Extracellular Elastin Molecule Modulates Alzheimer’s Aβ Dynamics <i>In Vitro</i> and <i>In Vivo</i> by Affecting Microglial Activities

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Jul 2021Extracellular Elastin Molecule Modulates Alzheimer's Aβ Dynamics In Vitro and In Vivo by Affecting Microglial Activities Jingjing Li†, Yao Sun†, Yingxia Liang†, Jun Ma, Bo Li, Chao Ma, Rudolph E. Tanzi, Hongjie Zhang, Kai Liu and Can Zhang Jingjing Li† State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun , Yao Sun† State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun , Yingxia Liang† Genetics and Aging Research Unit, McCance Center for Brain Health, Mass General Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA , Jun Ma *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun , Bo Li State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun , Chao Ma School of Engineering and Applied Sciences & Department of Physics, Harvard University, MA , Rudolph E. Tanzi Genetics and Aging Research Unit, McCance Center for Brain Health, Mass General Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA , Hongjie Zhang State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun Department of Chemistry, Tsinghua University, Beijing. , Kai Liu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun Department of Chemistry, Tsinghua University, Beijing. and Can Zhang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Genetics and Aging Research Unit, McCance Center for Brain Health, Mass General Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA https://doi.org/10.31635/ccschem.020.202000330 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Alzheimer's disease (AD) is a neurodegenerative disorder, and the etiology of AD has not been completely elucidated. It remains unknown how the components from the brain's extracellular matrix (ECM), particularly fibrous entities, may influence the pathogenesis of AD. Herein, we report that treatment with elastin-like polypeptides (ELPs), a component of the brain ECM, significantly increases the extracellular levels of AD-related amyloid-beta (Aβ) peptides and decreases intracellular Aβ levels in human microglial cell model HMC3 cells (HMC3). Furthermore, treatment with ELPs in animals demonstrates activation of microglia in the hippocampus of brain slices in a dose-dependent manner. Our results strongly suggest that the simplistic elastin molecule is associated with AD-related pathological changes via modulation of microglial activation and phagocytosis. These findings present a new aspect for Alzheimer's amyloidosis event associated with ELPs-related neuroinflammatory reactions. Furthermore, our results suggest this revelation of the diverse functions of ELPs and other ECM may pave the way to uncover new specific microglia-targeted approaches related to AD and potentially other neurological disorders. Download figure Download PowerPoint Introduction Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the major cause of dementia in the elderly.1 The well-known hallmark of AD is abnormal amyloid-beta (Aβ) aggregation and deposition in the brain.2,3 However, the etiology of AD is complex and the mechanistic elucidation of AD pathogenesis remains inconclusive. Recently, a variety of studies have focused on the role of microglial function in AD.4–7 Microglia are the resident macrophages of the central nervous system (CNS) and perform essential roles in diverse aspects such as neurodevelopment, immunity, homeostasis, and neural repair.8–12 Early in the neurodegenerative process, microglia activates and triggers a series of neuroinflammatory reactions that are responsible for neuronal death.13,14 Increased numbers of morphologically reactive microglia are a well-characterized histological observation indicator from AD brains. Several interactions have been reported regarding the function of AD risk genes to modulate microglial activities.15–21 However, it is challenging to clearly illustrate pathological cues that impact microglial morphology and function. Recently, increased findings on brain functions arise from morphofunctional activity of neuroglial networks that strictly interact with the blood–brain barrier and extracellular matrix (ECM).22–25 The ECM in the brain provides microenvironments for both neurons and glial cells, meanwhile these cells secrete diverse molecules that, in turn, contribute to the dynamics of the ECM.26,27 The macromolecules from the perineuronal nets and neural interstitial matrix of the ECM, such as tenascin R and proteoglycan, have been studied in the context of neurodegeneration due to their maintenance features.28–30 In terms of age-related AD pathology, however, little attention has been paid to the fibrous proteins (e.g., elastin) from the basement membranes of ECM. There are reports31,32 that elastin can be substantially fragmented and released with aging in AD pathogenesis, yet the effects of these released elastin polypeptides on microglial remain elusive. In our previous study, the levels of AD-related Aβ peptides was strongly elevated by elastin-like polypeptide (ELP) both in vitro and in vivo, which can be possibly attributed to the overexpression of γ-secretases when dosing with ELP.33 However, the relationship of ELPs to other major players in AD pathology, such as microglia, remains elusive. Herein, we demonstrate that a recombinant ELP, derived from ECM native elastin, plays a previously unrecognized role in microglial activation. We found ELP-induced upregulation of extracellular Aβ levels in a previously established human microglial cell model of HMC3 cells (HMC3). Furthermore, ELP-treated HMC3 cells showed a significant decrease in intracellular Aβ levels. Importantly, microglial activation was detected in the brain slices of mice at 1 or 2 months post-ELPs injection. Our results strongly suggest that ELPs are important risk proteins involved in AD development through a dual pathway. On the one hand, ELPs antagonize microglial phagocytosis of Aβ and induce positive feedback of microglial activation. On the other hand, ELPs promote the cleavage of amyloid-β protein precursor (APP) in microglia and the release of Aβ. These findings might increase the insight of neuroinflammatory reactions and pave the way for new specific microglial-targeted therapeutic strategies for AD. Experimental Methods Materials Reagents for ELP expression, including LB medium, salts, antibiotics, as well as isopropyl-β-d-thiogalactoside, were purchased from Sigma–Aldrich and used as received. Reagents for cell culture were ordered from Gibco. Anti-Aβ1-16 mouse monoclonal antibody (6E10; BioLegend, 803001) was used at a ratio of 1∶1000 for Western blotting analysis. Anti-amyloid precursor protein (APP) A4 mouse monoclonal antibody (22C11, Merck Millipore, MAB348) was used at a ratio of 1∶500 for IHC. GAPDH mAb (60004-1-lg; ProteinTech, Chicago, Illinois, USA) was used at a ratio of 1∶10,000 for Western blot. Anti-Iba1 rabbit monoclonal antibody (EPR16588, ab178846; Abcam Inc, USA) was used at a ratio of 1∶1000 and 1∶3000 for IHC and Western blot, respectively. Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) antibody (111-035-144; Jackson ImmunoResearch, USA) was used at a ratio of 1∶100,000 for Western blot. Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) antibody (115-035-003; Jackson ImmunoResearch, USA) was used at a ratio of 1∶100,000 for Western blot. All solvents used were of analytical reagent grade. Protein expression and purification The plasmid encoding for ELP90 (from addgene.org, #68392) and GFP-ELP90 were transformed into chemically competent Escherichia coli BLR (DE3) cells (Novagen). The protocols for polypeptide production were the same as previously described.33 Simplistically, protein was enriched by Ni-sepharose chromatography, followed by anion-exchange chromatography to remove endotoxin. Protein was lyophilized and stored at −80 °C. MesoScale Aβ analysis 4G8 MesoScale Aβ 3-plex kits were used to measure Aβ peptides levels in 96 well-based assays. In detail, the plates were blocked with diluent and incubated with shaking for 1 h. About 25 μL of detection antibodies were added to the 96-well plates. Next, 25-μL MesoScale protein standards and their samples were added into the above plates, and the plates were incubated on the shaker for 2 h. Next, electrochemi-luminescence signals of the standard and protein samples were read by the MesoScale SQ 120 system (Meso Scale Diagnostics LLC, USA). Finally, the Aβ42 levels were analyzed by MesoScale protein standards. Animal studies Animal experiments were approved by the Institutional Animal Care and Use Committee of Jilin University. ELP90 were administrated via intravenous injection (i.v.) and intracerebroventricular injection (i.c.v.), and four groups of elevated doses were administrated by PBS, 50, 200, and 800 μg·mL−1, and three 4–8 week male C57BL/J mice were employed in each group. In the i.v. group, 100 μL PBS or ELP90 solution were injected for three consecutive days. In the i.c.v. group, all mice were anesthetized by 10% (w/v) sodium pentobarbital, locating the hippocampal area with the stereotactic positioning system and drilling a litter hole on the skull of the right hemisphere, then slowly inserting the