Gonadal hormones impart male-biased behavioral vulnerabilities to immune activation via microglial mitochondrial function.

There is a strong male bias in the prevalence of many neurodevelopmental disorders such as autism spectrum disorder. However, the mechanisms underlying this sex bias remain elusive. Infection during the perinatal period is associated with an increased risk of neurodevelopmental disorder development. Here, we used a mouse model of early-life immune activation that reliably induces deficits in social behaviors only in males. We demonstrate that male-biased alterations in social behavior are dependent upon microglial immune signaling and are coupled to alterations in mitochondrial morphology, gene expression, and function specifically within microglia, the innate immune cells of the brain. Additionally, we show that this behavioral and microglial mitochondrial vulnerability to early-life immune activation is programmed by the male-typical perinatal gonadal hormone surge. These findings demonstrate that social behavior in males over the lifespan are regulated by microglia-specific mechanisms that are shaped by events that occur in early development.

Microglia and sexual differentiation of the developing brain: A focus on ontogeny and intrinsic factors.

Sexual differentiation of the brain during early development likely underlies the strong sex biases prevalent in many neurological conditions. Mounting evidence indicates that microglia, the innate immune cells of the central nervous system, are intricately involved in these sex-specific processes of differentiation. In this review, we synthesize literature demonstrating sex differences in microglial number, morphology, transcriptional state, and functionality throughout spatiotemporal development as well as highlight current literature regarding ontogeny of microglia. Along with vanRyzin et al. in this issue, we explore the idea that differences in microglia imparted by chromosomal or ontogeny-related programming can influence microglial-driven sexual differentiation of the brain, as well as the idea that extrinsic differences in the male and female brain microenvironment may in turn impart sex differences in microglia.

Neuron Derived Cytokine Interleukin-34 Controls Developmental Microglia Function.

Neuron-microglia interactions dictate the development of neuronal circuits in the brain. However, the factors that support and broadly regulate these processes across developmental stages are largely unknown. Here, we find that IL34, a neuron-derived cytokine, is upregulated in development and plays a critical role in supporting and maintaining neuroprotective, mature microglia in the anterior cingulate cortex (ACC) of mice. We show that IL34 mRNA and protein is upregulated in neurons in the second week of postnatal life and that this increase coincides with increases in microglia number and expression of mature, homeostatic markers, e.g., TMEM119. We also found that IL34 mRNA is higher in more active neurons, and higher in excitatory (compared to inhibitory) neurons. Genetic KO of IL34 prevents the functional maturation of microglia and results in an anxiolytic phenotype in these mice by adulthood. Acute, low dose blocking of IL34 at postnatal day (P)15 in mice decreased microglial TMEM119 expression and increased aberrant microglial phagocytosis of thalamocortical synapses within the ACC. In contrast, viral overexpression of IL34 early in life (P1-P8) caused early maturation of microglia and prevented microglial phagocytosis of thalamocortical synapses during the appropriate neurodevelopmental refinement window. Taken together, these findings establish IL34 as a key regulator of neuron-microglia crosstalk in postnatal brain development, controlling both microglial maturation and synapse engulfment.

Hofbauer cells and fetal brain microglia share transcriptional profiles and responses to maternal diet-induced obesity.

Maternal immune activation is associated with adverse offspring neurodevelopmental outcomes, many mediated by in utero microglial programming. As microglia remain inaccessible throughout development, identification of noninvasive biomarkers reflecting fetal brain microglial programming could permit screening and intervention. We used lineage tracing to demonstrate the shared ontogeny between fetal brain macrophages (microglia) and fetal placental macrophages (Hofbauer cells) in a mouse model of maternal diet-induced obesity, and single-cell RNA-seq to demonstrate shared transcriptional programs. Comparison with human datasets demonstrated conservation of placental resident macrophage signatures between mice and humans. Single-cell RNA-seq identified common alterations in fetal microglial and Hofbauer cell gene expression induced by maternal obesity, as well as sex differences in these alterations. We propose that Hofbauer cells, which are easily accessible at birth, provide insights into fetal brain microglial programs and may facilitate the early identification of offspring vulnerable to neurodevelopmental disorders.

Hofbauer cells and fetal brain microglia share transcriptional profiles and responses to maternal diet-induced obesity.

Maternal immune activation is associated with adverse offspring neurodevelopmental outcomes, many mediated by in utero microglial programming. As microglia remain inaccessible throughout development, identification of noninvasive biomarkers reflecting fetal brain microglial programming could permit screening and intervention. We used lineage tracing to demonstrate the shared ontogeny between fetal brain macrophages (microglia) and fetal placental macrophages (Hofbauer cells) in a mouse model of maternal diet-induced obesity, and single-cell RNA-seq to demonstrate shared transcriptional programs. Comparison with human datasets demonstrated conservation of placental resident macrophage signatures between mice and humans. Single-cell RNA-seq identified common alterations in fetal microglial and Hofbauer cell gene expression induced by maternal obesity, as well as sex differences in these alterations. We propose that Hofbauer cells, which are easily accessible at birth, provide novel insights into fetal brain microglial programs, and may facilitate the early identification of offspring vulnerable to neurodevelopmental disorders in the setting of maternal exposures.

Protocol to measure endotoxin from opaque tissues in mice using an optimized kinetic limulus amebocyte lysate assay.

Endotoxin accumulation has been widely noted in several pathologies ranging from metabolic dysregulation to bacterial infection. Using limulus amebocyte lysate (LAL) assays to detect endotoxin load has been the only reliable way to assess endotoxin accumulation, but assays optimized for detection in opaque tissues are still lacking. We optimized a sensitive Kinetic LAL assay for endotoxin detection from murine tissues. In this protocol, we describe tissue collection and homogenization, followed by the procedure to run the assay and data analysis. For complete details on the use and execution of this protocol, please refer to Ceasrine et al. (2022).

Dietary fat: a potent microglial influencer.

Poor nutrition, lack of exercise, and genetic predisposition all contribute to the growing epidemic of obesity. Overweight/obesity create an environment of chronic inflammation that leads to negative physiological and neurological outcomes, such as diabetes, cardiovascular disease, and anxiety/depression. While the whole body contributes to metabolic homeostasis, the neuroimmune system has recently emerged as a key regulator of metabolism. Microglia, the resident immune cells of the brain, respond both directly and indirectly to dietary fat, and the environment in which microglia develop contributes to their responsiveness later in life. Thus, high maternal weight during pregnancy may have consequences for microglial function in offspring. Here, we discuss the most recent findings on microglia signaling in overweight/obesity with a focus on perinatal programming.

Maternal diet disrupts the placenta-brain axis in a sex-specific manner.

High maternal weight is associated with detrimental outcomes in offspring, including increased susceptibility to neurological disorders such as anxiety, depression and communicative disorders. Despite widespread acknowledgement of sex biases in the development of these disorders, few studies have investigated potential sex-biased mechanisms underlying disorder susceptibility. Here, we show that a maternal high-fat diet causes endotoxin accumulation in fetal tissue, and subsequent perinatal inflammation contributes to sex-specific behavioural outcomes in offspring. In male offspring exposed to a maternal high-fat diet, increased macrophage Toll-like receptor 4 signalling results in excess microglial phagocytosis of serotonin (5-HT) neurons in the developing dorsal raphe nucleus, decreasing 5-HT bioavailability in the fetal and adult brains. Bulk sequencing from a large cohort of matched first-trimester human samples reveals sex-specific transcriptome-wide changes in placental and brain tissue in response to maternal triglyceride accumulation (a proxy for dietary fat content). Further, fetal brain 5-HT levels decrease as placental triglycerides increase in male mice and male human samples. These findings uncover a microglia-dependent mechanism through which maternal diet can impact offspring susceptibility for neuropsychiatric disorder development in a sex-specific manner.

Prenatal environmental stressors impair postnatal microglia function and adult behavior in males.

Gestational exposure to environmental toxins and socioeconomic stressors is epidemiologically linked to neurodevelopmental disorders with strong male bias, such as autism. We model these prenatal risk factors in mice by co-exposing pregnant dams to an environmental pollutant and limited-resource stress, which robustly activates the maternal immune system. Only male offspring display long-lasting behavioral abnormalities and alterations in the activity of brain networks encoding social interactions. Cellularly, prenatal stressors diminish microglial function within the anterior cingulate cortex, a central node of the social coding network, in males during early postnatal development. Precise inhibition of microglial phagocytosis within the anterior cingulate cortex (ACC) of wild-type (WT) mice during the same critical period mimics the impact of prenatal stressors on a male-specific behavior, indicating that environmental stressors alter neural circuit formation in males via impairing microglia function during development.

Primetime for microglia: When stress and infection collide.

Inflammation during critical windows of development contributes to behavioral affect later in life. In this of Neuron, Cao et al. (2021) demonstrate a novel mechanism through which early life Tlr4-dependent inflammation in microglia permanently alters neuronal function and leaves male mice susceptible to stress-induced depressive-like behaviors.

Tamoxifen Improves Glucose Tolerance in a Delivery-, Sex-, and Strain-Dependent Manner in Mice.

Tamoxifen, a selective estrogen-receptor modulator, is widely used in mouse models to temporally control gene expression but is also known to affect body composition. We report that tamoxifen has significant and sustained effects on glucose tolerance, independent of effects on insulin sensitivity, in mice. IP, but not oral, tamoxifen delivery improved glucose tolerance in three inbred mouse strains. The extent and persistence of tamoxifen-induced effects were sex and strain dependent. These findings highlight the need to revise commonly used tamoxifen-based protocols for gene manipulation in mice by including longer chase periods after injection, oral delivery, and the use of tamoxifen-treated littermate controls.

Adrb2 controls glucose homeostasis by developmental regulation of pancreatic islet vasculature.

A better understanding of processes controlling the development and function of pancreatic islets is critical for diabetes prevention and treatment. Here, we reveal a previously unappreciated function for pancreatic ß2-adrenergic receptors (Adrb2) in controlling glucose homeostasis by restricting islet vascular growth during development. Pancreas-specific deletion of Adrb2 results in glucose intolerance and impaired insulin secretion in mice, and unexpectedly, specifically in females. The metabolic phenotypes were recapitulated by Adrb2 deletion from neonatal, but not adult, ß-cells. Mechanistically, Adrb2 loss increases production of Vascular Endothelial Growth Factor-A (VEGF-A) in female neonatal ß-cells and results in hyper-vascularized islets during development, which in turn, disrupts insulin production and exocytosis. Neonatal correction of islet hyper-vascularization, via VEGF-A receptor blockade, fully rescues functional deficits in glucose homeostasis in adult mutant mice. These findings uncover a regulatory pathway that functions in a sex-specific manner to control glucose metabolism by restraining excessive vascular growth during islet development.

Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake.

Improved understanding of lipoproteins, particles that transport lipids throughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome. Apolipoproteins are a key component of lipoproteins. Apolipoproteins are proteins that structure lipoproteins and regulate lipid metabolism through control of cellular lipid exchange. Constraints of cell culture and mouse models mean that there is a need for a complementary model that can replicate the complex in vivo milieu that regulates apolipoprotein and lipoprotein biology. Here, we further establish the utility of the genetically tractable and optically clear larval zebrafish as a model of apolipoprotein biology. Gene ancestry analyses were implemented to determine the closest human orthologs of the zebrafish apolipoprotein A-I (apoA-I), apoB, apoE and apoA-IV genes and therefore ensure that they have been correctly named. Their expression patterns throughout development were also analyzed, by whole-mount mRNA in situ hybridization (ISH). The ISH results emphasized the importance of apolipoproteins in transporting yolk and dietary lipids: mRNA expression of all apolipoproteins was observed in the yolk syncytial layer, and intestinal and liver expression was observed from 4-6 days post-fertilization (dpf). Furthermore, real-time PCR confirmed that transcription of three of the four zebrafish apoA-IV genes was increased 4 hours after the onset of a 1-hour high-fat feed. Therefore, we tested the hypothesis that zebrafish ApoA-IV performs a conserved role to that in rat in the regulation of food intake by transiently overexpressing ApoA-IVb.1 in transgenic larvae and quantifying ingestion of co-fed fluorescently labeled fatty acid during a high-fat meal as an indicator of food intake. Indeed, ApoA-IVb.1 overexpression decreased food intake by approximately one-third. This study comprehensively describes the expression and function of eleven zebrafish apolipoproteins and serves as a springboard for future investigations to elucidate their roles in development and disease in the larval zebrafish model.