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Pathogenic role of RAGE in tau transmission and memory deficits

Open AccessPublished:October 29, 2022DOI:https://doi.org/10.1016/j.biopsych.2022.10.015

      ABSTRACT

      Background

      In tauopathies, brain regions with tau accumulation strongly correlates with clinical symptoms and spreading of misfolded tau along neural network leads to disease progression. However, the underlying mechanisms by which tau proteins enter neurons during pathologic propagation remain unclear.

      Methods

      To identify membrane receptors responsible for neuronal propagation of tau oligomers, we established a cell-based tau uptake assay and screened cDNA expression library. Tau uptake and propagation were analyzed in vitro and in vivo, particularly using microfluidic device and stereotaxic injection. The cognitive function of mice was analyzed by behavioral tests.

      Results

      From a genome-wide cell-based functional screening, RAGE (receptor for advanced glycation end products) was isolated to stimulate the cellular uptake of tau oligomers. Rage deficiency reduced neuronal uptake of pathological tau prepared from rTg4510 mouse brains or cerebrospinal fluids from Alzheimer’s disease patients, and slowed tau propagation between neurons cultured in a three-chamber microfluidic device. RAGE levels were increased in the brains of rTg4510 mice and tau oligomer-treated neurons. Rage knockout decreased tau transmission in the brains of nontransgenic mice after injection with Alzheimer’s disease patient-derived tau and ameliorated memory loss after injection with GFP-P301L tau AAV. Treatment of RAGE antagonist FPS-ZM1 blocked transsynaptic tau propagation and inflammatory responses, and alleviated cognitive impairment in rTg4510 mice.

      Conclusions

      These results suggest that RAGE on neurons and microglia binds to pathological tau, and facilitates neuronal tau pathology progression and behavioral deficits in tauopathies.

      Keywords

      INTRODUCTION

      The distribution of pathological tau inclusions widens during disease progression and strongly correlates with clinical stages in tauopathies including Alzheimer’s disease (AD) (
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ). Recent advances in tau imaging enabled the prediction of patient-specific patterns of tau spreading along neural connectivity (
      • Brown J.A.
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      • Sible I.J.
      • Sias A.C.
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      • et al.
      Patient-Tailored, Connectivity-Based Forecasts of Spreading Brain Atrophy.
      ,
      • Vogel J.W.
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      • Smith R.
      • Ossenkoppele R.
      • Strandberg O.T.
      • et al.
      Four distinct trajectories of tau deposition identified in Alzheimer’s disease.
      ). Previous studies have focused on demonstrating transsynaptic propagation of various tau species in vitro (
      • Takeda S.
      • Wegmann S.
      • Cho H.
      • DeVos S.L.
      • Commins C.
      • Roe A.D.
      • et al.
      Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer’s disease brain.
      ,
      • Wu J.W.
      • Herman M.
      • Liu L.
      • Simoes S.
      • Acker C.M.
      • Figueroa H.
      • et al.
      Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons.
      ) and in vivo (
      • Clavaguera F.
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      • Abramowski D.
      • Frank S.
      • Probst A.
      • et al.
      Transmission and spreading of tauopathy in transgenic mouse brain.
      ,
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      • Polydoro M.
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      • William C.
      • Adamowicz D.H.
      • Kopeikina K.J.
      • et al.
      Propagation of Tau Pathology in a Model of Early Alzheimer’s Disease.
      ,
      • Guo J.L.
      • Narasimhan S.
      • Changolkar L.
      • He Z.
      • Stieber A.
      • Zhang B.
      • et al.
      Unique pathological tau conformers from Alzheimer’s brains transmit tau pathology in nontransgenic mice.
      ,
      • Wegmann S.
      • Maury E.A.
      • Kirk M.J.
      • Saqran L.
      • Roe A.
      • DeVos S.L.
      • et al.
      Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity.
      ). It has placed emphasis on macropinocytosis-mediated tau internalization in neurons (
      • Wu J.W.
      • Herman M.
      • Liu L.
      • Simoes S.
      • Acker C.M.
      • Figueroa H.
      • et al.
      Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons.
      ,
      • Frost B.
      • Jacks R.L.
      • Diamond M.I.
      Propagation of Tau Misfolding from the Outside to the Inside of a Cell.
      ), mediated in large part by heparin sulfate proteoglycans (HSPGs), which bind to extracellular tau aggregates and promote their cellular uptake (
      • Holmes B.B.
      • DeVos S.L.
      • Kfoury N.
      • Li M.
      • Jacks R.
      • Yanamandra K.
      • et al.
      Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds.
      ). Low-density lipoprotein receptor-related protein 1 (LRP1) operates cooperatively with HSPGs to control tau entry into neurons (
      • Rauch J.N.
      • Luna G.
      • Guzman E.
      • Audouard M.
      • Challis C.
      • Sibih Y.E.
      • et al.
      LRP1 is a master regulator of tau uptake and spread [no. 7803].
      ), while its contribution to tau pathogenesis has not been determined.
      In addition, dynamin-dependent endocytosis pathway was proposed to selectively regulate internalization of P301S tau aggregates into human iPSC-derived neurons (
      • Evans L.D.
      • Wassmer T.
      • Fraser G.
      • Smith J.
      • Perkinton M.
      • Billinton A.
      • Livesey F.J.
      Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.
      ). BIN1/Amphiphysin2, the genetic risk factor for late-onset AD, was also shown to modulate clathrin-mediated endocytosis of P301L tau aggregates and their transsynaptic propagation in vitro (
      • Calafate S.
      • Flavin W.
      • Verstreken P.
      • Moechars D.
      Loss of Bin1 Promotes the Propagation of Tau Pathology.
      ). Given that tau strains found in various tauopathies are highly heterogeneous (
      • Clavaguera F.
      • Akatsu H.
      • Fraser G.
      • Crowther R.A.
      • Frank S.
      • Hench J.
      • et al.
      Brain homogenates from human tauopathies induce tau inclusions in mouse brain.
      ,
      • Kaufman S.K.
      • Sanders D.W.
      • Thomas T.L.
      • Ruchinskas A.J.
      • Vaquer-Alicea J.
      • Sharma A.M.
      • et al.
      Tau Prion Strains Dictate Patterns of Cell Pathology, Progression Rate, and Regional Vulnerability In Vivo.
      ), there is an urgent need to identify the diverse modulators that are responsible for pathologic propagation of toxic tau forms. Although tau aggregates are considered to be the major pathological form to be transmitted between neurons, the precise mechanism by which they enter neurons largely remains unclear.
      In the present study, we performed cell-based assays to isolate membrane receptors responsible for cellular uptake of tau oligomers. We identified a receptor for advanced glycation end products (RAGE, also called AGER) from the screen and found that RAGE mediates neuronal uptake of pathological forms of tau. Rage deficiency reduced transsynaptic tau propagation in vitro and in vivo, and suppressed microglial inflammatory responses. Moreover, RAGE was required for tau-induced memory loss and blocking the interaction between RAGE and tau oligomers ameliorated cognitive impairment in rTg4510 mice. Overall, these results suggest the role of RAGE in tau pathogenesis.

      METHODS AND MATERIALS

      Cell-based tau uptake receptor screen using cDNA expression library

      SH-SY5Y cells were co-transfected for 24 h with pRFP-N1 and each cDNA encoding human and mouse transmembrane proteins in the mammalian expression vectors (total 1,523). The cDNAs tested for cell-based tau uptake screen are listed in Table S1. The pcDNA3 and SDC1 cDNA were used as negative and positive controls, respectively. Cells were treated for 6 h with 500 nM DyLight 488-tau aggregates, washed with PBS and extracellular DyLight 488 signals were quenched with 0.05% trypan blue. Cellular uptake of tau aggregates was automatically visualized using INCell Analyzer 2000 (GE Healthcare). Images were obtained from two random fields of each well and intracellular DyLight 488 signal intensities in the RFP-positive cells were measured using ImageJ.

