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Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

Abstract

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation1, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation2,3,4 and downregulated in the aged brain5,6,7,8. In addition to revitalizing other aged tissues9,10,11,12,13, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters14,15,16, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown17. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

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Figure 1: Human cord plasma enhances plasticity in the aged brain.
Figure 2: Cord plasma improves neuronal function within aged hippocampus.
Figure 3: Protein microarray analysis identifies putative pro-plasticity factors.
Figure 4: Systemic TIMP2 treatment improves neuronal function within aged hippocampus and is critical for normal spatial memory and cognitive effects of cord plasma.

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Acknowledgements

We thank clinical staff for human blood-plasma collection/coordination, C. Guenthner, L. Luo for TRAP-FOS breeders, T. Rando for discussion, Stanford Translational Applications Service Center/Protein and Nucleic Acid facilities for whole-genome microarrays, H. Zhang for mice for depletion experiments. This work was funded by the Jane Coffin Childs Postdoctoral Fellowship-Simons Foundation (J.M.C), Veterans Affairs (T.W.-C.), anonymous (T.W.-C.), the Glenn Foundation for Medical Research (T.W.-C.), the Stanford Brain Rejuvenation Project, and the National Institute on Aging (K99AG051711 (J.M.C.), AG045034 (T.W.-C.), DP1AG053015 (T.W.-C.) and AG040877 (K.I.M.)).

Author information

Authors and Affiliations

Authors

Contributions

J.M.C. and T.W.-C. designed research. J.M.C., K.I.M., D.B., and J.C.S. performed protein microarray experiments. J.M.C., R.J.A., and A.A.M. performed behaviour, staining/microscopy. J.M.C. performed biochemical assays; I.V.H. developed silver stain protocol. J.M.C. and M.L.J. performed radiolabelling/autoradiography experiments. J.M.C., B.Z., and X.S.X. performed LTP experiments. M.T. and M.S.A. provided human samples. J.M.C. analysed data and wrote the manuscript. T.W.-C. supervised study.

Corresponding author

Correspondence to Tony Wyss-Coray.

Ethics declarations

Competing interests

T.W.-C. is co-founder of Alkahest, Inc. T.W.-C., J.M.C., M.S.A. are Alkahest shareholders. Stanford filed patent applications covering a method treating aging-associated conditions by young plasma (PCT/US2014/068897; co-inventors: T.W.-C., J.M.C., M.S.A.) or TIMP2 (PCT/US2016/036032; co-inventors: T.W.-C., J.M.C.).

Additional information

Reviewer Information Nature thanks H. Eichenbaum and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Expression of common plasma proteins differs according to donor group.

a, Basic demographic characteristics of human subjects providing EDTA-plasma for corresponding quantitative analysis in b, and used for injections in Fig. 1. For umbilical cord plasma samples from neonates, gestational age reflects full-term births, and the APGAR is a composite health score reflecting appearance, pulse, grimace, activity, and respiration 5 min after birth. Mini-mental state examination (MMSE) is a 30-point neurological assessment of cognitive impairment (0-30, where 30 is least impairment). b, Heat map from unsupervised hierarchical cluster analysis of multiplexed plasma protein markers for cord, young adult, or elderly subjects (blue and yellow shades represent lower and higher relative expression, respectively; asterisks denote factors for which scatter plots are shown in ce). ce, Multiplex quantification of plasma α-fetoprotein (AFP), human chorionic gonadotropin (CGB5), and immunoglobulin-M (IgM) demonstrating significant donor-dependent differences. One-way ANOVA followed Tukey’s post hoc test; ****P < 0.0001, ***P < 0.001; n = 7 (cord), n = 12 (young), n = 11 (elderly); mean ± s.e.m.

Extended Data Figure 2 Immunodeficient NSG mice exhibit age-dependent hippocampal dysfunction independent of sensory function.

