Neurodegeneration refers to the progressive and selective atrophy and loss of the structure and function of neurons, which subsequently leads to cognitive and memory dysfunction. Several well-established pathways promote neurodegeneration, including mitochondrial dysfunction (Norat et al., 2020; Pandya et al., 2021; Bustamante-Barrientos et al., 2023; Bartman et al., 2024), oxidative stress (Barnham et al., 2004; Kim et al., 2015; Pandya et al., 2021), and pathological protein aggregation (Ross and Poirier, 2004, 2005; Monaco and Fraldi, 2020; Moda et al., 2023). Recent data suggest that blood-derived molecules, including extracellular hemoglobin (Hb) and heme (ferriprotoporphyrin IX), may also play crucial roles. Hb and heme within red blood cells (RBCs) are vital for the transport of gas molecules (NO, CO, and CO₂) and nutrients into the peripheral circulation. However, when these molecules are exposed to the extracellular environment, they acquire secondary functions, such as mediating inflammation (Ganz, 2012; Meegan et al., 2020; Qin et al., 2022; Sharma et al., 2022), cytotoxicity (Hsia and Everse, 1996; Jeney et al., 2002; Wang et al., 2002; Etzerodt et al., 2013), cellular apoptosis (Kim et al., 1995; Meguro et al., 2001; Qin et al., 2022), and disruption of the blood–brain barrier (BBB) (Butt et al., 2011; Bamm et al., 2015; Drvenica et al., 2022) and may be involved in the pathology of neurodegenerative diseases (NDs). The increase in BBB permeability, which is a characteristic of natural aging (Schaer et al., 2013; Thomsen et al., 2013) or a consequence of neurodegenerative pathologies (Strijkova-Kenderova et al., 2022), may allow the passage of neurotoxic Hb and heme to the brain parenchyma, altering the phenotype of the supporting neuroimmune cells that contribute to the progression of NDs.

Accumulating evidence has indicated an association between extracellular Hb/heme and Alzheimer’s disease (AD) and Parkinson disease (PD) development (Lara et al., 2009; Abbott et al., 2012; Chuang et al., 2012; Altinoz et al., 2019; Drvenica et al., 2022; Pal and Dey, 2023; Campomayor et al., 2024). Delaying the progression of these diseases remains a challenge because of their ambiguous pathological mechanisms. To date, studies using different approaches have been conducted to identify novel molecular pathways of pathogenesis in an attempt to identify new therapeutic targets for AD and PD. This review elaborates on the molecular insights into the oxidative and pro-inflammatory activities of extracellular Hb and heme. Furthermore, we present an updated overview of the molecular mechanisms by which Hb and heme contribute to neuropathological changes in AD and PD, and propose potentially rational targeted therapies that warrant further investigation.

Extracellular Hb and heme may originate from the intravascular hemolysis of RBCs, transfusions of Hb-based blood substitutes, or hematological diseases (Rifkind et al., 2015; Chen et al., 2023). The average lifespan of RBCs may vary from 70 to 140 days (Franco, 2012), and even under physiological conditions, RBC lysis occurs prior to the removal of aged cells from the circulation (Franco et al., 2013). In this context, extracellular Hb and heme production may be amplified in pathological conditions caused by blood-related disorders, such as hemolytic anemia, sickle cell anemia, and thalassemia, which are characterized by an increase in RBC hemolysis (Nagababu et al., 2008; Zhou et al., 2011; Rifkind et al., 2015). Under normal conditions, cells maintain a delicate balance between reactive oxygen species (ROS) production and neutralization by the antioxidant system. Abnormal ROS generation or inefficient antioxidant defense mechanisms can lead to oxidative stress, contributing to cellular damage. The binding and transport of oxygen by Hb result in the production of ROS in the form of superoxide (O2^•−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH). Free Hb and heme contribute to oxidative stress and inflammation through several mechanisms.

The redox cycle of Hb involves the reversible binding and release of oxygen, facilitated by the cycling of the iron atom in the heme group between the Fe(II)-Hb and Fe(III)-Hb states (Fig. 1A). Additionally, free Hb can undergo autoxidation, leading to the formation of methemoglobin (metHb) and O2^•−. (Rifkind et al., 2015). The intracellular enzymes of RBCs in the microcirculation reduce oxidized Hb back to the oxygen-binding Fe(II)-Hb form. Moreover, the cellular antioxidant system of RBCs, which is primarily located in the cytoplasm, neutralizes ROS (Gwozdzinski et al., 2021). Non-neutralized ROS bind to the cell membrane, becoming less accessible to the antioxidant system of the cell, and may cause oxidative damage to the RBC membrane at sufficient concentrations.