      Mice

      The rTg4510 mice were obtained by crossing the human P301L tau responder line (The Jackson Laboratory, #015815) to a tetracycline-controlled transactivator (tTA) line (The Jackson Laboratory, #016198). Rage-/- mice on the C57BL/6 background were kindly provided by Dr. A. M. Schmidt (New York University School of Medicine) and Dr. S. Vogel (University of Maryland School of Medicine) (
      • Liliensiek B.
      • Weigand M.A.
      • Bierhaus A.
      • Nicklas W.
      • Kasper M.
      • Hofer S.
      • et al.
      Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response.
      ). The littermates of WT and Rage-/- mice were used in stereotaxic injection of Tau PHFs from AD brains.

      Generation of Ager knockout (Rage KO) mice

      Ager-deficient mice were generated from the embryos (EM ID: 02352, LEXKO-2071) that were obtained from the European Mouse Mutant Archive (EMMA). The received embryos had deletion between exons 2 through 4 of the Ager gene, leaving only one LoxP site. The corresponding nucleotide sequence for LoxP excision region was validated by direct-sequencing analysis (Bionics Co., Ltd., Seoul, Korea). The genotyping for targeted allele was performed by PCR analysis using the following primers: forward 5′-AGT GTC CTC AGG TCG GGT GA-3′ and reverse 5′-CCA TCT AAG TGC CAG CTA AGG GTC-3′. Mice were backcrossed and maintained on a C57BL/6N background.

      Tau uptake assay in primary cultured cells

      Mouse primary cortical neurons at 7 days in vitro (DIV 7) were incubated for 24 h with 500 nM DyLight 488-tau oligomers. HSPG-mediated tau internalization was blocked by co-treatment with 15 U/ml heparin (Sigma-Aldrich). The effect of RAGE antagonist was assessed by co-treatment with 1 μM FPS-ZM1 (Calbiochem). To examine cellular uptake of pathology-associated tau, neurons were incubated for 24 h with PBS-soluble rTg4510 brain extracts containing 50 ng/ml human tau or with 1:20 diluted CSF prepared from human AD patients. Mouse primary microglia and astrocytes (DIV 14) were incubated for 24 h with 100 nM DyLight 488-tau oligomers. Cells were washed with PBS, fixed with 4% paraformaldehyde, and processed for immunocytochemistry. Images were obtained using confocal laser scanning microscopy LSM700 (Carl Zeiss) and intracellular tau signal intensities were measured using ImageJ.

      Purification and stereotaxic injection of Tau PHFs from AD brains (AD-tau)

      AD-tau was purified from human brain tissues of AD patients (Harvard Brain Tissue Resource Center, McLean Hospital, Massachusetts) as previously described (
      • Guo J.L.
      • Narasimhan S.
      • Changolkar L.
      • He Z.
      • Stieber A.
      • Zhang B.
      • et al.
      Unique pathological tau conformers from Alzheimer’s brains transmit tau pathology in nontransgenic mice.
      ). The detailed procedure is described in the Supplemental Information. Purified AD-tau was confirmed by western blotting using PHF-1 antibody, negative staining EM images before and after sonication and total tau and Aβ1-42 ELISA (ThermoFisher Scientific), which contains minimal Aβ1-42. Four-month-old C57BL/6 WT or Rage-/- mice were deeply anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). PBS or AD-tau (4 μg/site) was unilaterally injected into dorsal hippocampus and overlying cortex with the following coordinates: anteroposterior = −2.5 mm, mediolateral = +2.0 mm, dorsoventral = −2.4 mm and −1.4 mm, respectively from bregma. After the injection, the needle was maintained for an additional 5 min for a complete absorption of the solution. Animals were monitored and post-surgical care was provided. For immunohistochemical analysis 6 months after injection, mice were perfused with PBS and 4% paraformaldehyde and brains were removed, followed by fixation in 4% paraformaldehyde overnight and transfer to 30% sucrose for cryoprotection. The microglia of WT and Rage-/- mice were enriched from the brain and subjected to immunoblotting and quantitative PCR analysis.

      In vivo tau propagation using AAV system

      The 3-month-old mice (male) were anesthetized with a mixture of tiletamine/zolazepam (30 mg/kg) and xylazine (10 mg/kg), and intracranially injected with 5 μl GFP-P301L tau AAV (6.5x1010 ifu/ml) into the left hippocampus (stereotaxic coordinates: anteroposterior = −2.1 mm, mediolateral = 1.8 mm, and dorsoventral = −2.0 mm from bregma) using 30-gauge Hamilton microsyringe at the rate of 0.5 μl/min. After 20 weeks, mice were analyzed with behavioral tests. For neuronal RAGE reconstitution experiment, full-length (FL) and ΔV mutant RAGE were subcloned into the AAV vector with CaMKIIa promoter and their AAVs were purified (KIST Virus Facility). After 3 weeks of GFP-P301L tau AAV injection, WT mice were subsequently injected with 10 μl Control AAV (1.8x1013 GC/ml), and Rage KO mice were injected with 10 μl RAGE FL AAV (1.93x1013 GC/ml) or RAGE ΔV AAV (2.13x1013 GC/ml) into the cerebral ventricle. Mice were then anesthetized and perfused with PBS containing 10 U/ml heparin and 4% paraformaldehyde. Brain sections (40 μm) were prepared from the hippocampus and processed for immunohistochemistry. Images were obtained using confocal laser scanning microscopy LSM700.

      Study approval

      All mice used in this study were maintained in a specific pathogen-free animal facility. All housing, breeding, and procedures were performed according to the Korean Ministry of Food and Drug Safety (MFDS) and NIH Guide for the Care and Use of Experimental Animals, and approved by Seoul National University and Johns Hopkins University Animal Care and Use Committee. Frozen human brain tissues of AD patients were provided by Harvard Brain Tissue Resource Center (McLean Hospital, Massachusetts). This study was approved by the ethics committee of Seoul National University Bundang Hospital and Seoul National University. All participants gave informed consent to the use of clinical data for research purposes.
      More detailed methods and materials used in the study are described in the Supplemental Information.