microsyringe (catalog 80383; Hamilton, Switzerland, needle size 30 s) into the brain at a coordinate of x:1.5 mm, y:−1.7 mm, z:1.8 mm from the bregma. Then, 1 μL PBS or ELP90 was injected into the CA1 region of the hippocampus and pulled out of the needle slowly and sutured wound. The mice were placed in a warm environment until Palin esthesia. All animals were housed in individually ventilated cages in the animal experimental platform at the core facilities for life science at Jilin University, which were maintained three per cage and subjected to 20 °C to −22 °C on a 12 h dark/light cycle in 40–60% humidity. Additional experimental details are included in the Supporting Information. Results and Discussion ELPs are a family of recombinant engineered biopolymers comprised of repeats of canonical sequences derived from ECM native elastin. The remarkable feature of ELPs is its thermal-related phase-transition behavior. ELPs can be switched from water-soluble forms to hydrophobic proteinaceous aggregates in response to increasing temperature. This transition is completely reversible and is also known as lower critical solution temperature behavior. The inverse phase transition temperature (Tt) can be influenced by changing the amino acid sequence of the pentapeptide unit, the molecular weight, protein concentration, and so on.34,35 The primary structure of ELPs consists of repetitive pentapeptide units (VPGVG)n with low complexity.36–38 ELP90, which has 96 (VPGVG) repeats and a molecular weight of 39.68 kDa, was used in this study ( Supporting Information Figure S1). The protein was purified by chromatography and characterized via polyacrylamide gel electrophoresis (PAGE) (Figure 1). The Tt of ELP90 was quantitatively evaluated using protein solutions at concentrations of 50, 100, and 200 μg·mL−1, respectively.33 The Tt for the group with a concentration of 200 μg·mL−1 was ∼37 °C, whereas the Tt increased to higher than 37 °C when the solution was 100 or 50 μg·mL−1. The Tt for ELP90 solutions was shown to be concentration-dependent, with an increased temperature at lower concentrations. Thus, ELP90 solutions with concentrations of 200 μg·mL−1 and lower remained soluble at physiological temperatures ( Supporting Information Figure S2). Figure 1 | Overview of ELPs and microglia in brain. Plasmid-expressing ELPs was transformed into BL21 (DE3) E. coli cells. The SDS-PAGE analysis of purified ELP90. M, TransplusII prestained protein ladder. The protein purity was analyzed by ImageJ. The results showed 96% purity of ELP90. Download figure Download PowerPoint HMC3 cells were employed to further investigate the effects of ELPs on microglial phagocytosis in vitro. HMC3 cells were treated with or without ELP90 in the presence of exogenous Aβ42 peptides, which were set to mimic the abnormal extracellular environment in Aβ pathology. Then we assayed the levels of intracellular APP and extracellular product resulting from hydrolase-catalyzed APP cleavage, including neurotoxic Aβ42 and soluble N-terminal ectodomain of APP (sAPPα) (Figures 2a–c). We showed that the group treated with 100 μg·mL−1 of ELP90 had elevated extracellular sAPPα and Aβ42 levels compared with the other two groups (Figures 2a and 2b). It suggested that ELP90 promoted both α- and β-secretase pathways for APP cleavage, followed by releasing the product to extracellular space, and antagonized microglial phagocytosis of HMC3 cells to prevent outside Aβ42 clearance. However, the levels of intracellular APP did not change significantly with ELP90 treatment (Figure 2c and Supporting Information Table S1). It is speculated that this may be based on the compensatory effects of the increased synthesized APP by cells, which is consistent with our previous findings.33 Figure 2 | The effects of ELP90 on Aβ levels and APP levels in human microglial HMC3 cells. (a) Western blotting analysis of the extracellular levels of AD-related proteins in HMC, including sAPPα, Aβ42, and APP. Control: cells treated with PBS; Aβ42: cells treated with 2 µg·mL−1 of Aβ42; Aβ42 + ELP90: cells treated with 2 µg·mL−1 of Aβ42 and 100 µg·mL−1 of ELP90. Aβ42 and sAPPα were detected with 6E10 antibody and APP with G12A. Quantifications of the immunoreactivities to the antibodies in each panel are shown in (b) and (c). Y-axis values represented relative signal intensities (standardized by β-actin levels) of each band (**P < 0.01). (d) Confocal fluorescence microscopy analysis of GFP-ELP90 in previously reported 7PA2 AD model cells, a Chinese hamster ovary cell model that stably expresses the V717F human APP mutation.33,39 Cells were incubated with PBS (control) and 