Pharmacology & Therapeutics 225 (2021) 107840

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Purinergic signaling in nervous system health and disease:

Focus on pannexin 1
Juan C. Sanchez-Arias ⁎,1, Emma van der Slagt 1, Haley A. Vecchiarelli, Rebecca C. Candlish, Nicole York,
Penelope A. Young, Olga Shevtsova, Afnan Juma, Marie-Ève Tremblay, Leigh Anne Swayne ⁎
Division of Medical Sciences, University of Victoria, Victoria, British Columbia V8P 5C2, Canada

a r t i c l e i n f o a b s t r a c t

Available online 19 March 2021 Purinergic signaling encompasses the cycle of adenosine 5′ triphosphate (ATP) release and its metabolism into
nucleotide and nucleoside derivatives, the direct release of nucleosides, and subsequent receptor-triggered
downstream intracellular pathways. Since the discovery of nerve terminal and glial ATP release into the neuropil,
Keywords: purinergic signaling has been implicated in the modulation of nervous system development, function, and dis-
Pannexin ease. In this review, we detail our current understanding of the roles of the pannexin 1 (PANX1) ATP-release
Purinergic signaling channel in neuronal development and plasticity, glial signaling, and neuron-glial-immune interactions. We addi-
Nucleotides tionally provide an overview of PANX1 structure, activation, and permeability to orientate readers and highlight
Nervous system recent research developments. We identify areas of convergence between PANX1 and purinergic receptor ac-
Neurodevelopment
tions. Additional highlights include data on PANX1's participation in the pathophysiology of nervous system de-
Neurological diseases
velopmental, degenerative, and inflammatory disorders. Our aim in combining this knowledge is to facilitate the
movement of our current understanding of PANX1 in the context of other nervous system purinergic signaling
mechanisms one step closer to clinical translation.
© 2021 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. PANX1 in the healthy nervous system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. PANX1 in neurological disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Funding sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Abbreviations: AD, Alzheimer's disease; ADO, adenosine; ADP, adenosine 5′ diphosphate; APP, amyloid precursor protein; Arg, arginine; ARP2/3, actin-related protein 2/3 protein com-
plex; Asp, aspartate; ATP, adenosine 5′ triphosphate; C, carboxyl; CD, cluster of differentiation; COVID-19, coronavirus disease of 2019; CRMP2, collapsin response mediator protein 2;
CX3CR1, CX3 chemokine receptor 1; ENT1, equilibrative nucleoside transport 1; ECL1/2, extracellular loop 1/2; GFAP, glial fibrillary acidic protein; HIV, human immunodeficiency
virus; ICL1, intracellular loop 1; IL, interleukin; KO, knockout; N, amino; N2a, Neuro-2a; NBTI, Nitrobenzylthioinosine; NMDA, N-methyl-D-aspartate; PANX1, pannexin 1; PANX1CT,
pannexin 1 carboxy-terminus; poly(I:C), polyinosinic:polycytidylic acid; PS1, presenilin 1; Tyr, tyrosine; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TM1-4, transmem-
brane 1–4; TNF-α, tumour necrosis factor-α; Trp, tryptophan; UDP, uridine 5′ diphosphate; UTP, uridine 5′ triphosphate..
⁎ Corresponding authors.
E-mail addresses: juansa@uvic.ca (J.C. Sanchez-Arias), lswayne@uvic.ca (L.A. Swayne).
1
Equal contribution.

https://doi.org/10.1016/j.pharmthera.2021.107840
0163-7258/© 2021 Elsevier Inc. All rights reserved.
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al. Pharmacology & Therapeutics 225 (2021) 107840

1. Introduction
Pannexin 1 (PANX1) is a nervous system-enriched protein with di-
verse physiological and pathophysiological roles exerted throughout
the body. PANX1 is the most widely studied of the three-membered
pannexin family (for a general review see Boyce, Epp, Nagarajan, &
Swayne, 2018). PANX1 monomers oligomerize into heptameric chan-
nels permeable to small and large anions (such as chloride and adeno-
sine 5′ triphosphate (ATP)) as well as cations (e.g., Narahari et al.,
2021; see BOX 1 for additional details and references in Supplementary

Box 1
PANX1 channel structure and pharmacology.
Structure
PANX1 monomers oligomerize to form heptameric channels with extracellular, intracellular and transmembrane domains systematic mutagenesis
study revealed that conserved residues in this loop also play important roles in CBX function, potentially by (Deng et al., 2020; Michalski et al.,
2020; Mou et al., 2020; Navis, Fan, Trang, Thompson, & Derksen, 2020; Qu et al., 2020; Ruan, Orozco, Du, & Lü, 2020). Each PANX1 subunit con-
tains an amino (N)-terminal loop, four membrane spanning helices (TM1- 4), two extracellular loops (ECL1/2), an intracellular loop (ICL1), and a
carboxy- terminus (PANX1CT) (Deng et al., 2020; Michalski et al., 2020; Mou et al., 2020; Penuela et al., 2007; Qu et al., 2020; Ruan et al.,
2020; Sosinsky et al., 2011). Both the C-terminus and N-terminus are intracellular, and the main pore is positively charged at both ends (Deng
et al., 2020; Mou et al., 2020; Ruan et al., 2020). Seven positive residues pairs (Trp74-Arg75) line the narrowest portion (less than 5 Å) of the chan-
nel near the extracellular domain (Michalski et al., 2020; Mou et al., 2020; Qu et al., 2020; Ruan et al., 2020). Computational models and electro-
physiology data suggest that PANX1 channels contain seven narrow side tunnels within the intracellular domain at the inter-subunit interface that
provide an additional pathway for small ion flux (López et al., 2020; Ruan et al., 2020).

Activation
Under normal physiological conditions the main pore of the PANX1 channel is blocked by the PANX1CT (Y.-H. Chiu, Schappe, Desai, & Bayliss, 2018;
Deng et al., 2020; Ruan et al., 2020; Sandilos et al., 2012). Individual PANX1CT regions can be displaced in a step-wise manner leading to progres-
sive increases in channel activity and allowing for fine-tuning of channel flux (Y. H. Chiu et al., 2017). This phenomenon has been observed during
reversible a1-adrenoceptor activation of the channel, though it remains unclear if the PANX1CT are physically or allosterically displaced (Y. H. Chiu
et al., 2017). Irreversible activation of the channel has been demonstrated to occur during apoptosis via caspase-mediated cleavage at a caspase
cleavage site (Asp378; Yang et al. 2015) on the PANX1CT (Chekeni et al., 2010; Y.-H. Chiu et al., 2018; Ruan et al., 2020; Sandilos et al.,
2012). Additional stimuli have been proposed to activate PANX1 channels, such as changes in membrane potential, increased intracellular calcium,
increased extracellular potassium, mechanical stretching, and phosphorylation by Src family kinases (at residues Tyr198 and Tyr308; reviewed by
(Y.-H. Chiu et al., 2018; Whyte-Fagundes & Zoidl, 2018)). In a recent report, Lopez et al. (2020) identified two additional putative PKA phosphory-
lation sites (at residues Tyr302 and Ser328) that regulate stretch induced PANX1 activation. Given that these residues are located near the inter-sub-
unit interface, their phosphorylation may affect ion movement through the channel’s side tunnels. Further studies are required to fully understand the
mechanisms by which PANX1 is modulated under various physiological and tissue-specific contexts.
Selectivity
Both structural and functional data support that activated PANX1 channels favour the passage of anions and large anionic molecules such as ATP and
glutamate (Y. H. Chiu et al., 2017; Ma et al., 2012; Narahari et al., 2021). For example, the positively charged Arg75 residues in the main pore of the
channel form a cation-π interaction with Trp74 that constrain the pore and play a role in anion permeability (Deng et al., 2020; Michalski et al., 2020;
Ruan et al., 2020). In a recent study by Yang et al. (2020), increased PANX1 expression levels and membrane localization after TNF-a stimulation was
correlated with increased intracellular Ca2+ in endothelial cells. Molecular dynamics modelling revealed negatively charged residues (Asp and Glu) lo-
cated in the channel tunnels that could facilitate the transport of cations or Ca2+ (Sanchez Arias et al., 2020). Moreover, under caspase-mediated
activation of the channel, positively charged metabolites and dyes can slowly flux through the channel, supporting a role for PANX1 in the release
of signaling metabolites during apoptosis, such as spermidine (Medina et al., 2020; Narahari et al., 2021).