Figure 1. Hypothetical scheme illustrating the pro-oxidative and pro-inflammatory effects and the clearance of extracellular Hb and heme. (A) Various reactions occur with extracellular Hb, heme, and other blood-derived products. (B) In addition to inducing ROS production, free Hb and heme interact with TLR2/4 to initiate inflammatory cascades in macrophages and neutrophils. (C) Clearance of Hp and Hpx, demonstrating the binding of Hb by Hp followed by CD163-dependent endocytosis and detoxification, with parallel breakdown to produce heme, bound by Hpx with LRP1/CD91-dependent endocytosis and detoxification. Images are constructed using Biorender. CD91, Cluster of differentiation 91; CD163, Cluster of differentiation 163; Hb, Hemoglobin; Hp, Haptoglobin; Hpx, Hemopexin; LRP1/CD91, Low-density lipoprotein (LDL) receptor-related protein-1; ROS, Reactive oxygen species; TLR2/4, Toll-like receptors 2 and 4.

The Fenton reaction, first described by H.J.H. Fenton in 1894, involves the catalytic conversion of H₂O₂ into highly reactive •OH in the presence of transition metals, typically iron (Fe²+) ion released from free Hb (Ovalle, 2022). Although ROS have been shown to be involved in signal transduction, differentiation, and gene expression (Canton et al., 2021), prominent ROS are also potent inducers of oxidative stress and inflammation, which are closely linked (Pizzino et al., 2017; Checa and Aran, 2020; Ramos-González et al., 2024). Given that redox and Fenton reactions greatly contribute to ROS production, an important fact to consider is that NADPH oxidase and mitochondria are the intracellular originators of ROS (Canton et al., 2021). Extracellular Hb, heme, and ROS influence the differentiation of monocytes into tissue-resident macrophages and/or their activation by inflammatory stimuli (Fig. 1B). Accordingly, Hb/heme acts as a ligand for various receptors, including the Toll-like receptor 2/4 (TLR2/4) of macrophages, and initiates inflammatory signaling (Cox et al., 2007; Figueiredo et al., 2007; Quero et al., 2017; Bozza and Jeney, 2020). These interactions have been observed in mouse macrophages after TLR4 ligand stimulation, in which the intracellular levels of labile heme and heme oxygenase-1 (HO-1), an inducible enzyme responsible for heme degradation, were upregulated (Sudan et al., 2019). Additionally, heme shows a synergistic effect on macrophages with other foreign molecules, enhancing the ROS-dependent production of inflammatory cytokines (Fernandez et al., 2010). These data suggest that heme is an upstream signaling molecule required for HO-1 regulation and may function through interactions with heme-sensitive receptors, further enhancing the detrimental effects of ROS. Similar findings were observed in neutrophils (Fig. 1B), wherein Hb-derived heme was described as a chemoattractant that induces the migration of immune cells, ROS generation, and pro-inflammatory stimuli (Porto et al., 2007; Bozza and Jeney, 2020). Independently, ROS can activate redox-sensitive transcription factors, such as nuclear factor-kappa B (NF-κB), which regulate the expression of pro-inflammatory genes, including cytokines, chemokines, and adhesion molecules (Korashy and El-Kadi, 2008; Lingappan, 2018). ROS has been shown to activate NF-κB through alternative phosphorylation and degradation of IκBα, allowing the nuclear translocation of NF-κB and the subsequent transcription of inflammatory mediators (Morgan and Liu, 2011). Inflammatory processes can upregulate enzymes such as NADPH oxidase, which further enhance intracellular ROS production (Mittal et al., 2014; Vermot et al., 2021). The interplay of the Fenton reaction, oxidative stress, and inflammation culminates in significant cellular damage and cytotoxicity.

Autoxidation of Hb in plasma forms metHb that non-enzymatically dissociates into Hb dimers (αβ-globin dimers) and heme. The immune system has developed a scavenging mechanism to counteract the toxic and oxidative effects of these molecules. Haptoglobin (Hp) and hemopexin (Hpx) are scavenger proteins that selectively and efficiently bind to free Hb and heme, respectively (Fig. 1C) (Schaer et al., 2013). Hp is a glycoprotein synthesized in hepatocytes and parenchymal cells of the liver and is released into circulation. To a lesser extent, Hp is synthesized in the lungs, spleen, kidneys, thymus, and heart (Kalmovarin et al., 1991). Hp forms a complex with the α-globin chain of human Hb dimers (McCormick and Atassi, 1990). Hp-Hb binding is irreversible; this complex binds to the cluster of differentiation 163 (CD163) receptors and is internalized and degraded by tissue macrophages (Thomsen et al., 2013). CD163 is a member of the cysteine-rich scavenger receptor family and is exclusively expressed in cells of the monocyte/macrophage lineage (Van Gorp et al., 2010). Decreased free Hp levels are indicative of the intravascular hemolysis of RBCs (Schaer et al., 2013). Conversely, Hp is also considered a reactant showing increased levels in response to elevated levels of pro-inflammatory mediators such as interleukin (IL)-1 and IL-6 (Pfefferlé et al., 2020).

