Astrocyte adaptation in Alzheimer’s disease: a focus on astrocytic P2X7R

Abstract Astrocytes are key homeostatic and defensive cells of the central nervous system (CNS). They undertake numerous functions during development and in adulthood to support and protect the brain through finely regulated communication with other cellular elements of the nervous tissue. In Alzheimer’s disease (AD), astrocytes undergo heterogeneous morphological, molecular and functional alterations represented by reactive remodelling, asthenia and loss of function. Reactive astrocytes closely associate with amyloid β (Aβ) plaques and neurofibrillary tangles in advanced AD. The specific contribution of astrocytes to AD could potentially evolve along the disease process and includes alterations in their signalling, interactions with pathological protein aggregates, metabolic and synaptic impairments. In this review, we focus on the purinergic receptor, P2X7R, and discuss the evidence that P2X7R activation contributes to altered astrocyte functions in AD. Expression of P2X7R is increased in AD brain relative to non-demented controls, and animal studies have shown that P2X7R antagonism improves cognitive and synaptic impairments in models of amyloidosis and tauopathy. While P2X7R activation can induce inflammatory signalling pathways, particularly in microglia, we focus here specifically on the contributions of astrocytic P2X7R to synaptic changes and protein aggregate clearance in AD, highlighting cell-specific roles of this purinoceptor activation that could be targeted to slow disease progression.


Introduction
Neuropathological changes in Alzheimer's disease (AD) include the progressive deposition of senile plaques and neurofibrillary tangles (NFTs), alongside extensive and complex glial alterations, vascular changes, synapse and neuron loss, leading to cognitive impairment and dementia [1].
Astrocytes are a subpopulation of glial cells derived from neuroepithelial progenitors that account for 20-40% of total glial cells in humans, depending on the brain region [2,3]. Single-cell transcriptomics revealed considerable molecular heterogeneity among astrocyte populations in rodent brain [4] as well as a complex stratified architecture across cerebral layers [5] that is likely more diverse in human brain, which in addition contain several forms of astrocytes absent in other mammals [6].
Astrocytes perform critical functions in the developing and adult CNS [7]. For example, during development, astrocytes remodel neuronal circuits, participating in the formation and pruning of synapses [8,9]. Astrocytes are functionally integrated with synapses, with all astrocytic compartments found to abut synapses in adult mouse hippocampus [10]. A particularly high density of pre-synaptic terminals and/or dendritic spines contact astrocytic branches and leaflets [10,11] which may result from these being amongst the most dynamic of astrocytic structures in response to neuronal signals [10]. Astrocytes, Figure 1. Potential roles of astrocytic P2X 7 R in synaptic function and protein clearance in AD In AD brain, high levels of ATP could activate P2X 7 R in astrocytes and contribute to defects in neurotransmission. The opening of P2X 7 R channels allows calcium influx which modulates the release of glutamate to the synaptic cleft, where it could bind to NMDA-Rs at the post-synapse. P2X 7 R could also participate in the regulation of inhibitory synapses, by modulating the release of GABA from astrocytes. In addition to their intimate association with synapses, astrocytes also play important roles in the maintenance of protein homeostasis through the internalisation and degradation of Aß and tau aggregates. In AD, astrocytic P2X 7 R could alter protein clearance pathways via HSPB1-mediated autophagy or the regulation of HSPG expression, which might influence astrocyte uptake and clearance of tau species.
sensing through one of these receptors, the purinergic P2X 7 receptor, may contribute to alterations in synaptic and endolysosome-related functions of astrocytes in AD ( Figure 1).

