Research Article

Microglia monitor and protect neuronal function through specialized somatic purinergic junctions

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Science  31 Jan 2020:
Vol. 367, Issue 6477, pp. 528-537
DOI: 10.1126/science.aax6752
  • Fig. 1 Microglia contact specialized areas of neuronal cell bodies in the mouse and the human brain.

    (A) Single image plane (upper panel) and 3D reconstruction (lower panel) from an in vivo 2P Z-stack showing a neocortical neuron (red) being contacted by microglial processes (green). (B) In vivo 2P time-lapse imaging showing temporal dynamics of microglia–neuron contacts. (C) Analyzed trajectories of microglial processes contacting the neuron in (B). The lifetimes of somatic contacts were significantly longer than those of dendritic contacts. (D) 3D reconstruction from high-resolution CLSM Z-stack showing that microglial processes (yellow) contact GABA-releasing (red) and glutamate-releasing (cyan) boutons as well as the neuronal cell body (Kv2.1 labeling, magenta). (E) CLSM images showing P2Y12 receptor+ microglial processes (yellow) contacting an SMI32+ neuronal cell body (magenta) in human neocortex. (F) Quantitative analysis of contact prevalence between microglial processes and different neuronal elements confirming that microglia contact most neuronal cell bodies independently from neurochemical identity, whereas only a small fraction of synapses receive microglial contact. (G) CLSM image showing a neuronal cell body contacted by a microglial process at a Kv2.1 to Kv2.2 cluster. (H) The integrated fluorescent density of both Kv2.1 and Kv2.2 signal is significantly higher within the contact site than elsewhere. (I) Most microglia–neuron junctions expressed both Kv2.1 and Kv2.2 clusters. (J) Microglial processes contact Kv2.1-transfected HEK cells at the clusters, but not those transfected with a dominant-negative mutant. (K) Overlaid images showing microglial P2Y12 receptor (green for CLSM and cyan for STORM) and neuronal Kv2.1 (red for CLSM and yellow for STORM) clusters overlapping. Arrows show borders of Kv2.1 clusters. P2Y12 receptor clustering depends on the contact with neuronal cell body. Bar graphs show STORM localization point (LP) density (top) and density of identified P2Y12 receptor clusters (bottom) on different parts of microglia (for statistics, see table S1). P2Y12R, P2Y12 receptor. For statistical details, see the supplementary text for Fig. 1 and table S1.

  • Fig. 2 Microglia–neuron junctions have a specialized nanoarchitecture and molecular machinery optimized for purinergic cell-to-cell communication.

    (A) Transmission electron micrograph showing the area of the neuronal cell body (neu.) contacted by a P2Y12 receptor–immunogold (black grains)–labeled microglial process (mic.). The junction has a specific ultrastructure with closely apposed mitochondria (mito., cyan), reticular membrane structures (green), and intracellular tethers (red). A mitochondria-associated vesicle (blue, marked by white arrowhead) is also visible. The nucleus (n) of the neuron is purple. (B) A 0.5-nm-thick virtual section of an electron tomographic volume (left) and 3D model (right) showing the special nanoarchitecture of a somatic microglia–neuron junction [colors represent the same structures as in (A)]. Note the specific enrichment of P2Y12 receptor labeling at the core of the junction. (C and D) P2Y12 receptor density negatively correlates with the distance between microglial and neuronal membranes within the junctions. (E) P2Y12 receptor density is highest at those surfaces of microglial processes that are in direct contact with the neuronal cell bodies (P2Y12 receptor labeling is white; b, bouton). (F) CLSM maximal intensity projection (M.I.P.) showing microglial processes (yellow) contacting neuronal somata (magenta) with adjacent mitochondria (green). (G) Neuronal mitochondria are enriched at microglial junction sites. (H) Transmission electron micrographs showing TOM20-immunogold labeling in neocortical neurons. Immunogold labeling (black grains) is specifically associated with outer mitochondrial membranes, whereas TOM20-positive vesicles can also be observed (arrowheads). Some immunogold particles can be found on the PM of the neurons (arrows), suggesting the exocytosis of mitochondria-derived vesicles. (I) vNUT-labeled vesicles are enriched at microglial junction sites. (J) 3D reconstruction of high-resolution confocal Z-stack showing parts of two neuronal cell bodies (magenta), both contacted by microglial processes (yellow). The vNUT signal (cyan) was concentrated between the junctions and closely positioned mitochondria (green). For statistical details, see the supplementary text for Fig. 2.

  • Fig. 3 Neuronal mitochondrial activity and purinergic signaling are involved in microglia–neuron communication.

