Gut inflammation triggers C/EBPβ/δ‐secretase‐dependent gut‐to‐brain propagation of Aβ and Tau fibrils in Alzheimer’s disease

Article14 July 2021free access Source DataTransparent process Gut inflammation triggers C/EBPβ/δ-secretase-dependent gut-to-brain propagation of Aβ and Tau fibrils in Alzheimer’s disease Chun Chen Chun Chen orcid.org/0000-0003-3041-3263 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Yunzhe Zhou Yunzhe Zhou orcid.org/0000-0002-5482-5767 Innovation Center for Neurological Disorders, Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China Search for more papers by this author Hualong Wang Hualong Wang Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Microbiology, Immunology & Molecular Genetics, University of Kentucky, Lexington, KY, USA Search for more papers by this author Ashfaqul Alam Ashfaqul Alam Department of Neurology, The First Hospital of Hebei Medical University, Brain Aging and Cognitive Neuroscience Laboratory of Heibei Province, Shijiazhuang, China Search for more papers by this author Seong Su Kang Seong Su Kang Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Eun Hee Ahn Eun Hee Ahn orcid.org/0000-0002-1833-6720 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Xia Liu Xia Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Jianping Jia Jianping Jia orcid.org/0000-0002-1829-7776 Innovation Center for Neurological Disorders, Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China Search for more papers by this author Keqiang Ye Corresponding Author Keqiang Ye [email protected] orcid.org/0000-0002-7657-8154 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Chun Chen Chun Chen orcid.org/0000-0003-3041-3263 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Yunzhe Zhou Yunzhe Zhou orcid.org/0000-0002-5482-5767 Innovation Center for Neurological Disorders, Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China Search for more papers by this author Hualong Wang Hualong Wang Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Microbiology, Immunology & Molecular Genetics, University of Kentucky, Lexington, KY, USA Search for more papers by this author Ashfaqul Alam Ashfaqul Alam Department of Neurology, The First Hospital of Hebei Medical University, Brain Aging and Cognitive Neuroscience Laboratory of Heibei Province, Shijiazhuang, China Search for more papers by this author Seong Su Kang Seong Su Kang Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Eun Hee Ahn Eun Hee Ahn orcid.org/0000-0002-1833-6720 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Xia Liu Xia Liu Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Jianping Jia Jianping Jia orcid.org/0000-0002-1829-7776 Innovation Center for Neurological Disorders, Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China Search for more papers by this author Keqiang Ye Corresponding Author Keqiang Ye [email protected] orcid.org/0000-0002-7657-8154 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Author Information Chun Chen1, Yunzhe Zhou2, Hualong Wang1,3, Ashfaqul Alam4, Seong Su Kang1, Eun Hee Ahn1, Xia Liu1, Jianping Jia2 and Keqiang Ye *,1 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA 2Innovation Center for Neurological Disorders, Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China 3Microbiology, Immunology & Molecular Genetics, University of Kentucky, Lexington, KY, USA 4Department of Neurology, The First Hospital of Hebei Medical University, Brain Aging and Cognitive Neuroscience Laboratory of Heibei Province, Shijiazhuang, China **Corresponding author. Tel: +1 404 712 2814; E-mail: [email protected] The EMBO Journal (2021)40:e106320https://doi.org/10.15252/embj.2020106320 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Inflammation plays an important role in the pathogenesis of Alzheimer's disease (AD). Some evidence suggests that misfolded protein aggregates found in AD brains may have originated from the gut, but