Among the known heme-binding proteins, Hpx has the highest binding affinity for heme (Karnaukhova et al., 2021). Similar to Hp, this plasma protein is primarily expressed in hepatocytes. Hpx binds to free heme to form a heme-Hpx complex that maintains the solubility of heme while preventing or limiting its oxidative and pro-inflammatory effects (Smeds et al., 2017). The heme-Hpx complex enters hepatocytes through receptor-mediated endocytosis via lipoprotein receptor-related protein-1 receptor (LRP). During endocytosis, the complex dissociates and releases Hpx into the plasma. Heme is then transported into the cytoplasm by internal heme-binding membrane proteins, and heme oxygenase (HO) facilitates iron removal. The rate of heme catabolism is controlled by HO, which exists in two main isoforms, inducible HO-1 and constitutive HO-2, that catalyze the degradation of heme to biliverdin, free iron, and carbon monoxide (Karnaukhova et al., 2021).

Both Hp and Hpx have a minimal half-life of approximately 3.5-7 days in the circulation and are further reduced in complex with free Hb and heme (Pfefferlé et al., 2020; Karnaukhova et al., 2021). The depletion of these plasma proteins does not result in an increase in protein production. Hence, it affects the efficiency of extracellular Hb and heme clearance in the system.

The BBB is a highly selective semipermeable border that prevents toxins, pathogens, and fluctuations in plasma composition in the peripheral circulation from non-selectively crossing into the extracellular fluid of the central nervous system (CNS). In the context of brain hemorrhage, the movement of Hb, heme, and other blood-derived molecules from the blood vessels into the brain parenchyma involves multiple complex pathways. These processes include passage through blood vessels, the Virchow-Robin space (VRS), the luminal surface of the BBB, and eventually into the brain parenchyma. The VRS, also known as the perivascular space, is a fluid-filled space surrounding the blood vessels in the brain parenchyma (Montaser-Kouhsari et al., 2022). The VRS acts as a conduit for the exchange of fluids, solutes, and immune cells between the cerebrospinal fluid and brain interstitial fluid. Following a hemorrhagic event, Hb, heme, and other blood-derived damage-associated molecular patterns (DAMPs) have been suggested to enter the VRS through paracellular and transcellular pathways (Laksitorini et al., 2014; Roh and Sohn, 2018). Increased permeability of blood vessel walls due to inflammation and oxidative stress may allow Hb and heme to penetrate the EC junctions into the VRS. Alternatively, active transport mechanisms involving ECs facilitate the movement of Hb and heme into the VRS. For instance, heme carrier protein 1 (HCP1) transports heme across the ECs into the VRS (Le Blanc et al., 2012). The pro-oxidative and inflammatory properties of extracellular Hb and heme have been shown to be involved in the disruption of the cellular and molecular mechanisms of pathophysiological cascades that occurs within the BBB complex.

The response of ECs to blood-derived neurotoxicity promotes oxidative stress, activation of inflammatory pathways, and BBB disruption (Fig. 2A, 2B). Extravasation of blood following hemolytic conditions or hemorrhagic events releases degradation products (e.g., oxyhemoglobin, excess heme, and iron) that contribute to oxidative stress. The increased production of free radicals results in cellular damage to ECs by promoting lipid peroxidation, protein breakdown, and DNA fragmentation (Kadry et al., 2020; Solár et al., 2022). Hb-derived heme intercalates into the lipid membranes of ECs to generate ROS. Moreover, heme is recognized by membrane-bound toll-like receptors (TLRs) in ECs, which initiate an inflammatory response (Janciauskiene et al., 2020). The binding of Hb/heme to TLR2/4 activates the myeloid differentiation primary response 88 (MyD88)-dependent pathway, leading to the activation of NF-κB (Nagyoszi et al., 2010; Solár et al., 2022; Wei et al., 2024). Subsequently, NF-κB translocates to the nucleus and promotes the transcription of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukins (IL-1β, IL-6), and chemokines (CCL2, CXCL8) (Park et al., 2021; Rong et al., 2022; Guo et al., 2024).

