CX3CL1/CX3CR1 signaling targets for the treatment of neurodegenerative diseases

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Abstract

Neuroinflammation was initially thought of as a consequence of neurodegenerative disease pathology, but more recently it is becoming clear that it plays a significant role in the development and progression of disease. Thus, neuroinflammation is seen as a realistic and valuable therapeutic target for neurodegeneration. Neuroinflammation can be modulated by neuron-glial signaling through various soluble factors, and one such critical modulator is Fractalkine or C-X3-C Motif Chemokine Ligand 1 (CX3CL1). CX3CL1 is produced in neurons and is a unique chemokine that is initially translated as a transmembrane protein but can be proteolytically processed to generate a soluble chemokine. CX3CL1 has been shown to signal through its sole receptor CX3CR1, which is located on microglial cells within the central nervous system (CNS). Although both the membrane bound and soluble forms of CX3CL1 appear to interact with CX3CR1, they do seem to have different signaling capabilities. It is believed that the predominant function of CX3CL1 within the CNS is to reduce the proinflammatory response and many studies have shown neuroprotective effects. However, in some cases CX3CL1 appears to be promoting neurodegeneration. This review focusses on presenting a comprehensive overview of the complex nature of CX3CL1/CX3CR1 signaling in neurodegeneration and how it may present as a therapeutic in some neurodegenerative diseases but not others. The role of CX3CL1/CXCR1 is reviewed in the context of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), ischemia, retinopathies, spinal cord and neuropathic pain, traumatic brain injury, amyotrophic lateral sclerosis, multiple sclerosis, and epilepsy.

Introduction

A substantial body of evidence supports the hypothesis that neuroinflammation contributes to degeneration in many neurological diseases including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and other neurodegenerative disease. Elevations in adaptive and innate immune markers have been widely reported in both animal models and human subjects and correlate with disease progression (Ardura-Fabregat et al., 2017; Hopperton et al., 2018; Novellino et al., 2020; Pajares et al., 2020; Valadao et al., 2020). Modulation of inflammation in neurodegenerative diseases represents a realistic and valuable therapeutic target for reducing disease progression. Neuroinflammation can be modulated by neuron-glial signaling through various soluble factors, such as cluster of differentiation 200, 22, 47 (CD200, CD22, CD47) and CX3CL1, making these molecules of particular interest for therapeutic intervention.

CX3CL1 is unique among chemokines in a number of aspects. Firstly, CX3CL1 is the sole member of the CX3C family of chemokines. Secondly, CX3CL1 has only one reported receptor, CX3CR1, unlike other chemokines and cytokines which can interact with multiple receptors (Nomiyama, Osada, & Yoshie, 2013). Lastly, CX3CL1 is one of two known transmembrane anchored chemokines (CXCL16 being the other), which can be proteolytically processed to a soluble chemokine (Abel et al., 2004). CX3CL1 is produced in neurons with in the central nervous system and is critical for neuron-glial signaling.

The maintenance of normal CX3CL1/CX3CR1 signaling appears to be critical for normal brain function. Mice deficient in Cx3cr1 (Cx3cr1−/−) demonstrate cognitive deficits in both hippocampal- and cerebellar-dependent learning tasks, in conjunction with synaptic deficits such as decreased synaptic function and abnormalities in synaptic pruning (Cook et al., 2001; Paolicelli et al., 2011; Rogers et al., 2011; Zhan et al., 2014). In aging, CX3CL1 levels were found to be decreased in the brains of aged wild type mice when compared to younger controls (Wynne et al., 2010). Altogether these results suggest that a dysregulation in fractalkine signaling with age and or disease could result in cognitive dysfunction. In this review, we will further discuss the known literature on the involvement of CX3CL1 (fractalkine) and its signaling in neurodegeneration.

Chemokines are a class of peptides that were first identified by their chemoattractant properties towards bone marrow derived cells. Chemokines are divided into four families: C, CC, CXC, and CX3C, according to the number and spatial organization of conserved cysteine residues in their N-terminus (de Munnik et al., 2015). The protein fractalkine (CX3CL1) is the sole member of one of the CX3C family. CX3CL1 is the exclusive ligand of CX3CR1, a 7 transmembrane domain class A G-protein coupled receptor (GPCR) that is expressed primarily on microglia, monocytes, natural killer (NK) cells, T cells and smooth muscle cells (Sheridan & Murphy, 2013).