2
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al. Pharmacology & Therapeutics 225 (2021) 107840

PANX1 inhibitors: common uses, other targets and structures

Inhibitor Common Use Other Receptor Targets References Structure

Probenecid Gout P2X7 receptor (Dahl et al., 2013; Díaz et al., 2019; Silverman et al.,
treatment 2008; Willebrords et al., 2017)

C13H19NO4S
PubChem Identifier:
CID 4911.
Spironolactone Hypertension Nuclear receptor subfamily (Good et al., 2018)
treatment 3 group C member 2 (Navis et al., 2020)

C24H32O4S
PubChem Identifier:
CID 5833.
Trovafloxacin Antibiotic DNA gyrase (Garg et al., 2018; Poon et al., 2014)
Topoisomerase IV

C20H15F3N4O3
PubChem Identifier:
CID 62959.
Carbenoxolone Gap channel Hemichannels (connexin (Michalski et al., 2020; Michalski & Kawate, 2016;
(CBX) inhibitor 26, connexin 38) Ruan et al., 2020; Willebrords et al., 2017)
Gap junctions

C34H50O7
PubChem Identifier:
CID 636403.
10
Panx Peptide Hemichannels (connexin (Pelegrin & Surprenant, 2006; Wang, Ma, Locovei,
46) Keane, & Dahl, 2007)

C58H79N15O16
Amino acid sequence:
WRQAAFVDSY
PubChem Identifier:
CID 44233736.
Brilliant Blue Food dye Voltage gated sodium (Jo & Bean, 2011; Wang, Jackson, & Dahl, 2013)
FCF channels

C37H34N2Na2O9S3
PubChem Identifier:
CID 19700.

material). PANX1 channels can be activated by a number of different
mechanisms and blocked by several drugs (BOX 1). Their well-
characterized role in ATP release implicates PANX1 channels as new
players in the intricate game of purinergic signaling within the nervous
system (BOX 2). In line with the ever-expanding ways that nervous sys-
tem cell and tissue development and function are controlled by the vast
repertoire of purinergic signaling players (e.g., ATP-release channels,
nucleotide P2X & P2Y receptors, extracellular nucleotidases, nucleoside
transporters, and nucleoside P1 receptors, see BOX 2 for summary and
for reviews see Burnstock, 2020; Parkinson et al., 2011; Illes, Xu, &
Tang, 2020; Young, Yao, Sun, Cass, & Baldwin, 2008), PANX1 has
emerged as a key convergence point of multiple signaling pathways in-
volved in nervous system health and disease (reviewed in Yeung, Patil,
& Jackson, 2020).
In this review we focus on recent advances in our understanding of
PANX1 regulation of neuronal morphological and functional plasticity,
glial biology, behaviour, and neurological disease. We highlight exam-
ples of similarities and differences between PANX1 and the spectrum
of purinergic signaling players, and examine emerging links to inflam-
mation and infection within the nervous system.

