Targeting innate immunity for neurodegenerative disorders of the central nervous system

Abstract

Neuroinflammation is critically involved in numerous neurodegenerative diseases, and key signaling steps of innate immune activation hence represent promising therapeutic targets. This mini review series originated from the 4th Venusberg Meeting on Neuroinflammation held in Bonn, Germany, 7-9th May 2015, presenting updates on innate immunity in acute brain injury and chronic neurodegenerative disorders, such as traumatic brain injury and Alzheimer disease, on the role of astrocytes and microglia, as well as technical developments that may help elucidate neuroinflammatory mechanisms and establish clinical relevance. In this meeting report, a brief overview of physiological and pathological microglia morphology is followed by a synopsis on PGE2 receptors, insights into the role of arginine metabolism and further relevant aspects of neuroinflammation in various clinical settings, and concluded by a presentation of technical challenges and solutions when working with microglia and astrocyte cultures. Microglial ontogeny and induced pluripotent stem cell-derived microglia, advances of TREM2 signaling, and the cytokine paradox in Alzheimer's disease are further contributions to this article. Neuroinflammation is critically involved in numerous neurodegenerative diseases, and key signaling steps of innate immune activation hence represent promising therapeutic targets. This mini review series originated from the 4th Venusberg Meeting on Neuroinflammation held in Bonn, Germany, 7-9th May 2015, presenting updates on innate immunity in acute brain injury and chronic neurodegenerative disorders, such as traumatic brain injury and Alzheimer's disease, on the role of astrocytes and microglia, as well as technical developments that may help elucidate neuroinflammatory mechanisms and establish clinical relevance. In this meeting report, a brief overview on physiological and pathological microglia morphology is followed by a synopsis on PGE2 receptors, insights into the role of arginine metabolism and further relevant aspects of neuroinflammation in various clinical settings, and concluded by a presentation of technical challenges and solutions when working with microglia cultures. Microglial ontogeny and induced pluripotent stem cell-derived microglia, advances of TREM2 signaling, and the cytokine paradox in Alzheimer's disease are further contributions to this article.

Keywords: Alzheimer disease; Venusberg Neuroinflammation Meeting Bonn 2015; blood-brain barrier; innate immunity; macrophage; non-steroidal anti-inflammatory drugs (NSAIDs).

Figure 1. Microglial differentiation

Rounded yolk sac derived precursors invade the brain parenchyma in PU.1- and IRF8- dependent pathways and are regulated by CSF-1R, its ligand IL-34, and TGF-β to become fully differentiated microglial cells. During embryonic development, these cells progressively develop ramified morphology and constantly scan the brain microenvironment, eliminating neurons and pruning excessive synapses. In the mature healthy brain, surveilling microglia assist in maintaining brain homeostasis and function by regulating neuronal activity using special morphological features- bulbous endings, controlling synaptic plasticity by finger-like protrusions that wrap around dendritic spines, and engulfing cells undergoing apoptosis. These cells are highly ramified expressing CX3CR1, Iba-1 and P2YR12, are highly dynamic and dense to cover the tissue uniformly. During aging, microglia exhibit chronic mildly activated phenotype with increased expression of proinflammatory mediators, such as TNF-α, IL-1β, and IL-6 and exhibit reduced ramification with process shortening and thickening and some aberrant morphological features resembling dystrophy. Their coverage of the tissue is impaired. In Alzheimer’s disease (AD), microglia proliferate and accumulate with activated and dystrophic phenotypes around plaques of amyloid β (Aβ). Microglia cells surround the plaque with distinct morphological phenotypes. The first layer of more amoeboid cells are found in close proximity to the plaque and resemble activated microglia and/or infiltrating monocytes. Additional layers of microglial cells are located on the plaque edges, exhibiting decreased morphological complexity compared to the healthy mature microglia. Microglia cell coverage is impaired in APP-Tg mice, in which tissue space is left devoid of microglia processes. Considering the dynamic nature of the microglial processes, such a robust loss of branches during aging and disease may significantly impair the overall sensing capacity of microglia. Confocal z-stack images showing microglia process densities and spatial coverage area were adopted from (Baron, Babcock et al. 2014). Bars represent 50 μm.

Figure 2. Microglial ontogeny and implications for the use of pluripotent stem cell-derived macrophages for therapy

A precise understanding of microglial ontogeny in vivo may provide adequate methodologies for in vitro microglial differentiation from induced pluripotent stem cells. Findings in such in vitro models using stem cells will complement the knowledge gained in vivo. The choice of an adequate cell source for microglia will further contribute to a better understanding of microglial biology in health and disease, and will facilitate the refinement of accurate developmental and pathophysiological study models as well as drug screening systems for new microglia-targeted therapies.

Figure 3. Paradoxical effects of cytokine signaling in amyloid-β clearance

Amyloid-β induced microglial activation can be modified in the presence of anti-inflammatory cytokines (e.g. IL-4) or by genetic/pharmacologic blockade of TGFβ and/or IL-10 signaling to shift microglia from pro-inflammatory neurotoxic cells to activated pro-phagocytic cells with the capacity to reduce amyloid-β plaque load. In experimental systems, increased IL-1β expression can drive both a neurotoxic response (e.g. increased tau phosphorylation, indicated by interneuronal fibrils) as well as create an environment that supports pro-phagocytic microglial activation.

Figure 4. Mechanisms of action of inflammatory PGE₂ EP receptors in preclinical models of AD

The primary action of NSAIDs is inhibition of COX-1/COX-2, with one consequence being reduction of downstream PGE₂ production and signaling. The PGE₂ EP2 and EP3 receptors enhance inflammatory oxidative stress, pro-inflammatory gene expression and are pro-amyloidogenic. In contrast, EP4 signaling is anti-inflammatory and enhances Aβ peptide phagocytosis. Reduction of PGE₂ generation in response to NSAIDs may reduce deleterious EP2/EP3 signaling but also reduce beneficial EP4 signaling, which plays a beneficial role early in development of pathology in the APPSwe-PS1∆E9 model of familial AD.

Figure 5. L-Arginine pathways

L-arginine represents a branch point of metabolic pathways including arginine decarboxylase (ADC), arginases (ARG), glycine amidotransferase (AGAT), and nitric oxide synthases (NOS). Arginine is essential for protein synthesis and amino acid turnover and may serve as a sensor for amino acid deprivation and autophagy activation.

Figure 6. Microglia in LTP

Several factors stimulate microglia to adopt an inflammatory phenotype and, in most cases, inflammasome activation and the consequent production and release of IL-1β. IL-1β interacts with IL-1R1 activating signalling molecules like JNK and NFκB that exert an inhibitory effect on LTP. In addition, the age-related increase in IL-1RAcP at synapses results in an exaggerated effect locally that can also depress LTP. In age, neurons have been shown to release DAMPs that can feed back to enhance microglial activation, contributing to a damaging cycle of events.

Figure 7. Targeting dysregulated glial activation and innate immunity in TBI and AD

Top left panels show a conceptualized profile of the temporal changes in cytokines / chemokines, astrocytes, and microglia / macrophages following TBI, and how the neuroinflammatory responses are heightened and prolonged in the presence of beta-amyloid (Aβ) pathology (Webster, Van Eldik et al. 2015). In the figure, potential areas for therapeutic intervention targeting glial activation and innate immunity are highlighted in the yellow boxes. APP=amyloid precursor protein, BBB=blood-brain barrier, ROS/RNS=reactive oxygen species/reactive nitrogen species.

Figure 8. The Bioactive3D culture system supports the complex cell morphology of astrocytes and comes as a novel tool to study and modulate astrocyte activation in situations such as nerodegeneration, ischemia or neurotrauma

Upper panel. Astrocytes are highly complex cells with a multitude of cellular processes as demonstrated on this 3D reconstruction of adjacent astrocytes in the adult mouse hippocampus. Astrocytes were filled with either Alexa 568 or Lucifer yellow dyes. The gray matter is largely subdivided into individual astrocyte domains. Astrocytes are interconnected via gap junctions into a highly dynamic network, however in the healthy adult brain, astrocytes do enter domains of their astrocyte neighbors. Hence the limited overlap zone between adjacent astrocytes reflecting the interdigitation of fine cellular processes of adjacent astrocytes (shown in yellow). Reproduced from (Wilhelmsson, Bushong et al. 2006). Lower panel. Primary astrocytes cultured in the Bioactive3D system, which is composed of polyurethane nanofibers coated with poly-L-ornithin and laminin (Puschmann, Zanden et al. 2013), preserve some of the complex morphological and biochemical features of in vivo astrocytes that are normally lost upon 2D culture (compare the left and right panel). Space bar, 10 μm. The primary astrocytes were derived from mice expressing enhanced green fluorescein protein in their astrocytes (Nolte, Matyash et al. 2001).

Figure 9. The Bioactive3D culture system highly reduces the cellular stress seen in conventional 2D cell culture systems

A) and B) Western blot analysis and levels of intermediate filament proteins GFAP, nestin, synemin and vimentin, which are known to be upregulated upon astrocyte activation and cellular stress, in freshly isolated astrocytes and astrocytes cultured in a standard 2D and Bioactive3D system. The undesired baseline activation of astrocytes seen upon 2D culture is absent in Bioactive3D; ***, p<0.001. C) The levels of intermediate filament proteins nestin and vimentin in astrocytes initially cultured in a standard 2D and then transferred to Bioactive3D, decrease correspondingly, indicating that astrocyte cultures can be established and expanded in 2D cultures and move to Bioactive3D just prior to experimental manipulations; days, days after transfer from 2D to Bioactive3D. D) The expression of HSP70, a cellular stress marker, is highly reduced in astrocytes cultured in Bioactive3D. E) Western blot analysis of phosphorylated MapK/Erk1/2 in astrocytes maintained in standard 2D cultures or in Bioactive3D and treated for 1 hour with 100 μM ATP shows that astrocytes grown in Bioactive3D are highly responsive to activation. Modified from (Puschmann, Zanden et al. 2013).