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Cited by 422Neuroinflammation and Neurodegeneration MechanismsClusterin in disease pathologyExercise and Physiological Responses

Exercise plasma boosts memory and dampens brain inflammation via clusterin

Open access
De Miguel, Zurine, Khoury, Nathalie, Betley, Michael J., Lehallier, Benoit, Willoughby, Drew, Olsson, Niclas, Yang, Andrew C., Hahn, Oliver +15 more·2021·Nature
DOI 10.1038/s41586-021-04183-xPMC PMC9721468
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Abstract

Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration 1. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus 2-4, yet little is known about the factors and mechanisms that mediate these effects. Here we show that 'runner plasma', collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer's disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise.

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Crossref · 10.1038/s41586-021-04183-x

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Claims the paper makes

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  • Findingown data

    Plasma from running mice reduces experimentally induced brain inflammation.

    “Together, these results demonstrate that RP effectively antagonizes the neuroinflammatory response to LPS in the hippocampus.”
    Quote in the paperSubstantial claim
  • Findingown data

    Proteomic analysis of plasma found a coordinated rise in complement cascade inhibitors, including clusterin.

    “Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU).”
    Quote in the paperSubstantial claim
  • Findingown data

    Repeated retro-orbital saline injections under anaesthesia did not significantly change hippocampal cell proliferation or survival.

    “Importantly, repeated control injections of saline through the retro-orbital vein under anaesthesia did not significantly alter hippocampal cell proliferation and survival”
    Quote in the paperSubstantial claim
  • Findingown data

    Runner plasma improved Morris water maze performance, a measure of spatial learning and memory.

    “Similarly, RP improved their performance in the Morris water maze (Fig. 1e–g and Extended Data Fig. 2c)–a test of spatial learning and memory.”
    Quote in the paperSubstantial claim
  • Findingown data

    Runner plasma caused a twofold increase in surviving neural stem and progenitor cells relative to control plasma.

    “Furthermore, RP induced a twofold expansion of surviving NSPCs compared with CP.”
    Quote in the paperSubstantial claim
  • May combine two claims. 44
  • The quote only partly states this claim. 4
  • Classified as probably not a substantial claim. 32

  • May combine two claims.

    Findingown data

    Runner plasma did not alter auditory cue responses, light-dark arena activity, or anxiety levels in the activity chamber.

    “without affecting their response to auditory cues, activity in a light–dark arena or anxiety levels in the activity chamber”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Twenty-eight days of running increased overall cell survival, including neurons, neural stem and progenitor cells, and astrocytes, with the largest effect in 3-month-old mice.

    “We observed that 28 d of running was sufficient to increase overall cell survival (BrdU+ cells), including neurons(NeuN+BrdU+ cells), the number of neural stem and progenitor cells (NSPCs; Sox2+GFAP− cells) and astrocytes (GFAP+BrdU+ cells) and that this effect was most prominent in mice aged 3 months”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among the top 250 differentially expressed genes, downregulation of the acute inflammatory response was the most affected biological pathway, followed by upregulation of cell junction organization and negative regulation of proteolysis.

    “the top 250 DEGs pointed to a downregulation of the acute inflammatory response as the most significantly affected biological pathway, followed by upregulation of cell junction organization and negative regulation of proteolysis”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mass spectrometry identified 235 unique plasma proteins across runner and control conditions, with 23 downregulated and 26 upregulated in running mice.

    “We identified 235 unique proteins across the two experimental conditions; of these, 23 were downregulated and 26 were upregulated in the plasma of running mice”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In an independent experiment, plasma from mice with 28 days of wheel access reproduced the findings, while plasma from 7- or 14-day runners increased only neuroblast counts and did not increase cell proliferation or survival.

    “In an independent experiment, RP from mice that had access to a running wheel for 28 d replicated these findings, whereas RP from mice that had access to a running wheel for 7 d or 14 d increased only the neuroblast counts and did not increase cell proliferation and survival”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Runner plasma, but not control plasma, improved contextual learning and memory in fear conditioning.

