Mouse microglia express unique miRNA-mRNA networks to facilitate age-specific functions in the developing central nervous system

ARTICLE https://doi.org/10.1038/s42003-023-04926-8 OPEN Mouse microglia express unique miRNA-mRNA networks to facilitate age-specific functions in the developing central nervous system 1234567890():,; Alexander D. Walsh1,6, Sarrabeth Stone1, Saskia Freytag2,3, Andrea Aprico1, Trevor J. Kilpatrick1, Brendan R. E. Ansell 3,4,7 & Michele D. Binder 4,5,7 ✉ Microglia regulate multiple processes in the central nervous system, exhibiting a considerable level of cellular plasticity which is facilitated by an equally dynamic transcriptional environment. While many gene networks that regulate microglial functions have been characterised, the influence of epigenetic regulators such as small non-coding microRNAs (miRNAs) is less well defined. We have sequenced the miRNAome and mRNAome of mouse microglia during brain development and adult homeostasis, identifying unique profiles of known and novel miRNAs. Microglia express both a consistently enriched miRNA signature as well as temporally distinctive subsets of miRNAs. We generated robust miRNA-mRNA networks related to fundamental developmental processes, in addition to networks associated with immune function and dysregulated disease states. There was no apparent influence of sex on miRNA expression. This study reveals a unique developmental trajectory of miRNA expression in microglia during critical stages of CNS development, establishing miRNAs as important modulators of microglial phenotype. 1 The Florey Institute of Neuroscience and Mental Health, Parkville, Melbourne, VIC 3052, Australia. 2 Personalised Oncology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. 3 Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia. 4 Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. 5 Department of Anatomy and Physiology, University of Melbourne, Parkville, Melbourne, VIC 3052, Australia. 6Present address: Cognitive Neuroepigenetics Laboratory, Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia. 7These authors jointly supervised this work: Brendan R. E. Ansell, Michele D. Binder. ✉email: mbinder@florey.edu.au COMMUNICATIONS BIOLOGY | (2023)6:555 | https://doi.org/10.1038/s42003-023-04926-8 | www.nature.com/commsbio 1 ARTICLE M COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04926-8 icroglia are dynamic regulators of the central nervous system (CNS) where they support early development, adult homoeostasis and immune function. Following embryonic colonisation of the brain, microglia regulate neurogenesis, synaptogenesis and myelination via both the clearance (efferocytosis) of immature cells and synapses, and secretion of trophic factors1–4. In the adult brain, microglia continue to regulate existing neural networks, as well as supporting neurogenic niches and oligodendrocyte precursor cell pools5,6. Adult microglia adopt a ramified phenotype and extend processes to survey the local environment and maintain tissue homoeostasis7,8. Upon interaction with pathological stimuli, microglia can rapidly shift their phenotype to initiate apoptotic clearance and cytokine signalling to suppress inflammation and promote a neuroprotective environment9. Given the broad spectrum of phenotypes and activities of microglia in the healthy CNS, appropriate microglial function is heavily dependent upon precise regulation of gene expression. Consequently, genetic dysregulation of microglial biology is implicated in numerous neurological disorders. Chronically activated ‘neurotoxic’ microglia have been identified as a hallmark of neurodegeneration and autoimmunity, with key genes and signalling pathways explicitly linked to the pathology of multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD) and Huntington’s disease (HD)10. Perturbed microglial activity is also implicated in neurodevelopmental disorders including autism spectrum disorder (ASD), schizophrenia and epilepsy11–14. Multiple sequencing studies of microglial populations have captured genetic and epigenetic networks that regulate cell identity and define microglial phenotypes in the healthy and diseased brain15–19. In addition, comprehensive profiling has identified distinct programmes of gene expression that tightly regulate developmental phenotypes and age-specific microglial functions20,21. Pre-natal and early postnatal microglia are highly reactive and adopt an amoeboid morphology similar to that of adult-activated microglia during inflammation and disease. However, there is no strong gene expression overlap between these cell subsets, indicating that developmental microglia are a distinct cell population that uniquely contribute to CNS development. These studies highlight the multiplicity of transcriptional programmes which can be activated by microglia in response to normal development or pathological challenge. An important question then arises as to how these transcriptional programmes are controlled? One strong potential candidate is the class of small RNAs known as microRNAs (miRNAs). miRNAs are small (18–22 bp) non-coding RNAs that act as negative post-transcriptional regulators of gene expression22. Mature miRNAs are integrated into a catalytic complex that binds to target mRNAs and triggers degradation or stalled translation of the target mRNA transcript23. Ubiquitous in the mammalian genome, miRNAs regulate fundamental processes including cell differentiation, proliferation and immune homeostasis24,25. Tissue-specific sequencing of miRNAs has revealed that ubiquitously expressed miRNAs are less common than previously predicted and emphasise the importance of cell-specific studies to capture accurate miRNA profiles26. Normal specification of microglia is known to be reliant upon miRNAs. Conditional knockout of Dicer in microglia ablates the miRNA biogenesis pathway, which results in perturbation of microglia and induces hyper-responsiveness to inflammatory stimuli27. In addition, specific miRNAs have a profound influence on microglial biology. For example, miR-155 and miR-124 have been identified as ‘master regulators’ of activated and quiescent microglia, respectively28–30. However, much of the evidence for miRNAmediated regulation of microglia stems from studies of adult 2 mice, and thus the role of miRNAs in regulating developmental microglial functions are not as well understood. Further, studies of the miRNAome are often limited in their ability to predict transcriptional effects, as they rely on in silico predictions of miRNA–mRNA interactions rather than integrated network analyses. In this study, we have characterised the miRNAome of microglia in male and female mice at three developmental timepoints: postnatal days 6, 15 and 8 weeks. These timepoints represent critical stages in CNS development in the mouse. We identified a unique microglial miRNA profile c (...truncated)