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Stroke, or cerebral infarction, is one of the most serious complications of sickle cell anemia (SCA) in childhood, potentially leading to impaired development and life-long physical and cognitive disabilities. About one in ten children with SCA are at risk for developing overt stroke and an additional 25% may develop silent cerebral infarcts. This is largely due to underlying cerebral injury caused by chronic cerebral ischemia and vascular insult associated with SCA. We previously identified two elevated markers of cerebral injury, plasma brain-derived neurotropic factor (BDNF) and platelet-derived growth factor (PDGF)-AA, in children with SCA and high stroke risk. The objective of this study was to investigate whether neuregulin-1β (NRG-1), an endogenous neuroprotective polypeptide may also be elevated in children with SCA. Neuregulin-1β is involved in the preservation of blood brain barrier integrity and brain microvascular cell viability and is cytoprotective in conditions of heme-induced injury and ischemia. Since elevated plasma heme and ischemia are signature characteristics of SCA, we hypothesized that NRG-1 would be elevated in children with SCA, and that NRG-1 levels would also correlate with our biomarkers of cerebral injury. Plasma NRG-1, BDNF and PDGF-AA levels were measured in children with SCA and healthy Controls. Plasma NRG-1 was found to be nearly five-fold higher in those children with SCA compared to Controls. Neuregulin-1β was also positively correlated with both BDNF and PDGF-AA concentrations, but was not associated with degree of anemia, suggesting that NRG-1 production may be an endogenous response to subclinical cerebral ischemia in SCA warranting further exploration.

In malaria endemic countries, anemia in pregnant women occurs as a result of erythrocyte destruction by Plasmodium infections and other causes including malnutrition. Iron supplementation is recommended as treatment of iron-deficiency anemia. Erythrocyte destruction results in increased release of cytotoxic free heme that is scavenged by haptoglobin (Hp), hemopexin (Hx) and heme oxygenase-1 (HO-1). Paradoxically, iron supplementation in pregnant women has been reported to enhance parasitemia and increase levels of free heme. The relationship between free heme, heme scavengers, and birth outcomes has not been investigated, especially in women who are on iron supplementation. We hypothesized that parasite-infected pregnant women on routine iron supplementation have elevated heme and altered expression of heme scavengers. A cross-sectional study was conducted to determine the association between plasma levels of free heme, HO-1, Hp, Hx, and malaria status in pregnant women who received routine iron supplementation and their birth outcomes. Heme was quantified by colorimetric assay and scavenger protein concentration by ELISA. We demonstrated that iron-supplemented women with asymptomatic parasitemia had increased free heme (mean 75.6 µM; interquartile range [IQR] 38.8–96.5) compared with nonmalaria iron-supplemented women (mean 34.9 µM; IQR 17.4–43.8, P < 0.0001). Women with preterm delivery had lower levels of Hx (mean 656.0 µg/mL; IQR 410.9–861.3) compared with women with full-term delivery (mean: 860.9 µg/mL; IQR 715.2–1055.8, P = 0.0388). Our results indicate that iron supplementation without assessment of circulating levels of free heme and heme scavengers may increase the risk for adverse pregnancy outcomes.

Human cerebral malaria (HCM), a severe encephalopathy associated with Plasmodium falciparum infection, has a 20–30% mortality rate and predominantly affects African children. The mechanisms mediating HCM-associated brain injury are difficult to study in human subjects, highlighting the urgent need for non-invasive ex vivo human models. HCM elevates the systemic levels of free heme, which damages the blood-brain barrier and neurons in distinct regions of the brain. We determined the effects of heme on induced pluripotent stem cells (iPSCs) and a three-dimensional cortical organoid system and assessed apoptosis and differentiation. We evaluated biomarkers associated with heme-induced brain injury, including a pro-inflammatory chemokine, CXCL-10, and its receptor, CXCR3, brain-derived neurotrophic factor (BDNF) and a receptor tyrosine-protein kinase, ERBB4, in the organoids. We then tested the neuroprotective effect of neuregulin-1 (NRG-1) against heme treatment in organoids. Neural stem and mature cells differentially expressed CXCL-10, CXCR3, BDNF and ERBB4 in the developing organoids and in response to heme-induced neuronal injury. The organoids underwent apoptosis and structural changes that were attenuated by NRG-1. Thus, cortical organoids can be used to model heme-induced cortical brain injury associated with HCM pathogenesis as well as for testing agents that reduce brain injury and neurological sequelae.