Studies using transgenic mice (CX3CL1cherry:CX3CR1gfp), which express red and green fluorescent reporter genes under the respective control of the CX3CL1 and CX3CR1 promoters allowed specific localization of expression of CX3CL1 and CX3CR1 (Kim et al., 2011). Confirming previous studies, these results showed that within the central system (CNS) and during steady state, CX3CL1 expression was restricted to mature neurons (Harrison et al., 1998) specifically in the hippocampus, striatum, and cortical layer II, and was absent from the brainstem, midbrain, and cerebellum (Kim et al., 2011; White & Greaves, 2012). Neuronal synthesis of CX3CL1, but not its processing, was also shown to be induced by noradrenaline, through activation of β2 adrenergic receptors (Madrigal et al., 2017). At the periphery, CX3CL1 is primarily expressed by epithelial cells in lung, kidney and intestine and is increased in endothelial cells at sites of local inflammation (White & Greaves, 2012), but CX3CL1 expression is higher in the brain than in most other organs (Biber, Vinet, & Boddeke, 2008).

CX3CL1 is one of two unique chemokines in that it is expressed as a transmembrane protein that can be proteolytically processed to generate a soluble chemokine (Fig. 1). CX3CL1 is 373 amino acid length protein produced with an N-terminal chemokine domain (77 amino acid receptor binding domain), a long mucin-like stalk thought to possess adhesive properties, a single transmembrane domain and a short C-terminal cytoplasmic peptide (Fig. 1). The CX3CL1 molecule is subject to cleavage at the cell membrane by various sheddases such as A Disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10), an alpha secretase which acts constitutively (and also produces sAPPα), and ADAM 17 or Tumor necrosis factor-alpha converting enzyme (TACE) which is induced during inflammation and also processes tumor necrosis factor α (TNFα) to an active form (Garton et al., 2001; Hundhausen et al., 2003). Recent findings also show that CX3CL1 is a substrate for beta secretase (BACE1), and gamma secretase, which are also involved in the production of Aβ from amyloid precursor protein (APP) cleavage (Fan et al., 2019). Cathepsin S is also known to generate the soluble form of CX3CL1, but it appears to generate a smaller soluble peptide by cleaving within the mucin like stalk domain (Clark, Yip, & Malcangio, 2009; Fonovic, Jevnikar, & Kos, 2013).

This proteolytic processing of CX3CL1 appears to regulate its function. We have recently shown differential activity for different forms of CX3CL1 in the context of cognitive function. Cognitive impairment, hippocampal neurogenesis and long-term potentiation were recused in CX3CL1−/− mice after the expression of sCX3CL1 but not a membrane-bound CX3CL1 mutant (Winter et al., 2020). Similarly, we have observed neuroprotective effects of sCX3CL1 but not the membrane CX3CL1 in Parkinson's disease (Morganti et al., 2012; Nash et al., 2015). Other groups have seen differences in different diseases, which are all discussed in more detail below, but clearly demonstrate that proteolytic processing of CX3CL1 could be a regulatory mechanism for cell signaling within the CNS.

In the CNS, CX3CL1 is neuroprotective in both glutamate and NMDA induced neurotoxicity in primary cultures in vitro (Deiva et al., 2004; Lauro et al., 2008; Lauro et al., 2010; Lauro et al., 2015; Scianni et al., 2013). CX3CL1 stimulation of primary microglia and BV2 cells results in release of adenosine which with adenosine 1 receptor (A1R) was shown to contribute to the neuroprotective effects of CX3CL1 on neurons (Lauro et al., 2008; Lauro et al., 2010). Interestingly, CX3CL1 can modulate NMDA-mediated synaptic transmission in the hippocampal CA1 region through the activity of the A2AR and the release of d-serine from glia (Scianni et al., 2013). This activity through A2AR and extracellular d-serine contributes to the protective effect of CX3CL1 in NMDA neurotoxicity (Lauro et al., 2015).

In peripheral tissues, CX3CL1 has 4 primary functions. The soluble CX3CL1 (sCX3CL1) functions as a chemotactic peptide which forms a concentration gradient in extracellular matrix attracting leukocytes to sites of inflammation. In endothelial cells, the membrane bound CX3CL1 provides an adhesive function to capture circulating cells expressing CX3CR1. This leads to the infiltration of leukocytes into the tissues (Zhang & Patel, 2010). Once within the tissue, CX3CL1 can induce proliferation of the leukocytes. In addition, a more recently described function is to enhance the survival of the cells within the inflamed tissue. This latter function appears dependent upon the presence of full length CX3CL1, as the chemokine domain alone was not sufficient to promote the survival function in gene replacement studies of knockout mice (White & Greaves, 2012). In a number of disease conditions, such as atherosclerosis (Liu & Jiang, 2011), chronic pulmonary inflammation (Zhang & Patel, 2010) and rheumatoid arthritis (Jones, Beamer, & Ahmed, 2010), CX3CL1 signaling is thought to promote the maintenance of inflammatory states. Importantly, this impact is due to recruitment and maintenance of inflammatory leukocytes to the tissue.