    (A) CLSM image showing a microglial process (green) contacting Kv2.1 clusters (magenta) on a neuronal soma in the vicinity of a mitochondrion (Mito-R-Geco1, red) in a perfusion-fixed brain. (B) In vivo 2P imaging of CX3CR1+/GFP mice in utero electroporated with CAG-Mito-R-Geco1 construct. Dashed line shows the outline of the neuron. Green microglial processes touch the neuronal cell body where somatic mitochondria are present. Regions of interest 1 and 2 are enlarged to show the development of somatic junctions. (C and D) Representative samples from time-lapse imaging of microglia showing processes extending and contacting neuronal soma in CX3CR1+/GFP/P2Y12 receptor+/+ (C) and CX3CR1+/GFP/P2Y12 receptor−/− (D) mice. White arrow indicates the contact site of microglia. Differential interference contrast (DIC) images of the imaged neurons and the fluorescence signal of GFP (green) and NADH (dark cyan) of red outlined areas are shown. (E) Average (and standard deviation) of NADH intrinsic fluorescence of all neurons in P2Y12 receptor+/+ (red, n = 10) and P2Y12 receptor−/− (black, n = 11) mice. (F) CLSM image showing microglial process contacting a neuronal Kv2.1 cluster with closely apposed quinacrine-labeled ATP-containing vesicle and closely localized neuronal mitochondria. Quinacrine labeling colocalizes with the vNUT signal. (G) Images from CLSM in vitro time-lapse imaging showing that quinacrine-labeled ATP-containing vesicles (green) are released (red arrows) from the neuronal cell body (white dashed outline) after KCl stimulation (M.I.P. of Z stack, 2.5 μm). (H) Number of released quinacrine-positive vesicles plotted as a function of time after KCl or vehicle treatment. (I) Size distribution of quinacrine-labeled puncta. The smaller ones (vesicles) tend to be released and the larger ones (mitochondria) are retained. (J) KCl induces a robust ATP release in cultured neurons, which could not be inhibited by a mixture of the synaptic calcium-channel blockers ω-agatoxin and ω-conotoxin (SC), but was almost completely inhibited by the L-type calcium-channel blocker nimodipine (NIM) or the vNUT inhibitor clodronate (CLO). (K) CLSM image showing robust NTPDase1 expression on microglial processes within the somatic junctions. Electron microscopic insert shows NTPDase1-labeled (dark precipitate) microglial process contacting the neuronal cell body. Neuronal mitochondria (m), vesicles, and membrane structures (white arrowheads) are closely apposed to the contact site (black arrows) where NTPDase1 is expressed on the microglial membrane. For statistical data, see the supplementary text for Fig. 3.

  • Fig. 4 Physiological microglia–neuron communication at the somatic junction site is P2Y12 receptor dependent.

    (A) Outline of acute P2Y12 receptor–blockade experiments. i.c.m., intra–cisterna magna. (B) CLSM images showing examples of the recorded microglia–neuron contacts. Empty arrowheads point to dendritic contacts and full arrowheads mark somatic junctions. (C) Acute intra–cisterna magna administration of PSB significantly reduced somatic junction lifetime, but did not affect the lifetime of dendritic microglia–neuron contacts. n.s., not significant. (D) Neuronal activity induced a robust elevation of microglial process coverage of neuronal cell bodies in CNO-treated animals but not in DREADD+/cFos cells. (E) CNO-triggered neuronal activity could not induce an elevation of microglial process coverage of neuronal cell bodies in P2Y12 receptor−/− mice. (F) Outline of combined chemogenetic and acute P2Y12 receptor–blockade experiments. (G) Acute inhibition of microglial P2Y12 receptors prevented neuronal activity–induced increase of microglial process coverage. For statistical data, see the supplementary text for Fig. 4.

  • Fig. 5 Microglia protect neurons after acute brain injury in a P2Y12 receptor–dependent manner through altered somatic junctions.

    (A) CLSM images showing that stroke induces the fragmentation of mitochondria (magenta) in neuronal cell bodies (Kv2.1 labeling, cyan) in the penumbra. Mitochondrial area and mitochondrial major axis are both significantly decreased. (B) CLSM images of cortical neurons showing that, in parallel with the declustering of Kv2.1-channels (cyan), microglial coverage (yellow) is significantly increased after stroke in the penumbra. (C) 3D reconstruction from electron tomographic volume showing elevated microglial coverage and fragmentation of neuronal mitochondria. (D) Microglial coverage of neuronal cell bodies is robustly increased after stroke, whereas acute central blockade of P2Y12 receptors or activation of KATP channels completely abolishes the stroke-induced increase of coverage. (E) Stroke induces a 1.5-fold increase in somatic microglia coverage of human cortical neurons. (F) Topographical maps showing the area of pixels with a global connectivity (GC) score < 0.6 after ischemia. The sum of outlined pixels revealed higher dropdown of GC in PSB-treated animals after stroke. (G) Left panel: Topographical maps showing increased region of interest to GC of the contralateral HLs in PSB-treated mice 120 min after stroke. Right panel: Seed-to-seed connectivity is increased in PSB-treated animals after stroke. (H) In vivo 2P calcium imaging revealing a significant increase of neuronal calcium load during reperfusion after acute P2Y12 receptor inhibition with PSB. (I) Infarct volume is increased after acute central P2Y12 receptor inhibition, which is accompanied by a significantly worse neurological outcome. For statistical data, see the supplementary text for Fig. 5.

Supplementary Materials

  • Microglia monitor and protect neuronal function via specialized somatic purinergic junctions

    Csaba Cserép, Balázs Pósfai, Nikolett Lénárt, Rebeka Fekete, Zsófia I. László, Zsolt Lele, Barbara Orsolits, Gábor Molnár, Steffanie Heindl, Anett D. Schwarcz, Katinka Ujvári, Zsuzsanna Környei, Krisztina Tóth, Eszter Szabadits, Beáta Sperlágh, Mária Baranyi, László Csiba, Tibor Hortobágyi, Zsófia Maglóczky, Bernadett Martinecz, Gábor Szabó, Ferenc Erdélyi, Róbert Szipőcs, Michael M. Tamkun, Benno Gesierich, Marco Duering, István Katona, Arthur Liesz, Gábor Tamás, Ádám Dénes

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Supplementary Text for Main Figs. 1 to 5
    • Figs. S1 to S6
    • Tables S1 to S3
    • Captions for Movies S1 to S7
    • References

    Images, Video, and Other Media

    Movie S1
    In vivo 2P time-lapse imaging shows temporal dynamics of microglia-neuron contacts. A tdTomato expressing neocortical neuron (red) is being contacted by processes of a microglial cell (green) in CX3CR1+/GFP mouse electroporated in utero with pCAG-IRES-tdTomato. The analyzed trajectories of microglial processes contacting the neuron are shown on the right panel, warm colors label trajectories of somatic contacts, while cold colors label trajectories of microglial processes contacting neuronal dendrites. The middle panel shows the trajectories overlaid on the recording.
    Movie S2
    In vitro CLSM time-lapse imaging of cocultured HEK293 and microglial cells. HEK cells were transfected with GFP-coupled control Kv2.1 construct on the left panel, and with YFP-coupled dominant-negative (DN) Kv2.1 construct on the right panel. Microglia is visualized by Alexa594-conjugated Lectin (red). Microglial processes contact Kv2.1-transfected HEK-cells at the clusters, but not those transfected with a dominant-negative mutant.
    Movie S3
    Left: stack of electron tomographic 0.5 nm thick virtual sections shows the special nanoarchitecture of a somatic microglia-neuron junction with closely apposed mitochondria, mitochondria-associated membranes (MAMs) and cytoplasmatic structures. The silverintensified P2Y12R-immunogold grains are clearly visible at the cytoplasmatic surface of microglial membrane. Note that large number of gold particles are clustered exactly where the neuronal cytoplasmatic structure is anchored. Right: 3D model of the same tomographic volume, neuronal membrane is magenta, microglial membrane is green, immunogold particles white, mitochondria light blue, MAM light green, cytoplasmatic densities red, vesicle-like structures blue and microglial reticular membrane structures darker green.
    Movie S4
    Stack of electron tomographic virtual sections shows the special nano-architecture of the core of a somatic microglia-neuron junction. The silver-intensified P2Y12R-immunogold grains are clearly visible at the cytoplasmatic surface of microglial membrane. The tethers between the anchored mitochondria and MAM are clearly visible. The neuronal cytoplasmatic structure (mitochondria, MAM) is anchored to a membrane segment that is precisely facing the high density of P2Y12Rs on the microglial membrane. Note that distance between the neuronal and microglial membrane is the smallest exactly here, and intercellular tethers are also clearly visible.
    Movie S5
    Left: stack of electron tomographic virtual sections shows the special nano-architecture of a somatic microglia-neuron junction with closely apposed mitochondria, MAMs and cytoplasmatic structures. The silver-intensified P2Y12R-immunogold grains are clearly visible at the cytoplasmatic surface of microglial membrane that touches the neuronal cell body, but are present only at a lower density at membrane segments, where the microglia touches a perisomatic bouton. Right: 3D model of the same tomographic volume, neuronal membrane is magenta, microglial membrane is ocker, immunogold particles white, mitochondria light blue, MAM light green, bouton membrane vivid green.
    Movie S6
    In vivo 2P imaging of CX3CR1+/GFP mice in utero electroporated with CAG-Mito-R-Geco1 construct. Dashed lines show the outline of a neuron, green microglial processes touch neuronal cell body where somatic mitochondria (red) are present. White arrows indicate the contact sites of microglia.
    Movie S7
    In vitro CLSM time-lapse imaging of KCl-stimulation of quinacrine-loaded cultured neuron. Left panel shows transmitted channel and superimposed green channel (quinacrine-labeled ATPcontaining vesicles), right panel shows the green channel and the outline of the neuron (white dashed line). White arrows point to vesicles that are released spontaneously, red arrows point to vesicles that are released after 40 mM KCl stimulation. MIP of z-stack (z-range: 2.5 μm), frame dimension: 12.3 x 22.2 μm.

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