An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Schafer, Dorothy P.; Lehrman, Emily K.; Heller, Christopher T.; Stevens, Beth

doi:10.3791/51482-v

Cited by 11 publications

(15 citation statements)

References 21 publications

(31 reference statements)

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“…For microglia counts, images were processed with the ImageJ software. For microglial morphology and engulfed PSD-95 puncta analysis, 3D reconstruction images were processed as described previously (Schafer et al, 2014) using the Imaris 9.2.1 software (Bitplane).…”

Section: Immunostainingmentioning

confidence: 99%

Microglial Tmem59 Deficiency Impairs Phagocytosis of Synapse and Leads to Autism-Like Behaviors in Mice

et al. 2022

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Synaptic abnormality is an important pathologic feature of autism spectrum disorders (ASDs) and responsible for various behavioral defects in these neurodevelopmental disorders. Microglia are the major immune cells in the brain and also play an important role in synapse refinement. Although dysregulated synaptic pruning by microglia during the brain development has been associated with ASDs, the underlying mechanism has yet to be fully elucidated. Herein, we observed that expression of Transmembrane protein 59 (TMEM59), a protein recently shown to regulate microglial function, was decreased in autistic patients. Furthermore, we found that both male and female mice with either complete or microglia-specific loss of Tmem59 developed ASD-like behaviors. Microglial TMEM59-deficient mice also exhibited enhanced excitatory synaptic transmission, increased dendritic spine density, and elevated levels of excitatory synaptic proteins in synaptosomes. TMEM59-deficient microglia had impaired capacity for synapse engulfment both in vivo and in vitro. Moreover, we demonstrated that TMEM59 interacted with the C1q receptor CD93 and TMEM59 deficiency promoted CD93 protein degradation in microglia. Downregulation of CD93 in microglia also impaired synapse engulfment. These findings identify a crucial role of TMEM59 in modulating microglial function on synapse refinement during brain development and suggest that TMEM59 deficiency may contribute to ASDs through disrupting phagocytosis of excitatory synapse and thus distorting the excitatory-inhibitory (E/I) neuronal activity balance.

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Section: Immunostainingmentioning

confidence: 99%

Microglial Tmem59 Deficiency Impairs Phagocytosis of Synapse and Leads to Autism-Like Behaviors in Mice

et al. 2022

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“…Some z‐stacks also had the “De‐speckle” and/or “Remove Outliers” function applied (choice guided by the type of background noise present). To facilitate microglia surface rendering (Schafer et al, 2012, 2014), a mean filter (size of 1.5) was additionally applied to Iba‐1 channels. Preprocessed images were transferred from FIJI to Imaris (Oxford Instruments, UK) for quantitative analysis using the “Image from FIJI” function of the ImarisXT Imaris‐ImageJ/FIJI Bridge.…”

Section: Methodsmentioning

confidence: 99%

An early enriched experience drives targeted microglial engulfment of miswired neural circuitry during a restricted postnatal period

Rogerson‐Wood,

Goldsbury,

Sawatari

et al. 2024

Glia

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Brain function is critically dependent on correct circuit assembly. Microglia are well‐known for their important roles in immunological defense and neural plasticity, but whether they can also mediate experience‐induced correction of miswired circuitry is unclear. Ten‐m3 knockout (KO) mice display a pronounced and stereotyped visuotopic mismapping of ipsilateral retinal inputs in their visual thalamus, providing a useful model to probe circuit correction mechanisms. Environmental enrichment (EE) commenced around birth, but not later in life, can drive a partial correction of the most mismapped retinal inputs in Ten‐m3 KO mice. Here, we assess whether enrichment unlocks the capacity for microglia to selectively engulf and remove miswired circuitry, and the timing of this effect. Expression of the microglial‐associated lysosomal protein CD68 showed a clear enrichment‐driven, spatially restricted change which had not commenced at postnatal day (P)18, was evident at P21, more robust at P25, and had ceased by P30. This was observed specifically at the corrective pruning site and was absent at a control site. An engulfment assay at the corrective pruning site in P25 mice showed EE‐driven microglial‐uptake of the mismapped axon terminals. This was temporally and spatially specific, as no enrichment‐driven microglial engulfment was seen in P18 KO mice, nor the control locus. The timecourse of the EE‐driven corrective pruning as determined anatomically, aligned with this pattern of microglia reactivity and engulfment. Collectively, these findings show experience can drive targeted microglial engulfment of miswired neural circuitry during a restricted postnatal window. This may have important therapeutic implications for neurodevelopmental conditions involving aberrant neural connectivity.

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“…For quantification of myelin debris engulfment in microglia, images were acquired with an FV1200 laser scanning confocal microscope and analyzed by IMARIS (Bitplane, Switzerland) as previously described [ 20 , 21 ]. CD68 and Iba1 volumes were quantified by applying 3D surface rendering of confocal z-stacks in their respective channels.…”

Section: Methodsmentioning

confidence: 99%

TREM2 deficiency inhibits microglial activation and aggravates demyelinating injury in neuromyelitis optica spectrum disorder

You

Chen

Tang

et al. 2023

J Neuroinflammation

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Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disorder of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are activated and play a pivotal role in response to tissue injury. Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed by microglia and promotes microglial activation, survival and phagocytosis. Here, we identify a critical role for TREM2 in microglial activation and function during AQP4-IgG and complement-induced demyelination. TREM2-deficient mice had more severe tissue damage and neurological impairment, as well as fewer oligodendrocytes with suppressed proliferation and maturation. The number of microglia clustering in NMOSD lesions and their proliferation were reduced in TREM2-deficient mice. Moreover, morphology analysis and expression of classic markers showed compromised activation of microglia in TREM2-deficient mice, which was accompanied by suppressed phagocytosis and degradation of myelin debris by microglia. These results overall indicate that TREM2 is a key regulator of microglial activation and exert neuroprotective effects in NMOSD demyelination. Graphical Abstract

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An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Cited by 11 publications

References 21 publications

Microglial Tmem59 Deficiency Impairs Phagocytosis of Synapse and Leads to Autism-Like Behaviors in Mice

Microglial Tmem59 Deficiency Impairs Phagocytosis of Synapse and Leads to Autism-Like Behaviors in Mice

An early enriched experience drives targeted microglial engulfment of miswired neural circuitry during a restricted postnatal period

TREM2 deficiency inhibits microglial activation and aggravates demyelinating injury in neuromyelitis optica spectrum disorder

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