The Safeguarding Microglia: Central Role for P2Y12 Receptors

Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, China, International Collaborative Center on Big Science Plan for Purine Signalling, Chengdu University of Traditional Chinese Medicine, Chengdu, China, Rudolf Boehm Institute for Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom, Achucarro Centre for Neuroscience, IKERBASQUE, Basque Foundation for Science, Bilbao, Spain


INTRODUCTION
The brain is the most complex organ of human body composed of several highly specialised and heterogeneous population of cells, represented by neurones, neuroglia (astrocytes, microglia, oligodendrocytes) and cells of brain vasculature. Neurones and neuroglia form neural circuits; different types of glial cells contribute to shaping and maintaining synaptic connections, plasticity, homeostasis, and network level activity through dynamic monitoring and alteration of central nervous system (CNS) functional architecture (Kettenmann et al., 2013;Allen and Lyons, 2018;Verkhratsky and Nedergaard, 2018;Augusto-Oliveira et al., 2020). Microglial cells are scions of foetal macrophages invading the neural tube early in embryonic development (Ginhoux et al., 2013); after settling in the nervous tissue these cells undergo the most remarkable metamorphoses acquiring specific morphology (small soma with long, ramified motile processes) and physiology. In particular, microglial cells gain receptors to neurotransmitters and neuromodulators, while retaining the pattern recognition receptors from their immune heritage; this extended complement of receptors makes microglia arguably the most "receptive" cells in the CNS (Kettenmann et al., 2011;Garaschuk and Verkhratsky, 2019). Among these many receptors, microglia possess several types of purinoceptors, which are linked to microglial housekeeping, neuroprotective and defensive capabilities Tozaki-Saitoh et al., 2012). Purinergic signalling emerges as the key mechanism in the dynamic interactions between neurones and glial cells, with ATP being a classical neurotransmitter and a danger signal damage-associated molecular pattern (DAMP). This duality makes ATP and related purines versatile signalling molecules controlling microglial behaviours in both physiological and pathological context (Domercq et al., 2013;Illes et al., 2020).
The metabotropic P2Y 12 purinoceptor is of a particular relevance for microglia. First and foremost, the expression of this receptor distinguishes CNS resident microglia from peripheral macrophages (Sasaki et al., 2003;Haynes et al., 2006). Second, in the healthy brain P2Y 12 receptors are universally and specifically expressed in microglia in all brain regions and across different species from rodents to humans (Sasaki et al., 2003;Mildner et al., 2017); the P2Y 12 receptors are widely considered to be a signature of microglia in the healthy brain (Hickman et al., 2013;Bosco et al., 2018;Peng et al., 2019). Third, expression of P2Y 12 receptors is stable from foetal state and throughout human lifespan (Crain et al., 2009;Mildner et al., 2017). The P2Y 12 receptors share the seventransmembrane topology characteristic for G-protein coupled receptors of P2Y family (Burnstock and Verkhratsky, 2012). The preferred agonist for P2Y 12 receptors is adenosine diphosphate (ADP), which in the periphery acts as a major instigator of platelet aggregation and granule secretion thus supporting thrombogenesis (Liverani et al., 2014). In the CNS, microglial P2Y 12 receptors are activated by ADP deriving from enzymatic degradation of ATP released from neurones, astrocytes and oligodendroglia during their physiological activity or following tissue damage (Abbracchio et al., 2009;Zimmermann et al., 2012). Metabotropic P2Y 12 receptors are localised in the processes and in the somata of surveilling microglia, where they mediate various aspects of intercellular signalling targeting microglia (Table 1 and Posfai et al., 2019;Vainchtein and Molofsky, 2020).

MODES OF MICROGLIAL PATROLLING OF THE HEALTHY CNS: ROLE FOR P2Y 12 RECEPTORS
Microglial cells are indefatigable surveillants and overseers of the nervous tissue; their ramified processes are in constant move scanning CNS parenchyma (Davalos et al., 2005;Nimmerjahn et al., 2005) with a particular attention paid to neurones (Wake et al., 2009;Cserep et al., 2020). Microglial surveillance of the nervous tissue occurs in several distinct modes.

Microglia-Dendritic/Synaptic Patrolling
In the healthy brain microglial processes are constantly contacting synaptic contacts located on neuronal dendrites. These microglia-dendritic contacts are instrumental for synaptic pruning in early development, which removes silent, aberrant or redundant synapses by en passant phagocytosis (Sierra et al., 2010) thus contributing to shaping neuronal ensembles and supporting neuroplasticity (Kettenmann et al., 2013;Sakai, 2020). Synaptic pruning is controlled by neuronal complement system (Stevens et al., 2007;Schafer et al., 2012), which tags the synapses to be removed, and by neurone-derived chemokine CX3CL1 also known as fractalkine. Microglial cells specifically express fractalkine receptors, activation of which stimulates synaptic pruning by physiological phagocytosis (Paolicelli et al., 2011). At later developmental stages microglia can remove not only whole synapses but also synaptic fragments through the process known as trogocytosis (Weinhard et al., 2018).
Microglia-dendritic interactions are regulated by neuronal activity: an increase in neuronal firing increases the frequency and number of contacts between microglial processes and synapses (Li et al., 2013). Plastic remodelling of the nervous tissue involves substantial changes in microglial morphology, manifested in hyper-ramification of processes, decreased intrinsic motility of processes and increased number of contacts with synaptic sites. P2Y 12 receptors play a primary role in these preocesses; pharmacological and genetic occlusion of these receptors suppressed both microglial changes and neuronal plasticity, thus revealing contribution of microglia to experience-induced reshaping of neuronal networks (Sipe et al., 2016).

Microglia-Somatic Patrolling
The second distinct type of microglial patrolling is aimed at neuronal somata. In the cortex microglial processes frequently contact neuronal cell bodies. The microglia process-neuronal somata contacts (defined as somatic microglial junctions) last for tens of minutes and even up to 1 h, which is much longer compared to microglia-dendritic or microglia-synaptic contacts which usually last for several minutes only . Neuronal part of microglia-somatic junction contains mitochondria and secretory vesicles closely associated with plasmalemma; the microglial part of the junction was characterised by exceptionally high density of P2Y 12 receptors. The P2Y 12 receptors control formation of microglia-somatic junctions, as pharmacological blockade of these receptors halves the duration of microglia-somatic contacts. The microglia-somatic junctions seem to be particularly important for neuroprotection after ischemic attack: the stroke greatly increases microglial coverage of neuronal cell bodies; this increase requires operational P2Y 12 receptors. Inhibition of P2Y 12 -mediated signalling negatively impacts on neurones, which experience greater calcium load and increased functional disconnection. Signalling between neurones and microglial processes at the somatic level is supported by neuronal mitochondria and ATP exocytosis from vesicularnucleotide transporter (VNUT)-containing secretory vesicles: disruption of either impairs the microglia-somatic junction . To summarise, microglial P2Y 12 receptors provide for specialised interaction between neuronal cell bodies and microglial cells, interaction which appears to be critical for neuroprotection.

Microglia-Axonal Patrolling
Microglial processes establish intimate contacts with axon initial segments early in development and these contacts are maintained through adulthood probably supporting axonal structure (Baalman et al., 2015). Increased firing of the axon, reflective of neuronal hyperexcitability initiates further extension of microglial processes, which enwrap the axon and suppress axonal action potential generation, thus preventing excitotoxicity. Inhibition of microglial motility blocks this mechanism and facilitates neuronal death (Kato et al., 2016). Which microglial receptors are responsible for axonal patrolling remains unknown, although the involvement of fractalkine receptors has been excluded (Baalman et al., 2015).

Microglial Processes Converging Response-Counteracting Acute Lesions to the Nervous Tissue
Another type of microglial patrolling is associated with rapid convergence processes response, in which microglial processes swiftly move towards the site of potential injury. Thus response is regulated solely by P2Y 12 receptors that detect the source of ATP/ ADP as a potential damage signal (Davalos et al., 2005). The converging response of microglial processes represents a specific form of patrolling associated with primary defensive function of microglia. This response occurs at the initial stages of various neuropathologies. In particular, local cortical damage, associated with rapid increase in ATP/ADP instantly triggers microglial processes convergence towards the site of the lesion (Haynes et al., 2006). This directional extension of microglial processes involved activation of β1 integrin signalling cascade (Ohsawa et al., 2010). Microglial processes converge on axons after traumatic brain injury to reduce neuronal excitability (Benusa and Lafrenaye, 2020). Similarly, microglial processes move to and enwrap neurones and axons in experimental epilepsy, which  (2019) Rat/prelimbic cortex, central amygdala, perifornical lateral hypothalamic area, and dorsal raphe nucleus Immunocytochemistry; densitometry and cell counts; histology; qRT-PCR Sleep deprivation increased Iba1 staining, but did not affect immunoreactivity of P2Y 12 receptors and pro-inflammatory cytokines Hall et al. (2020) Humans, tissues and cells Humans/23-92 years/post-mortem tissue of MDD patients (5) and mentally healthy controls (5)/frontal lobe, temporal lobe, thalamus, subventricular zone Freshly isolated microglial cell suspension; purified with CD11b − assisted multiplexed single-cell mass cytometry. Immunocytochemistry A subpopulation of microglia from MDD brains have increased expression of P2Y 12 receptors, arguably associated with an increase in homeostatic and neuroprotective capacity of microglial cells in the diseased nervous tissue Bottcher et al. (2020) Humans/recent-onset schizophrenia patients (20) and 20 non-psychiatric controls (20)/myeloid cell Monocytes induced into microglia-like cells; RNA isolation and sequencing; mass cytometry; phagocytosis assay; immunocytochemistry and microscopy P2Y 12 receptors mRNA was enriched in a subpopulation of cells from schizophrenia patients Ormel et al. (2020) Human patients with MS/rats (8-11 weeks)/ tissue Experimental autoimmune encephalomyelitis in rats; human microglia isolation; immunocytochemistry; q-PCR; western blot; autoradiography P2Y 12 receptors were associated with an antiinflammatory phenotype; expression of P2Y 12 receptors was decreased in tissues with active MS lesions Beaino et al. (2017) (Continued) Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 627760 again counteracts hyperexcitability and potentially limits the seizures (Eyo et al., 2014). Mechanistically, excessive neuronal activity results in activation of NMDA receptors, which trigger ATP release that translates, through activation of P2Y 12 receptors, into converging microglial processes response (Dissing-Olesen et al., 2014) Genetic or pharmacological silencing of P2Y 12 receptors obliterates microglial processes converging response in all these pathological contexts (Haynes et al., 2006;Eyo et al., 2014).