Figure 2. Illustration of how the different BBB components react to the pro-oxidative and pro-inflammatory actions of extracellular Hb and heme. (A) In response to Hb/heme-induced TLR4 activation, the downstream signaling cascade triggers NF-kB and AP-1 activation, and the levels of pro-inflammatory mediators are increased. Alternatively, increased production of ROS results in the downregulation of TJ proteins, increasing BBB permeability. Extracellular Hb, heme, and ROS increase CypA production by the pericytes. Interaction of CypA and CD147 receptor activates NF-kB and caspase-9/3, leading to the additional production of cytokine. (B) Hb/heme-induced inflammatory signaling in astrocytes. ROS disrupt the extracellular matrix proteins of the basal membrane by activating matrix metalloproteinases such as MMP-9. Images are constructed using Biorender. AP-1, Activator protein-1; BBB, Blood-brain barrier; CD147, Cluster of differentiation 147; CypA, Cyclophilin A; Hb, Hemoglobin; MMP-9, Matrix metalloproteinases 9; NF-kB, Nuclear factor kappa-light-chain-enhancer of activated B; ROS, Reactive oxygen species; TJ proteins, Tight junction proteins; TLR4, Toll-like receptors 4.

Heme catabolism has been previously reported to release iron that catalyzes the Fenton reaction, producing ROS. Iron transporters, such as divalent metal transporter 1 (DMT1) and ferroportin, which are localized within the membrane of ECs, regulate intracellular iron levels (Skjørringe et al., 2015). However, excessive iron levels can overwhelm these regulatory mechanisms, leading to iron overload and subsequent oxidative damage. Consequently, they activate multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway, which is composed of extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinase (JNK), and p38 kinase (Lee et al., 2003; Kefaloyianni et al., 2006; Son et al., 2011). These kinases are crucial for regulating cellular responses to stress, including apoptosis, cytokine production, and cell survival. Specifically, p38 and JNK are heavily involved in the inflammatory response by promoting the expression of pro-inflammatory genes via activation of transcription factors, including NF-κB and activator protein 1 (AP-1) (Kefaloyianni et al., 2006). The combination of oxidative stress and inflammatory signals can trigger apoptosis via the intrinsic (mitochondrial) and extrinsic (death receptor) pathways (Elmore, 2007). ROS can induce mitochondrial dysfunction, leading to the release of cytochrome c and the activation of caspase-9, followed by caspase-3, resulting in apoptosis (Redza-Dutordoir and Averill-Bates, 2016; Villalpando-Rodriguez and Gibson, 2021).

The pro-oxidative and inflammatory actions of Hb and heme have been implicated in the breakdown of the junctional complex, which is localized to the apical membrane of ECs (Butt et al., 2011). The junctional complex between ECs is composed of transmembrane proteins (such as claudin, occludin, and junctional adhesion molecules) and cytoplasmic proteins linked to the cytoskeleton (Hartsock and Nelson, 2008). Data from a study using an animal model of intracerebral hemorrhage demonstrated that the intravenous injection of oxyglobin, an Hb-based oxygen-carrying fluid, altered the expression of zonula occludens 1 (ZO-1), claudin-5, and occludin in TJs (Ding et al., 2014). Additionally, pro-inflammatory cytokines like TNF-α and IL-1β were reported to disrupt occludin, claudins, and ZO-1, leading to increased BBB permeability and potential infiltration of inflammatory cells and molecules into the brain parenchyma (Versele et al., 2022; Zhao et al., 2022b). Secretion of cytokines and chemokines by ECs recruits immune cells, including neutrophils and monocytes, into the CNS, thereby contributing to neuroinflammation (Chen et al., 2024).

During hemorrhagic events, cell-free Hb, heme, and iron disrupt the basal membrane through oxidative stress and protease activation (Fig. 2A). ROS disrupt extracellular matrix (ECM) proteins, which are major components of the BBB basal membrane, by activating matrix metalloproteinases (MMPs), such as MMP-9 (Walter et al., 2020). In addition to basal membrane disruption, oxidative stress also induces the release of cyclophilin A (CypA) from pericytes. Intracellular CypA activates its receptor, CD147, and the downstream NF-κB inflammatory pathway to induce MMP9 expression and proteolytic functions in the degradation of endothelial TJ proteins and basal membranes (Kim et al., 2009; Wang et al., 2019). Pericytes express inducible nitric oxide synthase (iNOS) in response to Hb and ROS, which increases nitric oxide (NO) production and exacerbates oxidative stress and cellular damage (Underly and Shih, 2021). In contrast, astrocytes in the CNS respond robustly to extracellular Hb and heme-induced oxidative stress by activating TLR2/4 inflammatory signaling cascades. The interaction between TLR2/4 and its ligands activates the MyD88-dependent pathway, resulting in the recruitment and activation of interleukin-1 receptor-associated kinase (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6) (Muroi and Tanamoto, 2008). NF-κB and AP-1 translocate to the nucleus and promote the transcription of pro-inflammatory TNF-α, IL-1β, and IL-6 (Kwon and Koh, 2020; Li et al., 2024). Concurrently, the MAPK pathway amplifies the inflammatory response and contributes to astrogliosis, a process characterized by astrocyte proliferation and hypertrophy (Saha et al., 2020; Ding et al., 2021).