    “Consistent with the effects on neurogenesis, injection of RP, but not CP, increased the contextual learning and memory of mice in the fear conditioning paradigm”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Intravenously administered clusterin lowers neuroinflammatory gene expression in a mouse model of Alzheimer's disease.

    “We found that rCLU reversed many of the genes that are abnormally expressed in the BECs of APP transgenic mice”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Hippocampal RNA sequencing found 1,952 significant differentially expressed genes after runner plasma treatment, with 61% downregulated and 39% upregulated.

    “We observed 1,952 significant differentially expressed genes (DEGs), of which 61% were downregulated and 39% were upregulated after RP treatment”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pathway analysis of the plasma proteome indicated activation of the alternative complement pathway and endothelial cell differentiation.

    “Biological pathway analysis pointed to activation of the alternative pathway of the complement system and endothelial cell differentiation”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Mice receiving runner plasma had more total proliferating cells, DCX+ neuroblasts, and surviving cells than mice receiving control plasma.

    “Notably, recipient mice that received RP showed a significant increase in total proliferating cells, DCX+ neuroblasts and surviving cells compared with CP-recipient mice”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Intravenously administered clusterin lowers neuroinflammatory gene expression in a mouse model of acute brain inflammation.

    “administration of rCLU, but not saline, reversed the expression of multiple transcripts in the hippocampal BECs of mice that were systemically inoculated with LPS”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Clot formation assays showed a significant reduction in maximum clot in runner plasma versus control plasma, with no differences in clot lysis time, time to half lysis, or in vivo tail-clipping blood clotting.

    “assessment of fibrinolysis and measurement of clot formation over time showed a significant reduction in the maximum clot formed in RP compared with in CP, yet no differences in clot lysis time or the time to half lysis and in vivo blood clotting after tail clipping were found in runner and control mice”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Runner plasma, but not control plasma, reversed expression of many LPS-induced genes (r = −0.78), making the hippocampal transcriptome more similar to that of mice not given LPS.

    “Strikingly, treatment with RP, but not CP, reversed the expression of a large number of these genes (r = −0.78) such that the hippocampal transcriptome of RP-treated mice had greater similarity to that of control mice that did not receive LPS”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Runner plasma reduced expression of toll-like receptor signalling genes, interferon pathway genes, and Gfap, an astrocyte activation marker.

    “RP reduced the expression of genes–validated using quantitative PCR (qPCR) (Extended Data Fig. 3b and Supplementary Table 5)–of the toll-like receptor (TLR) signalling pathway (Myd88, Tlr1 and Tlr2, and Irak3), the interferon pathway (Irf9, Infgr1, Stat1 and Stat3) as well as glial fibrillary acidic protein (Gfap), a sensitive indicator of astrocyte activation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Removing CLU from runner plasma largely abolished its anti-inflammatory properties, while removing FH, PEDF, or LIFR had little or no effect.

    “Notably, the depletion of CLU largely abrogated the anti-inflammatory properties of RP, whereas depletion of FH, PEDF or LIFR had little or no effect”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TMT mass spectrometry of female plasma found 31 of 567 detected proteins significantly changed with running, including four complement/coagulation proteins: C4BPA, F12, and MASP1 downregulated and C1INH upregulated.

    “Analysis of female RP and CP using TMT MS identified that 31 out of 567 detected proteins were significantly changed with running (Extended Data Fig. 5a, b), with 4 belonging to the complement and coagulation pathways–C4BPA, F12 and MASP1 were downregulated and C1INH was upregulated with exercise”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Proteomic analysis showed increased CLU, complement factor H, complement 1 inhibitor, and a coordinated increase in C8 alpha and gamma chains.

    “Our proteomic analysis also showed increased levels of CLU, complement factor H (FH), complement 1 inhibitor (C1INH), and a concerted increase in the C8 alpha and gamma chains of the C8 component of the terminal pathway for the formation of the membrane attack complex”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the same patients, complement factor 3, coagulation factor II/prothrombin, uPA, and plasminogen decreased after exercise.

    “while complement factor 3 (C3), coagulation factor II (FII/prothrombin), uPA and plasminogen (PLG) were decreased”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The commercial aptamer-based proteomic platform accurately measures the targeted complement and coagulation proteins in human plasma.