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J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
Box 2
Cell type specific PANX1 expression and associated purinergic signaling systems in health and disease.
2. PANX1 in the healthy nervous system
2.1. Neural precursor cells and PANX1 in neuronal development
Neuronal development is comprised of a complex set of processes
occurring both embryonically and postnatally, beginning with neural
precursor cell proliferation, specification, migration, differentiation,
and integration into circuitry (for reviews see Castello & Gleeson,
2021; Jurkowski, Bettio, Woo, Patten, Yau, & Gil-Mohapel, 2020; Reh
et al., 2020). Neural precursor cells express PANX1 (Wicki-Stordeur
et al., 2016; Wicki-Stordeur, Dzugalo, Swansburg, Suits, & Swayne,
2012; Wicki-Stordeur & Swayne, 2013) and several different P2 recep-
tor proteins (reviewed in Franke & Illes, 2006; Oliveira, Illes, & Ulrich,
2016; Ulrich & Illes, 2014). Purinergic signaling, including ATP release
by PANX1 as well as both ionotropic and metabotropic purinergic (P2)
receptor signaling, regulates many aspects of neuronal development
(for reviews see Cavaliere, Donno, & D’Ambrosi, 2015; Oliveira et al.,
2016; Rodrigues, Marques, & Cunha, 2019; Swayne & Bennett, 2016).
With the established role of purinergic receptor signaling in neural
precursor cell biology and neuronal development, PANX1's role as an
ATP-release channel (first described by Bao, Locovei, & Dahl, 2004;
reviewed in Lohman & Isakson, 2014) provided the foundation for its
detection in neural precursor cells by our group (Wicki-Stordeur et al.,
2012). We observed decreased neural precursor cell proliferation in
the presence of the gout remedy and PANX1 blocker, probenecid (see
BOX 1; Silverman, Locovei, & Dahl, 2008), or the P2 receptor-blocker,
pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (Lambrecht
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Pharmacology & Therapeutics 225 (2021) 107840
et al., 1992). In follow-up work, (Wicki-Stordeur et al., 2016), we ob-
served selective loss of adult mouse brain ventricular zone neural pre-
cursor cells that underwent virus-mediated Panx1 knockout (KO),
suggesting PANX1 was critical for their maintenance. This finding was
expected since proliferation is a critical factor for maintaining cell pop-
ulation size. Our methods did not detect evidence for PANX1 regulation
of cell proliferation in vivo; albeit we cannot completely rule out this
possibility. In addition to comparing and contrasting the roles of
PANX1 and its purinergic receptor counterparts in neural precursor bi-
ology, a significant knowledge gap that remains is the potential
crosstalk between PANX1 and P2 receptors in these cells both in vitro
and in vivo. For example, as we have previously proposed (Wicki-
Stordeur et al., 2016), ATP released via PANX1 could act as a “don't eat
me” signal for neural precursor cells. Neural precursor cells mediate
phagocytosis for the clearance of cells during the early stages of neuro-
nal development by a non-canonical P2X7 receptor-dependent process
that is inhibited by ATP (Lovelace et al., 2015). It is tempting to speculate
that PANX1-mediated ATP release could repel roving phagocytic neural
precursors to prevent premature clearance via phagoptosis, death by
phagocytosis (Brown & Neher, 2012; Brown, Vilalta, & Fricker, 2015).
Another potential point of PANX1-P2X7 receptor crosstalk in neural
precursor cells, as well as in neurons, is ATP-induced PANX1 internaliza-
tion, a phenomenon we discovered (Boyce, Kim, Wicki-Stordeur, &
Swayne, 2015; Boyce & Swayne, 2017) using a Neuro-2a (N2a)
neuroblastoma-derived neural precursor cell model (Truding,
Shelanski, Daniels, & Morell, 1974). We proposed (Swayne & Boyce,
2017) that this process, which depends on an ATP-triggered interaction
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
between PANX1 and P2X7 receptors (Boyce & Swayne, 2017), could
provide fine-tuning of purinergic receptor regulation of neural precur-
sor cell proliferation and differentiation by downregulating PANX1
ATP-release channels when extracellular ATP levels are in excess. A re-
cent study by our group (Boyce et al., 2020) suggests that ATP-
induced PANX1 internalization likely occurs via macropinocytosis, and
furthermore, PANX1 itself could regulate macropinocytosis of ATP.
Additionally, we also found that PANX1 regulates neural precursor
cell differentiation (Wicki-Stordeur & Swayne, 2013). PANX1 protein
expression levels decreased significantly in N2a cells and primary neo-
natal ventricular zone neural precursor cells upon retinoic acid- and
low-serum-induced differentiation (Wicki-Stordeur & Swayne, 2013).
As follows, PANX1 disruption (via siRNA-mediated Panx1 knockdown
or probenecid) stimulated marked neurite formation in N2a cells and
primary neonatal ventricular zone neural precursor cells, while overex-
pression of PANX1 (transient transfection with PANX1-EGFP) decreased
neurite extension. Relatedly, others observed decreased expression of
P2X7 receptors with neuroblastoma-derived cell line and neural precur-
sor cell neuronal differentiation, and disruption of P2X7 receptors pro-
moted neuronal differentiation and neurite formation (Miras-Portugal,
Sebastián-Serrano, De Diego García, & Díaz-Hernández, 2017; Wu
et al., 2009). Notably, in the context of peripheral nerves, Panx1 KO
did not appear to affect their development in situ; however, Panx1 KO
increased neurite outgrowth from dorsal root ganglion explants, and
this effect was mimicked by application of apyrase (an enzyme that hy-
drolyzes extracellular ATP) and the PANX1 blocker, 10Panx (BOX 1;
Horton et al., 2017). Altogether, these findings suggest that PANX1 reg-
ulates neurite formation and/or stability, potentially through crosstalk
with purinergic signaling and through interactions with the cytoskele-
ton and its regulators.
Consistent with a role in neurite formation, our recent findings sug-
gest that PANX1 regulates dendritic spine development (Sanchez-Arias
et al., 2019; Sanchez-Arias, Candlish, van der Slagt, & Swayne, 2020). As
neurons mature and form physically and functionally connected net-
works, highly dynamic protrusions arise from dendritic shafts and ma-
ture into dendritic spines that express ionotropic postsynaptic
glutamate receptors and thereby receive excitatory synaptic input
(Berry & Nedivi, 2017). Using confocal microscopy to image fluorescent
lipophilic cationic indocarbocyanine dye-labeled neuronal membranes,
we detected increased cortical neuron dendritic spine density associ-
ated with Panx1 KO both in situ (layer 5 pyramidal neurons), as well
as in primary cortical neuron cultures at 12 to 13 days in vitro
(Sanchez-Arias et al., 2019). Accordingly, the expression levels of
ionotropic glutamatergic receptors (glutamate receptor 1 and gluta-
matergic receptor ionotropic, N-methyl-D-aspartate (NMDA) 2A) and
postsynaptic density protein-95 were elevated in cortical synaptosomes
from Panx1 KO mice. To determine if the Panx1 KO-mediated increase in
dendritic spine density was due to increased dendritic protrusion stabil-
ity, we developed new methods to measure dendritic protrusion dy-
namics in immature cortical neurons at 10 days in vitro (Sanchez-
Arias et al., 2020). At this relatively early in vitro developmental stage
(Takano, Xu, Funahashi, Namba, & Kaibuchi, 2015), Panx1 KO neurons
already exhibited increased dendritic protrusion density when com-
pared to wild-type controls. Transient expression of PANX1-EGFP in
both wild-type and KO cultures was associated with decreased dendritic
protrusion density (Sanchez-Arias et al., 2020). In terms of dynamics,
increased dendritic protrusion movement and turnover were observed
with transient PANX1-EGFP expression, while dendritic protrusions
were relatively stable in Panx1 KO cultures. Overall, these results sug-
gest that PANX1 acts as a developmental brake for the formation of den-
dritic spines.
Cytoskeletal dynamics play key roles in the processes involved in
dendritic spine development (Borovac, Bosch, & Okamoto, 2018; Dent,
2017). Our previous proteomic study identified two key cytoskeletal
regulating proteins as novel PANX1 interactors: actin-related protein 3
(ARP3, a component of the ARP2/3 complex) and the microtubule and
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Pharmacology & Therapeutics 225 (2021) 107840
actin-interacting protein collapsin response mediator protein 2
(CRMP2) (Frederiksen, Wicki-Stordeur, Sanchez-Arias, & Swayne,
2019; Wicki-Stordeur & Swayne, 2013; Xu et al., 2018). Probenecid
disrupted the PANX1-CRMP2 interaction, and increased microtubule
stability and neurite outgrowth in N2a cells (Xu et al., 2018). Notably,
CRMP2 is an important positive regulator of dendritic spines (Zhang
et al., 2016). Our current working model states that high levels of so-
matosensory cortical neuronal PANX1 at the beginning of the postnatal
critical period for dendritic spine development in mice (Schlaggar, Fox,
& O'Leary, 1993) allows PANX1 to physically sequester CRMP2 and
ARP2/3, thereby preventing aberrant dendritic spine stabilization and
synapse development. Developmental downregulation of PANX1,
through mechanisms under investigation, triggers the release of
CRMP2 and ARP2/3, allowing them to promote cytoskeletal changes
that enhance spine growth and stability. Whether purinergic receptor
crosstalk is involved in aspects of this mechanism, including perhaps
the downregulation of PANX1 through ATP-mediated internalization
involving ionotropic P2X7 receptors, as outlined above, remains to be
investigated.
2.2. PANX1 regulates synaptic plasticity
In addition to regulating the structural development of synapses, as
described above, PANX1 modulates hippocampal synaptic transmission
and consequently synapse strength and plasticity. In hippocampal brain
slices, PANX1 disruption (Panx1 KO and inhibition of PANX1 channels)
enhanced excitability: induction of long-term potentiation was facili-
tated, frequency of spontaneous excitatory postsynaptic currents was
increased, and induction of long-term depression was hindered
(Ardiles et al., 2014; Bialecki et al., 2020; Gajardo3 et al., 2018;
Prochnow et al., 2012), suggesting that PANX1 modulates hippocampal
synaptic plasticity mechanisms that play a critical role in learning and