The cerebral accumulation of Aβ is critical to the pathology of AD. This peptide is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by β- and γ-secretases (Chen et al., 2017). The consequences of genetic mutation of this transmembrane protein or its cleaving proteins include the production of excessive Aβ. Although extensive studies have been conducted on Aβ, the exact mechanism underlying the abnormal accumulation and deposition of cerebral Aβ peptides in AD remains inconclusive. Recent studies have demonstrated the interactions between Hb and Aβ. Hb levels are notably increased in brain areas most commonly affected by amyloid pathology, including the inferior temporal gyrus, cerebral parietal gray matter, and parietal white matter (Wu et al., 2004). Neuronal Hb has been shown to be upregulated in aging and mice that overexpress amyloid precursor protein and presenilin 1 (APP/PS1) transgenic mice (Oyama et al., 2000; Wu et al., 2004), and heme has been observed to co-localize with Aβ deposits in AD brain samples (Cullen et al., 2006). Stereotactic injection of human Hb into the hippocampi of APP/PS1 mice resulted in the formation of envelope-like structures composed of Aβ around Hb (Chuang et al., 2012). In addition, western blotting analysis showed co-incubation of both Hb and Aβ monomers (20 kDa) and dimers (24 kDa) that resulted to the formation of large Hb and Aβ (40 kDa) (Chuang et al., 2012). Furthermore, the iron-containing heme has been suggested to serve as the critical Aβ binding moiety of Hb (Chuang et al., 2012). The human-specific N-terminal sequence of the Aβ binds to the free heme groups, most likely via the histidine 13 (H13) subtype, that functions as heme ligands in a variety of peroxidases (Flemmig et al., 2018). The complex has been suggested to be formed by a 1:1 ratio of the Aβ peptide and heme (Pramanik and Dey, 2011; Lu et al., 2014). On the other hand, a 2:1 ratio, wherein one molecule of Aβ binds with heme iron via H13 while a second peptide provides amino acid R5 and Y10 for the subsequent H₂O₂ peroxidase activity, has been proposed (Chiziane et al., 2018; Flemmig et al., 2018). Hb components have also been identified to be associated with Aβ aggregation in senile plaques (Cullen et al., 2006; Ghosh et al., 2015; Flemmig et al., 2018). These observations indicate that Hb and heme may influence Aβ accumulation by physical binding and co-localizing with the protein that further suggest a link between Hb/heme and AD pathogenesis.

Tau is a microtubule-associated protein that stabilizes neuronal microtubules and is critical for the preservation of neuronal structure, generation, and intracellular transport (Cooper, 2000; Ori-McKenney and McKenney, 2024). In AD and other tauopathies, tau proteins undergo abnormal hyperphosphorylation, which is characterized by the addition of excess phosphate groups, leading to the formation of neurofibrillary tangles (NFTs) (Goedert et al., 1989; Casali and Reed-Geaghan, 2021; Kim et al., 2021). Hb levels in the brain decrease with age and AD (Biagioli et al., 2009; Ferrer et al., 2011; Freed and Chakrabarti, 2016). In fact, fluorescence labeling and confocal imaging revealed reduced Hbα- and β-globin chains in neurons with hyperphosphorylated tau deposits in the frontal cortex and hippocampus in AD (Ferrer et al., 2011). Similarly, both Hbα and Hbb-b1 chains have been observed to be decreased while the levels of phosphorylated tau (p-tau) and the p-tau/tau ratio were significantly increased in the cerebral cortex regions of mice that showed neurological deficits after exposure to a low-intensity open-field blast (Chen et al., 2018). Although these findings imply an association between the expression patterns of Hb and tau/p-tau in tauopathy-associated brain regions, the direct association or co-localization of these proteins remains to be characterized.

