Microglia contribute to the propagation of Aβ into unaffected brain tissue

Microglia appear activated in the vicinity of amyloid beta (Aβ) plaques, but whether microglia contribute to Aβ propagation into unaffected brain regions remains unknown. Using transplantation of wild-type (WT) neurons, we show that Aβ enters WT grafts, and that this is accompanied by microglia infiltration. Manipulation of microglia function reduced Aβ deposition within grafts. Furthermore, in vivo imaging identified microglia as carriers of Aβ pathology in previously unaffected tissue. Our data thus argue for a hitherto unexplored mechanism of Aβ propagation.

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Software and code
Policy information about availability of computer code Data collection Fluorescently labeled brain slices or cell cultures were acquired using a Zeiss fluorescent microscope (Axio Imager M2M) or an Olympus confocal microscope (Fluoview FV 1000). Organotypic brain slices were acquired using a confocal microscopy (Leica TCS SP5 II). In vivo imaging was performed with an Olympus FV1000 two-photon microscope with Mai Tai DeepSee Laser (Spectra-Physics, Newport Corporation, Franklin, MA, USA). 3D reconstruction was performed using IMARIS 8 software. Western blot membranes were visualized with ImageLab 4 software (Bio-Rad Laboratories).
Data analysis ImageJ 1.52a was used for all immunohistochemical analyses and for quantification of two-photon in vivo acquisitions. The cDNAs libraries for Bulk-RNA sequencing were quantified using the KAPA Library Quantification Kit -Illumina/ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA) and image analysis done by the Real Time Analysis Software (RTA) v2.4.11. Each library was sequenced on a NextSeq 500 instrument controlled by the NextSeq Control Software (NCS) v2.2.0. The resulting .bcl files were converted into .fastq files with the bcl2fastq v2.18 software. For the analysis of flow cytometry experiments the FlowJo software (Tree Star) was utilized. GraphPad Prism 7 (GraphPad Software, Inc) was used for statistical analysis and to generate graphs presented throughout the manuscript.
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October 2018
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Replication
All attempts of replication were successful.To analyze Aβ inside primary microglia, between 20 and 50 cells per mouse were quantified and replicated on at least 3 mice per group. The western blot analysis of freshly isolated cortical cells for grafting were performed on three independent preparations. Methoxy-XO4 FACS analysis of microglia isolated from young-adult and aged 5xFAD mice was performed in three repeated experiments using at least 3 mice per group in each experiment. For the Bulk-RNA sequencing, graft and control cortical region isolation was repeated three times and for each independent experiment 7-10 mice were used. The grafting experiments were repeated at least three times using, when possible, at least 2 mice per group per experiments. The organotypic brain slice experiments where replicated two times using one mouse per group in each experiment. The in vivo laser lesion experiments were performed in at least 4 repeated experiments per animal group using one mouse in each experiment.
Randomization For all experiments, mice were randomly allocated into each experimental group by PdE and CM.

Blinding
The investigator was not blinded during the intracerebral grafting experiments since the procedure was independently identical. The investigator was not blinded during two-photon imaging and analysis due to obvious differences in microglia morphology between groups/ genotypes. The investigator was blinded for staining experiments, microglia counts and assessment of Aβ (slides and in vitro samples were imaged and saved with random numbers to identify them) and flow cytometry experiments. Images were then quantified and unblinded to perform group statistics.

Validation
All antibodies used in this study were commercially available and validated by the vendors and previous studies performed by our laboratory or by others.

Animals and other organisms
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Microglia isolation experiments:
Microglia were isolated from 20-30 weeks or 50-60 weeks 5xFAD mice and aged matched C57BL/6N WT controls. Both males and females were used for these experiments.

Wild animals
No wild animals were used in the study.

Field-collected samples
No field-collected samples were used in the study.

Ethics oversight
All animal experiments were carried out in accordance with the policies of the state of Baden-Württemberg under license number G16-100.
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Flow Cytometry
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