memory (Takeuchi, Duszkiewicz, & Morris, 2014). Panx1 KO-
associated changes in synaptic plasticity were normalized by adenosine
supplementation (Prochnow et al., 2012), suggesting PANX1 regulates
synaptic adenosine receptor activity. Once in the extracellular space,
ATP is hydrolyzed into adenosine by extracellular nucleotidases
(Hamann & Attwell, 1996; Wieraszko, Goldsmith, & Seyfried, 1989)
resulting in activation of A1 and A2A adenosine receptors (BOX 2)
which inhibit neuronal activity (Dunwiddie & Masino, 2001). This ten-
tative mechanism is consistent with the modulatory effect of adenosine
on neuronal networks (Fredholm, Chen, Masino, & Vaugeois, 2005).
The studies revealing PANX1 regulation of synaptic plasticity build
on prior work demonstrating that ATP itself acts as an important
synaptic modulator (Fields & Stevens, 2000; Zhang et al., 2003). When
co-released with other neurotransmitters at nerve terminals or by as-
trocytes, ATP can enhance or decrease excitability via a variety of
purinergic signaling pathways, such as ionotropic P2X receptors, metab-
otropic P2Y receptors, and adenosine receptors (for examples see Lalo
et al., 2014; Lalo, Palygin, Verkhratsky, Grant, & Pankratov, 2016; Tan
et al., 2017; Zhang et al., 2003; and reviewed in Guzman & Gerevich,
2016; Hnasko & Edwards, 2012; Illes, Burnstock, & Tang, 2019;
Pankratov, Lalo, Krishtal, & Verkhratsky, 2009; Pankratov, Lalo,
Verkhratsky, & North, 2006; Pascual et al., 2005; Sebastião & Ribeiro,
2015; Zimmermann, 2016). Although, Panx1 mRNA levels are very
low in astrocytes and other glial cells (Zhang et al., 2014) under naïve
conditions, new, more highly-selective, astrocytic Cre lines, such as
Aldh1l1-CreERT2 (Srinivasan et al., 2016), will help to disentangle the
contributions of astrocytic and neuronal PANX1 to physiological and
pathophysiological (i.e., seizure) neuronal network activity. Rafael,
Cairus, Tizzoni, Abudara, and Vitureira (2020) found that neuronal-
specific, but not astrocytic, Panx1 KO impaired P2X7 receptor-
mediated presynaptic terminal expansion induced by chronic inactivity
(tetrodotoxin) in hippocampal cultures; however, compensatory ho-
meostatic synaptic plasticity changes to nerve terminal density (based
on vesicular glutamate transporter 1 puncta) depended on both
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
neuronal and astrocytic Panx1 KO. We recently found that global Panx1
KO primary cortical neuron cultures (with low astrocyte density) exhib-
ited larger and more abundant neuronal networks (Sanchez-Arias et al.,
2019), which could in part be explained by increased cortical dendritic
spine density observed both in situ and in vitro described above. More
recently, ablation of the Panx1a gene in zebrafish disrupted expression
of genes involved in visual system development, visual processing,
and pre- and post-synaptic dopaminergic signaling. This also resulted
in abnormal locomotion in the dark and locomotion responses and
brain wave frequency transitions in response to light, suggesting that
zebrafish Panx1a plays an important role in the development of neuro-
nal networks involved in supporting visual-motor functions (Safarian,
Whyte-Fagundes, Zoidl, Grigull, & Zoidl, 2020).
Together, these results suggest PANX1 plays an important role in
neuronal network development, and both neuronal and astrocytic
PANX1 can contribute to purinergic modulation depending on the phys-
iological or pathophysiological context.
2.3. Glial and endothelial cell PANX1
There are mixed reports of Panx1 gene expression in glial cells under
naïve conditions. According to the RNA-sequencing transcriptome and
splicing database of glia, neurons, and vascular cells of the mouse cere-
bral cortex (https://www.brainrnaseq.org/), PANX1 is strongly
expressed in oligodendrocyte precursor cells and ‘newly-formed oligo-
dendrocytes’ at postnatal day 17, while very low levels are found in
myelinating oligodendrocytes at this timepoint, and microglia and en-
dothelial cells at postnatal day 7 (Zhang et al., 2014). It should be
noted that the oligodendrocyte lineages for this database were obtained
from postnatal day 17 brains because it is the earliest timepoint at
which the full spectrum of these cells is present; additional information
about the procedures and timepoints at which the various cell types
were purified can be found in Zhang et al. (2014). Consistent with the
results of Zhang and colleagues, Panx1 transcripts and PANX1 protein
have also been detected at appreciable levels in primary oligodendro-
cyte cultures (Domercq et al., 2010; Huang, Grinspan, Abrams, &
Scherer, 2007). Additionally, Panx1 (and Panx2) mRNA were not de-
tected in glial fibrillary acidic protein (GFAP)-positive glia in adult rats
(Vogt, Hormuzdi, & Monyer, 2005), and Panx1 mRNA was not found in
mouse brain astrocytes in situ under naïve conditions (Zappala et al.,
2006). Yet, there are numerous reports that pannexins are expressed
in astrocytic cells in culture conditions. PANX1 was detected in an astro-
cyte cell line (1231-N1) (Pelegrin & Surprenant, 2006), as well as in pri-
mary astrocyte cultures (Huang et al., 2007; Iglesias, Dahl, Qiu, Spray, &
Scemes, 2009; Lai et al., 2007), where there is evidence of functional
PANX1 channels (Iglesias et al., 2009). In terms of location on hippo-
campal astrocytes, PANX1 was distributed on processes as discrete
puncta (Boassa, Nguyen, Hu, Ellisman, & Sosinsky, 2015). Sorted mi-
croglia from the spinal cord on embryonic day 13.5 also express
PANX1 (Rigato et al., 2012). In the blood-brain barrier, PANX1 has
been detected in smooth muscle (Burns, Phillips, & Sokoya, 2012),
though there are conflicting reports on endothelial cell PANX1 expres-
sion. As mentioned above, endothelial Panx1 transcripts are sparse ac-
cording to a brain cell-type specific transcriptomic database (Zhang
et al., 2014), and PANX1 was not detected in RBE4 immortalized cul-
tured endothelial cells from rat brain capillaries (De Bock et al., 2012).
Conversely, PANX1 was detected in the immortalized human endothe-
lial cell line (hCMEC/D3) (Kaneko et al., 2015), and endothelial cells of
the mouse retina (Dvoriantchikova, Ivanov, Panchin, & Shestopalov,
2006).
There are similarly mixed results in terms of PANX1 expression in
other types of glia, namely retinal and peripheral glia. Panx1 transcripts
were detected in cerebellar Bergmann glia (Zappala et al., 2006), which
are thought to be a type of neural precursor cell, consistent with the ex-
pression of PANX1 in ventricular zone and hippocampal neural
6
Pharmacology & Therapeutics 225 (2021) 107840
precursors. While Panx1 mRNA and PANX1 protein were not detected
in glial cells in the retina (Dvoriantchikova et al., 2006), the velocity
by which retinal microglial processes extended and retracted was re-
duced in the presence of probenecid (PANX1 blocker, see BOX 1)
(Fontainhas et al., 2011). Peripherally, Panx1 mRNA was not detected
in a Schwann cell-like cell line (RT4-B5) (Ray, Zoidl, Weickert, Wahle,
& Dermietzel, 2005); however, more recently, PANX1 protein was de-
tected in myelin and blood vessels (as well as in the axon) in sections
from peripheral (sciatic) nerves of adult mice (Horton et al., 2017). Ad-
ditionally, Panx1 transcripts and PANX1 protein were detected in pri-
mary mouse Schwann cell cultures, where they contributed to
hypotonicity-induced ATP release dependent on intracellular RhoA
and the cytoskeleton (Wei et al., 2021). Type II cells in the carotid
body, which resemble glia, express PANX1 ex vivo (Zhang, Piskuric,
Vollmer, & Nurse, 2012). Therefore, PANX1 has been detected in a spec-
trum of glial cell types both in the central and peripheral nervous
systems.
In terms of its functional role in glial cells, PANX1 has been impli-
cated in the release of ATP and other metabolites. For example, PANX1
regulates ATP and D-serine release from cultured cortical astrocytes in
response to P2X7 receptor activation (Iglesias et al., 2009; Pan, Chou,
& Sun, 2015). PANX1 and P2X7 receptor inhibition reduced propidium
iodide uptake (Kovács et al., 2018) and glucocorticoid-induced cytosolic
translocation and release of the high mobility group box-1 pro-
inflammatory protein in primary cortical astrocyte cultures (Hisaoka-
Nakashima et al., 2020). In the 1321-N1 astrocyte cell line transiently
expressing the rat P2X7 receptor, the PANX1 inhibitor, 10Panx, blocked
ATP-induced ethidium bromide dye uptake, a readout of large-pore
channel activity (Pelegrin & Surprenant, 2006); although it should be
noted that 10Panx has also been reported to affect channels formed by
connexin 46 (Patel, Zhang, & Veenstra, 2014; Wang, Ma, Locovei,
Keane, & Dahl, 2007) and could have other offsite targets. Calcium
waves in astrocyte cultures were reduced by siRNA-mediated Panx1
knock-down (Scemes, Suadicani, Dahl, & Spray, 2007), and elevated ex-
tracellular potassium activated PANX1 channels (potassium levels were
elevated to pathologically high levels ranging from 20 mM to 130 mM,
which are levels detected in the context of seizure, cortical spreading
depression or stroke (Ayata & Lauritzen, 2015; Ito, Ohno, Nakamura,
Suganuma, & Inaba, 1979))), leading to ATP release and subsequent
neuronal P2X7 receptor activation and increased neuronal excitability
(Silverman et al., 2009). Long term exposure of astrocytes to patholog-
ically elevated extracellular potassium reduced calcium waves in a
PANX1 dependent manner, particularly when P2X7 receptors were im-
plicated (Scemes & Spray, 2012). Notably, extracellular ATP feeds back
to inhibit PANX1-mediated ATP release from cultured cortical astro-
cytes, which is likely a homeostatic response to prevent ATP-mediated
cell death (Jackson, Wang, Keane, Scemes, & Dahl, 2014); elevated ex-
tracellular potassium was able to attenuate this homeostatic response.
Moreover, lactate dehydrogenase and caspase-3 were increased in re-
sponse to elevated extracellular potassium, but this did not occur in
Panx1 KO astrocytes (Jackson et al., 2014), confirming an important
role for PANX1 responses to pathological stimuli. In mouse spinal cord
astrocytes (cultured and acute slice), fibroblast growth factor-1 led to
calcium induced ATP release, P2X7 receptor activation and subsequent
PANX1-mediated ATP release (Garré et al., 2010). In primary cortical as-
