Prior research has shown that activation of the microglial P2Y₆ receptor (P2Y₆R) with uridine diphosphate (UDP) induces microglial phagocytosis. Also, young mice with knockout of the P2Y₆R gene (P2ry6⁻´⁻) were found to perform worse in memory tests compared to P2ry6⁺´⁺ mice, but P2ry6⁻´⁻ mice were protected from ageing-associated memory loss. Finally, in a chronic model of tauopathy, P2ry6⁻´⁻ mice were protected from both TAU-induced memory loss and tauopathy. The goal of this thesis was to investigate microglial-dependent mechanisms might protect P2ry6⁻´⁻ mice from ageing- and tauopathy-induced memory loss, as well as possible roles for P2Y₆R in brain development. However, as P2Y₆R is found on numerous peripheral cells throughout the body, it cannot be dismissed that the cause of the effects observed by P2Y₆R deficiency in mice may not solely be microglial-dependent.

I found that microglia from P2ry6⁻´⁻ mice had no significant changes in microglial migration, cytokine release, or phagocytosis of beads. However, P2Y₆R deficiency substantially reduced the phagocytosis of synaptosomes. 4-month-old (young) and 17-month-old (old) mice were investigated to study ageing-associated effects. There was also a large increase of synaptic material within microglial lysosomes with age of P2ry6⁺´⁺ mice, but this apparent ageing-associated phagocytosis of synapses was not present in aged P2ry6⁻´⁻ mice. Similarly, there was a loss of synapses with age in hippocampal CA1 stratum radiatum and the somatosensory cortex in P2ry6⁺´⁺ mice, but no such synaptic loss was observed in aged P2ry6⁻´⁻ mice. Together, these findings indicate that P2Y₆R mediates microglial phagocytosis of synapses with age.

Postnatal day 15 (P15) and P30 mice were investigated as active microglial pruning is occurring at this timepoint. Synaptic protein levels were observed to be dysregulated in P15 P2ry6⁻´⁻ mouse brain homogenates compared to P2ry6⁺´⁺ mice. P30 P2ry6⁻´⁻ mice, but not P15 P2ry6⁻´⁻ mice, showed higher levels of synapses compared to P2ry6⁺´⁺ in multiple regions associated with memory. This increase in synapses was coupled with reduced internalisation of synaptic material within CD68+ lysosomes in Iba1+ microglia in P30 P2ry6⁻´⁻ mice. This indicates that P2Y₆R-dependent phagocytosis of synapses contributes to synaptic loss late in development.

In vitro, P2Y₆R deficiency reduced microglial phagocytosis of myelin debris. In vivo, P15 P2ry6⁻´⁻ mice, but not P30 P2ry6⁻´⁻ mice, were observed to have reduced cortical myelination compared to P2ry6⁺´⁺, with no change in the internalisation of a myelin-associated protein within CD68+ lysosomes in Iba1+ microglia. 4-month-old P2ry6⁻´⁻ were observed to have reduced cortical myelination compared to 4-month-old P2ry6⁺´⁺ mice. This was coupled with increased internalisation of a myelin-associated protein within CD68+ lysosomes in Iba1+ microglia with 4-month-old P2ry6⁻´⁻ mice. This suggests that lack of P2Y₆R-dependent phagocytosis of myelin early in development results in a myelination defect.

Mice expressing P301S TAU have TAU hyperphosphorylation and reduced memory, and memory is rescued when crossed with P2ry6⁻´⁻ mice. However, there was no difference in synaptic density in multiple regions associated with memory, and no change in the internalisation of hyperphosphorylated TAU within microglial lysosomes in P2ry6⁻´⁻ mice with P301S TAU. Activation or inhibition of P2Y₆R did not affect lysosomal exocytosis by microglia in culture. P2Y₆R did not regulate microglial phagocytosis of TAU fibrils or cells with TAU fibrils in ways that could explain why P2Y₆R deficiency reduced tauopathy in mice. Thus, why P2Y₆R deficiency reduces tauopathy and the associated memory loss in mice remains unclear.

Overall, the research described in this thesis shows that the microglial phagocytic receptor P2Y₆R mediates microglial phagocytosis of synapses during development and ageing, and additionally affects myelination during development, summarised in the graphical abstract below. Together these changes may explain why P2Y₆R deficiency affects memory, and suggests that, after development, P2Y₆R may be a good target to prevent excessive synaptic loss in pathology and ageing.