MICROGLIAL P2Y 12 RECEPTORS IN NEUROLOGICAL DISEASES
Pathological insults to the CNS invariably stimulate and recruit microglia (Kettenmann et al., 2011;Savage et al., 2019), triggering reactive microgliosis (the commonly used term "activation" is somewhat misleading; microglial cells are activated by numerous signals in physiological context, whereas microgliosis represent response to pathology and hence should be defined as reactivity).
Purines and ATP are, as alluded earlier, classic damage-associated molecular patters (DAMP) conserved throughout the evolution (Verkhratsky and Burnstock, 2014). The P2Y 12 receptor is intimately involved in the early stages of microglial response to the lesion, as discussed in previous chapter, and to the early stages of microgliotic response ( Table 1). Stimulation of microglial P2Y 12 receptors triggers microgliotic transformation into various reactive phenotypes that ultimately climaxes in amoeboid phagocyting microglia (Hanisch and Kettenmann, 2007;Savage et al., 2019). Genetic deletion of P2Y 12 receptors results deficits in up-regulation of K + outward rectifying channels and in membrane ruffling and chemotaxis of amoeboid microglia (Swiatkowski et al., 2016). Reactive microgliosis however almost invariably results in down-regulation of expression of microglial P2Y 12 receptors (Zrzavy et al., 2017). Injection of LPS into the striatum triggers massive activation of microglial cells associated with almost complete disappearance of P2Y 12 receptors 4 days after the insult (Fukumoto et al., 2019); treatment of human induced pluripotent stem cells derived microglia with LPS likewise resulted in disappearance of P2Y 12 receptors (Banerjee et al., 2020). Similarly, experimental stroke induced gradual and almost compete disappearance of microglial P2Y 12 receptors (Kluge et al., 2019); down-regulation of P2Y 12 receptors have been observed in microglia in several chronic neurological diseases (Mildner et al., 2017;Zrzavy et al., 2017). Recent investigations however have found P2Y 12 receptors expression in microglia in several chronic neurological and neuropsychiatric conditions. The P2Y 12 -positive microglial cells were detected in the microglia freshly isolated from post-mortem brains of human patients suffering from major depressive disorder . Similarly microglia bearing P2Y 12 receptors were found in the outer regions of senile plaques in post-mortem tissues from Alzheimer's disease patients (Walker et al., 2020). These results indicate that P2Y 12 microglia populate diseased brains, which might be associated with rise of defensive, safeguarding microglial phenotypes, distinct from reactive microglia.

CONCLUSION
The P2Y 12 purinoceptors are signature receptors of microglia in the healthy brain. These receptors mediate patrolling behaviours of surveilling microglia and coordinate neuronal activity with operation of microglia. The P2Y 12 receptors are instrumental for microglial response to neuropathological lesion, and are responsible for the initiation of reactive microgliosis. Reactive microglia as a rule do not express P2Y 12 receptors, however in neurodegenerative and neuropsychiatric disease the population of P2Y 12 -bearing microglia (distinct form reactive microglia) remains; these cells arguably participate in defensive, safeguarding responses against neuropathology.

AUTHOR CONTRIBUTIONS
Conceptualisation: S-SL and AV; writing-original draft preparation: S-SL and AV; literature review, writing-review and editing: S-SL, YT, PI, and AV.