Research ArticleNEUROSCIENCE

Reduction of AMPA receptor activity on mature oligodendrocytes attenuates loss of myelinated axons in autoimmune neuroinflammation

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Science Advances  08 Jan 2020:
Vol. 6, no. 2, eaax5936
DOI: 10.1126/sciadv.aax5936
  • Fig. 1 The GluA4 AMPAR subunit is reduced in adult spinal cord oligodendrocytes from PLPcreER+;GluA4fl/fl compared to PLPcreER;GluA4fl/fl mice.

    Mice were tamoxifen-induced at 6 weeks of age and euthanized for immunofluorescence staining at 8 weeks of age. Representative confocal images of PLPcreER;GluA4fl/fl (WT) versus PLPcreER+;GluA4fl/fl (KO) mouse lumbar spinal cords. (A) Images taken in lateral funiculus white matter. Far-red (pseudocolored magenta) immunofluorescence represents anti-GluA4 (first column). Green immunofluorescence represents the mature oligodendrocyte marker anti-CC1 (second column). Blue fluorescence represents the nuclear marker bisbenzimide (third column). All images are merged in the final column (Merge). (B) Images taken in lateral funiculus white matter. Far-red (pseudocolored magenta) immunofluorescence represents anti-GluA4 (first column). Green immunofluorescence represents the astrocyte marker anti-GFAP (second column). All images are merged in the final column (Merge). (C) Images taken in ventral gray matter. Far-red (pseudocolored magenta) immunofluorescence represents anti-GluA4 (first column). Green immunofluorescence represents the neuronal marker NeuN (second column). Blue fluorescence represents the nuclear marker bisbenzimide (third column). All images are merged in the final column (Merge). Images are representative of n = 4 mice per genotype. Scale bars, 10 μm.

  • Fig. 2 AMPARs on oligodendrocytes in PLPcreER+;GluA4fl/fl;tdTomato+ mice are functionally impaired.

    (A) Representative images of tdTomato+ oligodendrocytes in PLPcreER+;GluA4+/+;tdTomato+ (WT) and PLPcreER+;GluA4fl/fl;tdTomato+ (KO) mouse optic nerves (left column) loaded with calcium indicator OGB-1 (middle column), and merged image (right column) during peak response to stimulation with glutamate and cyclothiazide (CTZ). Scale bars, 10 μm. (B) Representative traces of ΔF/F0 for 120 s representing relative Cai2+ concentration in tdTomato+ oligodendrocytes from PLPcreER+;GluA4+/+;tdTomato+ (WT; black circles) and PLPcreER+;GluA4fl/fl;tdTomato+ (KO; black triangles) mice treated with Glu + CTZ (solid lines), or WT (open circles) and KO (open triangles) mice treated with NBQX + Glu + CTZ (dashed lines). (C) Maximum ΔF/F0 of Cai2+ responses during treatment with Glu + CTZ or NBQX + Glu + CTZ. (D) Area under the curve (AUC) of ΔF/F0 over the first 120 s of treatment with Glu + CTZ or NBQX + Glu + CTZ. For (C) and (D), statistical differences were determined using one-way analysis of variance (ANOVA) (P < 0.0001 for each test) with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant. For (C) and (D), quantification includes n = 94 WT (Glu + CTZ) cells from three nerves, n = 88 KO (Glu + CTZ) cells from three nerves, n = 50 WT (NBQX + Glu + CTZ) cells from three nerves, and n = 86 KO (NBQX + Glu + CTZ) cells from two nerves. Mice were euthanized at P9.

  • Fig. 3 Inducible reduction of AMPAR signaling in mature myelinating oligodendrocytes attenuates EAE consistent with reduced demyelination.

