West Nile Virus-Induced Expression of Senescent Gene Lgals3bp Regulates Microglial Phenotype within Cerebral Cortex.

Microglia, the resident macrophages of the central nervous system, exhibit altered gene expression in response to various neurological conditions. This study investigates the relationship between West Nile Virus infection and microglial senescence, focusing on the role of LGALS3BP, a protein implicated in both antiviral responses and aging. Using spatial transcriptomics, RNA sequencing and flow cytometry, we characterized changes in microglial gene signatures in adult and aged mice following recovery from WNV encephalitis. Additionally, we analyzed Lgals3bp expression and generated Lgals3bp-deficient mice to assess the impact on neuroinflammation and microglial phenotypes. Our results show that WNV-activated microglia share transcriptional signatures with aged microglia, including upregulation of genes involved in interferon response and inflammation. Lgals3bp was broadly expressed in the CNS and robustly upregulated during WNV infection and aging. Lgals3bp-deficient mice exhibited reduced neuroinflammation, increased homeostatic microglial numbers, and altered T cell populations without differences in virologic control or survival. These data indicate that LGALS3BP has a role in regulating neuroinflammation and microglial activation and suggest that targeting LGALS3BP might provide a potential route for mitigating neuroinflammation-related cognitive decline in aging and post-viral infections.

Senescent-like microglia limit remyelination through the senescence associated secretory phenotype.

The capacity to regenerate myelin in the central nervous system (CNS) diminishes with age. This decline is particularly evident in multiple sclerosis (MS), which has been suggested to exhibit features of accelerated biological aging. Whether cellular senescence, a hallmark of aging, contributes to remyelination impairment remains unknown. Here, we show that senescent cells (SCs) accumulate within demyelinated lesions after injury, and their elimination enhances remyelination in young mice but not in aged mice. In young mice, we observed the upregulation of senescence-associated transcripts primarily in microglia after demyelination, followed by their reduction during remyelination. However, in aged mice, senescence-associated factors persisted within lesions, correlating with inefficient remyelination. We found that SC elimination enhanced remyelination in young mice but was ineffective in aged mice. Proteomic analysis of senescence-associated secretory phenotype (SASP) revealed elevated levels of CCL11/Eotaxin-1 in lesions, which was found to inhibit efficient oligodendrocyte maturation. These results suggest therapeutic targeting of SASP components, such as CCL11, may improve remyelination in aging and MS.

Synaptic injury in the inner plexiform layer of the retina is associated with progression in multiple sclerosis.

While neurodegeneration underlies the pathological basis for permanent disability in multiple sclerosis (MS), predictive biomarkers for progression are lacking. Using an animal model of chronic MS, we find that synaptic injury precedes neuronal loss and identify thinning of the inner plexiform layer (IPL) as an early feature of inflammatory demyelination-prior to symptom onset. As neuronal domains are anatomically segregated in the retina and can be monitored longitudinally, we hypothesize that thinning of the IPL could represent a biomarker for progression in MS. Leveraging our dataset with over 800 participants enrolled for more than 12 years, we find that IPL atrophy directly precedes progression and propose that synaptic loss is predictive of functional decline. Using a blood proteome-wide analysis, we demonstrate a strong correlation between demyelination, glial activation, and synapse loss independent of neuroaxonal injury. In summary, monitoring synaptic injury is a biologically relevant approach that reflects a potential driver of progression.

Microglia phagocytic mechanisms: Development informing disease.

Microglia are tissue-resident macrophages and professional phagocytes of the central nervous system (CNS). In development, microglia-mediated phagocytosis is important for sculpting the cellular architecture. This includes the engulfment of dead/dying cells, pruning extranumerary synapses and axons, and phagocytosing fragments of myelin sheaths. Intriguingly, these developmental phagocytic mechanisms by which microglia sculpt the CNS are now appreciated as important for eliminating synapses, myelin, and proteins during neurodegeneration. Here, we discuss parallels between neurodevelopment and neurodegeneration, which highlights how development is informing disease. We further discuss recent advances and challenges towards therapeutically targeting these phagocytic pathways and how we can leverage development to overcome these challenges.

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.

Microglia-astrocyte crosstalk regulates synapse remodeling via Wnt signaling.

Astrocytes and microglia are emerging key regulators of activity-dependent synapse remodeling that engulf and remove synapses in response to changes in neural activity. Yet, the degree to which these cells communicate to coordinate this process remains an open question. Here, we use whisker removal in postnatal mice to induce activity-dependent synapse removal in the barrel cortex. We show that astrocytes do not engulf synapses in this paradigm. Instead, astrocytes reduce their contact with synapses prior to microglia-mediated synapse engulfment. We further show that reduced astrocyte-contact with synapses is dependent on microglial CX3CL1-CX3CR1 signaling and release of Wnts from microglia following whisker removal. These results demonstrate an activity-dependent mechanism by which microglia instruct astrocyte-synapse interactions, which then provides a permissive environment for microglia to remove synapses. We further show that this mechanism is critical to remodel synapses in a changing sensory environment and this signaling is upregulated in several disease contexts.