      RESULTS

      Isolation of RAGE as a prominent receptor for tau oligomer uptake

      To identify membrane receptors responsible for neuronal propagation of tau oligomers, we established a cell-based tau uptake assay. Purified His-tagged human tau protein was labeled with DyLight 488 (DyLight 488-tau) and was allowed to form aggregates after incubation with heparin. Using fast protein liquid chromatography (FPLC), we found that tau aggregates occurred mostly as oligomeric (10-20 units) and fibrillar (≥ 40 units) forms (Figure S1A, C). We have collected mammalian expression complementary DNAs (cDNAs) encoding full-length human and mouse transmembrane proteins (total 1,523). For the assay, SH-SY5Y human neuroblastoma cells were transfected with each cDNA and incubated with DyLight 488-tau aggregates (Figure 1A).
      Figure thumbnail gr1
      Figure 1RAGE is required for the internalization of tau oligomers. (A) Schematic representation of the cell-based tau uptake screen. Cells were transfected with cDNA clones encoding membrane proteins and incubated with DyLight 488-tau aggregates. Extracellular DyLight 488 signals were quenched with trypan blue and cellular uptake of tau aggregates was visualized. (B and C) Overexpression of RAGE increases cellular tau uptake. From the screen, mean intracellular DyLight 488 intensities of putative positive clones were measured (n = 3). (D) RAGE internalizes tau oligomers. Primary cortical neurons were treated with DyLight 488-tau oligomers and tau uptake was visualized. (E) Quantification of the intracellular DyLight 488 signal intensities. Unpaired t-test, two-tailed, 30 cells per group, n = 4. LMW, ****P < 0.0001, t = 4.494; HMW, ****P < 0.0001, t = 4.259. (F) RAGE regulates tau uptake in a HSPG-independent manner. Primary cortical neurons were treated with DyLight 488-tau oligomers with or without 15 U/ml heparin and the intracellular DyLight 488 signal intensities were measured. Two-way ANOVA with Tukey test, 30 cells per group, n = 3. Interaction effect F(1, 116) = 0.8645, WT/heparin versus WT/heparin+, ****P < 0.0001; WT/heparin versus KO/heparin, ****P < 0.0001; WT/heparin+ versus KO/heparin+, ****P < 0.0001; KO/heparin versus KO/heparin+, ***P = 0.0005. (G) Rage deficiency reduces tau oligomer binding. Primary cortical neurons were treated with biotin-labelled LMW (left) or HMW (right) tau oligomers and cell-bound biotin signal intensities were measured. The dissociation constants (Kd) of the tau binding to RAGE (ΔRage Kd) was obtained by subtracting tau binding to Rage KO neurons from tau binding to WT neurons (LMW, n = 6; HMW, n = 4). (H) RAGE mediates neuronal accumulation of pathology-associated tau. Primary cortical neurons were incubated with PBS-soluble brain extracts from 12-month-old rTg4510 mice (50 ng/ml human tau). (I) Quantification of intracellular human tau intensities. Mann-Whitney test, two-tailed, 30 cells per group, n = 3. ***P = 0.0001, U = 198.5. (J) RAGE internalizes tau species present in CSF from AD patients. Primary cortical neurons were treated with AD CSF and the intracellular human tau intensities were measured. Mann-Whitney test, two-tailed, 30 cells per group, n = 3. ****P < 0.0001, U = 93. Scale bars in C, D, H, 10 μm. Data in E-G, I, J are represented as mean ± SEM.
      From the screen, we isolated a list of putative positive clones that enhanced cellular uptake of tau aggregates (Figure S2A-C). Among them, RAGE most efficiently mediated internalization of tau aggregates by the transfected cells (Figure 1B-C). In the assay, overexpression of RAGE or recently reported tau receptor LRP1 (
      • Rauch J.N.
      • Luna G.
      • Guzman E.
      • Audouard M.
      • Challis C.
      • Sibih Y.E.
      • et al.
      LRP1 is a master regulator of tau uptake and spread [no. 7803].
      ) did not affect the basal level of the other in SH-SY5Y cells (Figure S2D-F). Monomeric, oligomeric and fibrillar forms of biotin-labeled tau proteins (biotin-tau) were then prepared and assayed for their interaction with RAGE. The results revealed that RAGE bound to a broad array of tau forms, but with significant preference for tau oligomers (Figure S3A-B). In vitro co-immunoprecipitation assays also showed that RAGE primarily bound to tau oligomers (Figure S3C-F).
      Using FPLC, we further divided tau oligomer species into low-molecular-weight (LMW, 2-4 units) and high-molecular-weight (HMW, 10-20 units) forms (Figure S1B-C). Incubating primary cultured wild-type (WT) and Rage knockout (KO) neurons with tau oligomers revealed that cellular uptake of LMW and HMW tau oligomers by Rage KO neurons significantly diminished as compared to WT neurons (Figure 1D-E). Interestingly, the levels of LRP1 were a little increased in Rage KO neurons compared to WT neurons (Figure S4A-B). Thus, the decrease of tau uptake by Rage KO is LRP1-independent. HSPG-mediated macropinocytosis was recently highlighted as a mechanism for cellular tau uptake (
      • Holmes B.B.
      • DeVos S.L.
      • Kfoury N.
      • Li M.
      • Jacks R.
      • Yanamandra K.
      • et al.
      Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds.
      ). Indeed, antagonizing HSPGs using heparin reduced uptake of tau oligomers into WT neurons (Figure 1F and Figure S4C). Intriguingly, tau oligomer uptake into Rage KO neurons was efficiently blocked by heparin (Figure 1F and Figure S4C), indicating RAGE-mediated neuronal tau uptake is HSPG-independent. We then prepared biotin-tau oligomers, assayed for their interaction with RAGE by treating WT and Rage KO neurons, and subtracted tau binding to Rage KO neurons from tau binding to WT neurons. The dissociation constants (Kd) for LMW and HMW tau oligomers binding to RAGE were 43.47 nM and 24.96 nM equivalent of tau monomer, respectively (Figure 1G).

      Rage knockout reduces pathologic tau uptake in neurons and microglia

      Given that diverse tau strains are subject to neuronal propagation (
      • Clavaguera F.
      • Akatsu H.
      • Fraser G.
      • Crowther R.A.
      • Frank S.
      • Hench J.
      • et al.
      Brain homogenates from human tauopathies induce tau inclusions in mouse brain.
      ,
      • Kaufman S.K.
      • Sanders D.W.
      • Thomas T.L.
      • Ruchinskas A.J.
      • Vaquer-Alicea J.
      • Sharma A.M.
      • et al.
      Tau Prion Strains Dictate Patterns of Cell Pathology, Progression Rate, and Regional Vulnerability In Vivo.
      ), we assessed neuronal uptake of tau proteins prepared from rTg4510 mice, which express a human P301L mutant tau under the control of the Ca2+-calmodulin kinase II (CaMKII) promoter (
      • SantaCruz K.
      • Lewis J.
      • Spires T.
      • Paulson J.
      • Kotilinek L.
      • Ingelsson M.
      • et al.
      Tau Suppression in a Neurodegenerative Mouse Model Improves Memory Function.
      ). When treated with the phosphate-buffered saline (PBS)-soluble brain extracts from rTg4510 mice which contain LMW and HMW tau oligomers (
      • Takeda S.
      • Wegmann S.
      • Cho H.
      • DeVos S.L.
      • Commins C.
      • Roe A.D.
      • et al.
      Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer’s disease brain.
      ), human P301L tau proteins were observed inside the WT neurons, while it was significantly decreased in Rage KO neurons (Figure 1H-I). We also tested uptake of the tau proteins purified from the cerebrospinal fluid of patients with AD (AD CSF) containing phospho-tau181 and T22-reactive tau oligomer (
      • Gordon B.A.
      • Friedrichsen K.
      • Brier M.
      • Blazey T.
      • Su Y.
      • Christensen J.
      • et al.
      The relationship between cerebrospinal fluid markers of Alzheimer pathology and positron emission tomography tau imaging.
      ,

      Schoonenboom NSM, Reesink FE, Verwey NA, Kester MI, Teunissen CE, Ven PM van de, et al. (2012): Cerebrospinal fluid markers for differential dementia diagnosis in a large memory clinic cohort. Neurology 78: 47–54.

