stage of the brain (Armstrong et al., 1994; Jackson and Ceresa, 2017). Mice lacking EGFR die in utero or shortly after birth, or undergo significant neurodegeneration (Craig et al., 1996). Previous data in rodents revealed that EGFR promotes the progression of stem cells to proliferative progenitor cells (Kuhn et al., 1997), but plays different roles in distinct brain cell populations. In a mouse model of neonatal HX injury, activation of EGFR in glial progenitors promoted oligodendrocytes regeneration and timely developmental myelination, together with functional recovery (Scafidi et al., 2014). Conversely, administration of EGFR agonists negatively affected neurogenesis and neuroblast development (Koprivica et al., 2005; Kuhn et al., 1997). EGFR overexpression induced apoptosis in some neuronal cell lines through modification of the GluN2B subunit of the NMDA receptor (Tang et al., 2015). EGFR was also found to be highly expressed in brain malignancies; however, its overexpression was due to HX in the tumor core rather than to genetic alterations, which is consistent with the fact that human tumors that overexpress EGFR often lack a receptor mutation (Franovic et al., 2007).

Activation of the EGFR by auto-phosphorylation leads to binding of the adaptor protein Grb2 in the cytosol and activation of the Ras exchange factor Son of Sevenless (Sos). These molecular events result in activation of the Ras/MAPK signaling cascade, and downstream activation of nuclear mechanisms leading to Ca2+ influx in the nucleus itself, and CREB transcription and caspase-dependent cell death (Delivoria-Papadopoulos et al., 2008; Hognason et al., 2001). Our analysis in the developing piglet cerebral cortex shows that EGFR blockade before HX prevents injury-induced activation of nuclear enzymes. The activity of nuclear CaMKIV was enhanced by HX, but attenuated following EGFR inhibition, indicating that EGFR function also impacts nuclear function. In our previous studies, we demonstrated that inhibition of Src kinase following HX leads to attenuated neuropathological alterations and reduced activation of the nuclear enzymes (Kratimenos et al., 2017a, 2017b). Furthermore, decreased activation of CaMKIV is reflected in improved neuropathology in the neonatal brain (Kratimenos et al., 2017b). Based on these findings, it can be hypothesized that a functional cross talk between EGFR and Src kinases exists, ultimately resulting in downstream activation of nuclear CaMKIV.

The significant involvement of EGFR in HX-induced cortical injury and associated cellular outcomes highlights its importance as a potential target for therapeutic intervention. However, the precise molecular mechanisms that link HX to activation of EGFR and the ultimate cellular/neuropathological outcomes are still undefined. It has been shown that EGFR may interact with the SH2 homolog domain of the Src kinase leading to phosphorylation of the c-terminal of the molecule (Wagner et al., 2013). Conversely, Src can phosphorylate the EGFR kinase at a specific tyrosine residue (Y845), resulting in downstream activation of Ras/MAPK (Jackson and Ceresa, 2017). Therefore, targeting specific regulatory enzymes of the apoptotic pathway with small molecules may be an effective way to interrupt the neural cell death signaling cascade initiated by HX and EGFR activation.

Consistent with the notion of a crucial role of EGFR in HX-induced injury, we demonstrate that increased activation of EGFR kinase is reflected in significant neuropathological changes following HX of the newborn piglet brain, mainly in the deep layers of the cortex. We also show that EGFR blockade protects cortical neurons from HX-induced injury in deep cortical layers. It is hypothesized that the superficial layers of the cortex receive additional blood supply by the meningeal vessels, and thus become more resistant to HX. In agreement with our observations in human, the cortical layers III–V were more severely affected by HX, displaying highest levels of neuronal abnormalities, including cell death. Neurons of deep layers of the cerebral cortex highly express the Forebrain embryonic zinc finger 2 (Fezf2), which is important for the development of corticospinal projection neurons, as well as for differentiation of neuronal stem cells in the subventricular zone (Chen et al., 2005; Zuccotti et al., 2014). Our validated transcriptomic analysis indicated that Fezf2 was up-regulated in HX (by 2.5-fold, when compared with NX), whereas EGFR blockade totally prevented its up-regulation (Figure S3F). In the mature brain, Fezf2 has a significant role in neuronal signaling and plasticity, and in cell adhesion molecules and calcium signaling pathways (Le Pichon et al., 2013). In summary, EGFR may play an important functional role in injury-induced alterations of Fezf2 during a crucial developmental period, which may ultimately result in disrupted deep cortical neuronal signaling and plasticity of the developing brain.

