Young microglia restore amyloid plaque clearance of aged microglia

Alzheimer′s disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co‐culturing organotypic brain slices from up to 20‐month‐old, amyloid‐bearing AD mouse model (APPPS1) and young, neonatal wild‐type (WT) mice. Surprisingly, co‐culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.     

阿尔茨海默病中β淀粉样蛋白的生成及清除的调节

阿尔茨海默病（Alzheimer’s disease, AD）是一种慢性退行性神经系统疾病,临床主要表现为进行性认知能力下降、记忆力衰退、人格改变等。AD的标志性病理特征包括脑细胞外β淀粉样蛋白（β-amyloid protein,Aβ）沉积形成老年斑、细胞内神经纤维缠结(neurofibrillary tangles,NFT)、神经炎症增加以及神经元凋亡。β淀粉样蛋白主要在神经元产生,是淀粉样前体蛋白经过一系列酶解反应生成的由39～42个氨基酸组成的多肽,调节Aβ的生成和清除能够有效延缓甚至逆转阿尔茨海默病的进程,因而具有重大的研究价值。β-分泌酶（β-site APP cleaving enzyme 1,BACE1）为Aβ产生过程中的关键酶,其含量及活性的改变均能影响Aβ产生,在阿尔茨海默病的发生发展中发挥至关重要的作用;老年斑周围炎性细胞的聚集提示,AD与神经炎症高度相关,神经炎症相关细胞能够参与Aβ的清除,多种炎性因子也能调节Aβ的生成;非编码RNA虽很少直接参与Aβ的产生、沉积和清除,但其可以通过多种途径调节Aβ的产生。本文从β淀粉样蛋白生成及清除的机制着手,重点阐述了BACE1、神经炎症、非编码RNA对Aβ调控的重要作用,以期为AD发病机制的进一步研究提供思路,并对阿尔茨海默病早期干预及治疗提供理论参考。     

Increase in the density of resting microglia precedes neuritic plaque formation and microglial activation in a transgenic model of Alzheimer's disease

The formation of cerebral senile plaques composed of amyloid β peptide (Aβ) is a fundamental feature of Alzheimer''s disease (AD). Glial cells and more specifically microglia become reactive in the presence of Aβ. In a triple transgenic model of AD (3 × Tg-AD), we found a significant increase in activated microglia at 12 (by 111%) and 18 (by 88%) months of age when compared with non-transgenic (non-Tg) controls. This microglial activation correlated with Aβ plaque formation, and the activation in microglia was closely associated with Aβ plaques and smaller Aβ deposits. We also found a significant increase in the area density of resting microglia in 3 × Tg-AD animals both at plaque-free stage (at 9 months by 105%) and after the development of A plaques (at 12 months by 54% and at 18 months by 131%). Our results show for the first time that the increase in the density of resting microglia precedes both plaque formation and activation of microglia by extracellular Aβ accumulation. We suggest that AD pathology triggers a complex microglial reaction: at the initial stages of the disease the number of resting microglia increases, as if in preparation for the ensuing activation in an attempt to fight the extracellular Aβ load that is characteristic of the terminal stages of the disease.     

Microglial PD‐1 stimulation by astrocytic PD‐L1 suppresses neuroinflammation and Alzheimer’s disease pathology

Chronic neuroinflammation is a pathogenic component of Alzheimer’s disease (AD) that may limit the ability of the brain to clear amyloid deposits and cellular debris. Tight control of the immune system is therefore key to sustain the ability of the brain to repair itself during homeostasis and disease. The immune‐cell checkpoint receptor/ligand pair PD‐1/PD‐L1, known for their inhibitory immune function, is expressed also in the brain. Here, we report upregulated expression of PD‐L1 and PD‐1 in astrocytes and microglia, respectively, surrounding amyloid plaques in AD patients and in the APP/PS1 AD mouse model. We observed juxtamembrane shedding of PD‐L1 from astrocytes, which may mediate ectodomain signaling to PD‐1‐expressing microglia. Deletion of microglial PD‐1 evoked an inflammatory response and compromised amyloid‐β peptide (Aβ) uptake. APP/PS1 mice deficient for PD‐1 exhibited increased deposition of Aβ, reduced microglial Aβ uptake, and decreased expression of the Aβ receptor CD36 on microglia. Therefore, ineffective immune regulation by the PD‐1/PD‐L1 axis contributes to Aβ plaque deposition during chronic neuroinflammation in AD.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

Metabolic syndrome exacerbates amyloid pathology in a comorbid Alzheimer's mouse model