the mechanism underlying this phenomenon is not fully understood. C/EBPβ/δ-secretase signaling in the colon was investigated in a 3xTg AD mouse model in an age-dependent manner. We applied chronic administration of 1% dextran sodium sulfate (DSS) to trigger gut leakage or colonic injection of Aβ or Tau fibrils or AD patient brain lysates in 3xTg mice and combined it with excision/cutting of the gut–brain connecting vagus nerve (vagotomy), in order to explore the role of the gut–brain axis in the development of AD-like pathologies and to monitor C/EBPβ/δ-secretase signaling under those conditions. We found that C/EBPβ/δ-secretase signaling is temporally activated in the gut of AD patients and 3xTg mice, initiating formation of Aβ and Tau fibrils that spread to the brain. DSS treatment promotes gut leakage and facilitates AD-like pathologies in both the gut and the brain of 3xTg mice in a C/EBPβ/δ-secretase-dependent manner. Vagotomy selectively blunts this signaling, attenuates Aβ and Tau pathologies, and restores learning and memory. Aβ or Tau fibrils or AD patient brain lysates injected into the colon propagate from the gut into the brain via the vagus nerve, triggering AD pathology and cognitive dysfunction. The results indicate that inflammation activates C/EBPβ/δ-secretase and initiates AD-associated pathologies in the gut, which are subsequently transmitted to the brain via the vagus nerve. SYNOPSIS Inflammation plays an important role in the pathogenesis of Alzheimer's disease (AD). The misfolded protein aggregates found in AD brains have been suggested to originate in the gut in some previous studies. This study shows that inflammation can activate C/EBPβ/δ-secretase and initiate AD pathologies in the gut, which are subsequently transported to the brain via the vagus nerve. C/EBPβ/δ-secretase signaling is activated in the gut of AD patients and 3xTg mice in an age-dependent manner, initiating Aβ and Tau fibril formation that propagates to the brain. Dextran sodium sulfate (DSS) triggers gut leakage and facilitates AD-like pathologies in both the gut and the brain of 3xTg mice in a C/EBPβ/δ-secretase-dependent manner. Vagotomy attenuates C/EBPβ/δ-secretase signaling, diminishes Aβ and tau pathologies and rescues the learning and memory. Colonic injected Aβ or Tau fibrils or AD patient brain lysates spread from the gut into the brain via the vagus nerve, initiating AD pathology and cognitive disorder. Introduction Alzheimer's disease is a neurodegenerative disorder associated with the progressive decline in cognitive functions. AD is characterized by several pathological hallmarks, including extracellular β-amyloid (Aβ) plaques, intraneuronal neurofibrillary tangles (NFTs), chronic neuroinflammation, and neuronal loss. Aβ pathology arises from the improper sequential cleavage of the transmembrane amyloid precursor protein (APP) by BACE1 and γ-secretase, resulting in Aβ peptides that aggregate into oligomers and eventually into Aβ fibrils and plaques. NFTs are mainly composed of hyperphosphorylated and truncated tau, a protein that stabilizes microtubules (Holtzman et al, 2011). Although approximately 1% of AD cases arise from rare causative mutations in APP or γ-secretase, most AD is sporadic, with ApoE4 as the most well-characterized genetic risk factor (Mahley, 2016). While age is the most critical factor for AD, epidemiological studies show that some diseases and lifestyle factors also increase the risk of developing AD, including traumatic brain injury, diabetes, hypertension, obesity, and other metabolic syndromes, which are all associated with inflammation. Enhanced inflammation can be detected in the cerebrospinal fluid (CSF) and blood of AD patients (Kauwe et al, 2014; Monson et al, 2014). In blood, cognitively impaired patients with brain amyloidosis show higher levels of pro-inflammatory cytokines compared with patients without brain amyloidosis and