A microglial circadian clock sculpts synapses during neurodevelopment

Abstract

In a recent study, Routh and colleagues demonstrated that the circadian clock regulates microglia-directed synaptic refinement during a critical period of neurodevelopment in mice in a sex-dependent manner. Loss of the major circadian rhythm regulator Bmal1 in microglia induced a proinflammatory state and excessive pruning of synapses primarily in adolescent male mice.

During gestation, fetal circadian rhythms are highly regulated by maternal influences [1]. After parturition, the offspring establishes its own circadian rhythms, but exactly how and when this occurs in neurodevelopment is incompletely understood [1,2]. Previous research indicated that microglial function is regulated by circadian rhythms, as transcription and cellular activity fluctuate between dark and light phases [2–4]. Studies in animal models indicate that microglia play a critical role in synaptic refinement during adolescence, a process that is broadly influenced by sleep and circadian rhythms [3,5,6]. Characterizing how circadian rhythms regulate microglial function is therefore important for understanding how microglial activity is temporally organized during healthy brain development and how disrupting the molecular clock contributes to neurodevelopmental vulnerability. In a recent study, Routh and colleagues addressed a significant knowledge gap by identifying a time-of-day-dependent pattern of microglial transcriptional activity regulated by the molecular circadian clock in the developing brain [7].

Routh and colleagues utilized an inducible Tmem119-CreERT2 mouse line to knockout expression of the master circadian clock regulator Bmal1 in microglia [7]. Tamoxifen was administered beginning on postnatal day three, as previous literature indicates circadian regulation of microglial function as early as postnatal day one [2]. Early postnatal days represent a critical period during which circadian rhythms transition from alignment with maternal cues to individual offspring control [1]. Transcriptional analysis of isolated hippocampal microglia from adolescent wildtype animals revealed robust time-of-day-dependent expression differences that were largely lost in microglia from Bmal1 knockout mice, with many typically diurnal transcripts losing their rhythmicity [7]. Importantly, pathways related to synaptic development and phagocytic function exhibited time-of-day-dependent expression, suggesting that the molecular clock may influence when microglia interact with developing neural circuits. Changes in microglial transcription were accompanied by decreased object recognition and impaired social behavior in both male and female knockout animals [7]. These behavioral changes extend findings from a previous mouse model of reduced Bmal1 expression in myeloid lineage cells that resulted in dysregulated sleep behavior; a phenotype not only linked to disturbance of the circadian rhythm but also neurodevelopmental disorders (NDDs) [3,5]. Together, these findings suggest that the microglial clock helps temporally organize developmental processes such as synaptic refinement.

Routh and colleagues observed significant differences in phagocytic gene expression in isolated hippocampal microglia from adolescent mice [7], suggesting that microglial phagocytic pathways may be altered during this critical period of synaptic refinement. Histological analysis within the CA1 region revealed differences primarily in male knockout mice. Male knockout microglia displayed significantly less branching, increased synaptic phagocytosis, and fewer dendritic spines, driven by the loss of immature spine types [7]. Female knockout animals did not display robust changes in these measures. As discussed by the authors, this may reflect female-specific resilience. Alternatively, these findings may also indicate that males and females engage distinct mechanisms in response to microglial clock disruption. Together, these findings suggest that the microglial clock may influence multiple components of developmental synaptic refinement, including the transcriptional programs associated with pruning and synaptic engulfment.

Given the known circadian rhythms in hippocampal synaptic activity, Routh and colleagues next examined whether microglial Bmal1 knockout results in functional changes to hippocampal synapses using ex vivo electrophysiology. The authors identified a reduction in miniature excitatory postsynaptic current frequency, but not amplitude, in male knockout mice [7]. Stimulus-evoked excitatory postsynaptic currents were reduced in both male and female knockouts, suggesting that microglial clock disruption impacts hippocampal synaptic transmission in both sexes [7]. Thus, while structural changes were most pronounced in males, synaptic dysfunction extended to both sexes.

Bulk RNA sequencing of CA1 hippocampal tissue revealed further sex-dependent responses [7]. While convergent pathways were altered in male and female knockouts, the direction of change was often different. Knockout males showed an upregulation of pathways linked to synaptic structure and plasticity, whereas in females, non-synaptic pathways were upregulated instead [7]. These findings may explain why males exhibited more pronounced structural alterations, while females showed functional synaptic alterations in the absence of overt structural remodeling. Broadly, these findings illustrate how similar behavioral phenotypes can arise from distinct cellular and molecular mechanisms, a feature that may be particularly relevant to heterogeneous NDDs [8].

Further analysis of bulk hippocampal differentially expressed genes (DEGs) identified significant overlap between sex-dependent DEGs and NDD risk genes. Notably, DEGs that were upregulated in male knockouts tended to be downregulated in females [7]. Of note, MECP2 signaling emerged among genes shared across multiple NDD risk sets [7]. MeCP2 is a key regulator of chromatin remodeling and is essential for neurodevelopment and learning [9]. Interestingly, Mecp2 is expressed on the X chromosome, raising the possibility that sex-dependent regulation of MECP2 signaling may contribute to the sexually divergent responses to microglial clock disruption [9]. These findings further highlighted that circadian regulation of microglia may contribute to NDD risk with possible sex differences.

Previous studies have demonstrated that Bmal1 disruption in adult or aged microglia can reduce synaptic phagocytosis, whereas the study by Routh and colleagues observed increased synaptic phagocytosis in the hippocampus of adolescent male mice following microglial clock disruption [3,7]. This difference suggests that the consequences of microglial clock disruption may depend strongly on developmental state. As microglial functions change substantially across the lifespan, the same circadian machinery may regulate distinct cellular programs during development, adulthood, and aging. Therefore, circadian regulation of microglial functionality may be best understood as a dynamic interaction between the molecular clock and developmental stage.

In summary, Routh and colleagues demonstrate that hippocampal microglia exhibit time-of-day-dependent transcription during early adolescence and that disrupting the microglial molecular clock alters synaptic development and function in a sex-dependent manner. These findings extend previous work describing circadian regulation of microglial inflammatory and synaptic functions by demonstrating that the microglial clock is associated with developmental circuit refinement. Broadly, this work underscores the importance of considering how internal cellular clocks coordinate the timing of developmental processes across different cell types. Disruption of these finely-timed processes may represent an underappreciated contributor to neurodevelopmental vulnerability.