Representative images of young and aged NSG hippocampi stained for c-Fos (a; n = 7 mice per group; male; 4.1 ± 0.2 versus 11.1 ± 0.1 months old), newborn neuron marker doublecortin (b; DCX) (female mice at 4.1 ± 0.1 months old (n = 8) and 10.9 ± 0.1 months old (n = 7)), and microgliosis (c), as indicated by confocal microscopy (yellow) of Iba1+CD68+ staining (female mice at 4.1 ± 0.1 months old (n = 8) and 10.9 ± 0.1 months old (n = 7); for ac, scale bars are 100 μm). df, Quantification of indicated areas in ac. g, Schematic diagram of modified Barnes maze (left) and contextual fear-conditioning tasks (right). h, Four-day trial time course for modified Barnes maze showing escape hole latency for young and aged NSG mice (n = 4 per group; 4.5-month-old versus 11.2-month-old). i, Change in escape latency (acquisition rate) between indicated trials on day 4 of Barnes maze testing. jl, Freezing levels for young and aged NSG mice at baseline (j), during exposure to previously aversive fear-conditioning context (k), or during exposure to chamber with training cues but new context (l) (n = 8 mice per group; 3.1 ± 0.1 versus 12.1 ± 0.1 months old). m, Thresholds for various pain-related behaviours using foot-shock grid (n = 6 NSG mice per group; 4.1 ± 0.1 versus 12.3 ± 0.3 months old). n, o, Proportion of time spent on bench (safe) side (n) and mean latency before stepping over cliff side (o) during the visual cliff task to assess visual ability (n = 10 NSG mice per group; 3.6 ± 0.2 versus 13.3 ± 0.1 months old). p, Acoustic startle response at various sound intensities (n = 10 mice per group; 3.6 ± 0.2 versus 13.3 ± 0.1 months old). q, Four-day trial Barnes maze time course showing latency to target hole for young (n = 9) and aged (n = 10) WT (C57Bl/6) mice (3-month-old versus 21-month-old). r, Change in escape latency (acquisition rate) between indicated trials on day 4 of Barnes maze testing. Two-way repeated-measures ANOVA with Bonferroni’s post hoc test for time × group comparisons; two-way ANOVA for stimulus × group comparisons; Student’s t-test for two-group comparisons. *P < 0.05, ***P < 0.001, ****P < 0.0001; mean ± s.e.m.

Source data

Extended Data Figure 3 Human cord plasma treatment enhances c-Fos expression in the dentate gyrus but does not alter pCREB expression or the number of newborn neurons.

a, b, Relative gene expression by qPCR for confirmatory plasticity genes after treatment of aged NSG mice with young (a; n = 6) or elderly (b; n = 7) human plasma versus vehicle (n = 7) (13.9 ± 0.2 months old). c, Representative dentate gyrus images from aged NSG mice treated intravenously with human cord (n = 8 mice), young (n = 7 mice), or elderly (n = 7 mice) plasma or vehicle (n = 7 mice), highlighting c-Fos-positive cells (arrowheads; scale bar, 100 μm; quantification in Fig. 1e). d, e, Quantification of c-Fos-positive cells in lateral/basolateral amygdala (d) and retrosplenial/motor cortex (e) in treated NSG mice. f, g, pCREB expression (f; scale bar, 150 μm) and corresponding pCREB intensity quantification (g) in treated NSG mice. h, i, Newborn neurons visualized by doublecortin antibody staining (h; DCX, arrowheads; scale bar, 100 μm) and corresponding quantification (i) in treated NSG mice; (for ag, 13.9 ± 0.2 months old). j, k, Quantification of TRAPed c-Fos-activated recombined cells (visualized by TdTomato fluorescence) also expressing the pan-neuronal marker NeuN in dentate gyrus (j) and the CA1 subfield of hippocampus (k; overall activation/recombination in CA1 was very low) from cord plasma-treated (n = 4), elderly plasma-treated (n = 3), and vehicle-treated (n = 4) TRAP-FOS mice (8–9.5 months old). l, m, TRAPed c-Fos-activated recombined cells in lateral/basolateral amygdala (l) and retrosplenial/motor cortex (m) in mice from jk; one-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01; NS, not significant; mean ± s.e.m.