  • May combine two claims.

    Findingown data

    In brain, CLU is expressed in astrocytes and does not change after runner plasma treatment.

    “In the brain, CLU is expressed in astrocytes but does not change in response to RP”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In 20 veterans with amnestic mild cognitive impairment, plasma CLU increased significantly and FX decreased after 6 months of exercise.

    “CLU was significantly increased and FX decreased in the patients who exercised”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The commercial aptamer-based proteomic platform accurately measures the targeted complement and coagulation proteins in human plasma.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In mice that received control plasma or runner plasma, freezing behavior to fear-conditioning cues did not differ significantly.

    “Graphs show percentage of freezing behaviour in response to the light/tone cues associated with the fear stimulus in CP and RP recipient mice (n = 18–20 per group). Means ± s.e.m; Unpaired Student’s two-tailed t test, N.S. not significant.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Activity of the classical, alternative, and lectin complement pathways did not differ significantly between control and runner plasma.

    “Measuring the activity of the classical, alternative, and lectin complement pathways in control versus runner’s plasma. Plots show absorbance at 450nm indicative of levels of membrane C5b-9 attack complex formation induced by IgM, LPS, or Mannose Binding Lectin (MBL) respectively (n = 10–13 per group). Means ± s.e.m; Unpaired Student’s two-tailed t test; n.s., not significant.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Blood loss after tail clipping did not differ significantly between runner and control mice.

    “Amount of blood lost in runner and control mice after tail clipping as measured by the haemoglobin concentration (n = 7 per group). Means ± s.e.m; unpaired Student’s two-tailed t test; n.s., not significant.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Mice infused with control or runner plasma showed no significant differences in activity, velocity, or time spent in the light arena.

    “CP and RP infused mice show comparable activity and velocity in the activity chamber (n = 13–14 per group) and percentage of time spent in the light arena (n = 12 per group). Means ± s.e.m; unpaired Student’s two-tailed t test; N.S., not significant.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    LPS significantly regulates genes in specific GO categories in the cortex, and rCLU treatment changes their expression.

    “Representative GO enrichment categories of LPS significantly regulated genes. Lengths of bars represent negative ln-transformed P using two-sided Fisher’s exact test. Colours indicate gene-wise log2 fold-changes (log2(FC)) between (e) LPS and Saline treatment or (f) LPS+rCLU and LPS treatment. Numbers beside bars indicate DEGs induced by LPS in that GO category.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There are common genes increased by both acute LPS and chronic APP inflammation and reversed by CLU, and common genes reduced by both and reversed by CLU.

    “Venn Diagram showing the number of unique and common genes that are increased by LPS and APP and reversed by CLU and reduced by LPS and APP and reversed by CLU (Extended Data Fig. 8f,g and 9e).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    There is overlap between DEGs induced by LPS and LPS+rCLU in the cortex.

    “Venn diagrams depicting the overlap of DEGs (shown in panel a) in whole cortex of mice inoculated with LPS or LPS+rCLU, relative to the Saline-injected controls.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    LPS and LPS+rCLU treatments produce differentially expressed genes in the cortex relative to saline.

    “Number of DEGs (Wald test; P < 0.1) between Saline-treated mice (n = 4) and either LPS (n =8) or LPS+rCLU (n = 7) injected animals.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In brain endothelial cells, chronic APP-induced inflammation alters a set of genes, and CLU treatment reverses these changes.

    “Scatter plots show a list of selected genes altered in BECs (arterial, capillary, and venous) by chronic (APP) inflammation and reversed by CLU treatment. Coloured genes represent genes that pass the cutoff fold change of 1.1. Green: Genes increased by inflammation and reversed by CLU. Blue: Genes reduced by inflammation and reversed by CLU.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In mouse models of Alzheimer's disease and related conditions, prolonged voluntary exercise enhances learning and memory and lowers neuroinflammation.

    “Long-term voluntary exercise in mouse models of Alzheimer’s disease (AD) and related disorders improve learning and memory, and decrease neuroinflammation3,7,8.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [3] He, X. F. et al. Voluntary exercise promotes glymphatic clearance of amyloid beta and reduces the activation of astrocytes and microglia in aged mice. Front. Mol. Neurosci. 10, 144 (2017). source

      Not checked in the free run.

