Neuroinflammatory Triangle Presenting Novel Pharmacological Targets for Ischemic Brain Injury

Abstract

Ischemic stroke is one of the leading causes of morbidity and mortality globally. Hundreds of clinical trials have proven ineffective in bringing forth a definitive and effective treatment for ischemic stroke, except a myopic class of thrombolytic drugs. That, too, has little to do with treating long-term post-stroke disabilities. These studies proposed diverse options to treat stroke, ranging from neurotropic interpolation to venting antioxidant activity, from blocking specific receptors to obstructing functional capacity of ion channels, and more recently the utilization of neuroprotective substances. However, state of the art knowledge suggests that more pragmatic focus in finding effective therapeutic remedy for stroke might be targeting intricate intracellular signaling pathways of the 'neuroinflammatory triangle': ROS burst, inflammatory cytokines, and BBB disruption. Experimental evidence reviewed here supports the notion that allowing neuroprotective mechanisms to advance, while limiting neuroinflammatory cascades, will help confine post-stroke damage and disabilities.

Keywords: blood brain barrier; brain microvascular endothelial cell; cytokine; neuroinflammation; reactive oxidative species.

Figure 1

Post-stroke neuroinflammatory pathway of TNF. TNF released from activated microglia binds to the trimeric TNFR1, primarily present on infiltrating leukocytes. This allows the intracellular part of the receptor to complex with the adaptor proteins TRADD and FADD via its death domain. This domain activates cytoplasmic caspase-8 enzyme. Activated caspase-8 acts as a transcriptional factor, entering the nucleus to induce target genes. The expressed mRNA encodes proteins such as adhesion molecules, IL-1, nitric oxide synthase (nNOS), plasminogen-activating inhibitor-1, and apoptosis-inducing factors. This ‘mix’ contributes to the neuronal damage in ischemic tissue following stroke.

Figure 2

The neuroinflammatory cascades of IL-1β following ischemic insult. Secreted by microvascular endothelial cells, resident microglia, astrocytes, and infiltrating macrophages, IL-1β forms a complex with IL-1R1, aided by IL-1RAcP. This ligand-receptor complex triggers three key neuroinflammatory mechanisms. In the NF-κB pathway, ligand-receptor binding results in the cytoplasmic recruitment of three key adaptor proteins: MYD88, TOLLIP, and TRAF6. This leads to the activation of intermediate kinases, particularly NIK, which prevent IκB from inactivating NF-κB, indirectly allowing NF-κB-dimer formation. These dimers of NF-κB function as transcriptional factors, entering the nucleus to induce transcription of IL-1, TNF, and many other genes in various cell types. In the JNK pathway, downstream of the “death” signal, intracellular MAPKs phosphorylate c-Jun or related proteins, which heterodimerize with Fos proteins to form the transcriptional factor activator protein (AP-1). AP-1 regulates gene expression that mediates pro-inflammatory cellular processes, particularly proliferation and differentiation of infiltrating immune lineage as well as apoptosis of regional neurons. In the p38 MAPK pathway, ligand binding activates intracellular MKKK3 which in turn activates p38-MAPK, causing expression of diverse apoptotic signals, adhesion molecules for infiltration, and IL-1.

Figure 3

The neuroinflammatory cascades of INF-γ following ischemic insult. Resident astrocytes as well as infiltrating T lymphocytes are the key sources of INF-γ. Following release, INF-γ binds to transmembrane INFGR, which is further composed of two subunits, INFG1 and INFG2. Each receptor subunit of INFGR is further composed of two different proteins, with each bound to Jak1 and Jak2 respectively. INF-γ binding with INFG1 part brings INFG2 in to proximity with INFG1 in such a way that, intracellularly, Jak1 and Jak2 phosphorylate each other’s two receptor domains. These receptor domains now act as docking points for STATs to bind with respective JAKs. The activated STAT dissociates and forms a homodimeric complex functioning as a transcription factor inducing the expression of variety of protein signals in various cell types. In cerebral ischemia, upregulated proteins are adhesion molecules on a variety of cells that play their role in the disruption of the BBB and the infiltration of leukocytes, along with proliferation of pro-inflammatory cells.

Figure 4

Neuroinflammation downstream of IL-6 in stroke. IL-6 binds its receptor and proceeds two separate but related pathways: the classical and trans-signaling pathways. The classical pathway begins when IL-6 binds with IL-6R-α chain subunit assisted by gp130, an intra-cellular signal transducer. This binding allows intracellular, receptor-associated JAKs to phosphorylate each other on respective tyrosine domains. Phosphorylated JAKs further phosphorylate related tyrosine residues on the receptor, creating binding sites for proteins possessing SH2 domains. Cytoplasmic STATs then bind to these SH2 domains to be phosphorylated by respective JAKs, making them dissociate from the receptor. The dissociated STATs form dimers, which act as transcription factors. The dimers translocate to the cell nucleus to induce gene expression for subsequent protein signaling outcomes. STATs may also be tyrosine-phosphorylated directly by receptor tyrosine kinases - but since most receptors lack built-in kinase activity, JAKs are usually required for signaling. The trans-signaling pathway begins when IL-6 ligands with sIL-6R-α in the extracellular matrix. The high affinity drives this complex to bind with gp130 expressed overly on glial and neuronal cells following ischemic insult. This trio-binding results in the activation of intracellular tyrosine-associated JAKs. Phosphorylated tyrosine-sited allow the adaptor proteins (SoS and Grb-2) to bind and convert inactive cytoplasmic RAS to an active one, initiating a series of phosphorylations of a variety of molecules like Raf, MEK, and ERK. The activated ERK acts as a transcriptional factor. It enters the nucleus to express targeted genes for inflammatory protein signaling.