In addition to extensive effects of EGFR-mediated HX injury on neurons, our analysis also revealed a role for EGFR signaling in HX-induced glial cell activation in the piglet cerebral cortex after HX. However, HX had distinct effects on astrocytes and oligodendrocytes, as it promoted astrocyte proliferation, but reduced oligodendrocyte lineage cell proliferation and number. These findings are consistent with analysis in human brain showing that neonatal HX causes reactive gliosis, as well as reduced oligodendrocyte maturation and myelination (Dean et al., 2011; Bruce and Becker, 1991; Back, 2017). The opposite effects of HX on astrocytes and oligodendrocytes were reversed by EGFR blockade, demonstrating that the EGFR pathway plays different roles in distinct glial cell types. Future analysis will further investigate the intracellular signaling pathways associated with EGFR in these glial cell types, to define the molecular mechanisms leading to opposite cellular outcomes.

To elucidate the molecular underpinnings of the cellular effects induced by HX, and to gain further mechanistic insight into the functional role of EGFR, we performed high-throughput analysis of the piglet cerebral cortex under different experimental conditions. Our analysis revealed that HX-induced alterations related to calcium signaling, synaptogenesis, and neuronal cell proliferation and death were normalized by EGFR blockade. Furthermore, our molecular analysis revealed that genes involved in cell death pathways, including calcium, cAMP, erbB, Neuregulin, and G-protein signaling, were significantly altered during HX, and EGFR blockade prevented their differential expression. In particular, HX-induced EGFR phosphorylation mediated activation of the apoptotic pathway through a variety of regulatory molecules of calcium signaling, including CAMK2A, CAMKIV, CAMKK2, and CREB5. EGFR blockade completely reversed the differential expression of these genes, resulting in normalization of CAMKIV, Caspase-3, and TUNEL, and ultimately attenuated neuropathological alterations.

Importantly, BDNF was significantly altered by HX and fully reversed by EGFRi treatment. BDNF has been shown to be a main biomarker in human neonatal HX encephalopathy, with most studies suggesting lower levels of BDNF during HX (Dietrick et al., 2020; Korhonen et al., 1998; Liu et al., 2013). BDNF promotes the survival and differentiation of neurons, mediates axonal growth and pathfinding, and promotes dendritic growth and morphological maturation. BDNF also contributes to adaptive neuronal responses and synaptic plasticity, as well as homeostatic regulation of intrinsic neuronal excitability (Failla et al., 2016; Munoz et al., 2017).

Interestingly, other genes related with EGFR signaling were differentially expressed in HX. Among these, we identified a subset of genes that are known to be important for brain development, such as the dopamine D1 and D2 (DRD1 and DRD2) receptors. Dopamine receptors are known to be involved in brain development, neuronal cell migration, and formation of connectivity, as well as in neuropsychiatric diseases such as schizophrenia, or drug addiction and motor learning disabilities (Sillivan and Konradi, 2011; Bertran-Gonzalez et al., 2008). These results suggest that some of the long-term functional abnormalities observed after HX in the human brain might be, at least in part, mediated by alterations in dopamine receptor signaling.

In conclusion, our analysis of the developing piglet cerebral cortex defines many new exciting avenues of scientific exploration to further elucidate the beneficial impact of EGFR blockade on perinatal brain injury at the cellular and molecular levels. This analysis could potentially result in the identification of new therapeutic targets associated with EGFR signaling in the developing mammalian brain that are linked with specific long-term abnormalities caused by perinatal brain injury.

Limitation of the Study

We acknowledge several limitations to our study. Our main goal in this study was to understand mechanistically how EGFR phosphorylation (activation) during HX results in neuronal injury and whether inhibition of EGFR can prevent neuronal injury during HX. Therefore, we chose to administer the inhibitor 30 min before HX. Using a stepwise approach, now that we have demonstrated that HX-induced activation of EGFR is a mechanism involved in neuronal damage, our next goal is to study the effect of EGFR inhibition following HX at different time points after HX. We will address time points after induction of HX. We will also test EGFR inhibition in combination with therapeutic hypothermia for HX ischemia.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Vittorio Gallo (vgallo@childrensnational.org).

Material Availability

This study did not generate new unique reagents.

Data and Code Availability

The RNA sequencing data have been deposited to Sequence Read Archive (SRA) database (Submission ID: SUB8338144, BioProject ID: PRJNA670468, accession link: https://submit.ncbi.nlm.nih.gov/subs/sra/SUB8338144/overview). Histology and western blot and PCR data are available upon request by Dr. Gallo and Dr. Kratimenos. Enzyme activity data and biological experiment information are available at Dr. Delivoria-Papadopoulos’ lab at Drexel University.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.