Physiological Significance of P2RY12 in Microglial-neuronal Interactions and Behavior Across Sexes

Microglia are the primary resident immune cells in the brain parenchyma. These cells are known to play pivotal roles in maintaining brain homeostasis and physiology. These cells are sexually dimorphic at almost level of investigation — epigenome, transcriptome, proteome, metabolome, and morphology. Yet, this critical aspect is often overlooked in neuroscientific studies. Decades of research have revealed the dynamic interactions between microglia and neurons, but infrequently consider how this dynamism could be different with microglial sex differences, leaving a significant gap in our knowledge. This study focuses on P2RY12, a highly expressed microglial signature that mediates microglial-neuronal interactions. While some studies have pointed to P2RY12’s neuroprotective effects in conditions such as seizures and ischemic events, its role can be deleterious in different scenarios, including neuropathic pain, and remains ambiguous in a rodent model of Parkinson’s disease. This variability emphasizes the necessity for more in-depth research into the physiological relevance of microglia P2RY12 across both sexes, particularly given the observed sexual dimorphism in microglia. Consequently, the primary aim of this study is to ascertain the physiological significance of microglial P2RY12 and its influence on behavior across sexes.
Since we are investigating the significance of P2RY12 across sexes, we first wanted to determine if there is a sex difference in P2RY12 expression across sexes. Using CX3CR1-GFP/+ (phenotypic wildtype) and P2RY12-/-:CX3CR1-GFP/+ (phenotypic P2RY12 deficient) mice, we showed that female microglia have a greater expression of P2RY12, which is influenced by hormonal fluctuations during estrous cycle. We further demonstrate that a genetic deletion of P2RY12 results in sex-specific cellular perturbations with microglia and neurons in females more significantly affected. Specifically, female microglia were more de-ramified than their male counterparts in the cortex, hippocampal CA1, and the dorsal striatum, accompanied by concomitant alterations in spine density. Our bulk RNA sequencing of P2RY12-deficient male and female microglia revealed profound alterations in the transcriptome of male microglia. Together, these data indicate that female microglia may be more vulnerable morphologically, yet more resilient at the transcriptomic level, while their male counterparts are more susceptible at the transcriptomic level. Additionally, our findings suggest that P2RY12 may play critical roles in pathways that are differentially regulated in males and females.
Lastly, our behavioral analysis of P2RY12 deficient male and female mice reveal sexually dimorphic behavioral anomalies. Our findings reveal that P2RY12 deficient females exhibit locomotor hyperactivity and anxiety, while their male counterpart displayed social interaction deficit. Overall, our findings reveal sex-specific roles for microglial P2RY12 that could be relevant for central nervous system disorders with a sex bias.