Several studies have indicated that free heme toxicity may be associated with AD pathology (Wu et al., 2004; Ferrer et al., 2011; Yu et al., 2024). For instance, heme complexation by neuronal peptides/proteins such as Aβ and the resulting peroxidase activities are well documented (Wu et al., 2004; Chiziane et al., 2018; Pandya et al., 2021). The binding properties of heme to tau isoforms have also been investigated (Asher et al., 2009; Pirota et al., 2016). Both N-terminal free amine and N-acetylated tau isoforms formed 1:1 complexes with monomeric and dimeric heme, but with moderate affinity, most likely due to the electrostatic repulsion between acetylated tau chains (Pirota et al., 2016). In addition, the peroxidase activity of the heme-tau isoform complex is minimal (Pirota et al., 2016). However, under pathological conditions, such as heavy metal release usually observed in hemorrhagic events, excessive accumulation of heme derived from Hb may increase the aggregation propensity of tau isoforms, which may further contribute to peroxidase activity and neuronal oxidative stress.

Microglia are the primary immune cells of the CNS that are highly responsive to extracellular Aβ, p-tau, and other neuronal deposits, including Hb and heme. Various microglial innate immune receptors, including TLR2/4, cluster of differentiation 14 and 16 (CD14, CD36), and the receptor for advanced glycation end products (RAGE), recognize such extracellular deposits and facilitate microglial activation, internalization and clearance of neurotoxic materials, and the subsequent production of inflammatory mediators. A summary of the potential inflammatory pathways associated with Aβ, p-tau, and Hb/heme is presented in Fig. 3A.

Figure 3. Hypothetical scheme summarizing the potential inflammatory pathways related to extracellular Hb/Heme and pathological proteins associated with AD- and PD-induced responses in microglia. (A) The binding of Hb, heme, Aβ, and Tau with the microglial membrane receptors RAGE and TLR2/4 may result in NF-kB/AP-1 pathway activation through several identified mediators, and lead to the assembly of the NLRP3 inflammasome. These effects together may lead to the production of inflammatory mediators and free radicals in AD pathology. (B) α-Syn interacts with glial receptors such as CD14, TLR2/4, and CD36. On the other hand, Hb and heme interact with CD14 and TLR2/4 while inhibiting CD36. These cascades result in the activation of NF-kB/AP-1 pathways, transcription of pro-inflammatory mediators, and activation of the NLRP3 inflammasome. Increased production of ROS results in mitochondrial dysfunction that further enhances inflammation and necrosis. The microglia also degrade Hb, heme, and pathological proteins in a process, that if compromised, can exacerbate inflammatory signaling. The resulting immune response will affect neuronal integrity and may enhance further protein aggregation in surrounding cells, contributing to disease progression. Images are constructed using BioRender. Aβ, Amyloid beta; AD, Alzheimer’s disease; AP-1, Activator protein-1; α-Syn, Alpha-synuclein; CD14, Cluster of differentiation 14; CD36, Cluster of differentiation 36; Hb, Hemoglobin; NF-kB, Nuclear factor kappa-light-chain-enhancer of activated B; NLRP3, Nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3; PD, Parkinson’s disease; RAGE, Receptor for advanced glycation end products; ROS, Reactive oxygen species; Tau, Tubulin associated unit; TLR2/4, Toll-like receptors 2 and 4.

Ample data indicate that TLR2 and TLR4 physically interact with Aβ, tau, and DAMPs, including Hb and heme (McDonald et al., 2016; Janciauskiene et al., 2020; Dallas and Widera, 2021; Lua et al., 2021; Dutta et al., 2023). TLR activation triggers the phagocytic activity of microglia to remove pathogens and neuronal waste (Fiebich et al., 2018). TLR2/4 are dependently activated through the MyD88 and the Toll/interleukin-1 receptor- (TIR) domain-containing adaptor inducing interferon-β (TRIF) signaling pathways (Wu et al., 2022). TLR2/4 is associated with myeloid differentiation factor 2 (MD-2) and recruits TIR domain-containing adaptor protein (TIRAP) and MyD88 (Akira and Takeda, 2004; Kim et al., 2023a). The interaction between MyD88 and IRAK recruits the tumor necrosis factor receptor-associated factor 6 (TRAF-6) that further activates the complex resulting in the phosphorylation of IκB kinase (Dallas and Widera, 2021). Microglial stimulation phosphorylates and degrades IκB, activating NFκB and causing transcription and release of inflammatory molecules. Alternatively, TLR2/4 signaling activates ERK, JNK, and p38 kinases, inducing nuclear translocation of the transcription factor complex AP-1 (Whitmarsh and Davis, 1996; Alhamdan et al., 2024). The activation of NFκB and AP-1 concludes with the transcription of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α (Dallas and Widera, 2021).