Microglia: Activity-dependent regulators of neural circuits.

It has been more than a century since Pío del Río-Hortega first characterized microglia in histological stains of brain tissue. Since then, significant advances have been made in understanding the role of these resident central nervous system (CNS) macrophages. In particular, it is now known that microglia can sense neural activity and modulate neuronal circuits accordingly. We review the mechanisms by which microglia detect changes in neural activity to then modulate synapse numbers in the developing and mature CNS. This includes responses to both spontaneous and experience-driven neural activity. We further discuss activity-dependent mechanisms by which microglia regulate synaptic function and neural circuit excitability. Together, our discussion provides a comprehensive review of the activity-dependent functions of microglia within neural circuits in the healthy CNS, and highlights exciting new open questions related to understanding more fully microglia as key components and regulators of neural circuits.

Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment.

Engulfment of cellular material and proteins is a key function for microglia, a resident macrophage of the central nervous system (CNS). Among the techniques used to measure microglial engulfment, confocal light microscopy has been used the most extensively. Here, we show that autofluorescence (AF) likely due to lipofuscin (lipo-AF) and typically associated with aging, can also be detected within microglial lysosomes in the young mouse brain by light microscopy. This lipo-AF signal accumulates first within microglia and it occurs earliest in white versus gray matter. Importantly, in gray matter, lipo-AF signal can confound the interpretation of antibody-labeled synaptic material within microglia in young adult mice. We further show that there is an age-dependent accumulation of lipo-AF inside and outside of microglia, which is not affected by amyloid plaques. We finally implement a robust and cost-effective strategy to quench AF in mouse, marmoset, and human brain tissue.

A 4D transcriptomic map for the evolution of multiple sclerosis-like lesions in the marmoset brain.

Single-time-point histopathological studies on postmortem multiple sclerosis (MS) tissue fail to capture lesion evolution dynamics, posing challenges for therapy development targeting development and repair of focal inflammatory demyelination. To close this gap, we studied experimental autoimmune encephalitis (EAE) in the common marmoset, the most faithful animal model of these processes. Using MRI-informed RNA profiling, we analyzed ~600,000 single-nucleus and ~55,000 spatial transcriptomes, comparing them against EAE inoculation status, longitudinal radiological signals, and histopathological features. We categorized 5 groups of microenvironments pertinent to neural function, immune and glial responses, tissue destruction and repair, and regulatory network at brain borders. Exploring perilesional microenvironment diversity, we uncovered central roles of EAE-associated astrocytes, oligodendrocyte precursor cells, and ependyma in lesion formation and resolution. We pinpointed imaging and molecular features capturing the pathological trajectory of WM, offering potential for assessing treatment outcomes using marmoset as a platform.

A comparative analysis of microglial inducible Cre lines.

Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has particularly benefited because microglia historically have been difficult to transduce with virus or electroporation methods for gene delivery. Here, we investigate five of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency, cell-type specificity, and spontaneous recombination, depending on the Cre line and inter-loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency, particularly in microglia. There is increasing evidence that microglia are key regulators of neural circuits and major drivers of a broad range of neurological diseases. Reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field and the development of microglia-based therapeutics.

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.

Cytokines as emerging regulators of central nervous system synapses.

Cytokines are key messengers by which immune cells communicate, and they drive many physiological processes, including immune and inflammatory responses. Early discoveries demonstrated that cytokines, such as the interleukin family members and TNF-α, regulate synaptic scaling and plasticity. Still, we continue to learn more about how these traditional immune system cytokines affect neuronal structure and function. Different cytokines shape synaptic function on multiple levels ranging from fine-tuning neurotransmission, to regulating synapse number, to impacting global neuronal networks and complex behavior. These recent findings have cultivated an exciting and growing field centered on the importance of immune system cytokines for regulating synapse and neural network structure and function. Here, we highlight the latest findings related to cytokines in the central nervous system and their regulation of synapse structure and function. Moreover, we explore how these mechanisms are becoming increasingly important to consider in diseases-especially those with a large neuroinflammatory component.

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope.

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

Distinct Th17 effector cytokines differentially promote microglial and blood-brain barrier inflammatory responses during post-infectious encephalitis.

Group A Streptococcus (GAS) infections can cause neuropsychiatric sequelae in children due to post-infectious encephalitis. Multiple GAS infections induce migration of Th17 lymphocytes from the nose into the brain, which are critical for microglial activation, blood-brain barrier (BBB) and neural circuit impairment in a mouse disease model. How endothelial cells (ECs) and microglia respond to GAS infections, and which Th17-derived cytokines are essential for these responses are unknown. Using single-cell RNA sequencing and spatial transcriptomics, we found that ECs downregulate BBB genes and microglia upregulate interferon-response, chemokine and antigen-presentation genes after GAS infections. Several microglial-derived chemokines were elevated in patient sera. Administration of a neutralizing antibody against interleukin-17A (IL-17A), but not ablation of granulocyte-macrophage colony-stimulating factor (GM-CSF) in T cells, partially rescued BBB dysfunction and microglial expression of chemokine genes. Thus, IL-17A is critical for neuropsychiatric sequelae of GAS infections and may be targeted to treat these disorders.

Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment.

Engulfment of cellular material and proteins is a key function for microglia, a resident macrophage of the central nervous system (CNS). Among the techniques used to measure microglial engulfment, confocal light microscopy has been used the most extensively. Here, we show that autofluorescence (AF), likely due to lipofuscin and typically associated with aging, can also be detected within microglial lysosomes in the young mouse brain by light microscopy. This lipofuscin-AF signal accumulates first within microglia and increases with age, but it is not exacerbated by amyloid beta-related neurodegeneration. We further show that this lipofuscin-AF signal within microglia can confound the interpretation of antibody-labeled synaptic material within microglia in young adult mice. Finally, we implement a robust strategy to quench AF in mouse, marmoset, and human brain tissue.

The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy.

Microglial phagocytosis of apoptotic debris prevents buildup damage of neighbor neurons and inflammatory responses. Whereas microglia are very competent phagocytes under physiological conditions, we report their dysfunction in mouse and preclinical monkey models of stroke (macaques and marmosets) by transient occlusion of the medial cerebral artery (tMCAo). By analyzing recently published bulk and single cell RNA sequencing databases, we show that the phagocytosis dysfunction was not explained by transcriptional changes. In contrast, we demonstrate that the impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrient deprivation (OND), which led to reduced process motility, lysosomal exhaustion, and the induction of a protective macroautophagy/autophagy response in microglia. Basal autophagy, in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using pharmacological and transgenic approaches. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro, where it even had a detrimental effect on microglia, suggesting that modulating microglial autophagy to optimal levels may be a hard to achieve goal. Nonetheless, our results show that pharmacological interventions, acting directly on microglia or indirectly on the brain environment, have the potential to recover phagocytosis efficiency in the diseased brain. We propose that phagocytosis is a therapeutic target yet to be explored in stroke and other brain disorders and provide evidence that it can be modulated in vivo using rapamycin.Abbreviations: AIF1/IBA1: allograft inflammatory factor 1; AMBRA1: autophagy/beclin 1 regulator 1; ATG4B: autophagy related 4B, cysteine peptidase; ATP: adenosine triphosphate; BECN1: beclin 1, autophagy related; CASP3: caspase 3; CBF: cerebral blood flow; CCA: common carotid artery; CCR2: chemokine (C-C motif) receptor 2; CIR: cranial irradiation; Csf1r/v-fms: colony stimulating factor 1 receptor; CX3CR1: chemokine (C-X3-C motif) receptor 1; DAPI: 4',6-diamidino-2-phenylindole; DG: dentate gyrus; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HI: hypoxia-ischemia; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MCA: medial cerebral artery; MTOR: mechanistic target of rapamycin kinase; OND: oxygen and nutrient deprivation; Ph/A coupling: phagocytosis-apoptosis coupling; Ph capacity: phagocytic capacity; Ph index: phagocytic index; SQSTM1: sequestosome 1; RNA-Seq: RNA sequencing; TEM: transmission electron microscopy; tMCAo: transient medial cerebral artery occlusion; ULK1: unc-51 like kinase 1.

A comparative analysis of microglial inducible Cre lines.

Cre/LoxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. The field of microglial biology has particularly benefited from this technology as microglia have historically been difficult to transduce with virus or electroporation methods for gene delivery. Here, we interrogate four of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency and spontaneous recombination, depending on the Cre line and loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency in microglia, which could be extended to other cell types. There is increasing evidence that microglia are key regulators of neural circuit structure and function. Microglia are also major drivers of a broad range of neurological diseases. Thus, reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field of microglial biology and the development of microglia-based therapeutics.

Neuronal signal-regulatory protein alpha drives microglial phagocytosis by limiting microglial interaction with CD47 in the retina.

Microglia utilize their phagocytic activity to prune redundant synapses and refine neural circuits during precise developmental periods. However, the neuronal signals that control this phagocytic clockwork remain largely undefined. Here, we show that neuronal signal-regulatory protein alpha (SIRPα) is a permissive cue for microglial phagocytosis in the developing murine retina. Removal of neuronal, but not microglial, SIRPα reduced microglial phagocytosis, increased synpase numbers, and impaired circuit function. Conversely, prolonging neuronal SIRPα expression extended developmental microglial phagocytosis. These outcomes depended on the interaction of presynaptic SIRPα with postsynaptic CD47. Global CD47 deficiency modestly increased microglial phagocytosis, while CD47 overexpression reduced it. This effect was rescued by coexpression of neuronal SIRPα or codeletion of neuronal SIRPα and CD47. These data indicate that neuronal SIRPα regulates microglial phagocytosis by limiting microglial SIRPα access to neuronal CD47. This discovery may aid our understanding of synapse loss in neurological diseases.

Microglia are SYK of Aß and cell debris.

During neurodegenerative disease, resident CNS macrophages termed "microglia" assume a neuroprotective role and engulf toxic protein aggregates and cell debris. In this issue of Cell, two groups independently show how spleen tyrosine kinase (SYK) acts downstream of microglial surface receptors to propagate this neuroprotective program in vivo.