Entanglement of CCR5 and Alzheimer’s Disease

Although the mechanisms of Alzheimer’s disease are diverse and unclear, the past 20 years have witnessed the unprecedented development of the AD inflammation theory. As a key inflammatory receptor family, the C-C chemokine receptor family is a remarkable participant in the cause of Alzheimer’s disease; of this family, CCR5 is the most widely studied. CCR5 is an essential entrance when HIV infects immune cells and is also involved in other inflammatory and immune activities. New evidence on the inevitably intertwined link between Alzheimer’s disease and CCR5 indicates that CCR5 accelerates the development of Alzheimer’s disease, and few studies disputed it. The role of CCR5 in Alzheimer’s disease remains elusive. However, as the research progresses, this intricate relationship will gradually be uncovered.

INTRODUCTION C-C chemokine receptors 5 (CCR5) is a kind of cytokine belonging to the β chemokine receptor family of integral membrane proteins (Power et al., 1995). CCR5 is well known (or notorious) mainly because it collaborates with human immunodeficiency virus-1 (HIV-1) when the virus enters target immunological cells (Murphy, 2001;Ghalib, 2009). Intriguingly, most current anti-HIV drugs target CCR5. At the end of the 2018, the announcement of Chinese gene edited infants whose CCR5 genes were deleted by CRISPR-Cas9 again brought the debate about CCR5 to the forefront.
Our receptor of interest, the chemokine receptor, is involved in substantial inflammatory diseases of the central nervous system (CNS) and functions in the recruitment and immigration of immune cells. These receptors are mainly distributed in microglia and recruited peripheral blood monocytes in the CNS (El, 2010), while they are rarely expressed on neuronal cell membranes

Microglia as a Double-Edged Sword
Notwithstanding existing evidence indicating that peripheral immune cells can infiltrate brain tissue through the damaged blood-brain barrier (BBB) under pathological conditions and cause inflammation (Villeda Saul et al., 2011), native microglia and activated astrocytes, which widely reside everywhere throughout the brain, are the main contributors to neuroinflammation. Unlike neurons, astrocytes, and oligodendrocytes, microglia are considered resident immune cells from the mesoderm and function in some housekeeping work, including neurogenesis, the trimming and stimulation of synapses, the modulation of cognitive processes, and immunological surveillance. Gradual deterioration of the immune system increases vulnerability to infections and diseases as individuals age. Some altered expression of microgliarelated genes that could accelerate the progression of AD has been identified in AD patients (Cribbs et al., 2012). Microglia isolated from postmortem aging brains also showed significant transcriptome characteristics compared with those obtained from younger brains; genes associated with celladhesion axon-guided cell surface receptor expression and actin assembly were specifically affected (Galatro et al., 2017). Moreover, microglial cells from the aged cortex show many morphological abnormalities, including the formation of nodal processes and division processes of acellular globules (Streit Wolfgang et al., 2010).
The proteins that microglia produce exhibit dramatic changes during aging, neurodegeneration, and neuroinflammation. For example, secreted cytokines, displayed membrane proteins, and energy and metabolism protein needs differ drastically (Ritzel et al., 2015;Mrdjen et al., 2018). However, the complex relationship between microglia, tau, and amyloid beta protein (Aβ) remains controversial. On the one hand, many studies have indicated that microglia are capable of congregating around Aβ and phagocytosing it to attenuate the pathological load. On the other hand, activated microglia could damage neurons and vascular epithelial cells, which lead to additional impairment of cognitive function (Grathwohl and Kalin, 2010;Seabrook et al., 2010;Dagher et al., 2015;Olmos-Alonso et al., 2016;Spangenberg et al., 2016). The distinct subtypes of microglia contribute to the AD course in a diverse manner, and the heterogeneity of pro-and anti-inflammatory timing of microglia is a formidable obstacle (Martin et al., 2010;Heneka Michael et al., 2013;Medeiros et al., 2013;Chakrabarty et al., 2015;Marie-Victoire et al., 2015;Fu et al., 2016). Reactive microglia can accelerate the propagation of tau pathology, thereby causing a deterioration in inflammation (Yoshiyama et al., 2007;Sanchez-Mejias et al., 2016). Deposition of Aβ worsens neurodegeneration and triggers pro-inflammatory responses. Moreover, Aβ binds to inflammatory receptors (Yan et al., 1996;Deane et al., 2003;Shirong et al., 2012) and promotes immune-related transcriptional signals (Bonaiuto et al., 1997;Rodrigo et al., 2007) (Figure 1). When microglia and astrocytes clear Aβ through phagocytosis and intracellular degradation, many transmembrane receptors (e.g., LRP-1 and ATP-binding cassette transporter family) bind to Aβ, and most of these receptors are essential in immune and inflammatory responses (Anthony et al., 2013;Chuang et al., 2016).