    • cites [7] Horder, H. et al. Midlife cardiovascular fitness and dementia: a 44-year longitudinal population study in women. Neurology 90, e1298–e1305 (2018). source

      Not checked in the free run.

    • cites [8] da Costa Daniele, T. M. et al. Exercise effects on brain and behavior in healthy mice, Alzheimer’s disease and Parkinson’s disease model—a systematic review and meta-analysis. Behav. Brain Res. 383, 112488 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IGF-1, VEGF and PF4 raise hippocampal neurogenesis in young mice.

    “Factors including IGF-1 (ref. 9), VEGF10 and PF4 (ref.11) increase hippocampal neurogenesis in young mice”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [9] Trejo, J. L., Carro, E. & Torres-Aleman, I. Circulating insulin-like growth factor I mediates exercise-induced increases in the number of new neurons in the adult hippocampus. J. Neurosci. 21, 1628–1634 (2001). source

      Not checked in the free run.

    • cites [10] Fabel, K. et al. VEGF is necessary for exercise-induced adult hippocampal neurogenesis. Eur. J. Neurosci. 18, 2803–2812 (2003). source

      Not checked in the free run.

    • cites [11] Leiter, O. et al. Exercise-induced activated platelets increase adult hippocampal precursor proliferation and promote neuronal differentiation. Stem Cell Rep. 12, 667–679 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Exercise-related cognitive gains depend on greater hippocampal plasticity and less hippocampal inflammation.

    “The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus2–4”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [2] Gleeson, M. et al. The anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease. Nat. Rev. Immunol. 11, 607–615 (2011). source

      Not checked in the free run.

    • cites [3] He, X. F. et al. Voluntary exercise promotes glymphatic clearance of amyloid beta and reduces the activation of astrocytes and microglia in aged mice. Front. Mol. Neurosci. 10, 144 (2017). source

      Not checked in the free run.

    • cites [4] van Praag, H., Christie, B. R., Sejnowski, T. J. & Gage, F. H. Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc. Natl Acad. Sci. USA 96, 13427–13431 (1999). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Exercise generally benefits health in humans and animals and slows cognitive ageing and neurodegeneration.

    “Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Prakash, R. S., Voss, M. W., Erickson, K. I. & Kramer, A. F. Physical activity and cognitive vitality. Annu. Rev. Psychol. 66, 769–797 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    GPDL1 reverses age-related declines in neurogenesis and cognition in old mice.

    “GPDL1 rescues the age-related loss in neurogenesis and cognition in old mice12”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Horowitz, A. M. et al. Blood factors transfer beneficial effects of exercise on neurogenesis and cognition to the aged brain. Science 369, 167–173 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Plasma factors from running male mice are largely sufficient to reproduce running effects on hippocampal neurogenesis, improve learning and memory, and lower baseline inflammatory gene expression in the young adult hippocampus.

    “These findings indicate that plasma factors from running male mice are largely sufficient to reproduce the effects of running on hippocampal neurogenesis, improve learning and memory, and downregulate the baseline expression of inflammatory genes in the young adult hippocampus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The findings show that anti-inflammatory exercise factors can be transferred, act on the cerebrovasculature, benefit the brain, and are present in exercising humans.

    “These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The complement and coagulation protein changes seen after exercise in humans reflect the same biology as the changes observed in mice.

  • May combine two claims.

    Interpretationown data

    Exercise-induced plasma changes are sufficient to reproduce exercise benefits on neurogenesis and memory and to reduce hippocampal inflammation in non-exercised mice and mice with induced neuroinflammation.

    “We show that changes in plasma as a result of physical aerobic exercise are sufficient to recapitulate the beneficial effects of exercise on neurogenesis and memory, and to reduce hippocampal inflammation in non-exercised mice and mice with experimentally induced neuroinflammation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The complement and coagulation protein changes seen after exercise in humans reflect the same biology as the changes observed in mice.

  • The quote only partly states this claim.

    Interpretationown data

    The cortical gene expression changes support the hippocampal anti-inflammatory effect of clusterin.

    “supporting the hippocampal anti-inflammatory effect of this protein.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Exercise anti-inflammatory effects have previously been linked to muscle and fat cytokine secretion changes, regulatory T cell mobilization from lymphoid organs, and reduced inflammatory monocytes in blood.

    “Anti-inflammatory effects of exercise have previously been linked to changes in the secretion of immune-modulatory cytokines from muscle and fat tissue, the mobilization of regulatory T cells from lymphoid organs, and the reduction of inflammatory monocytes in the blood2.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Gleeson, M. et al. The anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease. Nat. Rev. Immunol. 11, 607–615 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    Sex differences in physical exertion and oestrous cycle effects on complement and coagulation pathways may explain some sex-based differences.

    “Differences in physical exertion between sexes17 and direct effects of the oestrous cycle on the complement and coagulation pathways18 may explain some of the differential effects based on sex.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [17] Hicks, A. L., Kent-Braun, J. & Ditor, D. S. Sex differences in human skeletal muscle fatigue. Exerc. Sport Sci. Rev. 29, 109–112 (2001). source

      Not checked in the free run.

    • cites [18] Yip, K. S., Suvorov, A., Connerney, J., Lodato, N. J. & Waxman, D. J. Changes in mouse uterine transcriptome in estrus and proestrus. Biol. Reprod. 89, 13 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The effects of exercise plasma are linked to complement and coagulation pathways including CLU, which can target brain endothelial cells and broadly reduce interferon signalling.

    “The effects are linked to complement and coagulation pathways including CLU, which can target BECs and broadly reduce interferon signalling.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The complement and coagulation protein changes seen after exercise in humans reflect the same biology as the changes observed in mice.

  • May combine two claims.

    Interpretationown data

    CLU may reduce brain inflammation by targeting the vasculature, although other mechanisms are probably involved.

    “CLU may similarly reduce brain inflammation by targeting the vasculature, although other mechanisms are probably also involved.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    GPDL1 is an exercise-induced plasma protein reported to improve cognition in old mice and is also produced in the liver.

    “Notably, GPDL1–which is an exercise-induced plasma protein that has been reported to improve cognition in old mice12–is also produced in the liver.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Horowitz, A. M. et al. Blood factors transfer beneficial effects of exercise on neurogenesis and cognition to the aged brain. Science 369, 167–173 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In neurodegeneration, the complement pathway is activated, and inhibiting C1q or C3 reduces neuroinflammation, Aβ plaques, and synapse loss.

    “In neurodegeneration, the complement pathway is activated, and inhibiting the complement proteins C1q or C3 results in less neuroinflammation, Aβ plaques and synapse loss29,30.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [29] Yin, C. et al. ApoE attenuates unresolvable inflammation by complex formation with activated C1q. Nat. Med. 25, 496–506 (2019). source

      Not checked in the free run.

    • cites [30] Hong, S. et al. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Behavioural experimenters were blinded to treatment.

    “The experimenters were blinded to the treatment.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Mice were randomly assigned to experimental conditions.

    “Mice were randomly assigned to the experimental conditions.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Immunofluorescence images were obtained, quantified, and analysed by an observer blinded to treatment.

    “Images of immunofluorescent sections were obtained, quantified and analysed by an observer who was blinded to the treatment.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Running mice were randomly housed in activity cages with unlocked wheels, and control mice were housed in standard cages.

    “Next, running mice were randomly housed in the activity cages in pairs with free access to unlocked wheels and control mice were housed in pairs in standard animal facility cages.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Complement and coagulation proteins, including CLU, are largely produced by hepatocytes.

    “Complement and coagulation proteins, including CLU, are largely produced by hepatocytes28”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Zhou, Z., Xu, M. J. & Gao, B. Hepatocytes: a key cell type for innate immunity. Cell. Mol. Immunol. 13, 301–315 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Plasma CLU levels are increased in Alzheimer's disease and mild cognitive impairment, possibly as a compensatory mechanism.

    “plasma CLU levels are increased in patients with AD and MCI32, possibly as a compensatory mechanism.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Hsu, J. L. et al. Plasma biomarkers are associated with agitation and regional brain atrophy in Alzheimer’s disease. Sci. Rep. 7, 5035 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Polymorphisms in CLU are linked to Alzheimer's disease.