Alzheimer's disease (AD) often coexists with other aging-associated diseases including obesity, diabetes, hypertension, and cardiovascular diseases. The early stage of these comorbidities is known as metabolic syndrome (MetS) which is highly prevalent in mid-life. An important cause of MetS is the deficiency of SIRT3, a mitochondrial deacetylase which enhances the functions of critical mitochondrial proteins, including metabolic enzymes, by deacetylation. Deletion of Sirt3 gene has been reported to result in the acceleration of MetS. In a recently published study, we demonstrated in the brain of Sirt3^−/− mice, downregulation of metabolic enzymes, insulin resistance and elevation of inflammatory markers including microglial proliferation. These findings suggested a novel pathway that could link SIRT3 deficiency to neuroinflammation, an important cause of Alzheimer's pathogenesis. Therefore, we hypothesized that MetS and amyloid pathology may interact through converging pathways of insulin resistance and neuroinflammation in comorbid AD. To investigate these interactions, we crossed Sirt3^−/− mice with APP/PS1 mice and successfully generated APP/PS1/Sirt3^−/− mice with amyloid pathology and MetS. In these comorbid AD mice, we observed exacerbation of insulin resistance, glucose intolerance, amyloid plaque deposition, markers of neuroinflammation, including elevated expression of IL-1β, TNF-α and Cox-2 at 8 months of age. There was also increased microglial proliferation and activation. Our observations suggest a novel mechanism by which MetS may interact with amyloid pathology during the cellular phase of AD. Therapeutic targeting of SIRT3 in AD with comorbidities may produce beneficial effects.     

Microglial transplantation increases amyloid-beta clearance in Alzheimer model rats

Immunization with amyloid-beta (Abeta) peptides, a therapeutic approach in Alzheimer's disease (AD), reduces brain Abeta, and microglial Abeta phagocytosis has been proposed as an Abeta-lowering mechanism. We transplanted rat microglia into the rat lateral ventricle just after intra-hippocampal Abeta injection, and then investigated the contribution of exogenous microglia to Abeta clearance. Migration of exogenous microglia from the lateral ventricle to Abeta plaque was detected by magnetic resonance imaging and histochemical analysis, and the clearance of Abeta was increased by transplantation. These results suggest the possible usefulness of exogenous microglia to the therapeutic approach in AD.     

Fibrillar Amyloid Plaque Formation Precedes Microglial Activation

In Alzheimer’s disease (AD), hallmark β-amyloid deposits are characterized by the presence of activated microglia around them. Despite an extensive characterization of the relation of amyloid plaques with microglia, little is known about the initiation of this interaction. In this study, the detailed investigation of very small plaques in brain slices in AD transgenic mice of the line APP-PS1(dE9) revealed different levels of microglia recruitment. Analysing plaques with a diameter of up to 10 μm we find that only the half are associated with clear morphologically activated microglia. Utilizing in vivo imaging of new appearing amyloid plaques in double-transgenic APP-PS1(dE9)xCX3CR1^+/- mice further characterized the dynamic of morphological microglia activation. We observed no correlation of morphological microglia activation and plaque volume or plaque lifetime. Taken together, our results demonstrate a very prominent variation in size as well as in lifetime of new plaques relative to the state of microglia reaction. These observations might question the existing view that amyloid deposits by themselves are sufficient to attract and activate microglia in vivo.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

Neuroprotective role of γ‐enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X

γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.     

Accelerated microglial pathology is associated with Aβ plaques in mouse models of Alzheimer's disease

Microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network. Here, we used a digital tool for the quantitative morphometric characterization of fine cortical microglial structures in mice, and the changes they undergo with aging and in Alzheimer's‐like disease. We show that, compared with microglia in young mice, microglia in old mice are less ramified and possess fewer branches and fine processes along with a slightly increased proinflammatory cytokine expression. A similar microglial pathology appeared 6–12 months earlier in mouse models of Alzheimer's disease (AD), along with a significant increase in brain parenchyma lacking coverage by microglial processes. We further demonstrate that microglia near amyloid plaques acquire unique activated phenotypes with impaired process complexity. We thus show that along with a chronic proinflammatory reaction in the brain, aging causes a significant reduction in the capacity of microglia to scan their environment. This type of pathology is markedly accelerated in mouse models of AD, resulting in a severe microglial process deficiency, and possibly contributing to enhanced cognitive decline.     

SCD1 activation impedes foam cell formation by inducing lipophagy in oxLDL‐treated human vascular smooth muscle cells

The formation of fat‐laden foam cells, which contributes to the fatty streaks in the plaques of atheromas, is an important process in atherosclerosis. Vascular smooth muscle cells (VSMCs) are a critical origin of foam cells. However, the mechanisms that underlie VSMC foam cell formation are not yet completely understood. Here, we demonstrated that oxidized low‐density lipoprotein (oxLDL) inhibited lipophagy by suppressing lipid droplet (LD)‐lysosome fusion and increased VSMC foam cell formation. Moreover, although oxLDL treatment inhibited lysosomal biogenesis, it had no significant effect on lysosomal proteolysis and lysosomal pH. Notably, through TMT‐based quantitative proteomic analysis and database searching, 94 differentially expressed proteins were identified, of which 54 were increased and 40 were decreased in the oxLDL group compared with those in the control group. Subsequently, SCD1, a protein of interest, was further investigated. SCD1 levels in the VSMCs were down‐regulated by exposure to oxLDL in a time‐dependent manner and the interaction between SCD1 and LDs was also disrupted by oxLDL. Importantly, SCD1 overexpression enhanced LD‐lysosome fusion, increased lysosomal biogenesis and inhibited VSMC foam cell formation by activating TFEB nuclear translocation and its reporter activity. Modulation of the SCD1/TFEB‐mediated lipophagy machinery may offer novel therapeutic approaches for the treatment of atherosclerosis.