trocyte cultures, pre-treatment with tumour necrosis factor-α (TNF-α)
and Thy-1 or β3 integrin increased PANX1 levels and altered its cellular
distribution (Lagos-Cabré et al., 2017). Further supporting a role for
PANX1 in ATP release from primary astrocyte cultures, ATP release
was reduced in primary optic nerve astrocyte cultures from Panx1 KO
mice (Beckel et al., 2014).
Whereas low levels of PANX1 were detected in mature astrocytes
and microglia, its functional relevance in these cells could be enhanced
in pathologies associated with injury and inflammation where in-
creased PANX1 activity and levels have been observed (e.g., stroke, for
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
review see Brocardo, Acosta, Piantanida, & Rela, 2019). Therefore, astro-
cytic and microglial cultures in which PANX1 levels may be elevated
compared to in situ, could still provide important insight into cellular
PANX1 cell biology related to pathological contexts. It is also important
to note that the low levels of astrocytic and microglial PANX1 in situ
under naïve conditions could still be functionally important. The puta-
tive role(s) of PANX1 in oligodendrocytes, where appreciable Panx1
transcripts are detected, is an area of future investigation with
important clinical relevance. Finally, it is important to note that our un-
derstanding of glial PANX1 will be greatly aided by the enhanced cell-
type specificity of several relatively new Cre lines (e.g., Aldh1l1-driven
Cre for astrocytic KO (Srinivasan et al., 2016; Winchenbach et al.,
2016))).
3. PANX1 in neurological disease
3.1. PANX1 in neurodevelopmental disorders
Adverse changes in synaptic function and dendritic spine structure
and density have been implicated in a range of developmental, degener-
ative and injury-based neuropsychiatric disorders (for reviews see
Forrest, Parnell, & Penzes, 2018; Nishiyama, 2019; Penzes, Cahill,
Jones, VanLeeuwen, & Woolfrey, 2011). Thus, given PANX1's role in
dendritic spine development and synaptic plasticity, it is perhaps not
surprising that Panx1 KO mice exhibited increased anxiety-like behav-
iours, impaired spatial memory and object recognition (Gajardo et al.,
2018; Prochnow et al., 2012), increased day-long wakefulness and
motor activity (Kovalzon, Moiseenko, Kurtenbach, Shestopalov, &
Panchin, 2017), as well as, reduced restraint stress-induced ATP release
within the hippocampus (Orellana et al., 2015). PANX1 and P2X7 re-
ceptors were also implicated in stress-induced astrocytic release
of high-mobility group box-1, a neuroinflammatory protein implicated
in major depressive disorder (Hisaoka-Nakashima et al., 2020). In
contrast to the relatively minor neurological phenotypes associated
with Panx1 KO in mice, a human germline PANX1 variant, PANX1
Arginine217Histidine (which results in reduced channel activity when
characterized in vitro in a heterologous expression system), was associ-
ated with multi-system dysfunction, including hearing loss and
neurodevelopmental delay (Shao et al., 2016). PANX1 single nucleotide
polymorphisms have also been linked to autism spectrum disorders
(Davis et al., 2012), but no association was found with schizophrenia
(Gawlik, Wagner, Pfuhlmann, & Stöber, 2016). PANX1 variants have
also been shown to affect platelet aggregation (Molica et al., 2015), oo-
cyte biology (Sang et al., 2019; Wang et al., 2021), and channel-
trafficking in melanoma tumors (Nouri-Nejad et al., 2021), but nervous
system implications for these are currently unknown. The basis for the
range in severity of phenotypes associated with mouse Panx1 KO and
different human variants is not yet known, but it is important to caution
against comparison between complete Panx1 KO (Panx1tm1.1Fama
(Anselmi et al., 2008)); Panx1tm1.2Vmd (Qu et al., 2011)); Panx1tm1.1Vshe
((Dvoriantchikova et al., 2012)) or partial protein loss (Panx1tm1a
(KOMP)Wtsi
(Seminario-Vidal et al., 2011); discussed in Cone, Ambrosi,
Scemes, Martone, & Sosinsky, 2013; Hanstein et al., 2013)) versus ex-
pression of full-length PANX1 variant at single amino acids. In the latter
case, the protein is still expressed and would likely still interact with key
proteins and signaling partners. The consequences of variants on
PANX1-interactions and its role as a signaling hub (Frederiksen,
Wicki-Stordeur, Sanchez-Arias, & Swayne, 2019; Wicki-Stordeur &
Swayne, 2014) remain to be determined.
3.2. PANX1 in neurodegenerative disorders
PANX1 has also been also implicated in Alzheimer's disease (AD). Al-
though PANX1 is downregulated postnatally in healthy brain, its ex-
pression was maintained at high levels in the amyloid precursor
protein (APP)/presenilin 1 (PS1) transgenic mouse model of familial
7
Pharmacology & Therapeutics 225 (2021) 107840
AD (Flores-Muñoz et al., 2020). Moreover, probenecid reversed synaptic
plasticity impairments and normalized dendritic spine densities in APP/
PS1 transgenic mice (Flores-Muñoz et al., 2020). In hippocampal slices
from APP/PS1 transgenic mice, PANX1 contributed (albeit relatively
less than did connexin 43) to dye uptake in astrocytes associated with
plaques (Yi et al., 2016). Notably, the broad spectrum anti-bacterial/
fungal/inflammatory agent minocycline (known to act on microglia, as-
trocytes, endothelial cells, and other immune cells) blocked this effect,
suggesting inflammation drives the opening of PANX1 channels. In cul-
tured astrocytes, amyloid-β increased surface PANX1 levels (Orellana
et al., 2011). Additionally, chronic inflammation associated with AD pa-
thology produced an excess of pro-inflammatory molecules, including
TNF-α (Decourt, Lahiri, & Sabbagh, 2016). It was recently discovered
that application of TNF-α upregulated PANX1 expression in peripheral
endothelial cells (Yang et al., 2020). Moreover, brain mast cells (which
store various pro-inflammatory mediators in secretory granules, includ-
ing TNF-α; reviewed in Hendriksen, van Bergeijk, Oosting, & Redegeld,
2017) undergo rapid degranulation upon exposure to amyloid-β in a
PANX1-dependent mechanism (Harcha et al., 2015). These neuro-
inflammatory mechanisms and their effects in neuron-glial interactions
should be further investigated in the context of the role of PANX1 in the
AD brain. Similarly, chronic inflammation associated with AD pathology
increased extracellular ATP levels leading to purinergic receptor activa-
tion (reviewed in Cieślak & Wojtczak, 2018). The distribution and ex-
pression levels of several P2Y receptor subtypes, and the levels of
P2X7 receptors were reportedly altered in tissues from AD-affected
individuals and in several transgenic mouse models of AD expressing
different mutations associated with familial forms of the disease
(reviewed in Erb, Cao, Ajit, & Weisman, 2015; Francistiová et al.,
2020). Furthermore, within an APP/PS1 transgenic mouse background,
P2rx7 KO was associated with reduced amyloid-β plaque load, normal-
ization of cognitive and synaptic plasticity deficits, and modulation of
cluster of differentiation (CD)8-positive T cell recruitment, but had no
impact on microglial activation or cytokine production (Martin et al.,
2019). While the roles of glial PANX1 in AD remain largely elusive,
PANX1 blockers (probenecid and the PANX1 mimetic peptides 10Panx
and E1b) partially reversed amyloid-β induced dye uptake and current
in primary cortical microglia cultures, but not primary cortical astrocyte
cultures (Orellana, Shoji, et al., 2011). In the same study, exposure to
conditioned media from amyloid-β treated microglia induced astrocytic
connexin 43-mediated ATP and glutamate release that together trig-
gered neuronal PANX1 activation via P2 and NMDA receptor activation
(Orellana, Shoji, et al., 2011).
PANX1 is also altered in other neurodegenerative disorders. In pri-
mary cortical astrocyte cultures, α-synuclein (a protein linked to
Parkinson's disease (Stefanis, 2012)) stimulated PANX1-mediated ATP
release in a complex mechanism implicating cytokine and nitric oxide
production, as well as purinergic (P2X7 and/or P2Y1) and glutamatergic
receptor signaling (Díaz et al., 2019). Treatment of primary cultured as-
trocytes with α-synuclein also decreased intracellular PANX1 levels
(Díaz et al., 2019). Selective purinergic and adenosine receptor inhibi-
tion is used to provide symptomatic relief in Huntington's and
Parkinson's disease (Oliveira-Giacomelli et al., 2018). In particular, inhi-
bition of adenosine A2A receptors in Huntington's disease is associated
with symptom mitigation (Blum et al., 2003, 2018; Oliveira-Giacomelli
et al., 2018). Though A2A receptors were downregulated in
Huntington's disease, their downstream signaling pathways were am-
plified, suggesting these receptors or their signaling pathways could
be targeted for therapeutic intervention (Glass, Dragunow, & Faull,
2000; Varani et al., 2001). The precise mechanistic details of the
crosstalk between PANX1 and receptor systems associated with
purinergic signaling in AD and Huntington's disease, as well as in
other neurodegenerative diseases associated with abnormalities in den-
dritic spines represent clinically-relevant knowledge gaps to bridge in
future work (reviewed in Penzes et al., 2011; Forrest et al., 2018;
Nishiyama, 2019).
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
3.3. PANX1 in stroke, seizure, and traumatic brain injury
In accordance with its modulation of synaptic plasticity and its up-
regulation during disease states, PANX1 is also important acutely during
cerebral ischemia, as well as in the short- and long-term changes that
occur in the brain after stroke and seizure. The first indication of a link
between brain injury and PANX1 came with the discovery of the activa-
tion of PANX1 channels under ischemic and excitotoxic conditions
(Thompson, 2006; Thompson et al., 2008). NMDA receptor activation
of Src kinases results in PANX1 C-terminus phosphorylation at tyrosine
(Tyr)308, thereby promoting channel activation (Weilinger et al., 2016;
Weilinger, Tang, & Thompson, 2012). Disrupting PANX1 by blocking
PANX1 channels (e.g., probenecid) or via genetic ablation (global KO
or endothelial-specific KO) reduced infarct volume in models of ische-
mic (Bargiotas et al., 2011; Freitas-Andrade, Bechberger, MacVicar,
Viau, & Naus, 2017; Good et al., 2018; Xiong et al., 2014) and hemor-
rhagic (Zhou et al., 2018) stroke, and prevented cell death in in vitro