Identified as a TLR2 and 4 co-receptors, CD14 has been implicated in the phagocytic activity of immune cells in AD. Interactions of Aβ fibrils through surface receptors CD14, TLR2 and TLR4 initiate intracellular signaling cascades and microglial phagocytotic responses (Reed-Geaghan et al., 2009). Accordingly, Aβ-induced activation of p38 MAPK requires the collaborative activation of TLR2/4 and CD14 (Reed-Geaghan et al., 2009). The inhibition of p38 evades Aβ-induced ROS production and reduces the induction of microglial phagocytosis (Reed-Geaghan et al., 2009). Additionally, microglial CD14, TLR4, and TLR2 deficiency showed no IκBα phosphorylation following Aβ-induced stimulation (Reed-Geaghan et al., 2009). Similarly, neutralization and genetic deficiency of CD14 significantly reduced Aβ-induced microglial activation and toxicity (Fassbender et al., 2004; Liu et al., 2005; Ciesielska et al., 2021). Previous studies have reported the role of CD14 in Hb/heme-induced inflammation and uptake. CD14 facilitates the uptake of Hb and Hb-based oxygen carriers by immune cells (Nimz et al., 2023). Heme-induced inflammation is attenuated by the inhibition of CD14 (Janciauskiene et al., 2020). These observations indicate the role of CD14 in Aβ and Hb/heme uptake and inflammatory cascade initiation.

CD36 is a scavenger receptor with structural and functional similarities to CD14 (Ruysschaert and Lonez, 2015). CD36 has been suggested to recruit TLR2/4 heterodimers to lipid rafts, which are platforms that gather receptors, allowing for activation and signal transduction (Schmitz and Orsó, 2002). Free heme, a lipophilic molecule, intercalates into the cell membrane and induces the sequestration of CD36 during intracellular storage (Banesh et al., 2022). In AD, Hb and heme reduced the expression of CD36, reducing the Aβ uptake and phagocytotic capacity of immune cells (Sankar et al., 2018). To date, evidence of a direct physical interaction between CD36 and p-tau has not been obtained. Nevertheless, microglia that express this scavenger receptor may play a role in the absorption of tau proteins.

RAGE facilitates microglial recognition of Aβ, tau, and Hb/heme. Multiple lines of evidence indicate that RAGE is a key cellular factor for Aβ and tau-mediated dysfunction in AD. For instance, the expression levels of RAGE are correlated with AD severity, as indicated by the clinical score of Aβ accumulation (Lue et al., 2001). Aβ_1-42 treatment has been shown to induce higher RAGE expression in cultured microglia from AD in comparison with brain samples without dementia (Lue et al., 2001). Similarly, overexpression of RAGE in the microglia of transgenic mice expressing mutant APP displayed Aβ accumulation and enhanced expressions of IL-1β and TNF-α (Fang et al., 2010). Microglial RAGE expression has been demonstrated to be involved in p-tau-mediated inflammation. In vitro and in vivo analyses revealed that RAGE deficiency reduces microglial tau uptake and enhances tau propagation between neurons (Sbai et al., 2022). Additionally, RAGE knockout results in decreased tau uptake and clearance in mouse brains pretreated with tau derived from patients with AD (Kim et al., 2023b). Similarly, Hb/heme may also induce the pro-inflammatory polarization phenotype of microglia via RAGE. Previous studies have identified Hb and Hb-derived heme as DAMPs that form complexes with high-mobility group box 1 (HMGB1) protein (Fan et al., 2020; Kim et al., 2023b), a DAMP molecule released by ferroptotic cells in an autophagy-dependent manner (Yuan et al., 2020). Immunofluorescence data have revealed an increase in the number of HMGB1-positive glial cells after HMGB1 treatment (Fan et al., 2020). Furthermore, increased expression of TNF-α and iNOS has also been detected (Fan et al., 2020). Upon stimulation, RAGE activates intracellular cascades such as ERK and p38 MAPK to activate transcription of the pro-inflammatory cytokine NF-kB (Shanmugam et al., 2003). Alternatively, activation of AP-1 via the RAGE-JNK-dependent pathway has also been proposed (Bianchi et al., 2010). Inhibition of RAGE reduces Aβ transport from the cellular surface to mitochondria, restores mitochondrial functionality, and mitigates Aβ toxicity. Furthermore, Aβ shuttling into mitochondria promotes Drp1 activation and exacerbates mitochondrial dysfunction, thereby inducing NLRP3 inflammasome activation (Sbai et al., 2022). These findings suggest that RAGE plays a role not only in the microglial transport and clearance system of Aβ, p-tau, and Hb/heme but also in the inflammatory response in AD. Taken together, these findings indicate that microglia respond to extracellular Hb/heme, Aβ, and p-tau via activation of CD14-TLR2/4, CD36-TLR2/4 or RAGE-mediated signaling mechanisms.