The expression of CX3CR1 in the CNS is still somewhat controversial. Some studies using primary cell cultures from rat (Meucci et al., 2000) or human embryonic primary neurons (as well as human neuroblastoma SK-N-SH) (Deiva et al., 2004) suggest expression of CX3CR1 in neurons. This expression was attributed to the neuroprotective effects of CX3CL1 with signaling through protein kinase B (Akt) and Extracellular signal-regulated kinase (ERK) (Deiva et al., 2004; Meucci et al., 2000). Wang et al. (2018) observed in permanent middle cerebral artery occlusion (pMCAO) elevated CX3CR1 expression in the peri-infarct, hippocampus, and striatum of the ipsilateral hemisphere at 24 h after occlusion (Wang et al., 2018b). This increase was associated with increased co-labeling with the neuronal marker NeuN. Contrary to these finding, other studies using the transgenic mouse expressing green florescent protein (GFP) in place of the Cx3cr1 gene (Cx3cr1GFP/GFP) do not show expression of GFP in neurons suggesting microglial restrictive expression (Cardona et al., 2006; Jung et al., 2000; Kim et al., 2011; Limatola & Ransohoff, 2014; Mizutani et al., 2012). Whether this inconsistency is due to in vitro culturing in the absence of coculture, or expression due to stress/insult should be further studied.

Much of the work on CX3CL1/CX3CR1 in brain has taken advantage of Cx3cl1−/− and Cx3cr1−/− mouse models (Table 1). These mice demonstrate increased neurotoxicity in a number of neuroinflammation and neurodegeneration models including systemic lipopolysaccharide (LPS) administration, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity, and degeneration in the SOD1G93A model of amyotrophic lateral sclerosis (Cardona et al., 2006; Garcia et al., 2013; Morganti et al., 2012). In the absence of CX3CR1, the increased toxicity was interpreted to be due to elevated microglial proinflammatory activity, since CX3CL1 signaling decreases the overproduction of inducible nitric oxide synthase, interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6 (Garcia et al., 2000; Maciejewski-Lenoir et al., 1999; Ransohoff, Liu, & Cardona, 2007; Ré & Przedborski, 2006; Zujovic et al., 2000; Zujovic et al., 2001) (Fig. 2).

Impairment in CX3CR1-CX3CL1 signaling leads to aberrant microglial activation in various animal models of CNS diseases. There are six documented naturally occurring genetic variants of CX3CR1 gene, namely, E13D, T57A, V122I, V147I, V249I and T280M. Although many variants have been documented for CX3CR1 gene, individuals carrying single nucleotide polymorphisms (SNPs) V249I and T280M have impaired protein activity. In humans, most individuals carry CX3CR1 – Val249-Thr280, while 25–30% of the population carry the variant CX3CR1 – Ile249-Met280. These genetic variants have lower risk of developing cardiovascular diseases such as atherosclerosis, coronary endothelial dysfunction, and acute coronary syndrome (McDermott et al., 2003; Moatti et al., 2001). However, individuals with homozygous V249I and T280M genotypes are associated with more rapid progressions to Acquired immunodeficiency syndrome (AIDS) (Faure et al., 2000) and increased susceptibility for age-related macular degeneration (Tuo et al., 2004; Zhang et al., 2015). These genetic variants (CX3CR1 – Ile249-Met280) have higher adhesive capacity to the membrane bound form of CX3CL1 when compared to the wild type genotype (CX3CR1 – Val249-Thr280) and may explain the protective effects of these genetic variants against cardiovascular diseases (Daoudi et al., 2004). In Crohn's disease, patients homozygous for the CX3CR1-Met280 genotype have significantly increased frequency for intestinal stenoses and ileocolonic involvement when compared to patients carrying CX3CR1 – Ile249-Met280 or CX3CR1 – Val249-Thr280 (wild type) (Brand et al., 2006). The role of the CX3CR1 variants in neurodegenerative disorders is discussed in the different sections below.

Section snippets

Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease, which is characterized by the formation of extracellular amyloid plaques (or senile plaques) and intracellular neurofibrillary tangles resulting from tau protein hyperphosphorylation and aggregation (Soria Lopez, Gonzalez, & Leger, 2019). Neuroinflammation is also a hallmark of AD (Heneka et al., 2015), and is associated with activated microglia, increased expression of cell surface receptors, and secretion of cytokines and

Summary

CX3CL1/CX3CR1 signaling has been associated with a growing number of neurodegenerative diseases, because of its critically important role in the regulation of the microglial phenotype. Microglia are extremely pleiotropic and are constantly sensing the environment and respond to the molecular cues, including CX3CL1, in a manner dependent on the summation of those cues. However, the role of CX3CL1 is obviously more complex than we currently understand, as CX3CL1 signaling appears to lead to

Grant support

PCB is supported by a VA SRCS Salary Award 5IK6BX004214.

Declaration of Competing Interest

KRN and PCB were issued the patent (#10815285) titled Recombinant adeno-associated virus-mediated expression of fractalkine for treatment of neuroinflammatory and neurodegenerative diseases. MSS and AJA declare that there are no conflicts of interest.

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