      ) (Figure S4D). Similarly, neuronal uptake of tau proteins in AD CSF was active in WT neurons but was significantly impaired in Rage KO neurons (Figure 1J and Figure S4E). Moreover, Rage deficiency also blocked neuronal tau uptake in vivo when purified tau oligomers were injected into the hippocampus of WT and Rage KO mice (Figure S4F-H).
      Since RAGE is also expressed in microglia and astrocytes (
      • Lue L.-F.
      • Walker D.G.
      • Brachova L.
      • Beach T.G.
      • Rogers J.
      • Schmidt A.M.
      • et al.
      Involvement of Microglial Receptor for Advanced Glycation Endproducts (RAGE) in Alzheimer’s Disease: Identification of a Cellular Activation Mechanism.
      ,
      • Sasaki N.
      • Toki S.
      • Chowei H.
      • Saito T.
      • Nakano N.
      • Hayashi Y.
      • et al.
      Immunohistochemical distribution of the receptor for advanced glycation end products in neurons and astrocytes in Alzheimer’s disease.
      ), we tested whether RAGE mediates tau uptake into these cells. We found that primary cultured WT microglia and astrocytes both readily internalized extracellular tau oligomers (Figure S5A-B). In microglia, Rage KO reduced tau oligomer uptake as seen in neurons (Figure S5A). However, uptake of tau oligomers into astrocytes was unaffected by Rage deficiency (Figure S5B). Recent studies have shown that tau aggregates activate microglia, which promotes tau pathogenesis via releasing tau seeds and/or inflammatory cytokines (
      • Asai H.
      • Ikezu S.
      • Tsunoda S.
      • Medalla M.
      • Luebke J.
      • Haydar T.
      • et al.
      Depletion of microglia and inhibition of exosome synthesis halt tau propagation.
      ,
      • Wang C.
      • Fan L.
      • Khawaja R.R.
      • Liu B.
      • Zhan L.
      • Kodama L.
      • et al.
      Microglial NF-κB drives tau spreading and toxicity in a mouse model of tauopathy [no. 1].
      ). To examine whether RAGE-mediated tau uptake in microglia affects inflammatory responses, we generated Rage KO in BV2 mouse microglial cells (Figure S6A). Similarly seen in primary cultured microglia, tau uptake was significantly reduced in Rage KO BV2 cells (BV2/sgRage) compared to control BV2 cells (BV2/sgControl) (Figure S6B). In cell conditioned medium, the levels of IL-1β, but not TNF-α, were significantly increased by treatment with tau oligomers in BV2/sgControl cells, but this increase of IL-1β was reduced in BV2/sgRage cells (Figure S6C). These results indicate that RAGE plays a role in tau-mediated inflammatory responses in microglia.

      Rage knockout reduces neuron-to-neuron tau propagation in vitro and in vivo

      To assess the role of RAGE in the transsynaptic tau propagation in vitro, we used a three-chamber microfluidic device that enabled observation of neuron-to-neuron transfer via axon extension across the chambers (
      • Takeda S.
      • Wegmann S.
      • Cho H.
      • DeVos S.L.
      • Commins C.
      • Roe A.D.
      • et al.
      Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer’s disease brain.
      ,
      • Wu J.W.
      • Herman M.
      • Liu L.
      • Simoes S.
      • Acker C.M.
      • Figueroa H.
      • et al.
      Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons.
      ,
      • Calafate S.
      • Flavin W.
      • Verstreken P.
      • Moechars D.
      Loss of Bin1 Promotes the Propagation of Tau Pathology.
      ,
      • Park J.W.
      • Vahidi B.
      • Taylor A.M.
      • Rhee S.W.
      • Jeon N.L.
      Microfluidic culture platform for neuroscience research.
      ). For this assay, primary hippocampal neurons were cultured in the three chambers: WT neurons in the 1st chambers (C1), and WT or Rage KO neurons in the 2nd and 3rd chambers (C2 and C3) (Figure 2A). We applied a volume difference of 50 μl between chambers (lesser volume on the C1) to maintain fluidic isolation between the chambers, used an adenovirus to overexpress GFP-fused human tau (GFP-tau) in C1 neurons, and evaluated GFP-tau propagation from C1 to C2 and C3. Using dot blot assays, we confirmed robust formation of detergent-insoluble tau aggregates within neurons transduced with GFP-tau adenovirus compared to neurons treated with tau oligomers (Figure 2B). After 14 days of adenoviral transduction, GFP-tau aggregates showed transsynaptic spreading from the C1 to C3 in WT neurons (Figure 2C-D). On the other hand, Rage deficiency reduced neuronal GFP-tau propagation by ∼20% (Figure 2C-D).
      Figure thumbnail gr2
      Figure 2Rage deficiency reduces tau propagation in vitro and in vivo. (A) Schematic representation of the three-chamber microfluidic device used for in vitro tau propagation assays. Neurons in the C1 were transduced with a GFP-tau adenovirus for tau oligomer formation. (B) Primary hippocampal neurons transduced with GFP-tau adenovirus produce detergent-insoluble tau aggregates. Neurons were left untreated, transduced with GFP-tau adenovirus (GFP-tau AdV), or treated with tau oligomers. Formation of intracellular tau aggregates was assessed with dot blot assays. (C) Rage deficiency reduces transsynaptic tau propagation in vitro. The propagation of GFP-tau from C1 to C2 and C3 neurons was detected. Scale bar, 20 μm. (D) Quantification of the GFP signal intensities from the chambers. Unpaired t-test, two-tailed, n = 3. C2, *P = 0.0334, t = 3.183; C3, *P = 0.0117, t = 4.392. (E) Schematic illustrations of AD-tau injections with coronal planes (upper) and diagrams of experimental timetable (bottom). Red dot indicates the injection site. (F and G) Pathologic tau propagation is decreased in the Rage KO mouse brains. AT8 immunohistochemistry (F) and signal quantification (G) in the ipsilateral hippocampus and cortex at 6 months after AD-tau inoculation into mice (n = 5 per group). Scale bar, 100 μm. Two-way ANOVA, *P < 0.05, **P < 0.005, ***P < 0.001. (H) Diagram illustrating the distribution of AT8-positive tau pathology (red dots) in the brains from coronal sections. (I) The inflammatory responses in the microglia are reduced in Rage KO mice after the inoculation of AD-tau. Representative immunoblots of NF-κB p-p65, total NF-κB p65, Iba-1 and β-actin in the microglia from AD-tau-injected WT or Rage KO mouse brain. (J) Quantification of NF-κB p-p65 levels normalized to NF-κB p65 (left) and Iba-1 levels normalized to β-actin (right). Two-way ANOVA with Tukey’s post-hoc test, n = 3 biologically independent mouse brains. **P < 0.005, ***P < 0.0005. (K) Quantitative PCR analysis of Iba1, GFAP and NeuN in the microglia from AD-tau-injected WT or Rage KO mouse brain. Two-way ANOVA, n = 3 biologically independent mouse brains. ***P < 0.0005. Data in D, G, J, K are represented as mean ± SEM.
      Next, we tested whether RAGE plays a role in tau spreading in vivo using tau proteins prepared from the brains of patients with AD (AD-tau), which contains minimal Aβ and induces the propagation of pathological tau in nontransgenic (NonTg) mice (
      • Guo J.L.
      • Narasimhan S.
      • Changolkar L.
      • He Z.
      • Stieber A.
      • Zhang B.
      • et al.
      Unique pathological tau conformers from Alzheimer’s brains transmit tau pathology in nontransgenic mice.
      ) (Figure 2E). After 6 months of unilateral injection of AD-tau into the dorsal hippocampus and overlying cortex, we detected AD-tau-seeded AT8-positive pathological tau in the hippocampus and cortex of WT mice (Figure 2F-G). On the other hand, detection of AT8-positive pathological tau was significantly lower in the brains of Rage KO mice than in WT mice (Figure 2F-G). Furthermore, tau propagation to other brain regions, such as entorhinal cortex, corpus callosum and mammillary area, was active in WT mice but was remarkably reduced by Rage deficiency (Figure 2H). We also isolated microglia from the brains of WT and Rage KO mice after the inoculation of AD-tau and examined the changes of inflammatory responses. In the assay, inoculation of AD-tau increased NF-κB signaling in both WT and Rage KO microglia, but the NF-κB signaling was significantly lower in Rage KO microglia (Figure 2I-J). Similarly, the levels of Iba-1, a marker of microglia activation, was significantly reduced by Rage KO in response to AD-tau (Figure 2I-K). Together, these results imply that RAGE on microglia plays a role in microglia activation in response to tau and contributes to pathologic tau propagation together with RAGE on neurons.

      RAGE V-C1 domains bind to tau oligomers to facilitate tau uptake

      To characterize the interaction between RAGE and tau oligomers in detail, we generated several RAGE mutants lacking the extracellular V, C1, or C2 domain (Figure 3A). Overexpressing RAGE mutants in SH-SY5Y cells revealed that deletion of the V or C1 domain reduced cellular uptake of tau oligomers (Figure 3B-C). Thus, RAGE V and C1 domains are necessary for the internalization of tau oligomers. Importantly, the G82S polymorphism of RAGE in its V domain (Figure 3A) was reported to associate with the increased AD susceptibility (
      • Daborg J.
      • von Otter M.
      • Sjölander A.
      • Nilsson S.
      • Minthon L.
      • Gustafson D.R.
      • et al.
      Association of the RAGE G82S polymorphism with Alzheimer’s disease.
      ,
      • Li K.
      • Dai D.
      • Zhao B.
      • Yao L.
      • Yao S.
      • Wang B.
      • Yang Z.
      Association between the RAGE G82S polymorphism and Alzheimer’s disease.
      ) and to promote glycosylation of RAGE, which leads to structural change and enhances ligand binding (
      • Hofmann M.A.
      • Drury S.
      • Hudson B.I.
      • Gleason M.R.
      • Qu W.
      • Lu Y.
      • et al.
      RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response [no. 3].
      ,
      • Osawa M.
      • Yamamoto Y.
      • Munesue S.
      • Murakami N.
      • Sakurai S.
      • Watanabe T.
      • et al.
      De-N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts.
      ,
      • Park S.J.
      • Kleffmann T.
      • Hessian P.A.
      The G82S Polymorphism Promotes Glycosylation of the Receptor for Advanced Glycation End Products (RAGE) at Asparagine 81 COMPARISON OF WILD-TYPE RAGE WITH THE G82S POLYMORPHIC VARIANT.
      ). Notably, overexpressing G82S RAGE slightly increased tau binding (Figure 3D-E), suggesting the effect of RAGE glycosylation on tau binding and uptake.
      Figure thumbnail gr3
      Figure 3RAGE V-C1 domains interacts with tau oligomers and mediates tau uptake. (A) Schematic representations of RAGE full-length (FL), mutants lacking extracellular ligand-binding domains (ΔV, ΔC1 and ΔC2), and a functional single-nucleotide polymorphism (G82S). FPS-ZM1 binds specifically to the V domain. SP: signal peptide, V: Ig-like V-type domain, C1: Ig-like C2-type 1 domain, C2: Ig-like C2-type 2 domain, TM: transmembrane region, Cyto: cytoplasmic domain. (B) RAGE V-C1 domains mediate tau uptake. SH-SY5Y cells were transfected with RFP or RAGE-RFP and incubated with DyLight 488-tau oligomers. (C) Quantification of the intracellular DyLight 488 signal intensities. Data are represented as violin plots depicting median and quartiles. Kruskal-Wallis test with Dunn’s test, 180 cells per group, n = 3. ****P < 0.0001. (D) RAGE G82S polymorphism enhances tau binding. SH-SY5Y cells were transfected with RFP or RAGE-RFP and left untreated or treated with biotin-tau oligomers. (E) Cell-bound biotin signal intensities were measured and normalized to that from untreated cells. One-way ANOVA with Tukey test, n = 4. F(
      • Brown J.A.
      • Deng J.
      • Neuhaus J.
      • Sible I.J.
      • Sias A.C.
      • Lee S.E.
      • et al.
      Patient-Tailored, Connectivity-Based Forecasts of Spreading Brain Atrophy.
      ,
      • Wegmann S.
      • Maury E.A.
      • Kirk M.J.
      • Saqran L.
      • Roe A.
      • DeVos S.L.
      • et al.
      Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity.
      ) = 16.38, *P = 0.0179, ***P = 0.0008. (F) Blocking RAGE V domain using antagonist reduces tau uptake. Primary cortical neurons were treated with DyLight 488-tau oligomers in the presence of 1 μM FPS-ZM1. (G) Quantification of the intracellular DyLight 488 signal intensities. Unpaired t-test, two-tailed, 40 cells per group, n = 3. ****P < 0.0001, t = 4.799. (H) RAGE antagonist blocks cellular uptake of tau and Aβ42 oligomers. SH-SY5Y cells were treated with the increasing concentrations of FPS-ZM1 in the presence of 500 nM DyLight 488-tau or 125 nM FITC-Aβ42 oligomers and the intracellular DyLight 488 and FITC intensities were measured (n = 4). Scale bars in B, D, F, 10 μm. Data in E, G, H are represented as mean ± SEM.
      We then assessed the effect of RAGE antagonist FPS-ZM1 on tau uptake. FPS-ZM1 binds to the V domain and competitively inhibits the binding of RAGE ligands including Aβ42 oligomers (
      • Bongarzone S.
      • Savickas V.
      • Luzi F.
      • Gee A.D.
      Targeting the Receptor for Advanced Glycation Endproducts (RAGE): A Medicinal Chemistry Perspective.
      ,
      • Deane R.
      • Singh I.
      • Sagare A.P.
      • Bell R.D.
      • Ross N.T.
      • LaRue B.
      • et al.
      A multimodal RAGE-specific inhibitor reduces amyloid β-mediated brain disorder in a mouse model of Alzheimer disease.
      ) (Figure 3A). Co-treatment with FPS-ZM1 inhibited uptake of tau oligomers into primary cultured neurons (Figure 3F-G). Because RAGE is also known to interact with Aβ (
      • Deane R.
      • Du Yan S.
      • Submamaryan R.K.
      • LaRue B.
      • Jovanovic S.
      • Hogg E.
      • et al.
      RAGE mediates amyloid-β peptide transport across the blood-brain barrier and accumulation in brain.
      ,
      • Yan S.D.
      • Chen X.
      • Fu J.
      • Chen M.
      • Zhu H.
      • Roher A.
      • et al.
      RAGE and amyloid-β peptide neurotoxicity in Alzheimer’s disease [no. 6593].
      ), we compared RAGE interaction with tau oligomers to Aβ42 oligomers in SH-SY5Y cells. Co-treatment with FPS-ZM1 blocked cellular uptake of tau oligomers as efficiently as Aβ42 oligomers in a dose-dependent manner (Figure 3H). Therefore, RAGE antagonist FPS-ZM1 blocks cellular uptake of tau oligomers as well as Aβ42 oligomers.

      Tau oligomers upregulate RAGE expression in neurons

      To further investigate the role of RAGE in tau pathogenesis, we analyzed the pattern of RAGE expression in the brains of rTg4510 mice, which express a human P301L tau largely restricted to forebrain structures (
      • SantaCruz K.
      • Lewis J.
      • Spires T.
      • Paulson J.
      • Kotilinek L.
      • Ingelsson M.
      • et al.
      Tau Suppression in a Neurodegenerative Mouse Model Improves Memory Function.
      ). Remarkably, the hippocampal neurons of the rTg4510 mice showed increased RAGE expression compared to the neurons of the age-matched NonTg mice (Figure 4A-B). Neurons with higher pathologic tau accumulation showed higher RAGE expression (Figure 4B). Also, RAGE expression increased up to five-fold in the primary cultured neurons upon treatment with tau oligomers (Figure 4C-D). RAGE-ligand binding is known to activate MAPK/NF-κB pathway and leads to expression of RAGE itself (
      • Kierdorf K.
      • Fritz G.
      RAGE regulation and signaling in inflammation and beyond.
      ). Treatment of tau oligomers also induced nuclear translocation of NF-κB p65 and increased RAGE expression (Figure 4E-F), which was blocked by interfering RAGE-tau binding with FPS-ZM1 (Figure 4G-H). Thus, neuronal RAGE levels are upregulated by tau oligomers, which in turn likely enhances tau propagation in a vicious cycle.
      Figure thumbnail gr4
      Figure 4Tau oligomers increase neuronal RAGE expression via NF-κB pathway. (A) Neuronal RAGE level increases in the hippocampus of rTg4510 mice. Brain sections from the hippocampus of 4.5-month-old NonTg and age-matched rTg4510 mice were immunostained. Scale bar, 10 μm. (B) Quantification of neuronal RAGE and AT8 signal intensities. Scatter plot displaying correlation between RAGE and AT8 level in the hippocampal neurons from 4.5-month-old NonTg and age-matched rTg4510 mice. Data was analyzed with Pearson correlation test (NonTg, 35 cells; rTg4510, 40 cells; n = 5 per group). (C) Extracellular tau oligomers increase RAGE expression in neurons. Primary cortical neurons were treated for 48 h with tau oligomers and analyzed by immunoblotting. (D) The relative levels of RAGE (left) and human tau (right) were normalized to β-actin. Two-way ANOVA with Bonferroni test, n = 3. Interaction effect F(
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Guo J.L.
      • Narasimhan S.
      • Changolkar L.
      • He Z.
      • Stieber A.
      • Zhang B.
      • et al.
      Unique pathological tau conformers from Alzheimer’s brains transmit tau pathology in nontransgenic mice.
      ) = 52.72, ****P < 0.0001 (left). Interaction effect F(
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Guo J.L.
      • Narasimhan S.
      • Changolkar L.
      • He Z.
      • Stieber A.
      • Zhang B.
      • et al.
      Unique pathological tau conformers from Alzheimer’s brains transmit tau pathology in nontransgenic mice.
      ) = 227.9, ****P < 0.0001 (right). (E) Extracellular tau oligomers upregulate RAGE level via NF-κB pathway. SH-SY5Y cells were treated for 48 h with tau oligomers. Cytoplasmic RAGE expression and nuclear NF-κB p65 levels were analyzed by immunoblotting. (F) The relative level of cytoplasmic RAGE was normalized to β-actin (left) and the relative level of nuclear NF-κB p65 was normalized to Lamin A/C (right). One-way ANOVA with Tukey test, n = 3. F(
      • Brown J.A.
      • Deng J.
      • Neuhaus J.
      • Sible I.J.
      • Sias A.C.
      • Lee S.E.
      • et al.
      Patient-Tailored, Connectivity-Based Forecasts of Spreading Brain Atrophy.
      ,
      • Clavaguera F.
      • Bolmont T.
      • Crowther R.A.
      • Abramowski D.
      • Frank S.
      • Probst A.
      • et al.
      Transmission and spreading of tauopathy in transgenic mouse brain.
      ) = 20.39, *P = 0.0194, **P = 0.0018 (left). F(
      • Brown J.A.
      • Deng J.
      • Neuhaus J.
      • Sible I.J.
      • Sias A.C.
      • Lee S.E.
      • et al.
      Patient-Tailored, Connectivity-Based Forecasts of Spreading Brain Atrophy.
      ,
      • Clavaguera F.
      • Bolmont T.
      • Crowther R.A.
      • Abramowski D.
      • Frank S.
      • Probst A.
      • et al.
      Transmission and spreading of tauopathy in transgenic mouse brain.
      ) = 59.23, *P = 0.0320 ***P = 0.0009, ****P < 0.0001 (right). (G) RAGE antagonist blocks tau-induced RAGE upregulation. SH-SY5Y cells were treated for 48 h with tau oligomers in the presence of 1 μM FPS-ZM1. Cytoplasmic RAGE expression and nuclear NF-κB p65 levels were analyzed by immunoblotting. (H) The relative level of cytoplasmic RAGE was normalized to β-actin (left) and the relative level of nuclear NF-κB p65 was normalized to Lamin A/C (right). One-way ANOVA with Tukey test, n = 4. F(
      • Brown J.A.
      • Deng J.
      • Neuhaus J.
      • Sible I.J.
      • Sias A.C.
      • Lee S.E.
      • et al.
      Patient-Tailored, Connectivity-Based Forecasts of Spreading Brain Atrophy.
      ,
      • Wegmann S.
      • Maury E.A.
      • Kirk M.J.
      • Saqran L.
      • Roe A.
      • DeVos S.L.
      • et al.
      Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity.
      ) = 6.234, oTau/FPS-ZM1 versus oTau+/FPS-ZM1, *P = 0.0326; oTau+/FPS-ZM1 versus oTau+/FPS-ZM1+, *P = 0.0334 (left). F(
      • Brown J.A.
      • Deng J.
      • Neuhaus J.
      • Sible I.J.
      • Sias A.C.
      • Lee S.E.
      • et al.
      Patient-Tailored, Connectivity-Based Forecasts of Spreading Brain Atrophy.
      ,
      • Wegmann S.
      • Maury E.A.
      • Kirk M.J.
      • Saqran L.
      • Roe A.
      • DeVos S.L.
      • et al.
      Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity.
      ) = 6.249, oTau/FPS-ZM1 versus oTau+/FPS-ZM1, *P = 0.0380; oTau+/FPS-ZM1 versus oTau+/FPS-ZM1+, *P = 0.0287 (right). Data in D, F, H are represented as mean ± SEM.

      RAGE blockade ameliorates tau pathogenesis and behavioral abnormality

      We then prepared an adeno-associated virus for the expression of GFP-tau with P301L mutation (GFP-P301L tau AAV) in the hippocampus and examined whether RAGE takes part in tau-induced behavioral deficits. We performed behavioral tests after 5 months of unilateral GFP-P301L tau AAV injection into the hippocampus CA3 region of WT and Rage KO mice (Figure 5A). Interestingly, WT mice exhibited significant decline in spatial memory in the Y-maze test (Figure 5B), learning and memory deficits in the novel object recognition test (Figure 5C) and passive avoidance test (Figure 5D). On the contrary, Rage KO mice did not develop such cognitive impairment (Figure 5B-D). Without GFP-P301L tau AAV injection, WT and Rage KO mice showed no significant difference in spatial memory in the Y-maze test (Figure S7A). When examined for tau propagation, MC1-positive misfolded tau was found in the GFP-positive CA3 neurons in the injection area of WT mice and was also found in the GFP-negative CA1 neurons, showing transsynaptic tau propagation in the hippocampus (Figure 5E). However, MC1-positive misfolded tau was not found in the CA1 neurons of Rage KO mice (Figure 5E).
      Figure thumbnail gr5
      Figure 5RAGE is required for tau pathogenesis and behavioral deficits. (A) Schematic representation of in vivo tau propagation assays and diagrams of experimental timetable. The left hippocampus of 3-month-old mice (male) was intracranially injected with GFP-P301L tau adeno-associated virus (6.5x1010 ifu/ml, 5 μl). (B-D) Rage deficiency delays cognitive impairment induced by unilateral viral expression of GFP-P301L tau in the hippocampus. After 5 months of injection, mice were analyzed with the Y-maze test (B, unpaired t-test, two-tailed, n = 4 per group. NS, not significant, *P = 0.0296, t = 2.839), novel object recognition test (C, unpaired t-test, two-tailed, n = 3 per group. *P = 0.0402, t = 2.993) or passive avoidance test (D, paired t-test, two-tailed, n = 4 per group. NS, not significant, *P = 0.0380, t = 3.554). (E) Rage deficiency reduces neuronal tau propagation from hippocampus CA3 to CA1 neurons in vivo. After behavioral tests, brain sections from the hippocampus were immunostained. (F) Schematic representation of in vivo tau propagation assays and diagrams of experimental timetable. The hippocampus of 3-month-old mice (male) was intracranially injected with GFP-P301L tau AAV (6.5x1010 ifu/ml, 5 μl), followed by intracerebroventricular injection of Control AAV (1.8x1013 GC/ml) in WT mice, and RAGE FL AAV (1.93x1013 GC/ml, 10 μl) or RAGE ΔV AAV (2.13x1013 GC/ml, 10 μl) in Rage KO mice. (G) Neuronal RAGE mediates tau propagation from the hippocampal CA3 to CA2 neurons in vivo. After 8 weeks of the GFP-P301L tau AAV injection, brain sections prepared from the hippocampal tissues were immunostained and examined for GFP-P301L tau propagation (n = 3 per group). Arrowheads indicate GFP-tau. Scale bars in E, G, 50 μm. Data in B-D are represented as mean ± SEM.
      To assess whether neuronal RAGE is crucial for tau propagation, we injected GFP-P301L tau AAV into the hippocampus CA3 region of WT and Rage KO mice, and then expressed full-length (FL) or tau-binding defective ΔV mutant RAGE under CaMKIIa promoter in the neurons of Rage KO mice by injection of RAGE FL or ΔV AAV into the cerebral ventricle (Figure 5F and Figure S7B). After 8 weeks of GFP-P301L tau AAV injection, CP13-positive pathological tau was found in the GFP-positive CA3 neurons in the injection area of both WT and Rage KO mice (Figure 5G). The GFP-positive or CP13 pathological tau-positive neurons were found in the CA2 neurons of WT mice, showing transsynaptic tau propagation in the hippocampus (Figure 5G). Neuronal reconstitution of Rage KO mice with RAGE FL AAV rebuilt the active tau propagation comparable to WT mice, but Rage KO mice reconstituted with RAGE ΔV AAV did not show such tau propagation (Figure 5G). Therefore, RAGE-mediated transsynaptic tau propagation underlies tau-induced behavioral deficits.
      By assessing cognitive functions in rTg4510 mice, we further tested whether blocking the RAGE-tau interaction using RAGE antagonist could delay tau pathogenesis. The rTg4510 mice exhibit tau pretangles within their cortex as early as 2.5 months of age and then progressively form tau inclusions within the hippocampus, showing memory declines as they age from 2.5 to 4.5 months (
      • SantaCruz K.
      • Lewis J.
      • Spires T.
      • Paulson J.
      • Kotilinek L.
      • Ingelsson M.
      • et al.
      Tau Suppression in a Neurodegenerative Mouse Model Improves Memory Function.
      ,
      • Ramsden M.
      • Kotilinek L.
      • Forster C.
      • Paulson J.
      • McGowan E.
      • SantaCruz K.
      • et al.
      Age-Dependent Neurofibrillary Tangle Formation, Neuron Loss, and Memory Impairment in a Mouse Model of Human Tauopathy (P301L).
      ). We therefore began administration of FPS-ZM1 at 2 months of age and performed behavioral tests at 4.5 months of age (Figure 6A). As reported, 4.5-month-old vehicle-treated rTg4510 mice showed cognitive deficits in the Y-maze test (Figure 6B), novel object recognition test (Figure 6C) and passive avoidance test (Figure 6D). Notably, rTg4510 mice injected with FPS-ZM1 exhibited significant retention of spatial and learning memory (Figure 6B-D). Using AT8 immunohistochemistry, we found that rTg4510 mice developed significant less tau pathology in the hippocampus when treated with FPS-ZM1, while tau pathology in the cortex showed no significant difference (Figure 6E-G). In addition, treatment with FPS-ZM1 significantly decreased nuclear translocation of NF-κB p65, and the levels of IL-1β and TNF-α in the hippocampus of rTg4510 mice (Figure 6H-K). Together, these results suggest RAGE mediates tau pathology progression by promoting transsynaptic tau propagation and inflammatory responses, and blocking its function may delay memory impairment in tauopathies.
      Figure thumbnail gr6
      Figure 6RAGE antagonist ameliorates behavioral abnormality in rTg4510 mice. (A) Diagrams of experimental timetable. The 2-month-old mice (male and female) were intraperitoneally injected with vehicle or blood-brain barrier permeant RAGE antagonist FPS-ZM1 daily for 2.5 months. The mice were then analyzed with behavioral tests. (B-D) FPS-ZM1 treatment alleviates cognitive impairment in rTg4510 mice. The mice were injected with vehicle or FPS-ZM1, and analyzed with the Y-maze test [B, two-way ANOVA with Tukey test. n = 13 (8 males and 5 females), 13 (6 males and 7 females), 6 (2 males and 4 females), 9 (3 males and 6 females). Interaction effect F(
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Peng C.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      Protein transmission in neurodegenerative disease [no. 4].
      ) = 14.81, NS, not significant, *P = 0.0105, **P = 0.0093], novel object recognition test [C, two-way ANOVA with Tukey test. n = 15 (8 males and 7 females), 13 (6 males and 7 females), 8 (3 males and 5 females), 12 (5 males and 7 females). Interaction effect F(
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Lasagna-Reeves C.A.
      • Castillo-Carranza D.L.
      • Guerrero-Muñoz M.J.
      • Jackson G.R.
      • Kayed R.
      Preparation and Characterization of Neurotoxic Tau Oligomers.
      ) = 6.400, *P = 0.0400, **P = 0.0028, ***P = 0.0004] or passive avoidance test [D, Wilcoxon test, two-tailed. n = 14 (8 males and 6 females), 12 (6 males and 6 females), 9 (2 males and 7 females), 10 (4 males and 6 females). NS, not significant, NonTg/FPS-ZM1, ***P = 0.0001; NonTg/FPS-ZM1+, **P = 0.0015; rTg4510/FPS-ZM1+, **P = 0.0059]. (E-G) Tau pathology progression is decreased in the rTg4510 mice hippocampus after FPS-ZM1 treatment. AT8 immunohistochemistry (E) and signal quantification in the cortex (F, two-way ANOVA with Tukey test, n = 4 per group. Interaction effect F(
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Rauch J.N.
      • Luna G.
      • Guzman E.
      • Audouard M.
      • Challis C.
      • Sibih Y.E.
      • et al.
      LRP1 is a master regulator of tau uptake and spread [no. 7803].
      ) = 0.4094, NS, not significant, ****P < 0.0001) and hippocampus CA3 region (G, two-way ANOVA with Tukey test, n = 4 per group. Interaction effect F(
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Rauch J.N.
      • Luna G.
      • Guzman E.
      • Audouard M.
      • Challis C.
      • Sibih Y.E.
      • et al.
      LRP1 is a master regulator of tau uptake and spread [no. 7803].
      ) = 5.283, NonTg/FPS-ZM1 or NonTg/FPS-ZM1+ versus rTg4510/FPS-ZM1, ****P < 0.0001; NonTg/FPS-ZM1 versus rTg4510/FPS-ZM1+, **P = 0.0078; NonTg/FPS-ZM1+ versus rTg4510/FPS-ZM1+, **P = 0.0084; rTg4510/FPS-ZM1 versus rTg4510/FPS-ZM1+, *P = 0.0329) after treatment of FPS-ZM1 for 2.5 months. Scale bar, 100 μm. (H and I) Nuclear fractions of the hippocampal extracts were prepared form rTg4510 mice after treatment with vehicle or FPS-ZM1 for 2.5 months. Levels of nuclear NF-κB pSer536-p65 were analyzed by immunoblotting (H). The levels of nuclear NF-κB pSer536-p65 were normalized to Lamin A/C. Unpaired t-test, two-tailed, n = 3 per group. *P = 0.0119, t = 4.373 (I). (J and K) The levels of IL-1β (J) and TNF-α (K) were measured in the hippocampal extracts of 4.5-month-old rTg4510 mice after treatment with vehicle (-) or FPS-ZM1. n = 3 per group, X, not detected (J). Unpaired t-test, two-tailed, n = 5, 3. *P = 0.0258, t = 2.946 (K). Data in B-D, F, G, I-K are represented as mean ± SEM.

      DISCUSSION

      Recent studies of neurodegenerative diseases have aimed to shed light on the mechanisms underlying the propagation of protein aggregates (
      • Peng C.
      • Trojanowski J.Q.
      • Lee V.M.-Y.
      Protein transmission in neurodegenerative disease [no. 4].
      ,
      • Vaquer-Alicea J.
      • Diamond M.I.
      Propagation of Protein Aggregation in Neurodegenerative Diseases.
      ). In the present study, we investigated the mechanism of tau pathogenesis, focusing on the membrane receptor through which pathological tau proteins propagate between neurons. By establishing cell-based tau oligomer uptake assays and performing genome-wide gain-of-function screen, several membrane receptor candidates affecting tau uptake into neuronal cells were successfully isolated. An important question raised by the results of our screen is why there are a number of receptors responsible for tau uptake, including HSPG, LRP1 and RAGE. In this study, RAGE contributed to cellular uptake of highly phosphorylated and AD pathology-related tau proteins present in rTg4510 mice and the CSF of AD patients. While the biochemical properties of these pathogenic tau forms are not yet clear, it is conceivable that diverse tau strains are subject to protein propagation. Consequently, assessing the uptake of diverse tau strains may reveal various strain-specific receptors of different cell types and different tauopathies.
      RAGE is known to be tightly linked to amyloid pathology (
      • Cai Z.
      • Liu N.
      • Wang C.
      • Qin B.
      • Zhou Y.
      • Xiao M.
      • et al.
      Role of RAGE in Alzheimer’s Disease.
      ,
      • Yan S.S.
      • Chen D.
      • Yan S.
      • Guo L.
      • Du H.
      • Chen J.X.
      RAGE is a key cellular target for Abeta-induced perturbation in Alzheimer’s disease.
      ). Given that RAGE expression is upregulated in various cell types in the AD brain, it may have multiple functions that contribute to the pathogenesis (
      • Lue L.-F.
      • Walker D.G.
      • Brachova L.
      • Beach T.G.
      • Rogers J.
      • Schmidt A.M.
      • et al.
      Involvement of Microglial Receptor for Advanced Glycation Endproducts (RAGE) in Alzheimer’s Disease: Identification of a Cellular Activation Mechanism.
      ,
      • Sasaki N.
      • Toki S.
      • Chowei H.
      • Saito T.
      • Nakano N.
      • Hayashi Y.
      • et al.
      Immunohistochemical distribution of the receptor for advanced glycation end products in neurons and astrocytes in Alzheimer’s disease.
      ). For example, RAGE binding to Aβ induces neurotoxicity in neurons, triggers inflammatory responses in glial cells, and mediates Aβ transport across the blood-brain barrier in endothelial cells, all of which aggravate amyloid pathology in the central nervous system (
      • Deane R.
      • Du Yan S.
      • Submamaryan R.K.
      • LaRue B.
      • Jovanovic S.
      • Hogg E.
      • et al.
      RAGE mediates amyloid-β peptide transport across the blood-brain barrier and accumulation in brain.
      ,
      • Yan S.D.
      • Chen X.
      • Fu J.
      • Chen M.
      • Zhu H.
      • Roher A.
      • et al.
      RAGE and amyloid-β peptide neurotoxicity in Alzheimer’s disease [no. 6593].
      ,
      • Fang F.
      • Lue L.-F.
      • Yan S.
      • Xu H.
      • Luddy J.S.
      • Chen D.
      • et al.
      RAGE-dependent signaling in microglia contributes to neuroinflammation, Aβ accumulation, and impaired learning/memory in a mouse model of Alzheimer’s disease.
      ). RAGE also promotes tau hyperphosphorylation via GSK3β activation (
      • Esposito G.
      • Scuderi C.
      • Lu J.
      • Savani C.
      • Filippis D.D.
      • Iuvone T.
      • et al.
      S100B induces tau protein hyperphosphorylation via Dickopff-1 up-regulation and disrupts the Wnt pathway in human neural stem cells.
      ,
      • Li X.-H.
      • Lv B.-L.
      • Xie J.-Z.
      • Liu J.
      • Zhou X.-W.
      • Wang J.-Z.
      AGEs induce Alzheimer-like tau pathology and memory deficit via RAGE-mediated GSK-3 activation.
      ). In this study, we showed that RAGE is also linked to tau pathology by affecting transsynaptic tau propagation in neurons and inflammatory responses in microglia. Despite its lower expression in neurons than glial cells, tau-mediated induction of RAGE expression in neurons may provide a basis for its role in tau propagation. While Aβ may provide a platform that serves as a seed for the formation of tau aggregates (
      • Lasagna-Reeves C.A.
      • Castillo-Carranza D.L.
      • Guerrero-Muñoz M.J.
      • Jackson G.R.
      • Kayed R.
      Preparation and Characterization of Neurotoxic Tau Oligomers.
      ), the presence of Aβ accumulation may also upregulate RAGE expression and accelerate tau pathogenesis (
      • He Z.
      • Guo J.L.
      • McBride J.D.
      • Narasimhan S.
      • Kim H.
      • Changolkar L.
      • et al.
      Amyloid-β plaques enhance Alzheimer’s brain tau-seeded pathologies by facilitating neuritic plaque tau aggregation [no. 1].
      ,
      • Pooler A.M.
      • Polydoro M.
      • Maury E.A.
      • Nicholls S.B.
      • Reddy S.M.
      • Wegmann S.
      • et al.
      Amyloid accelerates tau propagation and toxicity in a model of early Alzheimer’s disease.
      ), thereby function as a pivotal receptor in AD pathogenesis.
      Lastly, our results show that knockout of RAGE or blocking its function ameliorates tau-induced cognitive impairment in nontransgenic mice and rTg4510 mice. Although the efficacy of RAGE antagonist on tau pathogenesis has to be further validated in other mouse models to exclude other factors from genomic disruption in rTg4510 mice (
      • Gamache J.
      • Benzow K.
      • Forster C.
      • Kemper L.
      • Hlynialuk C.
      • Furrow E.
      • et al.
      Factors other than hTau overexpression that contribute to tauopathy-like phenotype in rTg4510 mice.
      ), this study suggests a therapeutic approach that targets the process of tau propagation in AD or primary tauopathy. Previously, RAGE antagonist Azeliragon was the subject of a phase 3 clinical trial testing its ability to suppress amyloid-related pathology and inflammation in patients with AD; however, the trial was terminated with no significant relief in AD pathology. While there are several possible reasons for this failure, we believe that the clinical studies may have overlooked reductions in tau pathology due to a lack of suitable tau biomarkers. In addition, we observed that FPS-ZM1 treatment at early stages of the disease was protective of cognitive function in tauopathy model mice. It therefore may be worth evaluating efficacy of RAGE antagonists at early stages of the disease in patients with AD or primary tauopathy before there is widespread development of tau pathology.

      ACKNOWLEDGMENTS

      We thank Dr. A. M. Schmidt (New York University School of Medicine) and Dr. S. Vogel (University of Maryland School of Medicine) for providing Rage-/- mice, Dr. P. Davies (Albert Einstein College of Medicine) for providing tau antibodies, Dr. H. Lee (University of Ulsan College of Medicine) for providing AAV vectors, and Dr. N. L. Jeon (Seoul National University) for providing master mold of microfluidic device. This work was supported by the Korea Health Industry Development Institute (KHIDI) grant HU20C0334 (Y.-K.J.) and HI14C3331 (H.-W.L.); Samsung Science and Technology Foundation grant SSTFBA1401-16 (Y.-K.J.); Korea Centers for Disease Control and Prevention research fund 2018-ER6201-01 (S.K., Seoul National University Hospital Brain Bank).

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