P2X 7 R
The P2X 7 R protein consists of a short intracellular N-terminal domain, two transmembrane α-helixes, an extracellular loop enriched in N-glycosylation sites and a long cytoplasmatic C-terminal domain [56,57]. P2X 7 R is typically found in a resting/closed or apo-state conformation, with a narrow cavity through which ATP must access to reach the active binding pocket. When ATP binds to P2X 7 R, conformational rearrangements lead to the opening of an ion-permeable channel that allows the influx (i.e., Na + , Ca 2+ ) and efflux (i.e., K + ) of small cations, and upon channel dilatation into a larger pore, the entry of large hydrophilic molecules at a slower rate [58]. In contrast to other P2X subtypes, P2X 7 R does not undergo desensitisation after activation of the receptor due to permanent stabilization provided by a palmitoylated cysteine rich region in the cytoplasmatic domain [59]. This feature of P2X 7 R activation dynamics likely contributes to the hyperpolarised astrocyte membrane potential that is important for astrocyte physiology and functions [60].
The human P2X 7 R gene is highly polymorphic with more than 150 non-synonymous SNPs, the majority of which lead to amino acid substitutions in the extracellular loop or the cytoplasmic C-terminal tail [61], affecting agonist binding affinity [62], trafficking to membranes [63], ion channel activity [64] and permeability of the pore [65]. In humans, there are seven splice variants of P2X 7 R, two of which are predominantly expressed in the CNS and immune tissues including the full-length (P2X 7 R A) and a C-terminally truncated form with an early stop codon (P2X 7 R B) [66,67]. The latter gives rise to a receptor deficient in the formation of a large permeable pore that retains ion channel properties [67].

P2X 7 R in AD
While there is limited evidence that single nucleotide polymorphisms that alter P2X 7 R activity influence the risk of AD [97], converging studies show enhanced levels of P2X 7 R mRNA and P2X 7 R protein in AD post-mortem brains in comparison with non-demented controls suggesting an involvement of this purinoreceptor in AD [71,98,99]. This increase is similarly observed in several transgenic mouse models of familial AD that overexpress mutant forms of APP including Tg2576 [80], APP/PS1 [100] and J20 [99] mice as well as in tauopathy models carrying mutations in MAPT [63]. Higher levels of P2X 7 R were observed at 12 months relative to 3-month-old APP/PS1 mice suggesting that P2X 7 R expression increases with disease progression [100]. P2X 7 R protein increases are particularly apparent surrounding Aβ plaques in AD brain [71,99,101], and this plaque-associated up-regulation is recapitulated in transgenic rodent models of amyloidogenesis [80,99,100]. In addition to changes in P2X 7 R expression, pharmacological antagonism or genetic deletion of P2X 7 R protects against disease in mouse models harbouring Aß [62,92] or tau pathology [63,91,93] indicating that P2X7R-mediated functions contribute to the disease process.

P2X 7 R contributions to astrocyte driven synaptic changes in AD
Astrocytes are implicated in the deterioration of synaptic transmission in AD, affecting both excitatory (glutamatergic) and inhibitory γ-aminobutyric acid (GABA)-ergic synapses [14]. Aβ induces calcium dysregulation in astrocytes which can affect their ability to modulate neurotransmission [102,103]. For example, astrocytes can induce neuronal hyperactivity through the transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in response to Aβ, which potentiates the release of excitatory glutamate in APP/PS1 mice [104]. Reports describing an increase in the release of the N-methyl-D-aspartate receptor (NMDA-R) co-factor D-serine from these astrocytes are now questioned, since astrocytes do not produce D-serine but rather L-serine which can be shuttled to neurons to drive neuronal production of D-serine [105]. Compromised glucose metabolism is observed in prodromal stages of AD which correlates with disease progression [14,106]. Accordingly, reduced aerobic glycolysis is also observed in prodromal AD [107], one consequence of which is decreased L-serine synthesis by astrocytes [108]. This disrupts NMDA-R-mediated synaptic plasticity and cognitive function in AD mice, which can be recovered upon dietary L-serine supplementation [109].
In transgenic mice expressing AD-causing mutant forms of APP and PSEN1 (APP/PS1), astrocytes surrounding Aβ plaques have lower levels of EAAT2, which leads to an extra-synaptic accumulation of glutamate, neuronal hyperactivity [110] potentially mediated by neuronal NMDA receptors [111], and possibly neurotoxicity. However, whether or not this is true in human disease is uncertain since analysis of postmortem brain from AD cases with significant amyloid and tau pathology showed higher levels of astrocytic EAAT2 in comparison with non-demented cases carrying AD pathology, pointing towards a mechanism of astrocytic resilience against neuropathological changes in AD [112]. Astrocytic P2X 7 R could be activated by ATP, or potentially indirectly, by Aβ [113], in the vicinity of senile plaques to contribute to excess glutamate levels. Stimulation of P2X 7 R in hippocampal, spinal cord and substantia gelatinosa astrocytes using the potent broad agonist 2 (3 )-O-(4-benzoylbenzoyl)adenosine 5 -triphosphate (BzATP) induces glutamate release and stimulation of neighbouring NMDA-Rs through a Ca 2+ -dependent mechanism [114,115]. The opening of P2X 7 R pores could also mobilise other transmitter-containing vesicles following Ca 2+ entry, but the precise molecular pathways that mediate these events remain obscure [75]. Astrocytes can also influence neuronal inhibition by increasing GABA release at the synaptic cleft in mice expressing five familial mutations in APP and PSEN1 (5xFAD mice, [116]), and some GABA release from astrocytes is regulated by P2X 7 Rs, at least in stratum radiatum astrocytes proximal to interneurons in the hippocampus [117].