models (Jian et al., 2016; Weilinger et al., 2016; Wicki-Stordeur et al.,
2016). It is reasonable to speculate that ischemia-induced PANX1 chan-
nel opening could also trigger irreversible C-terminus caspase cleavage,
a phenomenon that has been observed in other non-nervous system
contexts (Chekeni et al., 2010; Medina et al., 2020; Sandilos et al.,
2012), but whether this occurs in the brain in the context of injury or
stroke remains to be confirmed. The increase in ATP released in the ex-
tracellular space following stroke was associated with activation of
P2X7 receptors, as well as excessive calcium influx and neuronal
death (Arbeloa, Pérez-Samartín, Gottlieb, & Matute, 2012; Martín,
Domercq, & Matute, 2018). The P2X7 receptor antagonist, Brilliant
Blue G, markedly reduced brain damage associated with transient
focal cerebral ischemia in a rat model (Arbeloa et al., 2012). Taken to-
gether, these findings suggest that in the context of brain injury, both
NMDA receptors and P2X7 receptors are linked to PANX1 channels
with dire consequences for the neurons involved.
In addition to affecting neuronal survival, several studies suggest
glial PANX1 is also important in stroke and seizure. For example,
ischemia-triggered PANX1-mediated ATP release from oligodendro-
cytes led to P2X7 receptor-dependent oligodendrocyte death
(Domercq et al., 2010). Several studies have shown that astrocyte and
microglia responses to stroke are tempered by blocking PANX1, which
could not necessarily be attributed to glial PANX1 per se. For example,
probenecid reversed transient ischemia/reperfusion increases in GFAP
and ionized calcium binding adaptor molecule 1 reactivity (Wei et al.,
2015), and in female Panx1 KO mice, there were fewer GFAP-positive
astrocytes and ionized calcium binding adaptor molecule 1-positive mi-
croglia (Freitas-Andrade et al., 2017). Furthermore, probenecid reduced
lactate dehydrogenase activity, cell death, reactive oxygen species
levels, NOD-, LRR-, and pyrin domain-containing protein 3 levels,
caspase-1 levels, aquaporin 4 levels, high mobility group BOX 1 levels,
and interleukin (IL)-1β secretion in primary astrocyte cultures follow-
ing oxygen and glucose deprivation (6 h) and reoxygenation (24
h) (Jian et al., 2016). Additionally, probenecid reversed focal cerebral
ischemia-induced increases in CD68-positive cells (microglia or macro-
phages), and partially normalized increased GFAP/aquaporin 4 levels
(Xiong et al., 2014). Despite low astrocytic and microglial PANX1 ex-
pression levels under naïve conditions, given that various types of injury
and inflammation have been associated with increases in PANX1 ex-
pression levels (e.g., inflammation (Yang et al., 2020)) and stroke
(Freitas-Andrade et al., 2017; Jiang et al., 2012)), one possible mecha-
nism for astrocytic and microglial PANX1 involvement in stroke/injury
and seizure could be a possible injury/inflammation-mediated PANX1
upregulation in these cells. Relatively higher PANX1 expression levels
were detected in GFAP-positive astrocytes in lesions from patients
with three types of focal cortical dysplasia compared to healthy controls
(Li et al., 2017). Astrocytic and neuronal PANX1 activation has also been
implicated in ictal-like activity in brain tissue from patients with epi-
lepsy and kainate-induced and kindling-induced seizures in rodents
8
Pharmacology & Therapeutics 225 (2021) 107840
(Dossi et al., 2018; Santiago et al., 2011; Scemes, Velíšek, & Velíšková,
2019; Wellmann, Álvarez-Ferradas, Maturana, Sáez, & Bonansco,
2018). Microglial PANX1 was upregulated by proinflammatory cyto-
kines (e.g., TNF-α, interferon-γ (Sáez et al., 2013)). Trovafloxacin, a
broad-spectrum antibiotic and PANX1 channel inhibitor (see BOX1), ex-
hibited anti-inflammatory and neuroprotective effects in mice suffering
from brain trauma (Garg et al., 2018). There was a reduction in pro-
inflammatory cytokine levels and fewer microglia localized to the site
of injury associated with trovafloxacin treatment (Garg et al., 2018).
The authors hypothesized that trovafloxacin-mediated inhibition of
microglial PANX1-mediated ATP release hindered the ability for microg-
lia to respond to injury (Garg et al., 2018). In a follow-up study,
microglial-specific conditional Panx1 KO reduced neuroinflammation
and other deleterious effects associated with traumatic brain injury
(Seo, Dalal, Calderon, & Contreras, 2020). While microglia express neg-
ligible levels of Panx1 transcripts in the naïve mouse brain (Zhang et al.,
2014), these findings suggest that inflammation and injury could upreg-
ulate PANX1 expression and functional protein levels (Seo et al., 2020).
Collectively, these studies implicate astrocytic and microglial PANX1 in
neurological diseases associated with neuroinflammation such as stroke
and seizure.
Nucleoside transporters have also been implicated in stroke and sei-
zure, particularly equilibrative nucleoside transporter 1 (ENT1). Expres-
sion levels of ENT1 peaked 12 h after middle cerebral artery occlusion in
the rat, and administration (30 min after stroke induction) of an ENT1
inhibitor, Nitrobenzylthioinosine (NBTI), reduced infarct volume, im-
proved neurological function (measured at 24 h and 72 h post-stroke in-
duction), and prevented neuronal apoptosis (and associated signaling
pathways), likely by elevating extracellular adenosine (Zhang et al.,
2020). Similarly, in the context of seizure, ENT1 protein levels were el-
evated in the temporal neocortex from patients with temporal lobe ep-
ilepsy, as well as in hippocampal lysates prepared from a rat model of
seizure (lithium chloride, followed by pilocarpine administration).
NBTI suppressed seizure susceptibility (longer onset latency and re-
duced seizure severity) in the rat model both when applied locally via
microinjection and when administered intraperitoneally (Xu et al.,
2015). NBTI also decreased rat hippocampal CA1 pyramidal neuron ex-
citability and mini excitatory postsynaptic currents in slices prepared
24 h after the onset of seizure (Xu et al., 2015). A follow-up study re-
vealed that the impact of seizure on ENT1 expression levels is regulated
by dynamic-related protein 1, a key protein involved in mitochondrial
fission (Luo et al., 2020). Selective inhibition of dynamic-related protein
1 function by intraperitoneal administration of mitochondrial division
inhibitor 1, 30 min prior to induction of status epilepticus, attenuated
seizures and prevented the increase in ENT1 expression levels (Luo
et al., 2020). The same group also showed that inhibition of p38
mitogen-activated protein kinase blocked seizure-induced expression
level increases in the A1 receptor and ENT1, reduced seizure onset la-
tency, and decreased the number of seizures in the same rat model
(Zhou et al., 2020).
The potential crosstalk between PANX1, purinergic receptors, aden-
osine receptors, and nucleoside transporters between various brain cell
types in stroke and seizure represents an important knowledge gap
with key translational implications.
3.4. PANX1 in pain
Consistent with its importance in shaping basal plasticity and
neuronal networks as well as its implication in maladaptive plasticity
in seizure, PANX1 also plays a role in neuropathic and mechanically-
induced chronic pain (Bravo, Maturana, Pelissier, Hernández, &
Constandil, 2015; von Hehn, Baron, & Woolf, 2012; Woolf, 2011). Vari-
ous central and peripheral mechanisms link PANX1 with biological pro-
cesses involved in hypersensitivity (mechanical allodynia) and central
sensitization. For instance, in dorsal root ganglia, ATP released from sen-
sory neurons activated satellite glial cell P2X7 receptors, eliciting
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
calcium entry and pro-inflammatory cytokine (TNF-α and IL-1β) re-
lease, thereby increasing neuronal network excitability (Mousseau
et al., 2018; Zhang, Chen, Wang, & Huang, 2007). Dorsal root ganglion
PANX1 expression levels were elevated in rodent models of neuropathic
and chronic pain, contributing to an increase in extracellular ATP. In
light of this increase in PANX1 associated with neuropathic and chronic
pain, it is perhaps not surprising that blocking PANX1 function de-
creased hyperalgesia, mechanical allodynia, and release of pro-
inflammatory cytokines (Bravo et al., 2014; Mousseau et al., 2018;
Zhang, Laumet, Chen, Hittelman, & Pan, 2015). Panx1 KO mice subjected
to sciatic nerve injury were protected from development of mechanical
allodynia, a process requiring PANX1 expression in circulating hemato-
poietic cells (Weaver et al., 2017). Spinal cord microglial PANX1 was re-
cently shown to be critically important for mediating aspects of pain
and responses to analgesics (Burma et al., 2017; Mousseau et al.,
2018). In a mouse model of arthritis, genetic deletion of microglial
PANX1 attenuated the development of mechanical allodynia
(Mousseau et al., 2018). Moreover, probenecid treatment suppressed
mechanical allodynia without affecting acute nociception (Mousseau
et al., 2018). Repeated morphine administration increased PANX1 ex-
pression levels in spinal cord microglia (Burma et al., 2017). Mice lack-
ing PANX1 in CX3 chemokine receptor 1 (CX3CR1)-positive microglia
showed attenuated morphine withdrawal symptoms and naloxone-
induced synaptic facilitation in the dorsal horn, and naloxone-induced
ATP release was mediated by PANX1 (Burma et al., 2017). Despite
these findings, CX3CR1-driven Panx1 KO microglia did not prevent the
development of opioid-induced tolerance or hyperalgesia (Burma
et al., 2017). Overall, these results implicate PANX1 in microglial mech-
anisms underlying the spinal cord processing of pain and suggest that
PANX1 pharmacological manipulation holds potential to treat these
complex disorders.
Although there is no evidence supporting a role for PANX1 in acute
nociception, PANX1's mechanosensitive properties, first described in
(Bao et al., 2004), raise the possibility that PANX1 could play a role in
touch sensation. Intriguingly, in Caenorhabditis elegans touch receptor
neurons, mouse PANX1 rescued touch sensation that was lost via KO
of the gene encoding for the uncoordinated-7 mechanosensitive
innexin hemichannel-forming protein (i.e., a half gap junction forming
a single membrane channel), suggesting PANX1 and its invertebrate
innexin relatives could play role(s) in neuronal mechanotransduction
under physiological conditions as well (Walker & Schafer, 2020).
3.5. PANX1 in nervous system cancers
In healthy tissues, extracellular ATP and adenosine concentrations
are typically at nanomolar levels (Blay, White, & Hoskin, 1997;
Falzoni, Donvito, & Di Virgilio, 2013; Yegutkin, 2014); however, their
concentrations in the tumour microenvironment are significantly
higher, reaching hundreds of micromolar levels (Alarcón et al., 2020;
Ohta et al., 2006; Pellegatti et al., 2008). Depending on their concentra-
tions and subsequent receptor subtype affinities, ATP and adenosine
play roles in the promotion and suppression of tumour growth and cy-
totoxicity, as well as in the activation of the host immune system (Di
Virgilio & Adinolfi, 2017; Di Virgilio, Sarti, Falzoni, De Marchi, &
Adinolfi, 2018). In support of the idea that PANX1 might play a role in
brain cancer, rat C6 glioma cells overexpressing PANX1 exhibited in-
creased tumorigenicity via accelerated assembly of three-dimensional
compact aggregates (Bao, Lai, Naus, & Morgan, 2012), although it should
be noted that PANX1 was previously suggested to be a tumour suppres-
sor in C6 glioma cells (Lai et al., 2007). In human glioblastoma U87-MG
cells, PANX1 levels were upregulated in mitotic cells and Panx1 knock-
down decreased cell proliferation (Wei, Yang, Shi, & Chen, 2015). Nota-
bly, coincident application of ATP in the presence of Panx1 knock-down
resulted in a greater reduction in cell proliferation (Wei, Yang, et al.,
2015), implying a level of divergence of PANX1 and ATP-dependent
mechanisms in cancer that could be dependent on the expression of
9
Pharmacology & Therapeutics 225 (2021) 107840
other purinergic signaling players. For example, sustained P2X7 recep-
tor activation has been associated with the formation of a high conduc-
tance pore causing cell death (for review see Khakh & Alan North,
2006), but there is also evidence that extracellular ATP promotes cancer
progression in myriad ways (Di Virgilio et al., 2018; Di Virgilio &
Adinolfi, 2017; Vultaggio-Poma, Sarti, & Di Virgilio, 2020). Further
support of the hypothesis that PANX1 may be involved in nervous
system cancers, comes from the emergent neural precursor-like
nature of nervous system tumour cells (Couturier et al., 2020; Jessa
et al., 2019) taken together with our previous discovery of PANX1
regulation of neural stem cell proliferation, maintenance and differenti-
ation (Wicki-Stordeur et al., 2012, 2016; Wicki-Stordeur & Swayne,
2013). We found that PANX1 overexpression increased N2a mouse
neuroblastoma-derived cell proliferation that was blocked by probene-
cid treatment (Wicki-Stordeur et al., 2012) and siRNA-mediated Panx1
knock-down and probenecid treatment resulted in neurite outgrowth
(Wicki-Stordeur & Swayne, 2013), reminiscent of the effects of retinoic
acid treatment on these cells (Shea, Fischer, & Sapirstein, 1985). Nota-
bly, retinoic acid is used clinically for the treatment of neuroblastoma
(Reynolds, Matthay, Villablanca, & Maurer, 2003), suggesting PANX1
could be an important therapeutic target for nervous system cancers
like neuroblastoma. More recently, PANX1 was detected in human
neuroblastoma tumour specimens and high-risk patient derived cell
lines and probenecid reduced neuroblastoma progression in vitro and
in vivo (Holland, 2020). We previously showed that ATP induced
PANX1-P2X7 receptor clustering and internalization in N2a cells
(Boyce et al., 2015; Boyce & Swayne, 2017). Building on that work, we
recently discovered that PANX1 may regulate macropinocytosis of ATP
in N2a cells (Boyce et al., 2020). Macropinocytosis of ATP confers drug
resistance to cancer cells by facilitating uptake of ATP and extracellular
debris (Jayashankar & Edinger, 2020; Qian et al., 2014) as a means of
obtaining additional energy (Di Virgilio et al., 2018; Recouvreux &
Commisso, 2017; Vultaggio-Poma et al., 2020), adding new metabolic
implications of PANX1 in cancer cell biology.
Cancer cells also use ectonucleotidases and nucleoside transporters
to increase adenosine concentrations in the extracellular milieu. Glioma
cells express high levels of membrane-bound ectonucleotidases, specif-
ically CD39 and CD73, whose sequential activity produces adenosine
from ATP (reviewed in Ceruti & Abbracchio, 2020). Additionally, rela-
tively low levels of ENT1 were detected in primary stem-like cells and
U87MG glioblastoma cells, further raising extracellular adenosine by
preventing its re-uptake (Alarcón et al., 2020). High levels of extracellu-
lar adenosine allow glioma cells to modulate the host immune response
(primarily through A1 and A2A receptors in peri-tumour tissue) and
boost their tumorgenicity, by promoting angiogenesis via A2 and A3 re-
ceptors (Ceruti & Abbracchio, 2020; Gessi, Merighi, Sacchetto, Simioni,
& Borea, 2011). Given the intricate connection between PANX1 and
purinergic receptors (in particular P2X7 receptors) in tumorigenicity
and associated host immune response (modulated by adenosine recep-
tors), further pharmacological or genetic manipulations that dampen
ATP release (e.g., by targeting PANX1) and signaling (e.g., by targeting
P2 receptors, ectonucleotidases and nucleoside transporters) represent
novel potential therapeutic approaches to cancer.
3.6. Highlight on PANX1 and purinergic signaling in neuron-microglial
interactions
Microglia play a critical role in maintaining homeostatic neuronal ac-
tivity during both physiological and inflammatory conditions (reviewed
in Illes, Rubini, Ulrich, Zhao, & Tang, 2020). Under naïve conditions,
microglial processes are highly dynamic and reach out to survey their
environment where they regulate synaptic pruning and network plas-
ticity (Tremblay et al., 2011). In the context of inflammation associated
with infection and several different neurological diseases, microglia are
triggered to move towards sites of injury to phagocytose dead or dying
cells and take part in antigen presentation and cytokine production
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
(reviewed by Bachiller et al., 2018). Increased extracellular ATP associ-
ated with inflammation acts on ionotropic P2X and metabotropic P2Y
receptors (see BOX2, reviewed by Illes, Rubini, Ulrich, Zhao, & Tang,
2020) on microglial membranes, contributing to their migration and in-
creased inflammatory function (Illes, Xu, & Tang, 2020; Tremblay et al.,
2011; Walz, Ilschner, Ohlemeyer, Banati, & Kettenmann, 1993). For in-
stance, expression of microglial P2Y12 receptors, associated with the
ramified microglial morphology observed under physiological condi-
tions (Sipe et al., 2016), is reduced significantly following microglial re-
activity to ATP (Haynes et al., 2006). Haynes et al. (2006) found that
microglia lacking P2Y12 receptors were unable to migrate or project
their processes towards extracellular nucleotides in vivo and in vitro,
suggesting that these receptors are important for microglia chemotaxis,
particularly in response to nucleotides (Kyrargyri et al., 2020; Madry
et al., 2018). It is tempting to speculate that neuronal, astrocytic, or oli-
godendroglial PANX1 channels could be potential sources of ATP release
for communication with microglial P2Y12 receptors. For instance, ATP
release induced by neuronal NMDA receptor activation following
kainate-induced seizure activity or glutamate application promoted
microglial process extension, which was abolished with P2ry12 KO or
probenecid treatment (Eyo et al., 2014). Notably, ATP and NMDA-
triggered microglial process extension was still observed with
carbenoxolone treatment (PANX1 and gap junction inhibitor, BOX1)
and Panx1 global KO (Dissing-Olesen et al., 2014); future investigations
using more selective pharmacological agents and cell-type specific KO
models could shed some light on the mechanisms underlying neuron-
glial communication. In addition to injury and inflammation, we de-
scribed how neuronal activity itself can result in changes to microglial
morphology and function (Tremblay, Lowery, & Majewska, 2010;
reviewed in Tremblay et al., 2011), in part by increasing local concentra-
tions of extracellular ATP (Badimon et al., 2020). Primary cultured neo-
natal microglia convert neuronally-released ATP to adenosine, eliciting
neuronal adenosine receptor-mediated activity suppression, thereby
creating a microglial-driven neuronal homeostasis negative feedback
loop (Badimon et al., 2020). Somewhat relatedly, Ent2 KO, which re-
sulted in elevated extracellular adenosine levels, provided resistance
to lipopolysaccharide-induced neuroinflammation (as assessed by mi-
croglia reactivity and astrogliosis), blood-brain barrier breakdown
(tight junction integrity, leakage) and neurotoxicity (based on IgG accu-
mulation and caspase-3 cleavage) via activation of A1 and A2A recep-
tors (Wu, Lee, Chou, Chern, & Lin, 2020). Altogether, these studies
highlight the importance of purinergic signaling and possibly PANX1 in-
volvement in the microglial dynamics associated with synaptic pruning
and inflammation.
3.7. Peripheral PANX1 signaling with implications for nervous system
diseases
Along with roles in development and cell function in the nervous
system, PANX1-associated processes in peripheral tissues have impor-
tant implications for nervous system function. These processes include
regulation of vascular tone, immune cell migration, and inflammatory
responses. For instance, α-1D-adrenergic receptor-mediated vasocon-
striction in vascular smooth muscle cells involves PANX1 activation
via phosphorylation at Tyr198 by Src kinases, and subsequent release
of ATP (Billaud et al., 2011, 2015; DeLalio et al., 2019), implicating
PANX1 in hypertensive vascular pathology (DeLalio et al., 2019).
Notably, PANX1 phosphorylation at Tyr198 has also been linked to
PANX1-mediated ATP release in venous endothelial cells following
TNF-α stimulation (Lohman et al., 2015). Under these inflammatory
conditions, PANX1 activation promoted leukocyte adhesion and trans-
migration through venous vessel walls, in part, by upregulating the
expression of vascular cell adhesion protein 1 in endothelial cells
and also by facilitating ATP release (Chen et al., 2006; Grassi, 2010).
ATP subsequently activated P2Y2 and A3 receptors on leukocytes