Source data

Extended Data Figure 4 Most c-Fos-expressing cells activated by cord plasma in dentate gyrus are granule cells.

a, b, Overlap (arrowheads) of c-Fos (red) and prox1 (green), and Gad67-expressing neurons (blue; arrows) in dentate gyrus from cord plasma-treated (n = 8), young plasma-treated (n = 7), elderly plasma-treated (n = 7), and vehicle-treated (n = 7) NSG mice by confocal microscopy (a) with corresponding counts of c-Fos+prox1+ neurons (b; 13.9 ± 0.2 months old; scale bar, 50 μm). c, The proportion of c-Fos-expressing cells quantified in dentate gyrus that were granule cells (prox1+), suggesting that nearly all the c-Fos+ cells detected in dentate gyrus were granule cells. d, Quantification of the number of c-Fos+Gad67+ (inhibitory neuron) cells in dentate gyrus from human plasma- and vehicle-treated NSG mice. e, Quantification of the number of Gad67+ cells in the dentate gyrus of treated NSG mice. One-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01; NS, not significant; mean ± s.e.m.

Source data

Extended Data Figure 5 Cord, but not young or elderly plasma, affects LTP in aged NSG hippocampi without altering synaptic strength and paired-pulse facilitation.

a, Input–output relationship in dentate gyrus synapses from brain slices taken from vehicle- or cord-plasma-treated aged NSG mice (n = 11 slices from n = 5 cord plasma-treated NSG mice; n = 12 slices from n = 5 vehicle-treated NSG mice; eight intravenous injections; 11.7 ± 0.2 months old), showing no difference in synaptic strength (basal synaptic transmission). b, c, Paired-pulse ratio before (b) and after (c) LTP protocol with various pulse intervals (in c, n = 10 slices per group were measured). df, Population spike amplitudes recorded in dentate gyrus granule cell layer by perforant path stimulation in slices from vehicle- or cord plasma-treated young NSG mice (d) with quantification of LTP maintenance phase (e), and input–output relationship (f) in dentate gyrus synapses from the slices (n = 11 slices per group from n = 5 mice per group; eight intravenous injections; 2.5 ± 0.1 months old). gi, Population spike amplitudes recorded in dentate gyrus granule cell layer by perforant path stimulation in slices from vehicle, young, or elderly human plasma-treated aged NSG mice (g) with quantification of LTP maintenance phase (h), and input–output relationship in dentate gyrus synapses (i) from the slices (n = 10, 11, and 11 slices from 5 mice per group (vehicle-treated, young plasma-treated, and elderly plasma-treated mice, respectively); eight intravenous injections; 12.0 ± 0.1 months old). j, k, Freezing levels for vehicle-treated (n = 9) or cord-plasma-treated (n = 7) aged NSG mice during baseline period (j) and cued fear-conditioning task (k; eight intravenous injections; 12.8 ± 0.2 months old). Student’s t-test for two-group comparisons; one-way ANOVA for h; two-way ANOVA for group × stimulus intensity comparisons (no significant interaction in a, f, i); NS, not significant; mean ± s.e.m.

Source data

Extended Data Figure 6 Systemic treatment with CSF2 improves learning and memory in aged mice.

