Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ42 Oligomers and Protect Synapses - PubMed (original) (raw)

doi: 10.3389/fimmu.2020.00049. eCollection 2020.

Songlin Li 1 2 3, Veronica J Garcia 5 6, Dieu-Trang Fuchs 3, Julia Sheyn 3, David A Daley 3, Altan Rentsendorj 3, Tania Torbati 7, Keith L Black 3, Ueli Rutishauser 3 6 8, David B Teplow 4, Yosef Koronyo 3, Maya Koronyo-Hamaoui 3 6

Affiliations

Songlin Li et al. Front Immunol. 2020.

Abstract

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ42) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ42 oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ42 oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ42 fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ42 removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1+-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115+-monocyte-grafted APPSWE/PS1ΔE9-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ42 oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD.

Keywords: Alzheimer's disease; amyloid-beta; immunomodulation therapy; neurodegeneration; regeneration; synaptogenesis.

Copyright © 2020 Li, Hayden, Garcia, Fuchs, Sheyn, Daley, Rentsendorj, Torbati, Black, Rutishauser, Teplow, Koronyo and Koronyo-Hamaoui.

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Figures

Figure 1

Figure 1

Impact of defined and stabilized Aβ42 oligomers vs. purified fibrils on synaptic integrity in primary cortical neurons. (A) SDS-PAGE image of cross-linked (XL+) and non-XL (XL−) Aβ42 oligomers. (B) Electron microscopy (EM) of preformed fibrillar (f)Aβ42 and (C) XL-oAβ42, consisting of a defined distribution of covalently linked oligomers that were characterized by negative EM stain. Spherical (arrows) and amorphous structures were observed for XL-oAβ42, whereas typical amyloid fibrils were seen in fAβ42. (D) Representative high-resolution images of pre-VGluT1 and post-PSD95 synapses in primary cortical neurons, treated with no (–)Aβ42 (vehicle), fAβ42 or XL-oAβ42. Areas within dashed boxes are magnified below. (E,F) Quantification of pre- and postsynapses in postnatal day 1 (P1) mouse primary cortical neurons. (E) VGluT1- and PSD95-immunoreactive area per neuron and (F) colocalized VGluT1/PSD95 synaptic puncta number in P1 cortical neurons treated with vehicle, fAβ42 or XL-oAβ42. ***P < 0.001, fAβ42 vs. vehicle or XL-oAβ42 vs. vehicle; **P < 0.01, fAβ42 vs. XL-oAβ42, by one-way ANOVA and Tukey's post-test. Data expressed as mean ± s.e.m.; n = 32 fields analyzed from 2 independent experiments; Scale bars = 100 nm (B,C) and 5 μm (D).

Figure 2

Figure 2

Oligomeric Aβ42 assemblies cause neuronal hyperactivity and altered extracellular spike waveforms. Microelectrode array recordings of spontaneous activity in P1 primary cortical neurons measured 24 and 48 h after addition of vehicle, 100 nM of fAβ42, or of XL-oAβ42. (A,B) Mean neuronal frequency of active neurons showing spontaneous activity during 5 min recordings. Neurons analyzed met a minimum threshold of 0.2 Hz firing rate. 24 h, *P < 0.05, vs. Vehicle or fAβ42; 48 h, *P < 0.05, ***P < 0.001, vs. Vehicle; Kruskal-Wallis analysis by Dunn's test. (C) Comparison of spontaneous activity measured from the same individual neuron populations over time, from untreated (time 0) to 24 h incubation with vehicle, fAβ42, or XL-oAβ42. ****P < 0.0001, vs. time 0; Kruskal-Wallis analysis by Dunn's test. (D) Representative extracellular waveforms from each condition (gray = overlays of multiple waveforms from a single neuron; black = fitted mean waveform for analysis), identifying the trough-to-peak width (blue) and repolarization slope (orange). Shaded blue box indicates trough-to-peak width observed in control wave forms, dotted blue lines indicate measurement in XL-oAβ42. Orange hashed lines indicate mean repolarization slope of each wave form. (E) Quantification of trough-to-peak width in P1 cortical neurons incubated with fAβ42 or XL-oAβ42 for 48 h. **P < 0.01 vs. Vehicle 48 h. (F–G) Quantification of repolarization slope in P1 neurons in each condition. *P < 0.05 vs. Vehicle 24 and 48 h or fAβ42 24 h, respectively. one-way ANOVA and Tukey's post-test. Data expressed as mean ± s.e.m., with individual data point from 5 independent experiments.

Figure 3

Figure 3

Activated MΦ effectively protect against oligomeric Aβ42-induced synaptic and neuritic arborization loss in primary cortical neurons. (A) Schematic of the in vitro experiments (timeline in days). P1 cortical neurons (treated with 100 nM XL-oAβ42, fAβ42, or vehicle for 12 h, respectively), bone marrow-derived MΦ (MΦBM), and GA-activated MΦBM (GA-MΦ) were cultured for 9 d. (B) Representative microphotographs of P1 neurons labeled with anti-Tuj1 and -NeuN serum (left), neuritic tracings with NeuriteTracer (83) (middle), and RGB merge tracings (right). Scale bar represents 20 μm. (C) Quantification of colocalized VGluT1/PSD95 synaptic puncta number in P1 neurons incubated with fAβ42, XL-oAβ42, or vehicle, and P1 neurons co-cultured with MΦ or with GA-MΦ. Note that fAβ42 and XL-oAβ42 both reduced the VGluT1/PSD95 synaptic density that was significantly preserved by co-culturing with MΦ. This effect was enhanced by co-culturing with GA-MΦ. (D) Quantification of neuritic length of P1 neurons incubated with fAβ42, XL-oAβ42, or vehicle, and P1 neurons co-cultured with MΦ or with GA-MΦ. Note that co-culturing with GA-MΦ significantly prevented decreases in neuritic length from fAβ42 or XL-oAβ42. Data expressed as mean ± s.e.m.; n = 48 fields analyzed from 3 independent experiments; *P < 0.05, **P < 0.01, comparisons as indicated by lines; #P < 0.05, vs. fAβ42 or XL-oAβ42 alone (no MΦ), by one-way ANOVA and Tukey's post-test. (E-H) Representative microphotographs of primary P1 neurons incubated with (E) vehicle, (F) XL-oAβ42, (G) fAβ42, and (H) co-cultured with GA-MΦ + XL-oAβ42. Scale bar = 20 μm.

Figure 4

Figure 4

Induced oligomeric Aβ42 clearance via GA activation of CD36/EEA1-intra- and MMP-9-extracellular mechanisms in MΦ, leading to neuronal preservation. (A–G) GA-activated (24 h) or naïve MΦ were exposed to 100 nM Aβ42 assemblies for 30 min, and co-labeled for Aβ (6E10, green), type B scavenger receptor CD36 (magenta), and early endosomal marker (EEA1, red). Cells were counterstained with DAPI (blue). Representative micrographs demonstrate elevated surface CD36 expression, intracellular Aβ, and Aβ colocalized within EEA1 endosomes in GA-MΦ vs. control MΦ, 30 min fAβ42 (A) and XL-oAβ42 (B) uptake assays. (C) Quantitative immunoreactive analysis of CD36 signals showed a substantial increase in CD36 in GA-MΦ. Basal levels of CD36 expression without Aβ42 were higher in GA-MΦ vs. control MΦ. This increase maintained its significance after exposure to XL-oAβ42. Regardless of GA effects, the presence of oligomeric Aβ42 substantially increased CD36 levels in both control MΦ and GA-MΦ. (D,E) Quantitative analysis of intracellular Aβ in MΦ following 30 min fAβ42 (D) and XL-oAβ42 (E). (F,G) Quantitative analysis of fAβ42 (F) and XL-oAβ42 (G) within EEA1-positive vesicles in early endosomes in GA-MΦ vs. control MΦ. Co-labeled 6E10/EEA1- immunoreactive puncta number per MΦ is displayed. (H) Increased MMP-9 signal in GA-MΦ after exposure to XL-oAβ42. (I) ELISA quantification of extracellular Aβ42 after 24 h incubation with fAβ42 or XL-oAβ42 compared to 30 min basal peptide levels. (J) Representative microphotographs of co-cultured P1 neurons and MΦ exposed to XL-oAβ42 for 24 h, stained with CD36, 6E10, Tubb3, and DAPI. P1 cortical neurons co-cultured with GA-MΦ vs. control MΦ exhibit increased neuritic outgrowth density with increased CD36-mediated Aβ uptake (arrows) and reduced extracellular Aβ42. Data expressed as mean ± s.e.m., with individual data point; n = 48 fields analyzed from 3 independent experiments; immunoreactive areas are normalized by cell number; **P < 0.01, ***P < 0.001, ****P < 0.0001, GA-MΦ vs. MΦ with fAβ42 or XL-oAβ42, and ### P < 0.001, #### P < 0.0001 for comparisons to MΦ without Aβ [(–) Aβ42], by one-way ANOVA with Tukey's test or two-tailed student _t_-test. Scale bar = 20 μm.

Figure 5

Figure 5

Cortical and hippocampal synaptic rescue by CD115+-monocyte blood enrichment and GA immunization in ADtg mice. (A,B) VGluT1/PSD95 synaptic quantification scheme in entorhinal cortex (ENT) and hippocampus (HIPPO). (A) (Middle) Representative crystal violet stained image of a coronal brain section at Bregma −2.65 mm. Scale bar = 1 mm. (Left) Representative microimage of HIPPO stained with the presynaptic (VGluT1, red) and postsynaptic (PSD95, green) markers. Note that 3 fields are chosen in each of the lateral and medial blade molecular layers (ML) of dentate gyrus (DG), as well as in the stratum lacunosum-moleculare (SLM), stratum radiatum (SR), and stratum oriens (SO) of CA1. (Right) Representative microphotograph of ENT immunolabled with the same pre- and postsynaptic markers. Note that ENT adjacent to piriform cortex (PIR) and 2 same fields are precisely selected from layers 2 and 3. Scale bar = 100 μm. (B) Representative microphotographs from the lateral blade ML immunostained with the same synaptic markers. Each high-magnification maximum intensity projection (MIP) image contains 15 Z-stack optical scanning (left). Scale bar = 10 μm. (Middle) Magnification for visualizing the proximity of pre- and postsynaptic signals. Scale bar = 2 μm. (Right) Schematic representation of pre- and post-terminal synapse. (C) Schematic of the in vivo experiments, where 10-month-old ADtg mice (all males) were injected with weekly s.c. GA immunization or monthly i.v. CD115+-MoBM injections for a duration of 2 months. Control groups were either naïve WT mice or monthly i.v. PBS injected ADtg mice. At the completion of the experiment, mice underwent behavioral testing, were euthanized, and brains were collected for analyses. (D,E) Quantification of presynaptic VGluT1 (D) and postsynaptic PSD95 (E) areas in ENT2/3 and hippocampal substructures of 13-month-old ADtg mice vs. age-matched WT littermates. (F,G) Quantification of VGluT1 (F) and PSD95 (G) immunoreactive areas in the entorhinal cortex, hippocampus, and whole brain of WT, GA-immunized, MoBM-injected, and PBS-control mice. Data expressed as mean ± s.e.m.; n = 6 mice per group; *P < 0.05, **P < 0.01, ***P < 0.001, and #P < 0.001, ## P < 0.0001, compared to ADtg control; @P < 0.05 compared to MoBM, ∧P < 0.05 compared to GA, one-way ANOVA with Tukey's test.

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