The P2X7 Receptor in AMD

  

    
dbSNP ID
    
Base change
    
Exon
    
Amino acid change
    
Function compared to WT  or    Disease Association 
    
Reference
  
  

    
rs35933842
    
151+1g → t
    
1
    
Null allele
    
 
      
associated with increased fracture risk and reduced    BMD in women
    
[92]
      
 
      
[100]
  
  

    
rs1752809
    
253T → C
    
2
    
Val76 to Ala
    
Partial inhibition
    
[45]
  
  

    
rs28360447
    
474G → A
    
5
    
Gly150 to Arg
    
loss-of-function-disrupted protein folding, no    pore formation
      
 
      
 
      
associated with increased susceptibility to AMD
      
 
      
associated with decreased hip BMD values
      
 
      
associated with decreased total hip BMD in women    and men combined
    
[44,45]
      
 
      
 
      
 
      
 
      
[35]
      
 
      
 
      
[102]
      
 
      
[100]
  
  

    
rs208294
    
489C → T
    
5
    
His155 to Tyr
    
gain-of-function
    
[45,93, 94]
  
  

    
rs7958311
    
835G → A
    
8
    
His270 to Arg
    
gain-of-function
    
[45]
  
  

    
rs7958316
    
853G → A
    
8
    
Arg276 to His
    
loss-of-function
    
[45]
  
  

    
rs28360457
    
946G → A
    
9
    
Arg307 to Gln
    
loss-of-function
      
 
      
associated with the rate of bone loss in    post-menopausal women
    
[95]
      
 
      
 
      
[101]
  
  

    
rs1718119
    
1068G → A
    
11
    
Ala348 to Thr
    
gain-of-function
      
 
      
 
      
enhanced interleukin-1ß secretion
      
 
      
associated    with a lower vertebral fracture incidence 10 years after menopause in post-menopausal women
      
 
      
associated with increased BMD values at the    lumbar spine
      
 
      
associated    with reduced fracture risk and increased BMD 
    
[94,96]
      
 
      
 
      
[45]
      
 
      
 
      
 
      
[101]
      
 
      
 
      
 
      
[102]
      
 
      
 
      
[100]
  
  

    
rs2230911
    
1096C → G
    
11
    
Thr357 to Ser
    
loss-of-function
    
[97]
  
  

    
rs2230912
    
1405A → G
    
13
    
Gln460 to Arg
    
small reduction, no major effect or    gain-of-function
      
 
      
 
      
 
      
 
      
a significantly decrease in risk of a lower BMD    T-score value
      
 
      
associated    with increased total hip BMD in women 
    
[44,45,93, 94]
      
 
      
 
      
 
      
 
      
 
      
[102]
      
 
      
 
      
[100]
  
  

    
rs3751143
    
1513A → C
    
13
    
Glu496 to Ala
    
loss-of-function; surface expression not affected
      
 
      
associated    with protection against bone loss in post-menopausal    women
      
 
      
associated with a decreased risk of IHD in    smokers as well as decreased risk of IS
      
 
      
significantly associated with increased    susceptibility to tuberculosis
      
 
      
associated with decreased hip BMD values
      
 
      
decreased    lumbar spine BMD in women and decreased total hip BMD in men 
    
[98]
      
 
      
 
      
 
      
[101]
      
 
      
 
      
[103]
      
 
      
 
      
 
      
[104]
      
 
      
 
      
 
      
[102]
      
 
      
 
      
[100]
  
  

    
rs1653624
    
1729T → A
    
13
    
Ile568 to Asn
    
loss-of-function
      
 
      
 
      
had    increased bone loss 
    
[98,99]
      
 
      
 
      
[101]
  

Table 1: Single Nucleotide Polymorphisms (SNPs) in P2X7Receptor.

By comparing amino acid sequences, it was found that P2X7R is more homologous to P2X4R (˜40%) than are the other P2X receptor subunits. Given their location adjacent to each other on human chromosome 12, and their overlap in tissue distribution [38,39], efforts have been made to identify if there is a physical and functional interaction between the two receptors. Several studies have found that P2X4R and P2X7R are co–expressed in immune cells and epithelial cells, and heteromerization can change both the functional and pharmacological properties of P2X receptors [40–43].

A loss–of–function polymorphism has been identified in human P2X4R Tyr315Cys (rs28360472) which is associated with increased susceptibility to AMD [35]. P2X7R 474G>A (Gly150Arg) (rs28360447) gene polymorphism leads to a single amino acid substitution from glycine to arginine at residue 150 (Figure 1), producing loss–of–function channel [44,45]. Haplotype analysis showed that the P2X4R 315–Cys minor allele was co–inherited with P2X7R 150–Arg 4–fold more often in patients with AMD than in normal control subjects [35]. Among 17 patients with AMD inheriting the haplotype of P2X4R315–Cys plus P2X7R 150–Arg, 14 were female [35]. Infiltrating macrophages within the choroid and microglia within a monkey neural retina were found to co–express P2X4R and P2X7R [35]. In mouse bone marrow–derived dendritic cells, it was found that expression of P2X4R is required for P2X7R–dependent IL–1β and IL– 18 release [46]. Based on Ca²⁺ influx triggered by ATP and BzATP was insensitive to suramin, we suggested that in addition to P2X7R, P2X4R could contribute to ATP– and BzATP–induced Ca2+ influx in the RPE [17]. Future studies are needed to test this hypothesis.

The P2X7R is a new scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP [47], suggesting the unstimulated P2X7R could have a beneficial role under physiological conditions. Whether the P2X7R is a new scavenger receptor in the RPE needs to be investigated. This is important, because one of the RPE’s essential functions is phagocytosis, removing photoreceptor outer segments (POSs) to maintain neural retina health [48]. Both human and mouse RPE express the P2X7R [17–19,49]. Moreover, P2X7R protein is expressed on both apical and basolateral membranes of mouse RPE monolayer in situ [19]. Compared to human macrophages, ARPE–19 cells (a human RPE cell line) are more efficient in clearing anoikic and UV–induced apoptotic cells [50], suggesting the importance of the RPE in clearance of dying cells and extracellular debris. The P2X7R expressed on apical membrane could participate in phagocytosis of POSs, while the P2X7R expressed on basolateral membranes could be important for clearance of apoptotic cells and against invading bacteria.

The P2X7R in the RPE

The P2X7R is expressed in epithelial cells [13–19], and up regulated by lipopolysaccharide and pro–inflammatory cytokines [18,51,52]. In the RPE, the expression of the P2X7R is also up regulated by aging [18]. Under normal physiological conditions, P2X7Ractivity is kept at a low level by the concentration of extracellular divalent cations [53,54] as well as by low micromolar range of extracellular ATP. Extracellular divalent cations appear to alter the affinity of ATP binding in an allosteric manner [55]. Low micromolar range of extracellular ATP is not favored for P2X7R activation, as the P2X7R is the least sensitive member of the P2X receptor family to activation by ATP with EC₅₀value of 0.1 to 1 mM compared with P2X1– 6receptors whose EC₅₀value is 1 to 10 µM [17,56–58]. This could avoid unnecessary cell permeability and pore formation [59]. Under stress conditions, both RPE cells and neural retina are capable of releasing ATP that can act on P2X receptors in RPE cells and⁄or photoreceptors in an autocrine or a paracrine manner [17,36]. Among seven P2X receptors, P2X7R mRNA and protein have been identified in the RPE by three independent research groups [17–19,49]. We detected not only P2X7R protein, but also P2X7R mRNA in human RPE cells [17]. Guha also detected both P2X7R mRNA and protein in mouse RPE cells, with the P2X7R protein expressed on both apical and basolateral membranes of mouse RPE monolayer in situ. Compared to wild–type mice, P2X7R mRNA in fresh RPE⁄choroid tissue was increased in ABCA4–/– mice, a mouse model of Stargardt’s retinal degeneration [19]. We also found that aging and inflammation up regulated the expression of P2X7R mRNA and protein in the RPE [18]. However, Gu [35], reported that P2X7R protein was not detected in retinal sections from an adult monkey (Macaca fascicularis) eye by using immunofluorescent labeling method. P2X7R mRNA was not reported in their study. The discrepancies between the report by Gu et al. [35] and other three independent research groups [17,19,49] could be explained by species difference. In addition, different sources of commercially available antibodies [60,61] used could play a role. Therefore, caution should be taken when interpreting P2X7R protein expression data obtained by immunocyto chemistry.

The P2X7R is functional in the RPE.ATP is an endogenous P2X7R agonist, while 2’,3’–O–(4–benzoylbenzoyl)–ATP (BzATP) is a synthetic, selective P2X7R agonist [62]. Both ATP and BzATP induce RPE apoptosis after 6 hr or 24 hr stimulation, and increase intracellular Ca²⁺ via extracellular Ca²⁺ influx rapidly in primary human RPE [17]. BzATP also raises intracellular Ca²⁺ in ARPE– 19cells [19]. However, ARPE–19 cells exposed to BzATP (50 or 100 µM) for 60 minutes did not release lactose dehydrogenase into the extracellular media [19], indicating that short exposure did not kill ARPE–19 cells. Both ATP and BzATP increased YO–PRO–1 (629 Da) dye uptake in a human RPE cell line, ARPE–19 cells [49]. BzATP also triggered a rapid and reversible elevation of Ca²⁺ in freshly isolated mouse RPE cells [19]. These data indicate that activation of P2X7R by ATP or BzATP not only opens a membrane channel permeable to Ca²⁺, but also leads to the formation of membrane pore permeable to YO–PRO–1, and cell death in RPE cells. Functional P2X7R in the RPE was further validated in the RPE by using P2X7R antagonists, oxidized ATP (oATP), brilliant blue G (BBG), and 1–[N,O–Bis(5– isoquinolinesulfonyl)–N–methyl–L–tyrosyl]–4–phenylpiperazine (KN– 62). Oxidized ATP significantly inhibited ATP– or BzATP–induced Ca²⁺ influx and apoptosis by the RPE. BzATP–induced RPE apoptosis was blocked or significantly inhibited by P2X7R antagonists BBG, KN–62, and oATP [17]. Reduction or removal of extracellular Ca²⁺ or the buffering of intracellular Ca²⁺ with BAPTA–AM significantly inhibited or blocked ATP–induced apoptosis [17]. These findings suggest that the P2X7R contributes to ATP– and BzATP–induced Ca²⁺ signaling and apoptosis in the RPE. Therefore, abnormal Ca²⁺ homeostasis and membrane pore formation through the activation of P2X7R could cause the dysfunction and apoptosis of RPE that underlie AMD.

One of major functions of the RPE is to degrade phagocytosed POSs. Guha et al. [19] demonstrate that stimulation of P2X7Rby 100 µMBzAT Palkalinizes lysosomes in ARPE–19 cells. Although P2X7R antagonistA438079 [63] had greater potency in blocking BzATP–induced intracellular Ca²⁺ increase in recombinant mouse, rat or human P2X7R–expressed human astrocytoma 1321N1 cells, compared with BBG [64], the potency of A438079 to inhibit BzATPinduced increase in lysosomal pH in ARPE–19 cells seemed lower, compared with BBG. A438079 at 10 µM and BBG at 1 µM suppressed BzATP–induced increase in lysosomal pH to a similar extent [19]. This BzATP–induced lysosomal alkalinization was dependent on extracellular Ca²⁺, because BzATP was unable to increase lysosomal pH in the absence of extracellular Ca²⁺ [19], indicating P2X7R plays a role in lysosomal alkalinization. Lysosomal enzymes function optimally at low pH. Thus, lysosomal alkalinization could impair lysosomal function. Indeed, they found that blockage of the P2X7R by BBG was able to reduce lipid oxidation and lipofuscin–like autofluorescence induced by POSs plus lysosomotropic agent chloroquine [19]. Cathepsin D is a major proteolytic enzyme participating in the lysosomal digestion of phagocytosed POSs [65,66]. BODIPY FLpepstatin A selectively binds to cathepsin D at pH 4.5 [67]. Lysosomal alkalinization induced by BzATP reduced BODIPY FL–pepstatin A binding to catheps in D [19], indicating the ability of catheps in D to digest proteins is compromised. Furthermore, stimulation of ARPE–19 cells with BzATP increased the ratio of LC3BII/LC3BI (autophagy markers), and decreased the level of p62 (autophagy adaptor protein), supporting impairment of autophagic flux [19]. Recently, [68] discovered LC3–associated phagocytosis in which the interplay between phagocytosis and autophagy within the RPE is required for degradation of POSs and the maintenance of retinoid levels to support optimal vision. Given that the P2X7R is involved in both autophagy [19,69] and phagocytosis [47], we anticipate that the P2X7R could play a role in this LC3–associated phagocytosis.

The P2X7R in AMD

Oxidative stress, inflammation, and cell death are implicated in AMD [8,70–73]. As activation of P2X7R induces Ca²⁺–dependent apoptosis and lysosomal alkalinization in the RPE [17,19], we propose that abnormal Ca²⁺ homeostasis, oxidative stress, inflammation, and cell death are important factors for the development of AMD (Figure 2). The RPE maintains a healthy environment for normal photoreceptor function. In dry or atrophic AMD, it appears that the RPE dies first, leading to dysfunction and death of photoreceptors and choriocapillaris; in neovascular or wet AMD, loss of choriocapillaris with an intact RPE monolayer in wet AMD has been observed, indicating that the loss of choroidal vasculature may be the initial insult to the RPE/Bruch’s membrane /choriocapillaris complex [74].

Figure 1b: Proposed model of the P2X7 receptor–mediated signaling that leads to age–related macular degeneration. Schematic diagram shows P2X7 receptormediated signaling pathways that lead to RPE senescence, RPE apoptosis and AMD [17,90,91; 19;37; 36]. Abbreviations Used: AMD, age–related macular degeneration; ATP, adenosine triphosphate; IL–8, interleukin 8; IL–18, interleukin–18; MCP–1, monocyte chemoattractant protein–1; NLRP3, nucleotide–binding domain and leucine–rich repeat containing family, pyrin domain containing 3; RPE, retinal pigmented epithelium; ROS,reactive oxygen species; VEGF, vascular endothelial growth factor.