Molecular Receptor Spectrum of Neuroinflammation
The theoretical framework regarding how inflammation interacts with the development of AD has been widened and explored on an unprecedented scale. Internationally renowned pharmaceutical companies have already started multiple clinical drug trials on the anti-inflammatory treatment of AD (Cuello, 2017). Despite cellular components (e.g., microglia, astrocytes, and endothelial cells), an increasing number of receptors, membrane proteins and metabolic enzymes have become candidates for further research and clinical trials. For example, positive modulation of a7 nicotinic acetylcholine receptors (a7nAChRs) had therapeutic potential for its anti-inflammatory effects (Echeverria et al., 2016). The lower dose of colonystimulating factor 1 receptor (CSF1R) inhibitors led to increased hippocampal-dependent spatial memory (Spangenberg and Green, 2017). Angiotensin II receptor blockers (ARBs) were considered a neuroprotective candidate against early damage factors to neurons, astrocytes, microglia, cerebrovascular endothelial, and BBB; consequently, ARBs prevent cognitive loss and dementia (Saavedra, 2016). Mimetics of glucagon-like peptide 1 (GLP-1) receptor agonists, first designed to treat diabetes, were capable of reducing inflammation, oxidative stress, and apoptotic signaling and protecting memory formation (Hölscher, 2018). In addition to membrane receptors, a substantial number of studies revealed that nuclear receptors could be potential interventional targets to suspend dementia progression in AD (Moutinho and Landreth, 2017). Notably, numerous studies have focused on various inflammatory receptors and diverse signaling pathways. Among these components, chemokine receptors, which are mainly studied in immunology, deserve further attention. The birth of Chinese gene edited infants has greatly enriched scientific theories because some studies showed that CCR5 deficiency could improve both prognosis and neuronal plasticity after stroke and traumatic injury, which encouraged an unprecedented CCR5 research boom (Joy et al., 2019).

CCR5
CCR5: Structure, Distribution, and Function CCR5 belongs to the seven transmembrane G-protein coupled receptors (GPCRs) that transmit signals via heterotrimeric G proteins (Pierce Kristen et al., 2002). CCR5 consists of 7 hydrophobic transmembrane domains with an extracellular N-terminus and cytoplasmic C-terminus, ranging in length from 340 to 370 amino acids (Oppermann, 2004). CCR5 is the receptor for nearly 10 chemotactic proteins of the β chemokine family, which are also named CC chemokines (Griffith et al., 2014).
CCR5 is widely known because it provides access for HIV-1 to invade host immune cells. The gp120 envelope protein is a chemokine mimic; thus, it can bind to CCR5 during HIV infection (Murphy, 2001). Additionally, CCR5 can accelerate the transmission of the R5-strain of the HIV-1 virus (Lieberman-Blum et al., 2008). CCR 32 is a 32-base-pair deletion that inserts a premature stop codon into the CCR5 receptor locus, leading to receptor dysfunction. Homozygous carriers of this mutation cannot be infected with M-tropics strains of HIV-1 (De and Stumpf, 2004;Gero et al., 2009;Rieger et al., 2010;Kay and Walker, 2014).
Several lines of cancer cells (e.g., breast and prostate cancer cells), instead of normal epithelial cells, express a high level of CCR5, which help the cancer transformation process (Velasco-Velázquez et al., 2012;Daniela et al., 2014). Furthermore, the chemotherapy resistance of cancer stem cells could be partly accredited to the expression of CCR5 (Jiao et al., 2018).

CCR5: Signal Pathway
CCR5 activates the cellular signaling pathway through G proteins, pertussis-sensitive heterotrimeric G proteins and G protein-independent pathways (Oppermann, 2004). The binding of a chemokine ligand to CCR5 results in conformational changes in G proteins, which will enable various signaling cascades, including those of the phosphoinositide-3 kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK), as well as calcium influx (Oppermann, 2004;Sudarshan et al., 2013). At present, G-protein-independent signaling pathways involving the Janus kinases (JAK), pyk2 kinase, and arrestin pathways have been identified (Corno et al., 2001;Mueller and Strange, 2004). These cascades activate various cellular functions, cytoskeleton recombination, and chemotaxis. Calcium flux is an essential procedure for sequent signal activation in the CNS. Given that various types of cells express CCR5, it is not impossible that its diverse biological effects require more than G-proteininitiated pathways. The different chemokine ligands that CCR5 binds to determine distinct signaling pathways accounting for the pleiotropic effects of chemokine signaling (Mueller et al., 2006;Jenny et al., 2015).
Additionally, blockage of CCR5 signaling could not only lead to rejection of HIV but also elicit neuroprotective mechanisms and promote neuron survival, which will alleviate HIV-associated dementia (Kaul and Lipton, 2006;Christoph and Pett Sarah, 2012;Martin-Blondel et al., 2016).