control subjects. A lower level of the anti-inflammatory cytokine IL-10 is observed in brain amyloidosis positive patients than in negative patients (Cattaneo et al, 2017). During aging, both the gastrointestinal tract epithelium and the blood–brain barrier become more permeable to small molecules, increasing the contribution of various microbiota metabolites to amyloid formation and dissemination (Marques et al, 2013; Montagne et al, 2015; Shoemark & Allen, 2015). Western diets high in saturated fats and sugars promote gut inflammation (Reichardt et al, 2017) and exacerbate brain neuropathological and associated behavioral deficits in animal models of AD and PD (Maesako et al, 2012; Rotermund et al, 2014; Busquets et al, 2017; Walker et al, 2017). Diarrhea, which is associated with gut inflammation, often affects older people and those with AD, and rapidly progressive dementia is one of the features in inflammatory bowel disease (IBD; Papathanasiou et al, 2014). Bacteria populating the gut microbiome produce amyloids, lipopolysaccharides (LPS), and other immunogenic compounds (Syed & Boles, 2014; Zhao et al, 2015). Enhanced inflammation, as a consequence of alterations in gut microbiota composition, is implicated in the initiation of α-synuclein (α-Syn) misfolding (Olanow et al, 2014), a hallmark of Parkinson’s disease (PD). Notably, the young 5xFAD mice which have not yet developed AD neuropathology do not have inflammation in GALT (gut-associated lymphoid tissues), and GALT of 5xFAD mice mirror the disease progression and reflect inadequate immune surveillance in the gut and lead to enhanced AD pathology (Saksida et al, 2018). Recently, we have reported that asparagine endopeptidase (AEP, gene name: LGMN) acts as a δ-secretase that cleaves both APP and Tau, promoting Aβ and Tau aggregation in AD brains. δ-secretase cuts APP at the N373 and N585 residues on the extracellular domain, facilitating BACE1 to produce Aβ more efficiently (Zhang et al, 2015). It also cleaves Tau at the N255 and N368 sites, accelerating Tau hyperphosphorylation and subsequent accumulation into NFTs (Zhang et al, 2014). Interestingly, δ-secretase cleavage also produces a truncated α-Synuclein (α-Syn) at N103 and promotes Lewy body formation in PD (Zhang et al, 2017). We have recently shown that α-Syn N103/Tau N368 fibrils spread from the gut into the brain via the vagus nerve, initiating PD pathogenesis (Ahn et al, 2019). δ-secretase is upregulated in the control and AD patient brains in an age-dependent manner. Notably, we identified that C/EBPβ, an inflammation-activated transcription factor (Magalini et al, 1995; Poli, 1998), dictates LGMN mRNA transcription and escalates δ-secretase abundance during aging (Wang et al, 2018b). Importantly, we showed that the C/EBPβ/δ-secretase pathway spatiotemporally mediates AD-like neuropathologies in 3xTg mice (Wang et al, 2018a). C/EBPβ mediates the learning and memory (Taubenfeld et al, 2001) and is implicated and upregulated in inflammation in various neurodegenerative diseases including AD (Li et al, 2004; Lukiw, 2004; Ejarque-Ortiz et al, 2007). In AD, poorly myelinated projection neurons with long axons are particularly prone to developing NFTs (Braak & Braak, 1996) and neurons affected by Tau pathology appear to be anatomically connected (Hyman et al, 1984). Intracellular Tau fibrils are directly released into the medium and can be taken up by co-cultured cells, and internalized Tau aggregates induce fibrillization of intracellular Tau in the naive recipient cells (Kfoury et al, 2012). The spread of Tau pathology between interconnected neurons is thought to occur via exosomes (Wang et al, 2017). Inoculation of Tau aggregates induces time-dependent spreading of Tau pathology from the inoculation site to brain regions with synaptic connection in transgenic mice overexpressing human tau or even in wild-type mice (de Calignon et al, 2012; Liu et