Role of P2Y12 receptor-mediated ADP signalling at microglia in the phagocytosis of neurons

Despite the prevalence of neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases, surprisingly little is known about the molecular mechanisms governing widespread neuronal loss observed in these conditions. Recent studies, however, have linked heightened microglial phagocytosis of live neurons under inflammatory stimuli such as amyloid-β and lipopolysaccharide (LPS) to this loss. The ADP-specific microglial receptor P2Y12 has been implicated in long-range communication between neurons and microglia, as well as in microglia chemotaxis preceding phagocytosis, but the direct role of this receptor in phagocytosis of live neurons has not been addressed. In addition, P2Y12 antagonists have already been established in the clinic as treatments for thrombosis, and whether these inhibitors could further serve a neuroprotective purpose by preventing phagocytosis was speculated. Upon examining the effect of P2Y12 inhibition using the competitive antagonist PSB0739, and P2Y12 genetic knockdown on phagocytosis of neuroblast-like, naïve PC12 targets by LPS-activated BV-2 microglia through a combination of flow cytometry and microscopy analyses, I show that P2Y12 perturbation does not affect microglial phagocytosis of neuronal targets under LPS stress. However, P2Y12 disruption led to significant decreases in phagocytosis of bead and neuronal targets under unstimulated conditions, which suggested that P2Y12 plays an important role in the phagocytic mechanisms of resting microglia. Additionally, fluorescent calcium response assays yielded significant decreases in acute calcium response to ADP in P2Y12-inhibited and knockdown cells, confirming that P2Y12 plays a major role in calcium mobilization due to ADP treatment in BV-2 microglia. Furthermore, examination of the role of ADP on the regulation of P2Y12 mRNA expression using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) led to the finding that P2Y12 expression is reduced in response to high concentrations of ADP. These findings highlight the novel ideas that 1) the P2Y12 receptor has functional roles in phagocytosis of basal microglia besides cell migration, and 2) persistent imbalance of ADP in the extracellular environment affects P2Y12 expression, which may underlie inflammatory stimuli-induced P2Y12 downregulation in the transition from resting to highly phagocytic, disease-associated microglia.

Name dc:type.qualificationname

Master of Philosophy (MPhil)

Level dc:type.qualificationlevel

Masters

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2018

Author dc:creator

• Inouye, Michiko O

Advisor dc:contributor.advisor

• Brown, Guy Charles

• microglia
• P2Y12
• ADP
• phagocytosis
• calcium signalling
• BV2
• PC12
• RT-qPCR
• Flexstation
• gene expression
• LPS

Licence

• https://www.repository.cam.ac.uk/bitstreams/3cc7bc37-2eb3-48fb-8738-32b1a3f57f30/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.31782

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/284409