100 µg·mL−1 of GFP-ELP90. Nucleus in blue fluorescence and APP in red (immunofluorescence was labeled by 22C11). The absence of GFP-specific fluorescence indicated that the cells have negligible uptake of ELPs. (e) Quantification of intracellular Aβ42 levels in HMC3 following treatment of 2 µg·mL−1 Aβ42, alone, or in combination with 100 µg·mL−1 of ELP90 for 3 h, using the electrochemiluminescence technology of the MSD platform. Aβ42 were detected with the monoclonal antibody 4G8. The MSD-Aβ analysis showed a significant reduction (P < 0.05) by 58.5% in the intracellular Aβ42 levels in the test group (ELP90/Aβ42) when compared with the group treated with only Aβ42. Download figure Download PowerPoint To elucidate the possible mechanism responsible for the effects on the Aβ levels and microglial phagocytosis, we carried out the fluorescence assay with GFP-ELP90, a recombinant protein generated by genetic fusion of green fluorescent protein (GFP) to the N-terminal of ELP90 for additional fluorescent functionality ( Supporting Information Figure S3). Intracellular fluorescence of HMC3 cells treated with the GFP-ELP90 sample was observed when incubated for 3 h and subjected to immunocytochemistry (ICC) analysis (Figure 2d). No GFP-specific fluorescence was found in the 7PA2 AD model cells, indicating the mechanisms of ELPs influencing cells occurred extracellularly and did not require cell entrance. The APP-specific red fluorescence signal by ICC further suggested unaltered 22C11-responsive APP levels, consistent with Figure 2c. The immunofluorescence assay was performed using anti-His antibodies, for further analysis ( Supporting Information Figure S4). It showed that ELP90 was hardly detected in the treated 7PA2 cells, which was consistent with the ICC test. To detect the effects of ELP90 on the intracellular Aβ42 levels, the electrochemiluminescence technology of the Meso Scale Discovery (MSD) platform was applied to quantitatively evaluate the levels of intracellular Aβ42. As shown in Figure 2e, we found a significant reduction in the intracellular Aβ42 levels in the test group (ELP90/Aβ42) when compared with the group treated with only Aβ42. These results demonstrate ELP90-modulated Aβ dynamics via decreasing intracellular levels and augmentation of extracellular levels. In summary, these results indicate that ELP90 antagonizes microglial phagocytosis of HMC3 to prevent outside Aβ42 clearance and promotes the cleavage of APP. The role of ELPs on microglia's activity in animals was investigated. Since we previously reported association of ELP90 in upregulation of amyloid pathology in vitro and in vivo,33 we hypothesized that ELP90 might induce the microglia activation in the brain. To verify this hypothesis, ELP90 was administrated to mice via i.v. and i.c.v., respectively. The ELP90 will aggregate when the concentration increases. Otherwise, it will remain soluble under this physiological temperature. Therefore, we chose concentrations of 50, 200, and 800 μg·mL−1 to simulate different states of ELP90 in vivo, which can help in understanding the effects of aggregation/solubilization of ELP protein to microglia/Aβ. Four groups of gradient doses were administrated as 0 (PBS), 50, 200, and 800 μg·mL−1. The endotoxins of all samples applied to animal experiments meet the requirement of preclinical research ( Supporting Information Figure S5). In the i.v.-treated groups, 100 μL of PBS or ELP90 solutions were injected for three consecutive days. In the i.c.v. groups, all mice were injected with 1 μL of PBS or ELP90 solutions directly into the cornu ammonis 1 (CA1) of the hippocampus. After 1 or 2 months, mice brains were extracted and divided into right and left hemispheres for slicing and homogenizing, respectively. These brain slices were imaged by immunohistochemistry (IHC) assay with anti-Iba1 antibody, which revealed microglia activation in the brain (Figure 3a). In comparison with the i.v. groups, the i.c.v. groups exhibited more obvious microglia activation and the microglial activation levels increased with elevated doses of ELP90 in both groups (Figure 3a). Furthermore, the steady-state levels of Iba1 were explored by western blotting analysis of the mice brain homogenate (Figure 3b). The levels of Iba1 were elevated in the i.c.v. group administered with 200 μg·mL−1 of ELP90 (Figures 3c and 3d), which was consistent with the tendency in the hippocampus, reflecting a strong upregulation effect of ELP90 on microglial activation in the brain (Figure 3a and Supporting Information Figure S6). Figure 3 | (a) IHC assay of mice (C57BL/6, 8-week-old male, 3 mice per group) brain slides after 1 month postadministration with 50, 200, or 800 μg·mL−1 of ELP90, respectively, via i.v. (upper panel) and i.c.v. (lower panel). Slides were stained with the Iba1 antibody for microglia. Brown pixels show Iba1-positive cells. Scale bar: 0.4 mm. (b) Quantifications of the immunoreactivities in (a). It showed that ELP90-induced activation of mouse microglia inside the hippocampus. (c) Western blotting analysis of the Iba1 expression levels in microglia in brain homogenate after 1 month postadministration with ELP90 samples. (d) Quantifications of the immunoreactivities to the antibodies in each panel. Y-axis values represented relative signal intensities (standardized by GAPDH levels) of each band. These results demonstrate that the levels of Iba1 were upregulated in the i.c.v. group administered with 200 μg·mL−1 of ELP90. Download figure Download PowerPoint Our results suggest that ELPs are important risk proteins involved in AD development through a dual pathway (Figure 4). On the one hand, the cleavage of APP in microglia or neuron33 is enhanced in the presence of increased ELPs, which can be attributed to the activation on transcription of intracellular γ-secretase and β-secretase by ELPs, instead of the overexpression of APP in cells. On the other hand, ELPs antagonize microglial phagocytosis of Aβ. Both of these effects lead to significant accumulation of extracellular Aβ, which then notably induces positive feedback of microglial activation, as shown by in vivo assays. These findings increase elastin regulatory function in CNS, especially for neuroinflammatory reactions, and might offer new specific microglial-targeted therapeutic strategies for AD. To further investigate the effects of ELP proteins on proliferation and differentiation of microglia, cell-loaded ELP90 3D constructs, which enable fine-tuned control over cell–material interactions, can be fabricated. Microglia or co-culture of microglia with other cells can be incubated in such constructs, followed by biochemical characterization, such as flow cytometry analysis, confocal microscopy, and so on. These results might open interesting opportunities for the detection and treatment of AD with specific ECM proteins as biomarkers or therapeutic targets. Figure 4 | Proposed schematic for ELPs-mediated regulation of microglial activity. Download figure Download PowerPoint Conclusion In summary, we have investigated the pleiotropic effects of ECM elastin molecules on microglial cells in vitro and in vivo. The in vitro test showed that ELP90 can antagonize microglial phagocytosis to prevent Aβ clearance and promote APP cleavage by its pathway, leading to accumulation of Aβ42 and APP cleavage products in extracellular space. Accumulation of Aβ42 may potentially aggravate AD pathology, including tau pathology and microglial activation-related neuroinflammation40,41, supported our in vivo results revealing microglia upregulation in the brain after ELP90 treatment. Collectively, the elastin from brain ECM can potentially induce the pathological changes in AD, implying that ELPs and other relevant biomacromolecules should be further investigated, which may serve as valuable model systems to better modulate and elucidate the pathogenesis of AD. Supporting Information Supporting Information is available. Conflict of Interest There is no conflict of interest to report. Funding Information This research was made possible as a result of a generous grant from National Key R&D Program of China (grant no. 2018YFA0902600), the Scientific Instrument Developing Project of the Chinese Academy of Sciences (grant no. ZDKYYQ20180001), the Jilin Province Science Fund for Excellent Young Scholars (20190103072JH), K.C. Wong Education Foundation (grant no. GJTD-2018-09), and the National Science Foundation of China (grant and and the Alzheimer's of Alzheimer's in the and the of Tanzi of the Alzheimer's on Microglial in Alzheimer's of Microglia in Alzheimer's as a in Both and in Brain Aging and Alzheimer's of in the and of and in and of the Brain to the of Zhang Zhang in and at and with Alzheimer's of for Alzheimer's of the of and with the AD in at and with Alzheimer's Jun and as and with Jun at and are with Alzheimer's at and are with Alzheimer's Cells in R of Brain are by Ma Ma Li Liu Zhang in Alzheimer's and of Liu of a and in of the Brain for Tanzi of Alzheimer's The of the in Aging and its to of Aging on Elastin in Ma Li Zhang Tanzi Liu Zhang of Alzheimer's in a by Elastin Liu by Li of the of an of of that and of Protein in of Aβ, sAPPα and and and in Information & Chinese

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