Journal: Cell Reports Medicine

Article Title: Synaptic injury in the inner plexiform layer of the retina is associated with progression in multiple sclerosis

doi: 10.1016/j.xcrm.2024.101490

Figure Lengend Snippet:

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-Bassoon (Enzo Lifesciences, ADI-VAM-PS003-F, 1:500), rabbit pAb Homer1 (Synaptic Systems, 160003, 1:1000), and guinea pig pAb α-VGluT1 (Sigma, AB5905, 1:2000).

Techniques: Recombinant, Software

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 1. Expression of LRP6-Val induces synaptic defects in cultured neurons. (A) Diagram of canonical Wnt signaling. Left: In the absence of Wnt, β-catenin is se- questered and degraded by the destruction complex preventing transcription of Wnt target genes. Right: Wnt, LRP6, and Fz receptors form a complex. Activation of the pathway results in disheveled (Dvl) recruitment to the plasma membrane and disassembly of the destruction complex. β-Catenin accumulates and translocates to the nucleus enabling Wnt target gene transcription. (B) Schematic of LRP6 showing the location of the Lrp6-val SNP (red asterisk and arrow) and the areas to which the Wnt antagonist Dkk1 and Wnt ligands bind. (C) Confocal images of vGlut1 (red) puncta on isolated axons of neurons expressing enhanced green fluorescent protein (EGFP)– actin alone or EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar, 5 μm. (D) WT LRP6 promoted the assembly of presynaptic sites but LRP6-Val did not. N = 4 independent cultures, 10 to 12 axons per culture. Kruskal-Wallis with Dunn’s post hoc test. (E) Top: Confocal images of Homer1 (red) and GFP (green) of neurons ex- pressing EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar, 21 μm. Bottom: Higher-magnification images show dendritic spines (GFP; green) and Homer1 (red) puncta along dendrites. Scale bar, 5 μm. (F) Left: Expression of LRP6-Val reduced spine density. Three independent cultures, 8 to 10 cells per culture. One-way analysis of variance (ANOVA) with Tukey’s post hoc test. Right: Expression of LRP6-Val failed to increase spine size. N = 3 independent cultures, 8 to 10 cells per culture. Kruskal- Wallis with Dunn’s post hoc. (G) LRP6-Val expression led to smaller and fewer Homer1 puncta. N = 3 independent cultures, 8 to 10 images per culture. One-way ANOVA with Tukey’s post hoc test. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques: Expressing, Cell Culture, Activation Assay, Clinical Proteomics, Membrane, Isolation

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 2. Lrp6-val mice exhibit synaptic defects at 7 to 9 months. (A) Sanger trace examples of WT, Lrp6-val heterozygous (Lrp6-val het), and Lrp6-val homozygous (Lrp6- val hom) KI mice. (B) Dendritic spines were analyzed in Lrp6-val hom KI mice crossed to a Thy1-GFP line. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar, 100 μm. (C) Confocal images of apical dendrites of CA1 pyramidal neurons of WT and Lrp6-val mice at 7 to 9 months. Scale bar, 25 μm. Insets show spines along a dendrite. Scale bar, 3 μm. (D) Lrp6-val mice display reduced spine head width. WT, N = 3; Lrp6-val N = 4. Unpaired t test. *P < 0.05. (E) Representative paired-pulse recordings of synaptic currents from WT and Lrp6-val brain slices at different ISIs. (F) Graph displays the mean PPR from all recorded cells. Lrp6-val increased the PPR at 50-ms ISIs. N = 11 to 21 cells recorded from four to five animals per genotype. Repeated measures one-way ANOVA with Tukey’s post hoc test. *P < 0.05. Data are represented as means ± SEM.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques:

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 3. Lrp6-val mice display postsynaptic defects and impaired basal synaptic transmission, synaptic vesicle release, and RRP size at 12 months. (A) Top: Dendritic spines were analyzed at 12 to 14 months in Lrp6-val KI mice crossed to a Thy1-GFP line. Scale bar, 25 μm. Bottom: Confocal images of regions of interest containing apical dendrites of CA1 pyramidal neurons in WT and Lrp6-val mice. Scale bar, 3 μm. (B) Lrp6-val mice have smaller and fewer spines. WT, N = 4; Lrp6- val, N = 4. Unpaired t test. (C) Representative traces of EPSCs elicited at increasing stimulation voltages with an average of three responses for each stimulus voltage. (D) I/O curves showing a significant reduction in EPSC amplitude in hippocampal slices from Lrp6-val mice. N = 12 to 13 cells from four animals per genotype. Repeated measure one-way ANOVA with Tukey’s post hoc test. (E) Representative traces of paired pulse evoked EPSCs at different ISIs using brain slices from WT and Lrp6-val mice. (F) Graph displays the mean PPR from all cells. Lrp6-val mice display increased PPR at 50- and 100-ms ISI. N = 13 to 14 cells from four animals per genotype. Repeated measure one-way ANOVA with Tukey’s post hoc test. (G) Representative traces of EPSCs elicited by a 20-Hz electrical stimulation for 3 s recorded from WT and Lrp6-val mice. (H) Graph showing reduced mean cumulative charge in Lrp6-val mice. N = 12 cells from four animals per genotype. Repeated measure one-way ANOVAwith Tukey’s post hoc test. (I) Graph displays the RRP size, obtained from all cells. Lrp6-val mice exhibited a reduced RRP. N = 12 cells from four animals per genotype. Unpaired Student’s t test. Data are represented as means ± SEM. *P < 0.05 and ***P < 0.001.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques: Transmission Assay

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 4. Presynaptic defects of Lrp6-val mice at 12 to 14 months. (A) Confocal images of vGlut1-labeled excitatory presynaptic terminals in the CA1 SR area of WT and Lrp6-val mice at 12 to 14 months. Scale bar, 2 μm. (B) Lrp6-val mice had fewer and smaller vGlut1 puncta. WT, N = 10; Lrp6-val, N = 9. Unpaired t test. *P < 0.05 and **P < 0.01. (C) EM images of an excitatory synapse of 12- to 14-month-old WT and Lrp6-val mice. Scale bar, 100 nm. (D) Lrp6-val mice had fewer synaptic vesicles, but no changes in PSD length were observed. N = 5, 19 to 25 images per animal. Mann-Whitney test. **P < 0.01. Data are represented as means ± SEM.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques: Labeling, MANN-WHITNEY

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 5. Lrp6-val mice exhibit synapse loss with age. (A, C, and E) Confocal images of the CA1 SR of WT and Lrp6-val mice labeled with Bassoon (green) and Homer1 (red) at 7 to 9 months (A), 12 months (C), and 16 to 18 months (E). Scale bars, 2.5 μm. Insets display high-magnification images of synapses. Scale bars, 2 μm. (B and D) Quantification of synapse number, based on the colocalization of pre- and postsynaptic puncta, showed no differences between WT and Lrp6-val mice at 7 to 9 months (B) or 12 months (D). N = 3 per genotype. Unpaired t test. (F) Synapse number was significantly reduced in Lrp6-val mice 16 to 18 months. WT, N = 8; Lrp6-val, N = 9. Unpaired t test. *P < 0.05. Data are represented as means ± SEM.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques: Labeling

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 6. Neurons from Lrp6-val mice fail to respond to Wnt7a and the presence of LRP6-Val affects the formation of the Wnt receptor complex and downstream signaling. (A) Diagram depicting hippocampal neuron isolation from WT and Lrp6-val mice. (B) Images of WT and Lrp6-val neurons treated with recombinant Wnt7a. vGlut1 (green), Homer1 (red), and MAP2 (Blue). Scale bar, 5 μm. (C) Wnt7a (200 ng/ml) increased synapse number in WT neurons but not in Lrp6-val neurons. N = 4 independent cultures. Two-way ANOVA with Games-Howell post hoc test. (D) Schematic of proximity ligation assay (PLA) to detect LRP6 and Fz5-HA interaction in close proximity (<40 nm). (E) Confocal images of HeLa cells expressing GFP (control), Fz5-HA, and WT LRP6 or LRP6-Val treated with control vehicle (Veh) or Wnt7a. GFP, green; PLA, red; and DAPI, blue. Scale bar, 10 μm. (F) The PLA signal intensity per cell was increased in cells expressing Fz5-HA and WT LRP6 or LRP6-Val compared to cells expressing GFP. N = 3 independent experiments. One-way ANOVA with Tukey’s post hoc test. (G) Wnt7a increased the PLA signal in cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6-Val and Fz5-HA. N = 3 independent experiments. Two-way ANOVA with Tukey’s post hoc test. (H) Wnt7a increased pLRP6 when normalized to total LRP6 in HeLa cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6-Val and Fz5-HA. WT LRP6 + Fz5-HA Veh, N = 50 cells; WT LRP6 + Fz5-HAWnt7a, N = 53 cells; LRP6-Val + Fz5-HAVeh, N = 71 cells; and LRP6-Val + Fz5-HAWnt7a, N = 61 cells from three independent experiments. Kruskal-Wallis with Dunn’s post hoc test. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. A.U. arbitrary units.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques: Isolation, Recombinant, Proximity Ligation Assay, Expressing, Control

Journal: Science advances

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

doi: 10.1126/sciadv.abo7421

Figure Lengend Snippet: Fig. 7. Lrp6-val exacerbates synapse loss around plaques in NL-G-F mice at 7 months. (A) Diagram shows synapse loss around an Aβ plaque (blue). (B) Confocal images of Bassoon (green) and Homer1 (red) at increasing distances from the center of an Aβ plaque (blue) in NL-G-F and NL-G-F;Lrp6-val mice or an equivalent point in WT and Lrp6-val mice, in the CA1 SR at 7 months. Scale bar, 4 μm. (C) NL-G- F mice displayed fewer synapses compared to WT mice or to Lrp6-val mice at 0 to 10 μm from the center of a plaque. Synapse number was reduced in NL-G-F;Lrp6- valmiceat 0 to 40 μm from the center of a plaque compared to NL-G-F mice. A sig- nificant reduction in synapse number was detected in NL-G-F;Lrp6-val micewhen compared to WT mice or Lrp6-val mice at all distances from the center of a plaque. WT = 17 slices from six animals, Lrp6-val = 18 slices from six animals, NL-G-F = 18 slices from seven animals, and NL-G-F;Lrp6-val = 19 slices from seven animals. Re- peated measure two-way ANOVA with Tukey’s post hoc test. *P < 0.05 and **P < 0.01. Data are represented as means ± SEM.

Article Snippet: 9, eabo7421 (2023) 13 January 2023 11 of 15 SC I ENCE ADVANCES | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 (Roche, 11867423001, RRID:AB_390918), Homer1, (Synaptic Systems, 160002, RRID:AB_2120990), Homer1, (Synaptic Systems, 160003, RRID:AB_887730), Homer1, (Synaptic Systems, 160006, RRID:AB_263122), LRP6 (Abcam, ab134146, RRID:AB_2895164), LRP6 (R&D Systems, AF1505, RRID:AB_2266025), LRP6 (Cell Signaling Technology, 2560, RRID:AB_2139329), LRP6 (Cell Signaling Technology, 3395, RRID:AB_1950408), pLRP6 (Cell Signaling Technology, 2568, RRID:AB_2139327), MAP2 (Abcam, ab5392, RRID:AB_2138153), MAP2 (Abcam, ab92434, RRID:AB_2138147), NeuN, (Cell Signaling Technology, 12943, RRID:AB_2630395), PSD-95 (Millipore, MAB1598, RRID:AB_94278), α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), vGlut1 (Millipore, AB5905, RRID:AB_2301751), and vinculin (Sigma-Aldrich, V4505, RRID:AB_477617).

Techniques:

Journal: bioRxiv

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1101/841601

Figure Lengend Snippet: ( A,B ) Coronal sections of the LGN from human controls (ctrl.) without neurological disease and from MS cases demonstrate a decrease in the density of ( Ai ) VGluT2 + -retinogeniculate presynaptic inputs (red) and ( Bi ) an increase in engulfment (arrows) by Iba1-labeled microglia/macrophages (green) in MS tissue. ( Ai ) n=5 for each condition, ( Bi ) n=3 ctrl./5 MS. Scale bars, 10 μm. ( C-F ) Representative images of the LGN from non-EAE or EAE animals with no detectable lesions (control, no lesions) and marmosets that developed EAE with demyelinating lesions in the optic nerve and tract (EAE with ON/OT lesions). ( Ci ) Similar to human tissue, there is a significant decrease in the density of VGluT2 + -inputs in the LGN from marmosets that developed ON/OT lesions following EAE compared to controls. ( Di ) Significant amounts of VGluT2 + -presynaptic RGC inputs were also detected within Iba1-labeled microglia/macrophages in the LGN of marmosets that developed ON/OT lesions following EAE compared to controls. ( D ) Inset shows phagocytic cups within Iba1 + -cell (green), which contain VGluT2 + -retinogeniculate synaptic material (red). No phagocytic cups were detected in controls. ( E,F ) VGluT1 staining reveals reduced density of ( E ) VGluT1 + -corticothalamic presynaptic inputs and ( F ) increased engulfment of VGluT1 + -synaptic material within Iba1 + -cells in the LGN of marmosets that developed ON/OT lesions following EAE. ( F ) Arrows denote engulfed VGluT1 + -corticothalamic inputs. ( C-F ) n=6. Scale bars, 10 μm. Data represent mean ± SEM, significant differences with ***P < 0.001, ****P < 0.0001, t-test. See also Fig. S1 & Table S1+2.

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669,1:100), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), rabbit pAb α-Factor H (Abcam, ab170036, 1:100), mouse mAb α-GFAP (clone clone G-A-5, Sigma, G3893, 1:500), chicken pAb α-EGFP (Abcam, ab13970, 1:500),rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019-19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8-18C5, Millipore MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6-1C9, Millipore, MAB1596, 1:100), rabbit pAb α-βIV-spectrin provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: Labeling, Staining

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: Synapse engulfment was analyzed in the LGN from the same mouse EAE tissue (A-C,F, see also Figs. 2,​,3,3, and S2,3) and the same mouse DTA tissue (D,E,G, see also Figs. 2, and S5) where synapse loss was observed. (A-C) Representative immunofluorescence images and 3D-surface rendering of P2RY12+-microglia (green) containing engulfed (A) VGluT2+-retinogeniculate inputs (red, inserts), (B) VGluT1+corticothalamic inputs (red, inserts), or PSD-95+-postsynaptic compartments (red, inserts) within CD68-labeled microglial lysosomes (blue) in the EAE model. (Ai-Ci) Quantification of presynaptic inputs within microglial lysosomes in EAE vs. CFA control mice. (D,E) Representative confocal images and 3D rendering of P2RY12 (green), CD68 (blue) and (D) VGluT2 (red) or (E) VGluT1 (red) in the DTA model. (Di-Ei) Quantification of presynaptic inputs within microglial lysosomes in the DTA model. (F) Representative images and 3D-surface rendering of ALDH1L1+-astrocytes (green) and VGluT2+-retinogeniculate inputs (red, inserts) within LAMP2-labeled lysosomes (blue) in the LGN of EAE and CFA control mice. (Fi) Quantification of VGluT2 engulfment within lysosomes of reactive astrocytes in the EAE model. (G,H) Quantification of VGluT2+-retinogeniculate inputs within LAMP2+-lysosomes of ALDH1L1-labeled astrocytes in the (G) mouse DTA model and (H) in the marmoset EAE model. (A-C,F) n=4 mice per condition from one experiment, (D,E,G) n= 6 ctrls./5 DTA mice per condition from two experiments, (H) n=6 marmosets per condition from two experiments. (A-F) Scale bars, 10 μm. Data represent mean ± SEM, significant differences with ****P< 0.0001, t-test. See also Figs. S2,3, and S2,3,5.

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: Immunofluorescence, Labeling

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: (A) Timeline of in vivo AAV-rescue experiments. (B-N) Analyses of adjacent LGN sections of onset EAE mice shown in Fig. 6. (B-D) Quantification of (B) MOG, MAG and MBP, (C) NeuN+-neuron, and (D) neurofilament+-axon density following intraocular AAV injection in the EAE model. (E-G) Quantification of (E) microglia soma size, (F) density of P2RY12+-microglia and (G) Clec7a intensity AAV-treated mice in the EAE model. Further quantification of (H) GFAP+-astrocytes, (I) CD3+-T cells and (J) CD45+-leukocytes in AAV-treated mice in the EAE model. (K) Representative images and 3D-surface rendering of P2RY12+-microglia (green) and engulfed VGluT2+-retinogeniculate inputs (red) within CD68-labeled microglial lysosomes (blue). (Ki) Quantification of engulfed VGluT2+-presynaptic inputs (red, inserts) and (L) VGluT1+-presynaptic inputs within microglial lysosomes. (M,N) Representative confocal images of the LGN of AAV-EGFP and AAV-Crry treated EAE mice immunostained against presynaptic (M) VGluT2, or (N) VGluT1 and (Mi,Ni) respective quantification. (O) Visual acuity measured as spatial frequency threshold (cycles/degree) in the optomotor test before the induction of EAE (three left columns) and at the onset of clinical symptoms (three right columns) of the same control and AAV-EGFP or AAV-Crry transduced mice as shown in B-N. (B-J) n=4, (K-N) n=5, (O) n=5–8 mice from one experiment. Scale bars, (K) 10 μm, (M,N) 5 μm. Data represent mean ± SEM, significant differences with *P<0.05, **P<0.01, ***P <0.001, ****P < 0.0001, ANOVA with Tukey’s post hoc test. See also Figs. 6, and S7.

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: In Vivo, Injection, Labeling

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: Recombinant, Electron Microscopy, Clone Assay, Plasmid Preparation, Multiplex Assay, In Situ, Sequencing, Generated, Software, Imaging

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TX, USA N/A Rabbit mAb α-CD3 (clone SP7) Abcam Cat. #ab16669 Mouse mAb α-CD8 (clone 8/114B) Thermo Fisher Scientific Cat. #MA5–13473 Rat mAb α-CD45 (clone IBL-3/16) BioRad Cat. #MCA1388 Mouse mAb α-CD68 (clone KP1) Abcam Cat. #ab955 Rat mAb α-CD68 (clone FA-11) AbD Serotec Cat. #MCA1957 Rat mAb α-Clec7a InvivoGen Cat. #mabg-mdect Rabbit pAb α-Clec12a a-Clec12a LSBio Cat. #LS-C377776 Rabbit pAb α-cleaved caspase-3 Cell Signaling Technologies Cat. #9661 Mouse mAb α-Crry (clone TLD-1C11) Santa Cruz Cat. #sc-53530 Chicken pAb α-EGFP Abcam Cat. #ab13970 Rabbit pAb α-EGFP Millipore Cat. #ab3080P Mouse mAb α-FoxP3 (clone 206D) BioLegend Cat. #320102 Goat pAb α-GAPDH Abcam Cat. #ab9483 Mouse mAb α-GFAP (clone G-A-5 Sigma Cat. #G3893 Rabbit pAb α-Homer1 Synaptic Systems Cat. #160003 Rabbit pAb α-Iba1 Wako Chemicals Cat. #019–19741 Goat pAb α-Iba1 (human/marmoset tissue) Abcam Cat. #ab5076 Rat mAb α-LAMP2 (clone GL2A7) Abcam Cat. #ab13524 Rat mAb α-Ly6C (clone HK1.4) BioLegend Cat. #128016 Mouse mAb α-MAG (clone 513) Millipore Cat. #MAB1567 Chicken pAb α-MAP2 (marmoset tissue) EnCor Biotechnology Cat. #CPCA-MAP2 Rat mAb α-MBP (clone 12) Millipore Cat. #MAB386 Mouse mAb α-MOG (clone 8–18C5 Millipore Cat. #MAB5680 Rabbit pAb α-Neurofilament 200 Sigma Cat. #N4142 Chicken pAb α-NeuN Millipore Cat. #ABN91 Rabbit pAb α-P2RY12 AnaSpec Cat. #AS-55043A Rat mAb α-P2RY12 (clone S16007D) BioLegend Cat. #848002 Mouse mAb α-PSD-95 (clone 6G6–1C9) Millipore Cat. #MAB1596 Guinea pig pAb α-RBPMS PhosphoSolutions Cat. #1832-RBPMS Rabbit pAb α-βIV-spectrin Matthew N. Rasband, Baylor College of Medicine, Houston.

Techniques: Recombinant, Electron Microscopy, Clone Assay, Plasmid Preparation, Multiplex Assay, In Situ, Sequencing, Generated, Software, Imaging