Figure 5

The ROS burst following ischemic insult and damage to biological molecules. Following hypoxia, brain cells witness a swift imbalance between ROS production and their neutralization mechanisms, particularly in neuronal cells, resulting in a constant rise in ROS levels, called ROS burst. One of the key sources of ROS burst inside the cell is mitochondria, others being cell membrane and peroxisomes. These ROS molecules damage cellular parts via protein oxidation, lipid peroxidation, and DNA damage. The neuronal nitric oxide synthase (nNOS) in the cytoplasm of hypoxic neuron produces excessive production of Nitric Oxide (NO) which after release reacts with cellular oxygen producing peroxinitrite (ONOO⁻), a powerful oxidant damaging cytoplasmic proteins and lipid composites. Another ROS-releasing source during hypoxia is the extracellular space-facing membrane bound enzyme called NADH oxidase. This enzyme releases ample quantities of superoxide molecules with respective damaging implications. A third major source of ROS release is the mitochondria. The external membrane-bound monoamine oxidase (MAO) releases a range of reactive molecules, primarily H2O2, which causes the production of hydroxyl radical (HO•) and hydroperoxyl radical (HO2⁻). These two radicals cause lipid peroxidation by selectively attacking carbon-carbon double bond (c=c) of saturated lipid compounds, releasing by-products such as 4-Hydroxynonenal (4-HNE), Hexanol, Propanol, and Malondialdehyde. Of these, the latter is a highly reactive organic compound, aggressively reacting nucleic material to cause DNA fragmentation. The key DNA fragmentation marker of this pathway is 8-Hydroxydeoxyguanosine (80HdG). The hypoxia-induced impaired mitochondrial functions, especially affected Complex I, III, and IV, which drive Electron Transport Chain (ETC), release abundant quantities of Superoxides (O2•⁻) into the cytoplasm. These superoxides further produce reactive species which have two-fold damaging effects. On the one hand, these inactivate chaperones result in increased levels of misfolded proteins and, on the other hand, cause carbonylation of cellular proteins. This carbonylation of proteins is evident by the elevated levels of carbonylation marker in stroke, such as 4-Hydroxy, 4-oxoneonenal.

Figure 6

Intrinsic apoptotic signaling and resultant neuronal death driven by ischemic-induced ROS burst. Ischemic events cause mitochondrial matrix of the neurons uptake extracellular calcium as a result of augmented mitochondrial respiratory burst. This calcium accumulation not only causes inactivation of anti-apoptotic proteins (BCL-2 and BCL-x) inside mitochondria but also results in an increase in the permeability of the outer and inner mitochondrial membranes. This permeation allows cytoplasmic Bax proteins to enter and make ‘tunnel-like’ complexes with mitochondrial Bar proteins, which then embed into the outer mitochondrial membrane as Bax-Bar pores. These specialized pores serve as channels to transport intra-mitochondrial pro-apoptotic proteins (AIF, Cy A, APAF-1, and Cyt C) into the cytoplasm. The AIF and Cy A make a complex (AIF-Cy A complex), directly entering the nucleus to inflict defragmentation. Similarly, APAF-1 binds with Cyt C to transform pro-Caspase 8 protein into an active form: Caspase 8. The Caspase 8 enters through the nuclear pores to activate Caspase Activated DNase (CAD) which defragments the nuclear material, a key apoptotic signaling outcome. This dual apoptotic signaling effect is primarily initiated by a respiratory burst in the affected neuron following ischemic insult.

Figure 7

Ischemic stroke and orderly compromisation of BBB. The brain microvascular endothelial cells (BMECs), Astrocytes, Pericytes, and Microglial cells make BBB. Compromised BBB integrity is the striking preliminary feature of the neuroinflammatory-triangle during the hypoxic pathophysiological state, where astrocytes in penumbra, pericytes, and BMECs start releasing ET-1 (disturbed balance of ET-1 and NO). This leads to the upsurge in the expression of a variety of adhesion molecules in/on BMECs such as ICAM-1, VCAM-1, and ELAM-1 to facilitate trans-endothelial migration of leukocytes. ET-1 also upregulates the expression and release of matrix metalloproteinases (MMP) from BMECs which lyse inter-endothelial connecting proteins such as Claudin 1, Claudin 5, Ocludin, Zona ocludin-1, junctional adhesion molecule-A (JAM-A), and others. This compromises the barrier’s integrity, otherwise tightly maintained by BMECs, thus allowing the release of inflammatory cytokines, infiltration of systemic immune cells, and fluid escape (brain edema). Inflammatory cytokines stimulate meningeal Mast Cells to release more inflammatory mediators to further the BBB shattering and causing long-term occlusion of blood to already starved brain tissue. Along with BMECs, the functionally compromised Pericytes and Astrocytes in penumbra put their respective neurodegenerative part (crossed red-mark in dotted boxes). The population of infiltrating immune cells and their pro-inflammatory secretions proceeds a vicious neuroinflammatory circle that only aggravates brain edema and infarct volume.