    “polymorphisms in CLU are linked to AD19,20”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] Harold, D. et al. Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer’s disease. Nat. Genet. 41, 1088–1093 (2009). source

      Not checked in the free run.

    • cites [20] Lambert, J. C. et al. Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer’s disease. Nat. Genet. 41, 1094–1099 (2009). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Runner plasma improves cognitive function and reproduces exercise effects on neural stem cell activity, possibly by reducing baseline negative effects of inflammation on adult neurogenesis and cognition.

    “Importantly, RP improves cognitive function and recapitulates the effects of exercise on neural stem cell activity possibly by reducing the baseline negative effects of inflammation on adult neurogenesis and cognition34,35.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [34] Lee, J. W. et al. Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation. J. Neuroinflammation 5, 37 (2008). source

      Not checked in the free run.

    • cites [35] Monje, M. L., Toda, H. & Palmer, T. D. Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760–1765 (2003). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Systemically administered CLU binds brain endothelial cells and reduces their inflammatory gene expression in experimentally inflamed mouse brains, partly by reducing interferon signalling.

    “systemically administered CLU binds to BECs and reduces their inflammatory gene expression in experimentally inflamed mouse brains, in part by reducing interferon signalling”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Dead cells were excluded from cell sorting by SYTOX Blue staining.

    “Dead cells were excluded by staining with SYTOX Blue Dead Cell Stain (1:2,000, Thermo Fisher Scientific, S34857).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    For APP samples, lower than anticipated cell yields from sorting led to loading all of the cell suspension, whereas other samples targeted 4,000 cells.

    “For the APP samples, all of the cell suspension was loaded after getting lower than anticipated yields from sorting. For the other samples, 4,000 cells were targeted.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Single cells were discriminated from doublets or aggregates using FSC-A and FSC-W gating.

    “FSC-A and FSC-W blotting was used to discriminate single cells from cell doublets/aggregates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The number of samples for the single-cell sequencing studies was set using prior studies and preliminary experiments that confirmed sufficient BECs would be available for statistical analysis.

    “To determine the samples size for the single-cell sequencing studies, we used previous studies13,26 and performed preliminary studies to confirm that we will obtain enough BECs for statistical analysis (not included in the manuscript).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13] Chen, M. B. et al. Brain endothelial cells are exquisite sensors of age-related circulatory cues. Cell Rep. 30, 4418–4432 (2020). source

      Not checked in the free run.

    • cites [26] Yang, A. C. et al. Physiological blood-brain transport is impaired with age by a shift in transcytosis. Nature 583, 425–430 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For TMT-based mass spectrometry, portions of each sample were checked for ion distributions and labelling efficiency.

    “Portions of each sample were used to check ion distributions and labelling efficiency.”
    • Also stated in: § MS analysis · plain
      “Portions of each sample were used to check ion distributions and labelling efficiency.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Low-quality cells and doublets were removed by retaining cells with at least 600 features, at most 6,000 features, and no more than 10% mitochondrial RNA.

    “Filtering criteria were applied to remove low-quality cells and doublets. Cells that had at least 600 features, a maximum of 6,000 features and 10% mitochondrial RNA were included in the downstream analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The quality of RNA used for hippocampal RNA-seq was validated with an Agilent 2100 Bioanalyzer.

    “RNA was isolated from hippocampal tissue as previously described and the quality was validated using the Agilent 2100 Bioanalyzer (Stanford PAN Facility).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    Human plasma samples were processed under Good Laboratory Practice for intrarun normalization and calibration and met all quality-control requirements.

    “The samples were processed under Good Laboratory Practice54 for intrarun normalization and calibration and passed all quality-control requirements.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] SOMAscan Technical White Paper (Somalogic, 2015); http://www.somalogic.com/wp-content/uploads/2016/08/SSM-002-Rev-3-SOMAscan-Technical-White-Paper.pdf

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Mice were housed in randomized groups of 2-4 before exercise or plasma injections.