    (A) EAE clinical scores (means ± SEM) of PLPcreER;GluA4fl/fl littermate controls (WT; circles; n = 20) and PLPcreER+;GluA4fl/fl (KO; triangles; n = 22) mice. Statistical differences were determined from disease onset (days 8 to 30 after EAE induction; bar above graph) using a two-tailed Mann-Whitney test, ***P < 0.0001. MBP staining of the thoracic (B) and lumbar (D) spinal cords. Quantification of OD of the thoracic (C) and lumbar (E) spinal cords. Statistical differences were determined using a two-tailed unpaired t test (P = 0.3188 for thoracic at ×5 magnification, *P = 0.0288 for lumbar at ×5 magnification, and *P = 0.0280 for lumbar at ×40 magnification). GFAP staining of thoracic (F) and lumbar (H) spinal cords. Quantification of percent area stained of the thoracic (G) and lumbar (I) spinal cords at ×5 magnification. Statistical differences were determined using a two-tailed unpaired t test (P = 0.9308 for thoracic and P = 0.1347 for lumbar). Iba1 staining of the thoracic (J) and lumbar (L) spinal cords. Quantification of percent area stained of the thoracic (K) and lumbar (M) spinal cords at ×5 magnification. Statistical differences were determined using a two-tailed unpaired t test (P = 0.7297 for thoracic and *P = 0.0275 for lumbar). Scale bars, 100 μm. All data are expressed as means ± SEM, including n = 5 to 6 WT and 7 to 8 KO mice induced with EAE and euthanized on day 35 after EAE induction, 1 to 4 spinal cords sections per mouse.

  • Fig. 4 Inducible reduction of AMPAR signaling in mature oligodendrocytes attenuates axonal loss in EAE.

    (A to C) Alpha-blended 3D representations of confocal z-stacks (112.71 μm by 112.71 μm by 9.00 μm) of spinal cord ventral funiculus from PLPcreER;GluA4fl/fl (WT; first column) and PLPcreER+;GluA4fl/fl (KO; second column) mice. Far-red (pseudocolored magenta) immunofluorescence represents anti–SMI-312 (neurofilament), and green immunofluorescence represents anti-MBP. Top-down view in (A) and (B) and angled view in (C). (D) Quantification of SMI-312+ axons per cubic millimeter (means ± SEM), n = 6 WT and 8 KO mice, 3 to 4 3D z-stacks from two sections per mouse euthanized 35 days after EAE induction. Statistical differences were determined using a two-tailed unpaired t test, *P = 0.0248.

  • Fig. 5 Inducible reduction of AMPAR signaling in oligodendrocytes attenuates axonal damage in EAE.

    (A) Anti–APP A4 staining of lumbar dorsal white matter. Scale bars, 50 μm. (B) Quantification of anti–APP A4+ puncta in dorsal white matter of lumbar spinal cords (means ± SEM), n = 6 PLPcreER;GluA4fl/fl (WT) and 8 PLPcreER+;GluA4fl/fl (KO) mice, 2 to 4 sections per mouse, euthanized 33 days after EAE induction. Statistical differences were determined using a two-tailed unpaired t test, *P = 0.0412. (C) Colocalization of anti–APP A4 with anti–SMI-312 (neurofilament) in WT mouse spinal cord white matter. Red (pseudocolored cyan) immunofluorescence represents anti–APP A4 (first column). Far-red (pseudocolored magenta) immunofluorescence represents anti–SMI-312 (second column). Anti–APP A4 and anti–SMI-312 are merged in the third column (Merge 1). Green immunofluorescence represents anti-MBP (fourth row). All images are merged in the final column (Merge 2). Images are representative of n = 3 WT mice. Scale bars, 10 μm. (D and E) Magnified images of APP+/SMI-312+ axons in the spinal cord white matter of WT mice. Scale bars, 5 μm.

  • Fig. 6 Inducible reduction of AMPAR signaling in oligodendrocytes reduces loss of myelinated axons in EAE.

    (A) Representative images of ventral funiculus in semithin lumbar spinal cord sections from PLPcreER;GluA4fl/fl (WT) and PLPcreER+;GluA4fl/fl (KO) mice stained with toluidine blue. White boxes outline inset areas. Scale bars, 10 μm. (B) Quantification of myelinated axons in toluidine blue–stained ventral funiculus of lumbar spinal cords from WT and KO mice, *P = 0.0175. (C) Quantification of total number of axons in transmission electron micrograph images of the ventral funiculus of lumbar spinal cords from naïve, WT EAE, and KO EAE mice. Naïve compared to WT EAE, ***P < 0.0001; naïve compared to KO EAE, ***P < 0.0001; WT EAE compared to KO EAE, #P = 0.0565. (D) Representative transmission electron micrograph images of ventral funiculus in ultrathin lumbar spinal cord sections from WT and KO mice. Scale bars, 2 μm. (E) Quantification of number of myelinated axons over total axons per square millimeter. Naïve compared to WT EAE, ***P < 0.0001; naïve compared to KO EAE, ***P < 0.0001; WT EAE compared to KO EAE, *P = 0.0431. (F) Quantification of percentage of myelinated axons over total axons in naïve, WT EAE, and KO EAE mice. Naïve compared to WT EAE, *P = 0.0144; naïve compared to KO EAE, P = 0.9930; WT EAE compared to KO EAE, *P = 0.0144. (G) Representative transmission electron micrograph images of ventral funiculus in ultrathin lumbar spinal cord sections from WT and KO mice. Asterisks denote unmyelinated axons. Scale bars, 2 μm. (H) Quantification of number of unmyelinated axons over total axons per square millimeter. Naïve compared to WT EAE, ***P < 0.0001; naïve compared to KO EAE, ***P < 0.0001; WT EAE compared to KO EAE, P = 0.5386. (I) Quantification of percentage of unmyelinated axons over total axons in naïve, WT EAE, and KO EAE mice. Naïve compared to WT EAE, *P = 0.0144; naïve compared to KO EAE, P = 0.9930; WT EAE compared to KO EAE, *P = 0.0144. All quantifications are expressed as means ± SEM and were performed on n = 6 naïve, 4 WT EAE, and 5 KO EAE mice, six fields per mouse, euthanized 30 days after EAE induction. Statistical differences were determined using a two-tailed unpaired t test (B) or one-way ANOVA with Holm-Sidak’s multiple comparison test (C, E, F, H, and I).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/2/eaax5936/DC1

    Fig. S1. AMPARs mediate most of the intracellular Cai2+ responses in primary cultured mature oligodendrocyte somas.

    Fig. S2. GluA4 expression is selectively reduced in CC1+ cells from PLPcreER+;GluA4fl/fl compared to PLPcreER;GluA4fl/fl mice.

    Fig. S3. AMPAR subunits GluA1-3 are not changed in adult spinal cord oligodendrocytes from PLPcreER+;GluA4fl/fl compared to PLPcreER;GluA4fl/fl mice.

    Fig. S4. GluA4 transcript expression is reduced in PLPcreER+;GluA4fl/fl mouse white matter.

    Fig. S5. Myelin is unaltered in naïve PLPCreER+:GluA4fl/fl mice.

    Fig. S6. Frequencies of myelin g-ratio and axon diameter are unchanged in naïve PLPCreER+:GluA4fl/fl mice.

    Fig. S7. TdTomato expression is predominately limited to oligodendrocytes in EAE PLPcreER+;tdTomato+ mice.

    Fig. S8. The GluA4 AMPAR subunit remains reduced in oligodendrocytes from PLPcreER+;GluA4fl/fl mice subjected to EAE.

    Movie S1. 3D representation of myelin and axons from a PLPcreER;GluA4fl/fl (WT) mouse.

    Movie S2. 3D representation of myelin and axons from a PLPcreER+;GluA4fl/fl (KO) mouse.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. AMPARs mediate most of the intracellular Cai2+ responses in primary cultured mature oligodendrocyte somas.
    • Fig. S2. GluA4 expression is selectively reduced in CC1+ cells from PLPcreER+;GluA4fl/fl compared to PLPcreER;GluA4fl/fl mice.
    • Fig. S3. AMPAR subunits GluA1-3 are not changed in adult spinal cord oligodendrocytes from PLPcreER+;GluA4fl/fl compared to PLPcreER;GluA4fl/fl mice.
    • Fig. S4. GluA4 transcript expression is reduced in PLPcreER+;GluA4fl/fl mouse white matter.
    • Fig. S5. Myelin is unaltered in naïve PLPCreER+:GluA4fl/fl mice.
    • Fig. S6. Frequencies of myelin g-ratio and axon diameter are unchanged in naïve PLPCreER+:GluA4fl/fl mice.
    • Fig. S7. TdTomato expression is predominately limited to oligodendrocytes in EAE PLPcreER+;tdTomato+ mice.
    • Fig. S8. The GluA4 AMPAR subunit remains reduced in oligodendrocytes from PLPcreER+;GluA4fl/fl mice subjected to AE.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). 3D representation of myelin and axons from a PLPcreER;GluA4fl/fl (WT) mouse.
    • Movie S2 (.mp4 format). 3D representation of myelin and axons from a PLPcreER+;GluA4fl/fl (KO) mouse.

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