Forebrain microglia from wild-type but not adult 5xFAD mice prevent amyloid-beta plaque formation in organotypic hippocampal slice cultures

The role of microglia in amyloid-beta (A beta) deposition is controversial. In the present study, an organotypic hippocampal slice culture (OHSC) system with an in vivo-like microglial-neuronal environment was used to investigate the potential contribution of microglia to A beta plaque formation. We found that microglia ingested A beta, thereby preventing plaque formation in OHSCs. Conversely, A beta deposits formed rapidly in microglia-free wild-type slices. The capacity to prevent A beta plaque formation was absent in forebrain microglia from young adult but not juvenile 5xFamilial Alzheimer's disease (FAD) mice. Since no loss of A beta clearance capacity was observed in both wild-type and cerebellar microglia from 5xFAD animals, the high A beta(1-42) burden in the forebrain of 5xFAD animals likely underlies the exhaustion of microglial A beta clearance capacity. These data may therefore explain why A beta plaque formation has never been described in wild-type mice, and point to a beneficial role of microglia in AD pathology. We also describe a new method to study A beta plaque formation in a cell culture setting

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies

Bone Marrow Cell Recruitment to the Brain in the Absence of Irradiation or Parabiosis Bias

<div><p>The engraftment of bone marrow-derived cells has been described not only during diseases of the central nervous system (CNS) but also under healthy conditions. However, previous studies pointing to an ample bone marrow cell engraftment used irradiation-induced bone marrow chimeras that evoked severe alterations of the CNS micromilieu including disturbances of the blood brain barrier (BBB), damage of endothelial cells and local induction of proinflammatory cytokines. On the other hand, parabiosis experiments using temporarily joined circulatory systems generally yielded low levels of myeloid cell chimerism thereby potentially underestimating bone marrow cell turnover with the CNS. To avoid these drawbacks we established a protocol using the alkylating agent busulfan prior to allogenic bone marrow transplantation from CX₃CR1^GFP/+ donors. This regimen resulted in a stable and high peripheral myeloid chimerism, significantly reduced cytokine induction and preserved BBB integrity. Importantly, bone marrow cell recruitment to the CNS was strongly diminished under these conditions and only weakly enhanced during local neurodegeneration induced by facial nerve axotomy. These results underscore the requirement of local CNS conditioning for efficient recruitment of bone marrow cells, establish busulfan as an alternative treatment for studying bone marrow chimeras and suggest a critical re-evaluation of earlier chimeric studies involving irradiation or parabiosis regimens.</p> </div

Reduced conditioning of the CNS after busulfan treatment.

<p><b>A)</b> Strongly diminished induction of proinflammatory cytokines and chemokines in busulfan-treated chimeras compared to irradiation protocols. Quantitative real-time PCR analysis of cytokine and chemokine induction in brains of busulfan-treated (grey columns) and whole-body irradiated (white columns) chimeras 24 hours (h), 7 days (d), 14 d and 16 weeks (w) after treatment. The mRNA expression was normalized to GAPDH and compared to untreated mice, indicated by the grey line. Data are shown as mean ± SEM. One out of two experiments is shown with three to six animals per group. Statistical significance is marked with asterisks (p<0.05 = *; p<0.01 = **; p<0.001 = ***). <b>B)</b> Largely preserved blood-brain-barrier (BBB) integrity after busulfan challenge. Direct fluorescence microscopic visualization depicting CD31⁺ endothelial cells (green), extravasal albumin (red) and nuclei (DAPI, blue). Arrow heads point to extravasated albumin. Representative pictures of cortices are shown. Scale bar = 100 µm. <b>C)</b> Albumin staining of the CNS parenchym is shown for both treated groups 24 hours (h), 7 days (d) and 14 d after treatment. Arrow heads point to extravasated albumin in superficial and deeper brain regions. Representative pictures are shown. Scale bar = 400 µm. Insert: Albumin staining of an untreated animal. Scale bar = 400 µm.</p

High peripheral blood myeloid chimerisms in busulfan-treated chimeras.

<p><b>A)</b> Representative FACS dot plots showing the expression of GFP in blood granulocytes, monocytes, B cells and T cells of busulfan-treated and irradiated CX₃CR1^GFP/+ → CX₃CR1^+/+ chimeras and untreated mice four weeks after bone marrow transfer. Percentages of the respective cell populations are indicated. CX₃CR1^+/+ mice served as untreated controls. <b>B)</b> Quantitative assessment of GFP⁺ expression in overall monocytes (SSC^loCD11b⁺) and the two monocyte subsets SSC^loCD11b⁺Ly6C^hi and SSC^loCD11b⁺Ly6C^lo out of all SSC^loCD11b⁺ cells. Data depict high reconstitution efficiencies and comparable chimerisms in both groups. One symbol represents one mouse (Busulfan-treated: filled circle; irradiated: filled squares). Mean ± SEM are shown. Six to seven animals per group were analysed. <b>C)</b> Photograph showing change of fur color 16 weeks after treatment. Irradiation leads to a loss of fur color, whereas busulfan-treated animals remain unaffected. Top: untreated animal, middle: whole-body irradiated animal, bottom: busulfan-treated mouse.</p

Engraftment of donor-derived GFP⁺ bone marrow cells into the brain strongly reduced in busulfan-treated animals.

<p>The number of engrafted GFP⁺Iba-1⁺ donor-derived phagocytes is strongly decreased in the busulfan-treated animals compared to irradiated mice. <b>A)</b> Immunohistochemistry of chimeric brains shows GFP⁺Iba-1⁺ cells in the cortex, hippocampus, thalamus and choroid plexus of busulfan-treated and irradiated chimeric mice 16 weeks after reconstitution. Arrow heads indicate representative ramified GFP⁺ (green) and Iba-1⁺ (red) cells of donor origin in the CNS. Nuclei are counterstained with DAPI (blue). Bars = 200 µm. <b>B)</b> High magnification images of donor-derived GFP⁺ Iba1⁺ reveal ramified morphology of engrafted cells in both groups. Sections are stained for Iba-1 (red), CX₃CR1 (GFP) and DAPI (blue). Scale bars = 50 µm. <b>C)</b> Quantification of ramified GFP⁺Iba-1⁺ cells in cortex, hippocampus, thalamus and choroid plexus. Each symbol represents one mouse. Data show significant differences in the number of engrafted GFP⁺Iba-1⁺ donor-derived cells in all investigated brain areas between the busulfan-treated (filled circle) and irradiated (filled squares) animals 16 weeks after bone marrow cell transfer. Asterisks indicate statistical significance (p<0.05 = *; p<0.01 = **; p<0.001 = ***). All graphs show mean ± SEM.</p

Recruitment of donor-derived GFP⁺ bone marrow cells into the lesioned brain depends on irradiation.

<p>Following facial nerve axotomy, the recruitment of GFP⁺Iba-1⁺ donor-derived bone marrow cells to the lesioned N. facialis is strongly diminished in busulfan-treated mice compared to irradiated animals. <b>A)</b> Representative FACS dot plots of peripheral blood in the SSC^loCD11b⁺ monocyte compartment in untreated, busulfan-treated and irradiated mice. <b>B)</b> Quantification of the peripheral blood chimerism shows sufficient and comparable reconstitution levels in busulfan-treated (filled circles) and irradiated mice (filled squares). One symbol represents one mouse. Chimerism was analyzed nine weeks after reconstitution. Six animals per group were analysed. All graphs show mean ± SEM. <b>C)</b> Immunohistochemistry of recruited GFP⁺Iba-1⁺ phagocytes in the lesioned facial nucleus two weeks following axotomy. Elevated numbers of ramified donor-derived GFP⁺ cells were found in the degenerating neurons (NeuN, red) of irradiated CX₃CR1^GFP/+ → CX₃CR1^+/+ mice compared to busulfan-treated CX₃CR1^GFP/+ → CX₃CR1^+/+ animals. GFP⁺ cells were in close proximity to NeuN-immunoreactive neurons in the facial nucleus. No GFP expressing cells were found on the control site in busulfan-treated mice, in contrast to irradiated mice where a few GFP⁺ cells could be found. Scale bars: overview = 200 µm; detail = 50 µm. Nuclei were stained with DAPI (blue). <b>D)</b> Quantification of engrafted GFP⁺ Iba-1⁺ cells 14 days after axotomy. A robust engraftment of donor-derived GFP⁺ cells was found in irradiated chimeric mice (filled squares), whereas only few GFP⁺ cells were detectable in busulfan-treated chimeric mice (filled circle). Symbols indicate individual mice. Data are expressed as mean ± SEM. Asterisks indicate statistical differences (p<0.001 = ***).</p

Seed-induced Abeta deposition impairs adult neurogenesis, triggers neurodegeneration and is modulated by microglia under environmental enrichment

Alzheimer´s disease (AD) is characterized by severe neuronal loss as well as the accumulation of amyloid- (Abeta) which ultimately leads to plaque formation. Although a decline of neurogenic capacity in the brain of AD patients and AD mouse models has been reported, our understanding of whether this impairment is specifically altered by Abeta plaques is limited. Here, we find that induced Abeta deposition (Abeta seeding), representing early stages of plaque formation, leads to a dramatic decrease in proliferation and neurogenesis. We further demonstrate that signs of neuronal cell death occur primarily in the vicinity of induced Abeta deposits. Notably, environmental enrichment and voluntary exercise not only revives adult neurogenesis but, most importantly, prevents Abeta seeding by activated, phagocytic microglia cells. Our work expands the current knowledge regarding Abeta seeding and the consequences thereof and attributes microglia an important role in diminishing Abeta seeding by environmental enrichment

Aβ oligomers trigger and accelerate Aβ seeding

Aggregation of amyloid-beta (A beta) that leads to the formation of plaques in Alzheimer's disease (AD) occurs through the stepwise formation of oligomers and fibrils. An earlier onset of aggregation is obtained upon intracerebral injection of A beta-containing brain homogenate into human APP transgenic mice that follows a prion-like seeding mechanism. Immunoprecipitation of these brain extracts with anti-A beta oligomer antibodies or passive immunization of the recipient animals abrogated the observed seeding activity, although induced A beta deposition was still evident. Here, we establish that, together with A beta monomers, A beta oligomers trigger the initial phase of A beta seeding and that the depletion of oligomeric A beta delays the aggregation process, leading to a transient reduction of seed-induced A beta deposits. This work extends the current knowledge about the role of A beta oligomers beyond its cytotoxic nature by pointing to a role in the initiation of A beta aggregation in vivo. We conclude that A beta oligomers are important for the early initiation phase of the seeding process

Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE