Image_1_Phagocytosis of Apoptotic Cells Is Specifically Upregulated in ApoE4 Expressing Microglia in vitro.TIF

Alzheimer’s disease (AD) is characterized by intracellular tau aggregates and extracellular deposition of amyloid-β (Aβ). The major genetic risk factor to develop AD is the Apolipoprotein E isoform 4 (ApoE4). ApoE4 may directly affect Aβ pathology, yet the exact role of ApoE4 in the progression of AD remains unclear. Although astrocytes are the main source of ApoE in brain tissue, other cell types might contribute to ApoE isotype-dependent effects. While ApoE expression does not play a relevant role in homeostatic microglia, we and others could recently show that ApoE expression is significant upregulated in disease-associated microglia including AD-mouse models and human AD. ApoE has been supposed to have an anti-inflammatory effect, with ApoE4 being less effective than ApoE3. However, ApoE-isotype specific effects on microglia function in disease have not been thoroughly investigated to date. In contrast to this, the role of ApoE2, the third most common major ApoE isoform, in neurodegeneration has not been characterized in detail, but it has been shown to delay the onset of disease in familial AD. To elucidate the differential roles of the three-major human ApoE isoforms on microglia function we each expressed the human ApoE isoforms in murine N9 microglia cells. We could show that ApoE4 specifically influences actin cytoskeleton rearrangement and morphology. In migration assays, ApoE4 significantly promotes cell motility. To quantify phagocytosis by microglia we established an uptake assay based on imaging flow cytometry. Although expression of ApoE4 led to significantly reduced uptake of Aβ in contrast to the other isoforms, we could show that ApoE4 specifically increased phagocytosis of apoptotic neuronal cells. Our findings show that ApoE4 intrinsically affects microglia physiology by upregulating motility and phagocytic behavior in vitro and may therefore specifically contribute to microglia dysregulation in AD.</p

Table_1_Phagocytosis of Apoptotic Cells Is Specifically Upregulated in ApoE4 Expressing Microglia in vitro.DOCX

Alzheimer’s disease (AD) is characterized by intracellular tau aggregates and extracellular deposition of amyloid-β (Aβ). The major genetic risk factor to develop AD is the Apolipoprotein E isoform 4 (ApoE4). ApoE4 may directly affect Aβ pathology, yet the exact role of ApoE4 in the progression of AD remains unclear. Although astrocytes are the main source of ApoE in brain tissue, other cell types might contribute to ApoE isotype-dependent effects. While ApoE expression does not play a relevant role in homeostatic microglia, we and others could recently show that ApoE expression is significant upregulated in disease-associated microglia including AD-mouse models and human AD. ApoE has been supposed to have an anti-inflammatory effect, with ApoE4 being less effective than ApoE3. However, ApoE-isotype specific effects on microglia function in disease have not been thoroughly investigated to date. In contrast to this, the role of ApoE2, the third most common major ApoE isoform, in neurodegeneration has not been characterized in detail, but it has been shown to delay the onset of disease in familial AD. To elucidate the differential roles of the three-major human ApoE isoforms on microglia function we each expressed the human ApoE isoforms in murine N9 microglia cells. We could show that ApoE4 specifically influences actin cytoskeleton rearrangement and morphology. In migration assays, ApoE4 significantly promotes cell motility. To quantify phagocytosis by microglia we established an uptake assay based on imaging flow cytometry. Although expression of ApoE4 led to significantly reduced uptake of Aβ in contrast to the other isoforms, we could show that ApoE4 specifically increased phagocytosis of apoptotic neuronal cells. Our findings show that ApoE4 intrinsically affects microglia physiology by upregulating motility and phagocytic behavior in vitro and may therefore specifically contribute to microglia dysregulation in AD.</p

Image_2_Phagocytosis of Apoptotic Cells Is Specifically Upregulated in ApoE4 Expressing Microglia in vitro.TIF

Alzheimer’s disease (AD) is characterized by intracellular tau aggregates and extracellular deposition of amyloid-β (Aβ). The major genetic risk factor to develop AD is the Apolipoprotein E isoform 4 (ApoE4). ApoE4 may directly affect Aβ pathology, yet the exact role of ApoE4 in the progression of AD remains unclear. Although astrocytes are the main source of ApoE in brain tissue, other cell types might contribute to ApoE isotype-dependent effects. While ApoE expression does not play a relevant role in homeostatic microglia, we and others could recently show that ApoE expression is significant upregulated in disease-associated microglia including AD-mouse models and human AD. ApoE has been supposed to have an anti-inflammatory effect, with ApoE4 being less effective than ApoE3. However, ApoE-isotype specific effects on microglia function in disease have not been thoroughly investigated to date. In contrast to this, the role of ApoE2, the third most common major ApoE isoform, in neurodegeneration has not been characterized in detail, but it has been shown to delay the onset of disease in familial AD. To elucidate the differential roles of the three-major human ApoE isoforms on microglia function we each expressed the human ApoE isoforms in murine N9 microglia cells. We could show that ApoE4 specifically influences actin cytoskeleton rearrangement and morphology. In migration assays, ApoE4 significantly promotes cell motility. To quantify phagocytosis by microglia we established an uptake assay based on imaging flow cytometry. Although expression of ApoE4 led to significantly reduced uptake of Aβ in contrast to the other isoforms, we could show that ApoE4 specifically increased phagocytosis of apoptotic neuronal cells. Our findings show that ApoE4 intrinsically affects microglia physiology by upregulating motility and phagocytic behavior in vitro and may therefore specifically contribute to microglia dysregulation in AD.</p

Image_3_Phagocytosis of Apoptotic Cells Is Specifically Upregulated in ApoE4 Expressing Microglia in vitro.TIF

Alzheimer’s disease (AD) is characterized by intracellular tau aggregates and extracellular deposition of amyloid-β (Aβ). The major genetic risk factor to develop AD is the Apolipoprotein E isoform 4 (ApoE4). ApoE4 may directly affect Aβ pathology, yet the exact role of ApoE4 in the progression of AD remains unclear. Although astrocytes are the main source of ApoE in brain tissue, other cell types might contribute to ApoE isotype-dependent effects. While ApoE expression does not play a relevant role in homeostatic microglia, we and others could recently show that ApoE expression is significant upregulated in disease-associated microglia including AD-mouse models and human AD. ApoE has been supposed to have an anti-inflammatory effect, with ApoE4 being less effective than ApoE3. However, ApoE-isotype specific effects on microglia function in disease have not been thoroughly investigated to date. In contrast to this, the role of ApoE2, the third most common major ApoE isoform, in neurodegeneration has not been characterized in detail, but it has been shown to delay the onset of disease in familial AD. To elucidate the differential roles of the three-major human ApoE isoforms on microglia function we each expressed the human ApoE isoforms in murine N9 microglia cells. We could show that ApoE4 specifically influences actin cytoskeleton rearrangement and morphology. In migration assays, ApoE4 significantly promotes cell motility. To quantify phagocytosis by microglia we established an uptake assay based on imaging flow cytometry. Although expression of ApoE4 led to significantly reduced uptake of Aβ in contrast to the other isoforms, we could show that ApoE4 specifically increased phagocytosis of apoptotic neuronal cells. Our findings show that ApoE4 intrinsically affects microglia physiology by upregulating motility and phagocytic behavior in vitro and may therefore specifically contribute to microglia dysregulation in AD.</p

Image_1_Reactive Astrocytes Contribute to Alzheimer’s Disease-Related Neurotoxicity and Synaptotoxicity in a Neuron-Astrocyte Co-culture Assay.pdf

Pathological hallmarks of Alzheimer’s disease (AD) include deposition and accumulation of amyloid- β (Aβ), neurofibrillary tangle formation, and neuronal loss. Pathogenesis of presymptomatic disease stages remains elusive, although studies suggest that the early structural and functional alterations likely occur at neuronal dendritic spines. Presymptomatic alterations may also affect different CNS cell types. However, specific contributions of these cell types as cause or consequence of pathology are difficult to study in vivo. There is a shortage of relatively simple, well-defined, and validated in vitro models that allow a straightforward interpretation of results and recapitulate aspects of pathophysiology. For instance, dissecting the AD-related processes (e.g., neurotoxicity vs. synaptotoxicity) may be difficult with the common cell-based systems such as neuronal cell lines or primary neurons. To investigate and characterize the impact of reactive astrocytes on neuronal morphology in the context of AD-related cues, we modified an in vitro co-culture assay of primary mouse neurons and primary mouse astrocytes based on the so-called Banker “sandwich” co-culture assay. Here, we provide a simple and modular assay with fully differentiated primary mouse neurons to study the paracrine interactions between the neurons and the astrocytes in the co-culture setting. Readouts were obtained from both cell types in our assay. Astrocyte feeder cells were pre-exposed to neuroinflammatory conditions by means of Aβ42, Aβ40, or lipopolysaccharide (LPS). Non-cell autonomous toxic effects of reactive astrocytes on neurons were assessed using the Sholl analysis to evaluate the dendritic complexity, whereas synaptic puncta served as a readout of synaptotoxicity. Here, we show that astrocytes actively contribute to the phenotype of the primary neurons in an AD-specific context, emphasizing the role of different cell types in AD pathology. The cytokine expression pattern was significantly altered in the treated astrocytes. Of note, the impact of reactive astrocytes on neurons was highly dependent on the defined cell ratios. Our co-culture system is modular, of low cost, and allows us to probe aspects of neurodegeneration and neuroinflammation between the two major CNS cell types, neurons, and astrocytes, under well-defined experimental conditions. Our easy-to-follow protocol, including work-flow figures, may also provide a methodological outline to study the interactions of astrocytes and neurons in the context of other diseases in the future.</p

Table_1_Reactive Astrocytes Contribute to Alzheimer’s Disease-Related Neurotoxicity and Synaptotoxicity in a Neuron-Astrocyte Co-culture Assay.xlsx

Pathological hallmarks of Alzheimer’s disease (AD) include deposition and accumulation of amyloid- β (Aβ), neurofibrillary tangle formation, and neuronal loss. Pathogenesis of presymptomatic disease stages remains elusive, although studies suggest that the early structural and functional alterations likely occur at neuronal dendritic spines. Presymptomatic alterations may also affect different CNS cell types. However, specific contributions of these cell types as cause or consequence of pathology are difficult to study in vivo. There is a shortage of relatively simple, well-defined, and validated in vitro models that allow a straightforward interpretation of results and recapitulate aspects of pathophysiology. For instance, dissecting the AD-related processes (e.g., neurotoxicity vs. synaptotoxicity) may be difficult with the common cell-based systems such as neuronal cell lines or primary neurons. To investigate and characterize the impact of reactive astrocytes on neuronal morphology in the context of AD-related cues, we modified an in vitro co-culture assay of primary mouse neurons and primary mouse astrocytes based on the so-called Banker “sandwich” co-culture assay. Here, we provide a simple and modular assay with fully differentiated primary mouse neurons to study the paracrine interactions between the neurons and the astrocytes in the co-culture setting. Readouts were obtained from both cell types in our assay. Astrocyte feeder cells were pre-exposed to neuroinflammatory conditions by means of Aβ42, Aβ40, or lipopolysaccharide (LPS). Non-cell autonomous toxic effects of reactive astrocytes on neurons were assessed using the Sholl analysis to evaluate the dendritic complexity, whereas synaptic puncta served as a readout of synaptotoxicity. Here, we show that astrocytes actively contribute to the phenotype of the primary neurons in an AD-specific context, emphasizing the role of different cell types in AD pathology. The cytokine expression pattern was significantly altered in the treated astrocytes. Of note, the impact of reactive astrocytes on neurons was highly dependent on the defined cell ratios. Our co-culture system is modular, of low cost, and allows us to probe aspects of neurodegeneration and neuroinflammation between the two major CNS cell types, neurons, and astrocytes, under well-defined experimental conditions. Our easy-to-follow protocol, including work-flow figures, may also provide a methodological outline to study the interactions of astrocytes and neurons in the context of other diseases in the future.</p

Deficiency in Serine Protease Inhibitor Neuroserpin Exacerbates Ischemic Brain Injury by Increased Postischemic Inflammation

<div><p>The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (<i>Ns^−/−</i>) mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in <i>Ns^−/−</i> mice. Our results show excessive microglial activation in <i>Ns^−/−</i> mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.</p></div

Activation of microglia is increased in the absence of neuroserpin.

<p>(<b>A</b>) Absolute numbers of brain microglia in the ischemic hemisphere of wild type, and <i>Ns^−/−</i> mice 3 days after MCAO. Cell counts were determined by flow cytometry analysis of the CNS-infiltrating cells using TrueCount tubes. Brain microglia cells were identified as CD11b⁺ CD45^intermediate. Representative dot plots show CD11b⁺ CD45^high and CD11b⁺ CD45^intermediate-gated populations identifying macrophages and microglia respectively. The graphs show means±SD of 9–12 animals per group analyzed three days after MCAO in three or four independent experiments. <i>t</i> test was used to assess statistical significance. (<b>B</b>) Immunohistochemical analysis of absolute numbers of Iba-1 positive brain microglia/macrophages in the ischemic hemisphere of wild type, and <i>Ns^−/−</i> mice 3 days after MCAO. The graphs show means±SD of 3 animals per group. <i>t</i> test was used to assess statistical significance. (<b>C</b>) Immunohistochemical analysis of the activation state (resting, bushy and amoeboid) of Iba-1 positive microglia in the ipsilesional hippocampus and penumbra area and contralesional hippocampus area 3 days following 1 h MCAO. The graphs show means±SD of 3 animals per group and the statistical analysis was assessed using one-way ANOVA with Bonferroni post hoc test (scale bar = 20 µm).</p

Deficiency in neuroserpin does not lead to alterations of the cellular post stroke infiltrate.

<p>(<b>A</b>) Absolute numbers of neutrophils (N), macrophages (Mφ), dendritic cells (DC) and lymphocytes (L) in the CNS-infiltrating cells of <i>wt and Ns^−/−</i> mice. Representative plots are shown for CD45^high and CD45^intermediate cells. Brain macrophages were identified as CD45^high, CD11b⁺, CD11c⁻ and distinguished from microglia by the higher expression of CD45. Dendritic cells were identified as CD45^high, CD11b⁺, CD11c⁺, neutrophils as CD45^high, CD11b⁺, Ly6G⁺ and lymphocytes as CD45^high, CD11b⁻, CD11c⁻. The graphs show the means±SD of 9 animals per group analyzed three days after MCAO in three or four independent experiments. One-way ANOVA with Bonferroni post-hoc test was used to assess statistical significance. (<b>B</b>) Immunohistochemical staining of T cells (CD3), neutrophils (Ly6G) and astrocytes (GFAP) in wt and <i>Ns^−/−</i> mice three days after MCAO (scale bar = 50 µm).</p

Deficiency in neuroserpin is detrimental in stroke.

<p>(<b>A</b>) TTC staining for evaluation of infarct volume at day three (left panel) and (<b>B</b>) neurological scores at days one and three (right panel) of wt and <i>Ns^−/−</i> mice after MCAO. Data are represented as means±SD of 10 wt and eight <i>Ns^−/−</i> animals. <i>t</i> test was used to assess statistical significance for infarct sizes and Mann-Whitney U test for neurological scores. (<b>C</b>) Survival rate of wt (n = 13) and <i>Ns^−/−</i> mice (n = 13). Survival was analyzed by the <i>χ</i>² test (survival rate). (<b>D</b>) In all mice subjected to MCAO, regional cerebral blood flow (rCBF) was measured with Laser Doppler. The decrease in rCBF of approximately 90% was similar between <i>Ns^−/−</i> and wt mice. Ten minutes after reperfusion rCBF was reconstituted to at least 60% of baseline levels and was unaltered between <i>Ns^−/−</i> and wt animals. (<b>E</b>) Accumulation of fibrin(ogen) in the infarcted and in the contralesional hemispheres of wt (n = 3) and <i>Ns^−/−</i> mice (n = 3). Fibrin(ogen) formation was analyzed by immunoblotting following fixation with 4% PFA (upper panel) or w/o 4% PFA-fixation (lower panel) using a rabbit polyclonal fibrin/fibrinogen-specific antibody 24 h following ischemia. Asterisks indicate additional bands representing fibrin degradation. <i>t</i> test was used to assess statistical significance.</p