(Chen et al., 2006; Grassi, 2010), suggesting that PANX1 is a positive
10
Pharmacology & Therapeutics 225 (2021) 107840
regulator of the venous endothelium inflammatory response. Similarly,
endothelial-specific deletion of Panx1 resulted in reduced infarct vol-
ume, leukocyte infiltration, and vascular tone (Good et al., 2018).
Given that integrity of the vascular system is paramount to sustain nor-
mal brain function and that inflammation and vascular pathology are
important contributors to neurodegenerative disease (Chitnis &
Weiner, 2017; Mangiafico, Sarnataro, Mangiafico, & Fiore, 2006;
Phillips & Mate-Kole, 1997; Sweeney, Kisler, Montagne, Toga, &
Zlokovic, 2018; Tasci et al., 2018), PANX1's role in purinergic and cyto-
kine signaling during neuroinflammatory responses marks PANX1
modulation as a possible treatment of cerebrovascular pathologies.
PANX1 channels are strongly associated with inflammation. PANX1
channels are, in part, responsible for inflammatory ATP release
(Chitnis & Weiner, 2017; Dahl, Qiu, & Wang, 2013; Linden, Koch-
Nolte, & Dahl, 2019; Makarenkova, Shah, & Shestopalov, 2018;
Pelegrin & Surprenant, 2006; Penuela, Gehi, & Laird, 2013) and PANX1
also regulates inflammasome activation and regulation of cytokine re-
lease in a variety of cell types (reviewed by Makarenkova, Shah, &
Shestopalov, 2018). Inflammasomes are intracellular protein complexes
that form in response to pathogen-associated molecular patterns or
danger-associated molecular patterns (reviewed by Zheng, Liwinski, &
Elinav, 2020) and induce caspase activation as well as an inflammatory
response, including pyroptosis, a type of inflammatory cell death dis-
tinct from apoptosis (Yang, He, Muñoz-Planillo, Liu, & Núñez, 2015;
Zheng et al., 2020). ATP promotes IL-1β secretion and release during
pathogen-associated molecular pattern-mediated responses through
activation of P2X7 receptors and by acting as an enhancer signal
(Crespo Yanguas et al., 2017; Di Virgilio, Dal Ben, Sarti, Giuliani, &
Falzoni, 2017; Makarenkova, Shah, & Shestopalov, 2018; Zhou, Green,
Bennet, Gunn, & Davidson, 2019). Nuclear factor-κβ activation follow-
ing TNF-α-triggered signaling, upregulated PANX1 expression levels
and increased trafficking of PANX1 channels to the membrane (Yang
et al., 2020). This led to increased intracellular calcium and subsequent
upregulation of IL-1β expression, processing, and release, suggesting
PANX1 signals in a feed-forward manner during sterile inflammation.
As mentioned above, probenecid reduced inflammasome activation,
IL-1β and high mobility group BOX 1 release, NOD-, LRR- and pyrin
domain-containing protein 3 and caspase-1 expression, and reactive ox-
ygen species production in response to inflammatory stimuli (Jian et al.,
2016), suggesting that inhibiting PANX1 might be therapeutic during
inflammatory conditions. Importantly, numerous studies have identi-
fied an association between PANX1 channels and P2X receptors both
upstream and downstream of inflammasome activation, though cell
specific mechanisms of activation are still unclear (Hung et al., 2013;
Jeong et al., 2020; Makarenkova, Shah, & Shestopalov, 2018; Silverman
et al., 2009). Given that PANX1-induced P2X7 receptor-mediated
inflammasome signaling is amenable to therapeutic intervention, fur-
ther understanding of the mechanism behind activation is paramount
to translate these findings into interventions. Understanding the mech-
anism will allow for treatment of heightened inflammatory states such
as: sepsis, ischemic stroke, cortical spreading depression in migraine
headache, chronic pain, and neuroimmune and neurodegenerative dis-
orders (Dvoriantchikova et al., 2012; Pronin et al., 2019; Yeung et al.,
2020; Zhang et al., 2015; Zhou et al., 2019).
Not only does PANX1 have a role in the host immune response, but it
also plays a critical role in viral infection itself. For example, human im-
munodeficiency virus (HIV)-binding to its receptors on CD4-positive T
lymphocytes and primary human peripheral blood mononuclear cells
stimulated a long-lasting and biphasic opening of PANX1 channels
(Orellana et al., 2013). HIV-binding to its cellular receptors has also
been found to induce ATP release via PANX1 which subsequently signals
P2Y2 receptors to induce viral uptake and initiate replication (Séror
et al., 2011). Of interest, Swartz, Esposito, Durham, Hartmann, and
Chen (2014) found that inhibition of P2X hindered HIV infection of
CD4-positive T lymphocytes. Notably, the elevation of circulating ATP
levels was recently identified as a biomarker for HIV-associated
J.C. Sanchez-Arias, E. van der Slagt, H.A. Vecchiarelli et al.
cognitive impairment (Velasquez et al., 2020). It has been speculated
that in the brain, particularly in glial cells, similar to the periphery
(Orellana et al., 2013), PANX1 facilitated purinergic receptor-
dependent cellular HIV infection (Malik & Eugenin, 2019). Once cells
are infected with HIV, HIV may interact further with PANX1 leading to
extracellular increases in glutamate and ATP, which drive downstream
effects of HIV infection including inflammation, cognitive deficits, and
excitotoxicity (Malik & Eugenin, 2019). In cultured astrocytes in a
PANX1-dependent manner, HIV altered their permeability and calcium
dynamics, and increased release of ATP and inflammatory mediators
(Gajardo-Gómez et al., 2020).
Stemming from the critical roles of PANX1 in viral infection and in-
flammation, we recently proposed PANX1 inhibition as a potential
treatment for the coronavirus disease of 2019 (COVID-19) (Swayne
et al., 2020). Though there is currently no evidence showing an interac-
tion between PANX1 and severe acute respiratory syndrome coronavi-
rus 2 (SARS-CoV-2), PANX1 activity has been implicated in the
propagation of inflammatory responses in tissues susceptible to
hyperinflammation following severe acute respiratory syndrome coro-
navirus 2 infection. In various models of injury, endothelial cell PANX1
disruption reduced inflammation (Good et al., 2018; Jankowski et al.,
2018; Sharma et al., 2018). As such, PANX1 inhibition could minimize
damaging effects of inflammation and reduce the cytokine storm seen
in COVID-19 patients (Hui et al., 2020). Given the role of PANX1 in the
regulation of neurological plasticity and its links to viral infection and
inflammation, PANX1 should also be investigated in the context of the
neurological effects of COVID-19 (Li, Bai, Hashikawa, & Yan-Chao Li,
2020).
Maternal infection during pregnancy and subsequent maternal im-
mune activation underlies a number of neurological disorders
(reviewed in Brown & Conway, 2019). Polyinosinic:polycytidylic acid
(poly(I:C)), a viral infection mimic that induces maternal immune acti-
vation as a model for autism spectrum disorders (Reisinger et al., 2015),
was used to investigate the role of P2X7 receptors in this process. Mater-
nal poly(I:C) injection produced an autism-like phenotype in offspring,
as well as increased levels of the pro-inflammatory cytokine, IL-6, in the
fetal brain (Horváth et al., 2019), and these effects were normalized by
genetic or pharmacological disruption of the P2X7 receptor. Addition-
ally, several adverse outcomes in poly(I:C)-treated mice were reversed
by administration of suramin, a non-selective purinergic antagonist, or
via downregulation of purinergic receptors (Naviaux et al., 2013).
PANX1's role in ATP release, pro-inflammatory cytokine signaling and
viral infection, together with the identification of PANX1 single-
nucleotide polymorphisms associated with risk for autism spectrum
disorders (Davis et al., 2012), support further investigation of a potential
link between PANX1 and maternal immune activation.
4. Future directions
As an ATP-release channel, it is not surprising that PANX1 partici-
pates in myriad physiological and pathophysiological nervous system
contexts. The recent high-resolution cryo-EM characterization of
PANX1 structure (BOX1; Deng et al., 2020; Jin et al., 2020; Michalski
et al., 2020; Mou et al., 2020; R. Qu et al., 2020; Ruan, Orozco, Du, &
Lü, 2020) will undoubtedly facilitate the design of PANX1-targeting
molecules with enhanced selectivity and specificity. Development of
improved PANX1 blockers will provide new options for treating chal-
lenging neurological disorders such as neurodegeneration,
neurodevelopmental disorders, stroke, and pain. Moreover, our grow-
ing appreciation of the contribution of PANX1 and other purinergic sig-
naling systems to nervous system cellular development throughout the
lifespan, under both healthy and pathological conditions, will further
serve to enable clinical translation related to PANX1 and purinergic sig-
naling. Improved, in-depth understanding of PANX1 and purinergic sig-
naling in nervous system cellular functions will also be facilitated by
transgenic mouse lines that selectively target neurons, interneurons,
11
Pharmacology & Therapeutics 225 (2021) 107840
astrocytes, or other glial cells (i.e., oligodendroglial lineage cells and mi-
croglia). Finally, further evaluation and validation of the PANX1 interac-
tome in neuronal and non-neuronal nervous system cells will allow us
to bridge important knowledge gaps in our understanding of the molec-
ular mechanisms underlying the roles attributed to PANX1 and other
purinergic signaling mechanisms in nervous system health and disease.
Funding sources
This work was supported by operating grants from the Canadian
Institutes of Health Research (CIHR Grant MOP142215) and from the
Natural Sciences and Engineering Research Council (NSERC) [RGPIN-
2017-03889] to L.A.S. L.A.S. was also supported by a Michael Smith
Foundation for Health Research (MSFHR) and British Columbia Schizo-
phrenia Society Foundation Scholar Award (5900). J.C.S.A. was sup-
ported by a University of Victoria Fellowship Graduate Award. P.Y.
was supported by an NSERC CGS-M. H.A.V. was supported by a CIHR
Postdoctoral Fellowship and a MSFHR Research Trainee Award. M.E.T.
is a Tier 2 Canada Research Chair in Neurobiology of Aging and
Cognition.
Author contributions
The manuscript idea was conceived by LAS in collaboration with
JCSA, EVDS, HAV and MET. JCSA and EVDS made equal contributions.
JCSA, EVDS, HAV, RCC and LAS were the primary writers. All authors re-
vised the manuscript.
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
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