P2X 7 R and astrocytic protein clearance pathways -implications for AD
Recent analysis showed that endolysosomal pathway components, fundamental for the uptake, processing, degradation and disposal of proteins and cellular debris, are down-regulated in AD astrocytes [42]. Indeed astrocytes, in addition to microglia, play central roles in the clearance of protein aggregates and other debris in degenerating AD brain [118]. By surrounding Aβ plaques, glia erect a physical and functional barrier to isolate and potentially clear proteinaceous aggregates from the affected neuropil [119].
Aß oligomers are observed within astrocytes in post-mortem AD brain [120] and mature healthy astrocytes engulf and degrade Aβ species in vitro and ex vivo [121,122]. Inhibition of reactive astrogliosis either increases [123] or reduces [124] levels of Aβ in APP/PS1 mice. The astrocyte-mediated internalisation of Aβ occurs in a ApoE-dependent manner, since ApoE deficient astrocytes are not capable of removing amyloid [125], with efficient Aβ uptake and clearance from astrocytes dependent on transcription factor EB (TFEB)-mediated lysosomal degradation [126]. Similarly, there is evidence that the Aβ sensor low density lipoprotein receptor-related protein 1 (LRP1) is critical for astrocytic uptake and degradation of Aß [47]. Astrocytes can also upregulate the expression of extracellular proteolytic enzymes that target Aβ including insulin degrading enzyme [127], released via an unconventional autophagy-dependent secretory pathway, and endothelin-converting enzyme-2 [128], they are efficient in autophagy and can potentially limit the accumulation of Aβ species in AD [118].
Astrocytes can also internalize modified forms of tau protein.
In a tauopathy mouse model in which tau was expressed specifically in entorhinal cortex neurons, tau aggregates that spread trans-synaptically to the dentate gyrus were detected in astrocytes [129]. These data indicate that astrocytes internalize and may contribute to tau propagation. Indeed, extracellular forms of fibrillar tau are taken up by astrocytes [46], including at synapses for redirection into lysosomal degradation pathways to regulate tau spread [130]. Data also implicates heparin sulphate proteoglycans (HSPGs) and LRP1 in tau uptake by astrocytes, with the efficiency of the uptake varying depending on disease-associated tau modifications [131][132][133][134].
While the direct contributions of astrocytic P2X 7 R to these processes have not been investigated in detail, several independent studies demonstrated that pharmacological blockade or genetic deletion of P2X 7 R is beneficial in mouse models of AD, reporting reduced amyloid plaque number and abundance of soluble Aβ species in mouse models of amyloidosis [71,135]. In tauopathy mouse models, decreases in tau phosphorylation at certain epitopes [72,101] or a reduction in the abundance of misfolded tau forms [136] were reported. Although some alterations in microglial morphology and functions including phagocytosis, migration and cytokine release were observed upon P2X 7 R inhibition [72,101], no consistent changes were detected between the different mouse models [71,136]. No alterations in protein degradation pathways were described although P2X 7 R induction in microglia is known to impair lysosomal function, increasing levels of the autophagosome membrane-associated form of microtubule-associated protein 1 light chain 3 (LC3)-II in a Ca 2+ -dependent manner, up-regulating the formation of autophagosomes and autophagolysosomes, and increasing the release of autophagosomes [137,138]. We suggest that further exploration of the potential contribution of astrocytic P2X 7 R to these processes is warranted since P2X 7 R activation also regulates autophagy in astrocytes [139,140]. Astrocytes are damaged in status epilepticus, and they form vacuoles containing lysosome-associated membrane protein (LAMP)-1 [141]. P2X 7 R antagonism was found to decrease astrocyte damage in the molecular layer of the dentate gyrus and frontoparietal cortex under these conditions [142] which could be caused by a prolonged induction of the molecular chaperone small heat-shock protein (HSP)B1, a HSP that facilitates the folding and removal of aberrant proteins, and, in turn, promotes astroglial autophagy [139]. Others have shown similar effects in P2X 7 R knockout mice, where P2X 7 R signalling to focal adhesion kinase (FAK) was found to regulate HSPB25 and fine tune autophagy [140]. Since, as we discuss above, astrocytes efficiently internalise Aβ and modified forms of tau in disease, these data may suggest that P2X 7 R-induced regulation of autophagy in astrocytes is important for limiting proteinaceous spread in AD and tauopathies. Finally, tau internalization and release may be mediated by HSPGs [143,144]. P2X 7 R also regulates HSPG expression and localization, at least in the cornea [145], and we suggest that exploration of the potential for P2X 7 R signalling to similarly affect HSPGs in astrocytes and alter tau clearance is warranted.
In summary, we provide an overview of synapse-related and protein clearance functions of astrocytes that can be modulated by P2X 7 R signalling in AD. P2X 7 R has gained much attention in recent years as a possible therapeutic target. Genetic deletion of P2X 7 R in APP/PS1 mice improved long-term synaptic plasticity, spatial learning and memory dysfunction relative to wild-type littermates [71], and P2X 7 R antagonism in mouse models of tauopathy harbouring the frontotemporal dementia (FTD)-causing MAPT mutations G272V and/or P301S ameliorates cognitive and behavioural deficits as well as synaptic dysfunction [72,101,136]. We suggest that further exploration of P2X 7 R-driven effects on biological processes linked with astrocyte contributions to AD may uncover novel targets for therapeutic intervention.

Summary
• Astrocytes play critical roles in maintaining a healthy brain environment. This is mediated through multiple homeostatic transporters, interactions with neurons and microglia, and functions at the blood brain barrier and synapses.
• Some astrocytes become 'reactive' in AD, while others show asthenia and loss of homeostatic functions. Astrocytes in AD reduce their support for synapses and show deficits in endolysosomal pathway components.
• There are increases in P2X 7 R mRNA and protein in AD, particularly in the vicinity of plaques. P2X 7 R activation in astrocytes influences synaptic activity and protein clearance pathways, and this may be one route by which P2X 7 R affects AD progression.
• Further exploration of the functional consequences of astrocytic P2X 7 R in AD may reveal novel cell-type specific targets for intervention.

Competing Interests
The authors declare that there are no competing interests associated with the manuscript.

Author Contribution
All authors wrote and edited the manuscript. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising.