Detection of Synaptic Proteins in Microglia by Flow Cytometry.

A growing body of evidence indicates that microglia actively remove synapses in vivo, thereby playing a key role in synaptic refinement and modulation of brain connectivity. This phenomenon was mainly investigated in immunofluorescence staining and confocal microscopy. However, a quantification of synaptic material in microglia using these techniques is extremely time-consuming and labor-intensive. To address this issue, we aimed to quantify synaptic proteins in microglia using flow cytometry. With this approach, we first showed that microglia from the healthy adult mouse brain contain a detectable level of VGLUT1 protein. Next, we found more than two-fold increased VGLUT1 immunoreactivity in microglia from the developing brain (P15) as compared to adult microglia. These data indicate that microglia-mediated synaptic pruning mostly occurs during the brain developmental period. We then quantified the VGLUT1 staining in microglia in two transgenic models characterized by pathological microglia-mediated synaptic pruning. In the 5xFAD mouse model of Alzheimer's disease (AD) microglia exhibited a significant increase in VGLUT1 immunoreactivity before the onset of amyloid pathology. Moreover, conditional deletion of TDP-43 in microglia, which causes a hyper-phagocytic phenotype associated with synaptic loss, also resulted in increased VGLUT1 immunoreactivity within microglia. This work provides a quantitative assessment of synaptic proteins in microglia, under homeostasis, and in mouse models of disease

Morphine withdrawal recruits lateral habenula cytokine signaling to reduce synaptic excitation and sociability.

The lateral habenula encodes aversive stimuli contributing to negative emotional states during drug withdrawal. Here we report that morphine withdrawal in mice leads to microglia adaptations and diminishes glutamatergic transmission onto raphe-projecting lateral habenula neurons. Chemogenetic inhibition of this circuit promotes morphine withdrawal-like social deficits. Morphine withdrawal-driven synaptic plasticity and reduced sociability require tumor necrosis factor-α (TNF-α) release and neuronal TNF receptor 1 activation. Hence, habenular cytokines control synaptic and behavioral adaptations during drug withdrawal

Analyses of circRNA expression throughout the light-dark cycle reveal a strong regulation of (Cdr1as), associated with light entrainment in the SCN

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that (Cdr1as), a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, (Cdr1as) has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that (Cdr1as) is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of (Cdr1as) in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation

Cross-talk between GABAergic postsynapse and microglia regulate synapse loss after brain ischemia.

Microglia interact with neurons to facilitate synapse plasticity; however, signal(s) contributing to microglia activation for synapse elimination in pathology are not fully understood. Here, using in vitro organotypic hippocampal slice cultures and transient middle cerebral artery occlusion (MCAO) in genetically engineered mice in vivo, we report that at 24 hours after ischemia, microglia release brain-derived neurotrophic factor (BDNF) to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the cornu ammonis 1 (CA1) in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75 neurotrophin receptor (p75 <sup>NTR</sup> ) and tropomyosin receptor kinase B (TrkB) receptors, respectively. After MCAO, we report that in the peri-infarct area and in the corresponding contralateral hemisphere, similar neuroplasticity occurs through microglia activation and gephyrin phosphorylation at serine-268 and serine-270 in vivo. Targeted deletion of the Bdnf gene in microglia or GphnS268A/S270A (phospho-null) point mutations protects against ischemic brain damage, neuroinflammation, and synapse downregulation after MCAO

Myeloid Metabolism as a New Target for Rejuvenation?-Comments on Restoring Metabolism of Myeloid Cells Reverses Cognitive Decline in Ageing. Nature. 2021 Feb;590(7844):122-128.

Research led by Katrin Andreasson suggests that fixing age-induced metabolic defects in myeloid cells would suffice to reverse cognitive impairment and to restore synaptic plasticity to the level of young subjects, at least in mice. This opens up the possibility to develop rejuvenating strategies by targeting immune dysfunction

Editorial: Assessing Microglial Function and Identity.

This work was supported by the Spanish Ministry of Science and Innovation CompetitivenessMCIN/AEI/10.13039/501100011033 (https://www.ciencia.gob.es/) and FEDER "A way to make Europe" (RTI2018-099267-B-I00 and RYC-201312817), a Tatiana Foundation Award (P-048-FTPGB 2018), and a Basque Government Department of Education project (PIBA 2020_1_0030; http://www.euskadi.eus/basque-government/department-education/) to AS; by grants from the Synapsis Foundation -Alzheimer Research Switzerland ARS, the Swiss National Science Foundation (SNSF 310030_197940) and the European Research Council (ERC StGrant REMIND 804949) to RCP

Microglia immunometabolism: From metabolic disorders to single cell metabolism.

Since the observation that obesity-associated low-grade chronic inflammation is a crucial driver for the onset of systemic metabolic disorders such as type 2 diabetes, a number of studies have highlighted the role of both the innate and the adaptive immune system in such pathologies. Moreover, researchers have recently demonstrated that immune cells can modulate their intracellular metabolic profile to control their activation and effector functions. These discoveries represent the foundations of a research area known as "immunometabolism", an emerging field of investigation that may lead to the development of new-generation therapies for the treatment of inflammatory and metabolic diseases. Most of the studies in the field have focused their attention on both circulating white blood cells and leukocytes residing within metabolic tissues such as adipose tissue, liver and pancreas. However, immunometabolism of immune cells in non-metabolic tissues, including central nervous system microglia, have long been neglected. In this review, we highlight the most recent findings suggesting that microglial cells play a central role in metabolic disorders and that interfering with the metabolic profile of microglia can modulate their functionality and pathogenicity in neurological diseases

A novel protocol to detect green fluorescent protein in unfixed, snap-frozen tissue.

The green fluorescent protein (GFP) is a powerful reporter protein that allows labeling of specific proteins or entire cells. However, as GFP is a small soluble protein, it easily crosses membranes if cell integrity is disrupted, and GFP signal is lost or diffuse if the specimen is not fixed beforehand. While pre-fixation is often feasible for histological analyses, many molecular biology procedures and new imaging techniques, such as imaging mass spectrometry, require unfixed specimens. To be able to use GFP labeling in tissues prepared for such applications, we have tested various protocols to minimize the loss of GFP signal. Here we show that, in cryocut sections of snap-frozen brain tissue from two GFP reporter mouse lines, leaking of the GFP signal is prevented by omitting the commonly performed drying of the cryosections, and by direct post-fixation with 4% paraformaldehyde pre-warmed at 30-37 °C. Although the GFP staining does not reach the same quality as obtained with pre-fixed tissue, GFP localization within the cells that express it is preserved with this method. This protocol can thus be used to identify GFP positive cells on sections originating from unfixed, cryosectioned tissue

Microglial metabolic flexibility: emerging roles for lactate.

Microglia, the resident macrophages of the central nervous system (CNS), play important functions in the healthy and diseased brain. In the emerging field of immunometabolism, progress has been made in understanding how cellular metabolism can orchestrate the key responses of tissue macrophages, such as phagocytosis and inflammation. However, very little is known about the metabolic control of microglia. Lactate, now recognized as a crucial metabolite and a central substrate in metabolic flexibility, is emerging not only as a novel bioenergetic fuel for microglial metabolism but also as a potential modulator of cellular function. Parallels with macrophages will help in understanding how microglial lactate metabolism is implicated in brain physiology and pathology, and how it could be targeted for therapeutic purposes