    “were purchased from Jackson Labs and housed in randomized groups of 2–4 mice, before any exercise or plasma injections.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    The diagnosis of amnestic mild cognitive impairment in the human participants was based on National Institute on Aging/Alzheimer's Association criteria.

    “Diagnosis was based on the National Institute on Aging/Alzheimer’s Association criteria52.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] Albert, M. S. et al. The diagnosis of mild cognitive impairment due to Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement. 7, 270–279 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    CD11b+ and CD45+ cells were gated out to exclude monocytes, macrophages and microglia during BEC sorting.

    “CD11b+ and CD45+ cells were gated to exclude monocytes/macrophages and microglia.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Single-cell RNA-seq libraries were deep-sequenced to achieve more than 20,000 reads per cell, as recommended.

    “Samples were sent to Novogene for sequencing, where they were first assessed for library quality on a single lane of the HiSeq System followed by deep sequencing on a NovaSeq S4 flow cell to ensure >20,000 reads per cell as recommended.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All experiments followed institutional guidelines approved by the VA Palo Alto Committee on Animal Research.

    “All of the experiments were performed in accordance with institutional guidelines approved by the VA Palo Alto Committee on Animal Research.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Sample size was determined from previous literature to estimate an optimal n for studies on runner plasma effects on neuroplasticity and behaviour.

    “Sample size was determined using previous literature4,10,12 to estimate an optimal n for our studies on the RP effects on neuroplasticity and behaviour.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [4] van Praag, H., Christie, B. R., Sejnowski, T. J. & Gage, F. H. Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc. Natl Acad. Sci. USA 96, 13427–13431 (1999). source

      Not checked in the free run.

    • cites [10] Fabel, K. et al. VEGF is necessary for exercise-induced adult hippocampal neurogenesis. Eur. J. Neurosci. 18, 2803–2812 (2003). source

      Not checked in the free run.

    • cites [12] Horowitz, A. M. et al. Blood factors transfer beneficial effects of exercise on neurogenesis and cognition to the aged brain. Science 369, 167–173 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    In RNA-seq differential expression analyses, P values were adjusted for multiple testing, and genes with adjusted P < 0.1 were considered significantly differentially expressed.

    “P values were adjusted for multiple testing and significant DEGs were defined by a cut-off of adjusted P < 0.1, as suggested for analyses using Deseq2 (ref. 39).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    The authors validated the gene expression changes caused by runner plasma infusion and the protein changes associated with running.

    “Validation of changes in gene expression in response to runner plasma infusions and validation of changes in proteins with running.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The proteins identified by shotgun-LC MS1 were confirmed by TMT-LC MS3 detection and analysis.

    “Validation of proteins captured with shotgun-LC MS1 using TMT-LC MS3 detection and analysis.”
    Quote in the paperNot a substantial claim
70 of 70

  • The paper relies on this without support.

    The rise in plasma CLU among patients after the exercise program is caused by the exercise itself rather than by other changes over time.

    “Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU.”

  • The paper relies on this without support.

    Before this study, changes in complement and coagulation cascades had not been implicated in exercise effects on brain neuroinflammation.

    “To our knowledge, changes in the complement and coagulation cascadeshave not been implicated in the effects of exercise and in reducing neuroinflammation within the brain.”

  • The paper relies on this without support.

    The commercial aptamer-based proteomic platform accurately measures the targeted complement and coagulation proteins in human plasma.

    “we measured 8 out of the top 13 complement/coagulation proteins, including CLU identified in mice, using a commercial aptamer-based proteomic platform in 20 veterans with amnestic mild cognitive impairment before and 6 months after a physical exercise intervention.”

  • The paper relies on this without support.

    The complement and coagulation protein changes seen after exercise in humans reflect the same biology as the changes observed in mice.

    “Consistent with the findings in mice, CLU was significantly increased and FX decreased in the patients who exercised”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Clusterin, a complement cascade inhibitor, can bind to receptor on brain endothelial cells and reduce expression of inflammatory genes in an AD mouse model.