al, 2012; Iba et al, 2015). Likewise, intracerebral injection of diluted, Aβ-containing brain extracts from AD patients or APP transgenic mice induces cerebral β-amyloidosis and associated pathology in APP transgenic mice in a time- and concentration-dependent manner (Kane et al, 2000; Meyer-Luehmann et al, 2006). Hence, both Aβ and Tau fibrils spread via prion-like mechanisms in AD (Walker, 2018). In the current study, we tested the hypothesis that inflammation triggers C/EBPβ/δ-secretase signaling, which initiates the onset of Aβ and Tau pathologies in the gut. The aggregated fibrils subsequently spread along the vagus nerve into the brain, initiating AD pathogenesis and cognitive defects. Results C/EBPβ/δ-secretase is age-dependently activated in the gut and brain, mediating AD pathologies The C/EBPβ/δ-secretase axis is activated in an age-dependent manner in different brain regions of the 3xTg AD mouse model, elevating δ-secretase-truncated APP N585 and Tau N368 and promoting senile plaques and NFT formation (Wang et al, 2018a). Moreover, δ-secretase also initiates α-Syn N103/Tau N368 aggregates spreading from the gut into the brain, triggering PD pathogenesis (Ahn et al, 2019). To explore whether this pathway is also activated in the gut and mediates AD-like pathology in the enteric nervous system, we conducted immunoblotting and monitored C/EBPβ/δ-secretase signaling and the downstream APP and Tau fragmentation. We found that p-C/EBPβ, a marker for its activation, and its total protein levels, as well as the total protein levels of δ-secretase and its cleaved form, which indicated its activation, were increased in the gut of 3xTg mice in an age-dependent manner (Fig 1A, top-4th panels). Consequently, the abundance of δ-secretase-truncated Tau and APP, two suggested downstream effectors of C/EBPβ (Kfoury & Kapatos, 2009), was also elevated (Fig 1, 4, 4th-bottom panels). Immunofluorescent (IF) co-staining with the gut tissues also demonstrated that expression of C/EBPβ and δ-secretase increased with age, mirroring the temporal appearance of APP C586 and Tau N368, which were distributed in MAP2-positive enteric neurons. In addition, hyperphosphorylated Tau and oligomeric Tau were evident as assessed by AT8 and T22 immunoreactivity, respectively. Notably, Aβ co-localized with Tau N368 and Thioflavin S (ThS) (Fig 1B). Enzymatic assay with gut lysates revealed that δ-secretase activity gradually increased over time (Fig 1C). Similar results were obtained using 3xTg brain lysates (Appendix Fig S1A and B). Thus, C/EBPβ/δ-secretase signaling is gradually activated in an age-dependent manner in both the gut and the brain of 3xTg mice, promoting AD pathogenesis. To examine whether the similar events take place clinically, we conducted IF co-staining in gut biopsy samples from AD patients. C/EBPβ and δ-secretase were detected in AD samples but not those from healthy control (HC) (Fig 1D). Consequently, truncated APP C586 and Tau N368 fragments were co-localized with δ-secretase in AD gut tissues (Fig 1 E and F), indicating that the C/EBPβ/δ-secretase pathway is activated in AD patient gut tissues, stimulating APP and Tau proteolytic cleavage and AD pathology. Figure 1. C/EBPβ/δ-secretase (AEP) is age-dependently activated in the gut, mediating AD pathologies A. Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and proteolytic processing in mouse colon. B. Immunofluorescent staining of C/EBPβ (red) and AEP (green), cleaved APP C586 (red) and AEP (green), cleaved Tau N368 (red) and AT8 (green), cleaved Tau N368 (red) and Aβ (green), cleaved APP C856 (red) and cleaved Tau N368 (green), cleaved APP C586 (red) and MAP2 (green), T22 (red) and AT8 (green), Aβ (red) and Thioflavin S (green) in colons of 3xTg mice. Scale bar: 20 μm. C. AEP activity assay with colon lysates from age-dependent 3xTg mice. Data represent the mean ± SEM; representative data of three samples; ***P = 0.0001, ****P < 0.0001 compared with control, one-way ANOVA. D. C/EBPβ and AEP immunofluorescent staining of colons from age-matched healthy control and AD patients. White arrows indicate that C/EBPβ and AEP were co-localized in AD patient’s colon. E. AEP and cleaved APP C586 fragment immunofluorescent staining of colons from age-matched healthy control and AD patient. White arrows indicate that AEP and cleaved APP C586 were co-localized in AD patient’s colon. F. AEP and cleaved Tau N368 fragment immunofluorescent staining of colons from age-matched healthy control and AD patient. Scale bar: 20 μm. White arrows indicate that APP C586 and AEP were co-localized in AD patient’s colon. Source data are available online for this figure. Source Data for Figure 1 [embj2020106320-sup-0002-SDataFig1.zip] Download figure Download PowerPoint DSS induces gut inflammation and C/EBPβ/δ-secretase pathway activation in 3xTg mice C/EBPβ is potently activated by pro-inflammatory cytokines, reactive oxygen species (ROS), or Aβ (Strohmeyer et al, 2014). To investigate whether the gut inflammation triggered by dextran sodium sulfate (DSS), a broadly utilized chemical that induces gut leakage, elicits C/EBPβ/δ-secretase signaling activation in the mouse gut tissues, we added 1% DSS to the drinking water of wild-type and 3xTg mice for 1 month. In wild-type mice, DSS robustly increased MPO (myeloperoxidase) and its truncation in the colon, confirming the gut inflammation. Total and p-C/EBPβ, as well as δ-secretase, and its active truncated form were augmented upon DSS stimulation. APP and Tau were also augmented, as were δ-secretase-cleaved APP and Tau fragments in the colon (Appendix Fig S2A). Similar but slighted less robust effects of DSS treatment were observed in wild-type mouse brains (Appendix Fig S2B). We observed even more prominent effects of DSS in both the gut and the brain of 3xTg mice, suggesting that DSS-induced gut leakage and inflammation strongly agitates C/EBPβ/δ-secretase signaling activation, resulting in augmented APP and Tau proteolytic fragmentation by active δ-secretase (Appendix Fig S2C and D). δ-secretase enzymatic assays with both gut and brain lysates corroborated its strong activation by DSS (Appendix Fig S2E and F). In alignment with the robust MPO activation, quantitative analysis with ELISA revealed that IL-6, TNFα, and IL-1β in the colon, but not the brain, were highly increased (Appendix Fig S2G). IF staining demonstrated that DSS treatment stimulated δ-secretase in the guts of both wild-type and 3xTg mice, but only elevated Aβ and p-Tau in the colon of 3xTg mice, and Aβ was detected in the cortex of young 3xTg mice but not wild-type mice. It is worth noting that Aβ amount in the hippocampus was substantially lower than the amount found in the cortex of 3xTg mice (Appendix Fig S2H–K). Thus, DSS-elicited gut inflammation strongly activates the C/EBPβ/δ-secretase pathway in the gut and brain of 3xTg mice, provoking AD pathology onset in the gut. To explore whether the C/EBPβ/δ-secretase pathway is responsible for the biological effects triggered by chronic DSS treatment (4 months), we employed 3xTg/C/EBPβ+/− and 3xTg/AEP−/− mice, as well as wild-type mice as control. Depletion of C/EBPβ or δ-secretase from 3xTg mice strongly attenuated DSS-provoked C/EBPβ/δ-secretase activation and its downstream effects in both the colon and the brain. Knockout of δ-secretase completely abolished APP N585 and Tau N368 cleavage and mitigated AT8 hyperphosphorylation (Fig 2A and B). As a result, DSS-stimulated δ-secretase enzymatic activity was significantly reduced in 3xTg/C/EBPβ+/− and below the level of detection in 3xTg/AEP−/− mice compared to 3xTg mice and wild-type mice (Fig 2C and D). Gut leakage assay with FITC-Dextran showed that DSS-elicited gut permeability was gradually reduced from 3xTg/C/EBPβ+/− to 3xTg/AEP−/− mice and wild-type mice as compared to 3xTg mice (Fig 2E). In addition, human Aβ42 concentration in the brain of 3xTg animals was decreased in a similar pattern (Fig 2F). Figure 2. DSS induces gut inflammation and C/EBPβ/δ-secretase pathway activation in 3xTg mice A. Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and proteolytic processing in mouse colon. B. Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and proteolytic processing in mouse brain. C. AEP activity assay with colon lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; ***P = 0.0002, ****P < 0.0001 compared with control, one-way ANOVA. D. AEP activity assay with brain lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; *P = 0.0103, ****P < 0.0001 compared with control, one-way ANOVA. E. Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; **P = 0.0071, ***P = 0.0009 (3xTg vs. 3xTg AEP KO), ***P = 0.0004 (3xTg vs. WT) compared with control, one-way ANOVA. F. Human Aβ1-42 concentration in brain lysates from DSS-treated mice. Data represent the mean ± SEM; representative data of three samples; **P = 0.0097, ***P = 0.0006 compared with control, one-way ANOVA. Source data are available online for this figure. Source Data for Figure 2 [embj2020106320-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint IF co-staining revealed that DSS stimulated demonstrable C/EBPβ/δ-secretase signaling in the brains of 1% DSS-treated 3xTg mice at the age of 6 months, which were decreased in 3xTg/C/EBPβ+/− and 3xTg/AEP−/− mice and wild-type mice. Accordingly, Tau N368/AT8, APP C586/Aβ, and T22/AT8 co-staining were demonstrable in 3xTg mice, and these effects were significantly abrogated in 3xTg/C/EBPβ+/− and 3xTg/AEP−/− mice and wild-type mice (Fig 3A–E). Thus, the C/EBPβ/δ-secretase pathway is indispensable for chronic DSS-induced AD-like neuropathology in the brain of young 3xTg mice. Electronic microscopic (EM) analysis revealed that synapse loss triggered by DSS was significantly rescued in 3xTg/C/EBPβ+/− and 3xTg/AEP−/− mice and wild-type mice as compared to 3xTg mice (Fig 3F and H). Golgi staining displayed that dendritic spine reduction evoked by DSS in 3xTg mice was also substantially restored in 3xTg/C/EBPβ+/−, 3xTg/AEP−/−, and wild-type mice (Fig 3G and I). Hence, inactivation of C/EBPβ/δ-secretase pathway alleviates DSS-induced AD pathologies in young 3xTg mice. Figure 3. DSS triggers C/EBPβ/δ-secretase activation in 3xTg mice, leading to synaptic degeneration A. Immunofluorescent staining of C/EBPβ (red) and AEP (green), Iba-1 (red) and AEP (green), cleaved Tau N368 (red) and AT8 (green), cleaved APP C856 (red) and Aβ (green), T22 (red) and AT8 (green) in cerebral cortex of brains from DSS-treated mice. Scale bar: 20 μm. White arrows indicate that C/EBPβ and AEP were co-localized in mouse brain. B. Quantitative analysis of AEP-positive cells and C/EBPβ-positive cells, respectively. The density of both AEP-positive cells and C/EBPβ-positive cells was significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P = 0.0390, ***P = 0.0002, ****P < 0.0001, one-way ANOVA. C. Quantitative analysis of AT8-positive cells (left) and TauN368-positive cells (right), respectively. The density of both AT8-positive cells and TauN368-positive cells was significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. n = 5 in each group, data are shown as mean± SEM. *P = 0.0194, ***P = 0.0001, ****P < 0.0001, one-way ANOVA. D. Quantitative analysis of Aβ-positive cells (left) and APPC586-positive cells (right), respectively. The fluorescence intensity of Aβ-positive cells and the density APPC586-positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P = 0.0107, **P = 0.0048 (3xTg vs. 3xTg AEP KO), **P = 0.0044 (3xTg vs. WT), ***P = 0.0006, ****P < 0.0001, one-way ANOVA. E. Quantitative analysis of AT8-positive cells (upper) and T22-positive cells (lower), respectively. The fluorescence intensity of both AT8-positive cells and T22-positive cells was significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. **P = 0.0014 (3xTg vs. 3xTg C/EBPβ+/−), **P = 0.0030 (3xTg vs. 3xTg AEP KO), ***P = 0.001, ****P < 0.0001, one-way ANOVA. F. Representative electron microscopy of the synaptic structures in hippocampus of brains from DSS-treated mice. Red stars indicate the synapses (scale bar: 1 μm). G. The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining (scale bar: 5 μm). H. Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA. I. Quantitative analysis of the spine density. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA. Download figure Download PowerPoint Vagotomy attenuates DSS-elicited AD pathologies in the brain of 3xTg mice DSS-elicited gut inflammation stimulates AD-like neuropathology in the gut, and it has been proposed that the pathology might get transmitted to the brain via the vagus nerve (Fulling et al, 2019). To test this possibility, we resected the right side of vagus nerve in 2-month-old 3xTg mice, followed by chronic 1% DSS treatment for 4 months. Immunoblotting analysis showed DSS-induced upregulation of C/EBPβ/δ-secretase pathway and its downstream proteolytic effects on APP and Tau by DSS treatment (Fig 4A and B). DSS-triggered AEP activities in the colon and the brain were abrogated by vagotomy (Fig 4C and D). Strikingly, the effects of DSS on C/EBPβ/δ-secretase pathway, Aβ and tau fragmentation and aggregation, gut leakage, neuroinflammation, and synapse/spine loss in both the colon and the brain were robustly attenuated in vagotomized mice (Fig 4E–K). These data strongly support a model in which DSS-induced AD pathologies originate in the gut and then are propagated into the brain via the vagus nerve. To further explore this possibility, we compared AD pathologies in colons from 3xTg mice with either reduced C/EPBPβ or δ-secretase, or vagotomy. DSS-induced Tau hyperphosphorylation and aggregation in the colon was significantly and similarly mitigated by vagotomy and in 3xTg/C/EBPβ+/− and 3xTg/AEP−/− mice (Appendix Fig S3). Figure 4. Vagotomy attenuates DSS-elicited AD pathologies in 3xTg mice A. Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and processing in mouse colon of 3xTg mice with neither DSS treatment nor single side vagotomy, with both DSS treatment and single side vagotomy, or with DSS treatment but no single side vagotomy. B. Immunoblot showing p-C/EBPβ, C/EBPβ, AEP, APP, and Tau expression and proteolytic processing in mouse brain. C. AEP activity assay in colon lysates from pre-vagotomy DSS-treated, DSS-treated, and vehicle-treated 3xTg mice. DSS treatment escalated AEP activity in colon of 3xTg mice, which was reversed by vagotomy. Data represent the mean ± SEM; representative data of three samples; ***P = 0.0002, **P = 0.0013 compared with control, one-way ANOVA. D. AEP activity assay with brain lysates from different brain hemispheres of single side pre-vagotomy DSS-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P = 0.0162 compared with control, one-way ANOVA. E. Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in pre-vagotomy DSS-treated, DSS-treated, and vehicle-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P = 0.0126, ***P = 0.0002 compared with control, one-way ANOVA. F. Pro-inflammatory cytokines IL-6, IL-1β, and TNFα concentrations in colon lysates of vehicle-treated 3xTg mice, DSS-treated single side vagotomy 3xTg and DSS-treated 3xTg, respectively. Representative data of three samples; data are shown as mean ± SEM. *P = 0.0372, **P = 0.0050, ***P = 0.0008, ****P < 0.0001 compared with control, two-way ANOVA. G. Immunofluorescent staining of AEP (red) and C/EBPβ (green), cleaved Tau N368 (red) and AT8 (green), cleaved APP C856 (red) and Aβ (green), T22 (red) and AT8 (green) and Iba-1(red) of the cerebral cortex in different brain hemispheres of pre-vagotomy DSS-treated 3xTg mice. Scale bar: 20 μm. White arrows indicate that C/EBPβ and AEP,