The Efficacy of CCR5 Antagonists
The promising development of CCR5 receptor inhibitors is largely based on CCR5's identity as a necessary pathway for AIDS infection. The only first-generation CCR5 antagonist that the Food and Drug Administration (FDA) approved, Maraviroc, was well tolerated and showed excellent repression of viral load in patients whose highly active antiretroviral therapy (HAART) failed but not in treatment-naïve patients (Lieberman-Blum et al., 2008;Sierra-Madero et al., 2010;Van Lelyveld et al., 2012). The high efficacy of maraviroc has set the tone for second-generation CCR5 antagonists, which account for the failures of aplaviroc and vicriviroc in clinical trials (Gulick et al., 2007;Leeson and Springthorpe, 2007;Stupple et al., 2011). Various modified and innovative second-generation CCR5 antagonists [e.g., piperidine amide compounds (Imamura et al., 2006;Ernst et al., 2008;Stupple et al., 2011), piperazines and diketopiperazines (Liu et al., 2008)] demonstrated guaranteed efficacy in I/II clinical trials, especially the dual CCR5/CCR2 antagonists (Meyer et al., 2013).
However, more data have revealed that CCR5 antagonists function in other disease courses. Dual CCR5/CCR2 antagonists and cenicriviroc could inhibit the progression of non-alcoholic fatty liver disease (Tacke, 2018;Ogawa et al., 2019). In addition, cenicriviroc could reduce liver injury in cholestatic rodents (Yu et al., 2018). Moreover, the inhibition of lymphocyte migration caused by CCR5 blockade could alleviate graft-versus-host disease (Reshef et al., 2019). Could the CCR5 blockade provide relief from AD?

AD AND CCR5
The definite relationship between AD and CCR5 remains an open debate. Disputable results from basic experimental studies have diverged into two opposed groups. Most of studies demonstrated that the expression of CCR5 contributes to the development of AD (Table 1), while the minority demonstrated that CCR5 could improve memory function in AD (Table 2). However, all existing epidemiologic studies confirmed that there was no Immunohistochemical study of the β-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains. Xia et al., 1998 Human Immunohistochemistry of brain tissue of post mortem human being.
CCR5−/− mice had less activation of microglia and astrocytes after injection of Aβ into lateral ventricle.
Role of the macrophage inflammatory protein-1alpha/CC chemokine receptor 5 signaling pathway in the neuroinflammatory response and cognitive deficits induced by beta-amyloid peptide.
CCR5 antagonists attenuated the neuroinflammation of sub cutaneous administration of lipopolysaccharide by decreasing the number of activated microglia and astrocytes.
Morphine induces the release of CCL5 from astrocytes: potential neuroprotective mechanism against the HIV protein gp120.

Avdoshina et al., 2010 Rats
Administration of CCR5 antagonist to rats preinjected with lipopolysaccharide.
CCR5 participated in the impairment of learning and memory in AD by activating microglia and promoting T cells transendothelial migration.
Peripheral T cells overexpress MIP-1α to enhance its transendothelial migration in Alzheimer's disease.

Man et al., 2007
Human and rats CCR5 detection of peripheral blood mononuclear cells from AD patients and healthy controls; Peripheral intravenous injection of Aβ in rats, followed by administration of CCR5 CCR5 antagonist (2D7 mAb).
The ligands of CCR5, CCL3 and CCL4, were upregulated in microglia isolated from AD patients' brain and stimulated with Aβ.
Gene expression profiling of amyloid beta peptide-stimulated human post-mortem brain microglia.

Walker et al., 2001
Human Gene array technology.
The CCR5 expression of PBMC from AD patients was significantly higher and in vitro PBMC culture with Aβ increased the CCR5 expression.
Peripheral chemokine receptors, their ligands, cytokines and Alzheimer's disease.

Reale et al., 2008
Human CCR5 detection of PBMC from AD patients and healthy controls.

The proportion of cells expressing CCR5 (Th1 cells and dendritic cells) was greater in AD patients.
Enhanced Chemokine Receptor Expression on Leukocytes of Patients with Alzheimer's Disease. Goldeck et al., 2013 Human CCR5 detection of PBMC from AD patients and healthy controls.
Mechanism of amyloid peptide induced CCR5 expression in monocytes and its inhibition by siRNA for Egr-1. Giri et al., 2005 Human CCR5 detection of PBMC from AD patients and healthy controls and in vitro administration of siRNA for inhibiting CCR5 relevant signal pathways.
The curcumin inhibited Aβ associated expression of CCR5 by preventing Egr-1 DNA binding to the promoter of CCR5.
Curcumin, the active constituent of turmeric, inhibits amyloid peptide-induced cytochemokine gene expression and CCR5-mediated chemotaxis of THP-1 monocytes by modulating early growth response1 transcription factor. Giri et al., 2005 Human Administration of curcumin into PBMC culture in vitro.
The CCR5 antagonist (DAPTA) of monocyte chemotaxis, was proved to reduce chronic neuroinflammation of AD.  association between the CCR5 32 allele and AD risk. Therefore, the entanglement of AD and CCR5 urgently needs to be addressed ( Table 3).
The Major Group: CCR5 Exacerbates AD (Table 1) Memory, learning and plasticity processes in hippocampal and cortical circuits involve electrochemical activity of neurons, including long-term potentiation (LTP), release of glutamate, activation of N-Methyl-D-aspartic acid (NMDA) receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and the generation of new dendritic spines and axons. Cai et al. (2016) reported that weakening the function of CCR5 in the mouse barrel cortex improved MAPK/CREB signaling, which resulted in enhanced spike-timing-dependent plasticity and experience-dependent plasticity (Figure 2). Consequently, LTP and hippocampus-dependent memory significantly improved, while neuronal CCR5 overaction led to memory deficits (Cai et al., 2016). This cornerstone study underlies the foundation of the relationship between memory and CCR5, which also implies a potential discoverable link between CCR5 and AD. The increase in both CCR5 and CCR3 on some reactive microglia was found in AD patients and associated with amyloid deposition (Xia et al., 1998). After the administration of Aβ into the lateral ventricle of CCR5−/− mice, the activation of microglia and astrocytes was decreased compared with that in CCR5 wild-type mice (Passos et al., 2009). CCR5−/− mice showed decreased astrocytosis and microgliosis in the hippocampus after Aβ injection, which resulted from decreased expression of cyclooxygenase-2 and inducible nitric oxide (NO) synthase, as well as reduced activation of nuclear factor-κB (NF-κB), activator protein-1 and cyclic AMP response element-binding protein (Passos et al., 2009). In humans, there were more widespread reactive astrocytes with CCR5 expression in AD than in healthy (Xia et al., 1998) controls. In addition, the impairment of memory and synaptic dysfunction caused by Aβ were alleviated in CCR5−/− mice (Passos et al., 2009). Similarly, both knockout of the CCR5 gene and administration of maraviroc (FDA-approved anti-HIV drug) could heighten plasticity in the premotor cortex proximal to the stroke site and upregulate CREB and DLK signaling in neurons, which finally aided in the formation of new connections in the contralateral premotor cortex (Joy et al., 2019). Although this conspicuous study did not connect CCR5 with AD, it suggested that CCR5 and its signaling pathways had a pivotal impact on the generation and regeneration of dendritic spines and synapses. Peripheral blood immune cells can filter into the CNS with the help of  CCR5 (Man et al., 2007). Man et al. (2007) suggested that CCR5 participated in the impairment of learning and memory in AD by activating microglia and promoting T cell transendothelial migration through the Rho/ROCH pathway. CCL3 and CCL4, as ligands for CCR5, were upregulated in microglia isolated from AD patient brains and stimulated with Aβ (Walker et al., 2001). In addition, peripheral blood mononuclear cell (PBMC) analysis suggested that the expression of both CCR5 and CCR2 were augmented in AD patients compared with those in control subjects (Reale et al., 2008). Further experiments suggested that acetylcholinesterase inhibitor (ACEI) was capable of decreasing the expression of CCR5 and CCR2 in the PBMCs of AD patients, while in vitro administration of Aβ increased their expression in PBMCs (Reale et al., 2008). Similar results were also obtained by Goldeck et al. (2013), who found that the distribution of cells expressing CCR4 (expressed on Th2 cells) and CCR5 (Th1 cells and dendritic cells) was also greater in patients and was more distinguished on CD4+ than CD8+ T cells. A CCR5 antagonist could reduce the number of active astrocytes and microglia after lipopolysaccharide injection, which implied that CCR5 was involved in neuroinflammation related to AD (Combarros et al., 2004). Giri et al. (2005) indicated that Aβ could increase CCR5 expression through cellular signaling of c-Raf, ERK-1/ERK-2, and c-Jun NH2-terminal kinase in PBMCs. As mentioned above, CCR5 is involved in neuroinflammation, wherein various CCR5 antagonists can attenuate the neuroinflammation associated with AD. D-Ala-peptide T-amide (DAPTA), a kind of CCR5 antagonist of monocyte chemotaxis, was shown to reduce chronic neuroinflammation by blocking the release of the proinflammatory cytokines TNF-α and IL-1 (Ruff et al., 2003;Rosi et al., 2005). After the administration of Egr-1 siRNA, Aβ-associated CCR5 expression and its concomitant ligands decreased significantly (Giri et al., 2005). Curcumin also inhibited the Aβ-associated expression of CCR5 by preventing Egr-1 DNA binding to the promoter of CCR5 (Giri et al., 2010).
The Minor Group: CCR5 Improves AD (Table 2) However, CCR5 expression was reduced, and both chemokine CCL3 and CCL4 chemokine expression levels were augmented in amyloid precursor protein plus presenilin-1 (APP/PS1) mice, which are a common model for AD studies (Obrador et al., 2019). Additionally, Hwang et al. (2016) showed that CCR5 reduction led to higher Aβ deposition and impairment of memory. Lipopolysaccharide injection of CCR5 knockout mice significantly increased astrogliosis and Aβ deposition compared with those in CCR5 wild-type mice (Hwang et al., 2016). Within the hippocampus and cerebral cortex of Tau-P201L mice, which is another frequently used model for AD, CCR5 gene expression was significantly reduced over time compared with that in wild-type mice, while no distinction was found between other brain regions (Andreassen et al., 2018). CCR5−/− mice showed a higher level of Aβ, which was related to astrocyte activation and CCR2 overexpression. These changes finally caused memory impairments .
No Epidemiological Link Between AD and CCR5 (Table 3) Despite the fact that experimental studies were full of controversies and the conclusive relationship between AD and CCR5 remained unknown, the epidemiological evidence was certain and negative. Several epidemiological studies confirmed that there was no differential distribution of the CCR 32 deletion in AD patients and controls in Italy (Balistreri et al., 2006). Moreover, no differences were observed by gender stratification, by the presence of the ApoE q4 allele, or by the age at onset in genotype distribution and allelic frequency (Balistreri et al., 2006). Identical results were found in Spanish and Iran: the CCR5 32 allele was not a correlative factor for AD (Galimberti et al., 2004;Onofre et al., 2004;Salvador et al., 2004).

CONCLUSION AND OUTLOOK
The definite mechanism of AD remains unclear, but our understanding of AD has far exceeded the two typical pathological manifestations-Aβ and tau. The neuroinflammation theory of AD is fascinating an increasing number of researchers. Currently, more than 5000 studies about AD and neuroinflammation are available on PubMed, and the number is expanding. CCR5 expression was strongly related to microglia and inflammation, which validated an inseparable relationship between inflammation, Alzheimer's disease, and CCR5 (Andreassen et al., 2018). As the most notable receptor among the chemokine receptor family, the accurate function and subsequent signaling pathways of CCR5 have been well studied. Available epidemiological evidence could provide no connection between the CCR5 32 gene and AD (Onofre et al., 2004;Salvador et al., 2004;Balistreri et al., 2006). However, given that all of the epidemiological evidence was based on genotype and performed more than a decade ago, potential links may lie deeper than the superficial genotype, such as translational modulation, proteomics, and epigenetics. Despite the controversy, it cannot be denied that CCR5 plays an important role in the process of LTP, cortical plasticity, learning and memory (Cai et al., 2016). We posited that the reason for experimental conflicts between CCR5 and AD are as follows: the animal models and the methods the authors used for acquiring AD models were varied. Furthermore, compared with administration of small interfering RNA (siRNA), maraviroc and other antagonists, the effect of total knockout of the CCR5 gene could lead to an enormous impact on cellular signal pathways inside the membrane. Further investigation should take these factors into account. Future studies of molecular mechanisms of subsequent intracellular signal pathways caused by CCR5 activation would shed new light on this entanglement between AD and CCR5.

AUTHOR CONTRIBUTIONS
TL contributed significantly on this review. JZ reviewed and proposed valuable modifications.

FUNDING
This work was supported by grants (2018YFA0107900, 31771491, and 2019CXJQ01) from Ministry of Science and Technology of China, the National Nature Science Foundation, and Shanghai Municipal Government.