    “Clusterin, one of the complement cascade inhibitors, can bind to the receptor on brain endothelial cells and reduce the expression of inflammatory genes in an AD mouse model.129”

    From Role of neuroinflammation in neurodegeneration development (2023) · cites this paper as [129]

  • Replenishing clusterin and platelet factor 4 ameliorates neuroinflammation, and oleoylethanolamide enhances microglial phagocytosis, all contributing to cognitive improvement and neuroprotection.

    “According to recent reports, replenishing clusterin and platelet factor 4 (PF4) ameliorates neuroinflammation,384,385 and oleoylethanolamide (OEA) enhances microglial phagocytosis,386 all of shese contribute to cognitive improvement and neuroprotection.”

    From Antiageing strategy for neurodegenerative diseases: from mechanisms to clinical advances (2025) · cites this paper as [384]

  • The benefits of GPLD1 and other exerkines on brain structure and function may relate to peripheral effects such as complement signalling or coagulation.

    “Hence, the benefit of GPLD1 (REF.148) and other exerkines on brain structure and function might relate to its peripheral effects, such as the complement signalling cascade149,158, or coagulation159.”

    From Exerkines in health, resilience and disease (2022) · cites this paper as [149]

  • Evidence is accumulating that factors released from non-neuronal tissue and delivered to the brain via the vasculature may have important roles in synaptic plasticity, memory function and mood regulation.

    “Evidence is now accumulating that factors released from non-neuronal tissue148–152 and delivered via the vasculature to the brain might have important roles in synaptic plasticity, memory function and mood regulation150.”

    From Exerkines in health, resilience and disease (2022) · cites this paper as [149]

  • Since 2020, studies transferring plasma from exercised animals to sedentary animals have produced subsequent improvements in cognitive function, supporting a transferable factor for cognitive improvement.

    “Since 2020, several studies have transferred plasma from exercised animals into sedentary animals, with subsequent improvements in cognitive function, supporting the presence of a transferable factor in improving cognitive function148,149.”

    From Exerkines in health, resilience and disease (2022) · cites this paper as [149]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • EdU timing differs between Methods and the figure legend

    Minor

    The Methods say EdU was administered 12 hours before euthanasia to measure proliferation, whereas the Extended Data Fig. 1 legend says it was administered 24 hours before sacrifice.

  • The same exercise factor has two names

    Minor

    The discussion calls the exercise-induced factor GPDL1, but the table names it GPLD1. The different letter order leaves the paper internally inconsistent about the factor's identifier.

  • Figure 4d is cited for incompatible analyses

    Minor

    The single-cell analysis Methods cite Fig. 4d for genes and pathways reversed by CLU, while the results identify Fig. 4d as showing plasma protein changes in exercising patients.

3 of 3

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Authors
De Miguel, Z., Khoury, N., Betley, M. J., Lehallier, B., Willoughby, D., Olsson, N., Yang, A. C., Hahn, O., Lu, N., Vest, R. T., Bonanno, L. N., Yerra, L., Zhang, L., Saw, N. L., Fairchild, J. K., Lee, D., Zhang, H., McAlpine, P. L., Contrepois, K., Shamloo, M., Elias, J. E., Rando, T. A., & Wyss-Coray, T.
Affiliations
  • Stanford Medicine (US)
  • VA Palo Alto Health Care System (US)
  • Stanford University (US)
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2021-12-08
Volume
600
Issue
7889
Pages
494-499
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-021-04183-x
PMC
PMC9721468
OpenAlex
W4200434404
Cited by
422
References
70
Citations by year
2026: 67 · 2025: 116 · 2024: 100 · 2023: 83 · 2022: 54 · 2021: 2
FWCI
23.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • U.S. Department of Veterans Affairs (I01 BX001319)
  • National Institute on Aging (F32AG067652, R01AG047820, P01AG036695, P30AG066515, R01AG071783)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Oncology › Clusterin in disease pathology
  • Health Sciences › Medicine › Rehabilitation › Exercise and Physiological Responses
Keywords
physical activity, plasma, clusterin, neuroinflammation, Alzheimer's disease, cognitive aging, hippocampus, plasma proteomics, brain endothelial cells, voluntary running, sedentary mice, cognitive impairment
MeSH terms
Alzheimer Disease, Animals, Encephalitis, Humans, Proteomics, Endothelial Cells, Clusterin, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-05.

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