ROS have critical physiological functions in the regulation of cellular homeostasis by mediating activities such as redox signaling, pathogen defense, and protein folding (Schieber and Chandel, 2014; Abramov et al., 2020; Smith et al., 2022). However, when reactive species reach cellular quantities, their detrimental effects outweigh their benefits, since antioxidant responses are overwhelmed. Pro-inflammatory microglia considerably affect brain energy metabolism and ROS production, resulting in impaired neuronal network function, neurodegeneration, and BBB dysfunction, all of which contribute to the etiology of AD and PD (Singh et al., 2019; Houldsworth, 2024). Microglia perform various activities, including phagocytosis and clearance of toxic protein aggregates, that demand high energy and are regulated by mitochondria (Fairley et al., 2023). Therefore, mitochondrial dysfunction may influence microglial immune activity. Abnormal production of redox-active metal ions, such as iron derived from heme, catalyzes the overproduction of ROS. Highly reactive hydroxyl radicals contribute to increased mitochondrial ROS production and oxidative damage (Manoharan et al., 2016). AD and PD are linked to oxidative stress and inflammation, resulting from reduced mitochondrial activity in the brain (Manoharan et al., 2016; Alqahtani et al., 2023). Mitochondrial dynamics have a significant impact on cellular necrosis via several mechanisms (Fig. 3A, 3B).

Oxidative stress is associated with programmed necrosis (necroptosis). Hb/heme and pathological proteins can induce chronic activation of microglia (Figueiredo et al., 2007; Bozza and Jeney, 2020) and trigger necroptosis (Weinlich et al., 2017). These processes can further induce the secretion of pro-inflammatory DAMPs and mediate inflammation (Rodríguez-Gómez et al., 2020). Necroptosis has been shown to be facilitated by two members of the receptor-interacting serine/threonine-protein kinase (RIP) family, RIP1 and RIP3. Accordingly, activated RIP1 interacts with RIP3 through interaction motifs, which phosphorylate RIP3 and form an RIP1/RIP3 complex known as the necrosome (Cho et al., 2009). RIP1 is crucial for ROS sensing and subsequent RIP1-2 complexation (Zhang et al., 2017). Therefore, ROS provide a positive feedback loop that ensures effective induction of necroptosis. In AD, the necrosome was shown to form a functional amyloid signaling complex resulting in TNF-induced necroptosis (Li et al., 2012). In PD, soluble α-syn can induce mitochondrial dysfunction by inhibiting sirtuin 3 (SIRT3), which assists mitochondria in maintaining metabolic stability and homeostasis (Park et al., 2020). Indeed, transduction of SIRT3 has shown promising therapeutic potential in patients with PD (Gleave et al., 2017). In contrast, the oxidative stress-responsive HO-1 isoenzyme protects against necrosis (Gozzelino et al., 2010). The mechanism underlying this cytoprotective action is based on the capacity of HO-1 to catabolize unbound heme and prevent it from sensitizing cells to programmed cell death. These findings suggest that Hb-derived heme and pathological proteins can individually induce oxidative stress and mitochondrial dysfunction, which further contribute to the inflammation associated with AD and PD.

In this review, we examined the multifaceted roles of extracellular Hb and heme in AD and PD, focusing on the potential mechanisms of systemic/neuroinflammation and other pathological features, including BBB disruption, neurotoxic protein accumulation, irregular iron metabolism, oxidative stress, and mitochondrial dysfunction. Although neuronal Hb and heme may show neuroprotective effects by maintaining mitochondrial homeostasis, they may also promote pathological protein aggregation, iron deposition, and necrosis. Co-localization and physical binding of free Hb/heme and toxic proteins occur in AD and PD brains. Although Hb and heme are substantially associated with the development of AD and PD, their exact roles in the pathogenesis of these diseases remain unclear. Further studies are required to elucidate the roles of extracellular Hb and heme. Although the pro-oxidative and inflammatory effects of Hb and heme were found to disrupt BBB integrity by targeting its components, further studies, including the identification of other membrane-bound protein receptors that physically interact with Hb/heme, may be helpful in mitigating BBB disruption. The signaling pathways involved in regulating the pro-oxidative and inflammatory effects of Hb/heme, as well as the toxic protein aggregates presented in this review, may also be similar to or different from those of other immune cells. Further studies elucidating these mechanisms would be beneficial for understanding AD- and PD-associated inflammation.

This study was supported by the National Research Foundation (NRF) of Korea (NRF-2021RlG1A1093620).

The authors declare that they have no competing interests.