The neuregulins Neuregulins, a family of EGF-like signaling molecules, are involved in cell-cell crosstalk and play an important role in development, maintenance and repair of the nervous system, heart, breast and other organs. Independent studies described a ligand for the oncogene ErbB2 (neu, Her2) and factors that stimulated proliferation of Schwann cells, as well as synthesis of receptors for acetylcholine by muscle. These ligands and factors are essentially products of the same gene, referred to by Marchionni M. as neuregulin (NRG-1) [1]. Besides NRG-1 gene, there are other genes that encode related proteins, such as NRG-2 (Don-1, NTAK), NRG-3 and NRG-4. There are few studies that describe NRG-2, −3 and −4 and a complete analysis of their function remains a challenge. However, NRG-2 was recently reported as a component of stress granules (SG), microscopically visible aggregates of translationally stalled messenger ribonucleoprotein complexes that are formed in response to direct stress conditions [2]. Furthermore, it was shown that NRG-2, secreted from astrocytes, bound to ErbB3 on neurons and promoted neuronal survival [3]. NRG-3 was shown to be associated with schizophrenia (SZ) in a Chinese population [4], with bipolar disorder (BD) [5] and with the risk and age of onset of Alzheimer disease (AD) [6]. In addition, NRG-3 has a key function in promoting early mammary morphogenesis and is involved in breast cancer (BC) [7]. NRG-4 expression was decreased in human inflammatory bowel disease samples and mouse models of colitis, suggesting that activation of ErbB4 is altered [8]. An interesting study [9] showed that NRG-4 overexpression prevents high fat diet-induced weight gain and fatty liver and reduces obesity-induced chronic inflammation. Considering the increasing interest and research focused on NRG-1 in the past years, this review summarizes what is currently known about NRG-1 and its impact on health and disease as well as its current and potential use(s) as a CNS anti-inflammatory agent against inducers of brain inflammation and injury as well as in the treatment of various neurological disorders. Neuregulin-1: Structure, function and signaling pathways: NRG-1 gene encodes 21 exons located on human chromosome 8p22–21. Alternative splicing of more than 30 exons in different parts of the NRG-1 transcript generates more than 30 isoforms that can be grouped into six types (I-VI) [10–12] (Figure 1). They are synthesized as transmembrane precursors consisting of either an immunoglobulin-like (Ig) domain or cysteine-rich domain (CRD), an EGF-like domain, a transmembrane domain and a cytoplasmic tail [13]. NRG-1 types I and II are released from the cell surface after protease-mediated proteolytic cleavage and may function as paracrine signals [14]. NRG-1 type III remains tethered to the cell membrane after cleavage and acts as a juxtacrine signal [15]. Full-length type IV spans 1.8 kb and encodes a putative protein of 590 amino acids with a predicted molecular mass of approximately 66 kDa [15]. Further-more, there are two major classes of NRG-1 identified as α and β isoforms. NRG-1α and NRG-1β contain related, but structurally distinct EGF-like domains composed of a common amino terminal segment followed by α or β variant sequences [16]. NRG-1β isoforms predominate in the nervous system while α isoforms are common in mesenchymal cells and are critically important for breast development [14]. Type I NRGα and β isoforms are the predominant isoforms expressed in early embryogenesis, whereas type II and type III are undetectable until the mid-gestation stage [17]. Type I or Heregulin is an acetylcholine receptor activator, type II is a Glial Growth Factor, type III is a sensory and motor neuron-derived factor and type IV is involved in neuronal activity regulation. Functions of types V and VI are not well known and their investigation remains a challenge. Types I, II and III of NRG-1 express in human peripheral blood in addition to neurons, while types IV and V express specifically in the brain [18]. Open in a separate window Figure 1: Neuregulin (NRG) and ErbB receptor structures. NRG gene products share a structural characteristic for the extracellular epidermal growth factor (EGF) domain and it differentiates this subfamily from other members of the EGF family. All EGF family members are ligands of ErbB receptors, although with varying affinities, with ErbB3 and ErbB4 being specific NRG-binding receptors. NRG-1 isoforms have been classified in types I-VI on the basis of differences in the NH2-terminal distal region [19].

Background Human cerebral malaria (HCM) is a severe form of malaria characterized by sequestration of infected erythrocytes (IRBCs) in brain microvessels, increased levels of circulating free heme and pro-inflammatory cytokines and chemokines, brain swelling, vascular dysfunction, coma, and increased mortality. Neuregulin-1β (NRG-1) encoded by the gene NRG1, is a member of a family of polypeptide growth factors required for normal development of the nervous system and the heart. Utilizing an experimental cerebral malaria (ECM) model (Plasmodium berghei ANKA in C57BL/6), we reported that NRG-1 played a cytoprotective role in ECM and that circulating levels were inversely correlated with ECM severity. Intravenous infusion of NRG-1 reduced ECM mortality in mice by promoting a robust anti-inflammatory response coupled with reduction in accumulation of IRBCs in microvessels and reduced tissue damage. Methods In the current study, we examined how NRG-1 treatment attenuates pathogenesis and mortality associated with ECM. We examined whether NRG-1 protects against CXCL10- and heme-induced apoptosis using human brain microvascular endothelial (hCMEC/D3) cells and M059K neuroglial cells. hCMEC/D3 cells grown in a monolayer and a co-culture system with 30 μM heme and NRG-1 (100 ng/ml) were used to examine the role of NRG-1 on blood brain barrier (BBB) integrity. Using the in vivo ECM model, we examined whether the reduction of mortality was associated with the activation of ErbB4 and AKT and inactivation of STAT3 signaling pathways. For data analysis, unpaired t test or one-way ANOVA with Dunnett’s or Bonferroni’s post test was applied. Results We determined that NRG-1 protects against cell death/apoptosis of human brain microvascular endothelial cells and neroglial cells, the two major components of BBB. NRG-1 treatment improved heme-induced disruption of the in vitro BBB model consisting of hCMEC/D3 and human M059K cells. In the ECM murine model, NRG-1 treatment stimulated ErbB4 phosphorylation (pErbB4) followed by activation of AKT and inactivation of STAT3, which attenuated ECM mortality. Conclusions Our results indicate a potential pathway by which NRG-1 treatment maintains BBB integrity in vitro, attenuates ECM-induced tissue injury, and reduces mortality. Furthermore, we postulate that augmenting NRG-1 during ECM therapy may be an effective adjunctive therapy to reduce CNS tissue injury and potentially increase the effectiveness of current anti-malaria therapy against human cerebral malaria (HCM).