a, Table of ranked plasma protein changes from protein microarray experiments. Shown in green are top hits of proteins decreasing in plasma in both mouse ageing (3, 6, 12, 18, 24 months) and human ageing (cord, young, elderly) arrays; shown in red are top hits of proteins increasing in plasma of aged mice sharing young blood (versus aged isochronic), while also decreasing in human ageing (cord, young, elderly) arrays. † Candidates selected for recombinant protein injections in aged mice to examine changes in c-Fos+ cell number. *Proteins appearing in both lists. b, Scatter plot of ELISA-based quantification of human plasma CSF2 (n = 15, 19, 16 subjects (left to right)), consistent with plasma elevations in aged mice sharing young blood via parabiosis by protein microarray. c, Number of c-Fos-positive cells in dentate gyrus from aged WT mice treated seven times systemically every other day with 50 μg kg−1 (intraperitoneal) recombinant CSF2 (n = 8 per group; 21-month-old). d, Barnes maze escape latency over 4-day trial time course for separate cohort of aged WT mice (20-month-old) treated systemically (intraperitoneal) eight times every other day with vehicle (n = 14) or 50 μg kg−1 of recombinant CSF2 (n = 15). e, Day 4 Barnes maze acquisition rate for indicated trial intervals. fh, Baseline freezing levels for the same CSF2- or vehicle-treated cohort in d and e (f), and freezing levels during exposure to previously aversive context (g) or during exposure to a new context chamber with training cues (h). i, Number of treated WT mice from dh exhibiting nesting behaviour within 24 h of placing intact nestlet under single-housing conditions. j, k, Representative dentate gyrus sections stained with doublecortin antibody (DCX, arrowheads; scale bar, 100 μm) in CSF2-treated versus vehicle-treated aged WT mice from di (j) and corresponding quantification of total newborn neuron number in dentate gyrus (k), suggesting that cognitive changes observed in d, e, and g are probably unrelated to neurogenesis changes. One-way ANOVA with Tukey’s post hoc test; two-way repeated-measures ANOVA with Bonferroni’s post hoc test for time × group comparisons; Student’s t-test for two-group comparisons; χ2 analysis for i. *P < 0.05, **P < 0.01, ****P < 0.0001; mean ± s.e.m.

Source data

Extended Data Figure 7 TIMP2 decreases early in plasma and hippocampus and enters the CNS following systemic treatment.

a, b, Representative immunoblot detecting TIMP2 from equal volumes of 1-month-old (n = 8), 3-month-old (n = 7), and 20-month-old (n = 8) WT mouse plasma with corresponding Ponceau S stain (a) and quantification (b). c, d, Representative immunoblot detecting TIMP2 and neuron-specific enolase loading control from hippocampal lysates (80 μg) from 1-, 12- and 20-month-old WT mice (c) with corresponding quantification (d; n = 7 per group). e, Representative confocal microscopy images from WT or TIMP2 KO mice demonstrating specificity of TIMP2 signal (green) in dentate gyrus (n = 4 mice per group; 3-month-old male mice; white arrowheads indicate TIMP2+ cells; scale bar, 100 μm). f, Representative confocal microscopy images showing TIMP2 (green), Iba1 (blue), and NeuN (red) staining in dentate gyrus/hilus of WT mice at various ages (C57Bl/6; National Institute on Aging colony; 1-month-old (n = 8), 2-month-old (n = 8), 6-month-old (n = 6), 12-month-old (n = 8), and 20-month-old mice (n = 9); scale bar, 100 μm). g, h, Quantification of TIMP2+Iba1+ cells (g) or TIMP2+ cells lacking Iba1 or NeuN staining (h) in the dentate gyrus/hilus of mice from f. i, Mean signal intensity per TIMP2+ cell for all counted cells within dentate gyrus/hilus in mice from f. j, NeuN+ cell counts per dentate gyrus/hilar area in the indicated ages. k, l, c-Fos-positive cell counts within dentate gyrus from WT mice treated once (k) or four times (l) with different rTIMP2 doses (n = 8 per group; 21-month-old). m, 64Cu-labelled BSA and 64Cu-labelled TIMP2 detected in blood of 21-month-old WT mice euthanized at the indicated time points following an injected dose of 64Cu-labelled BSA (n = 4 mice per time point (n = 3 mice at 24 h)) or 64Cu-labelled TIMP2 (n = 5 mice per time point). n, First-order elimination kinetics of 64Cu-labelled TIMP2 levels were analysed to approximate its blood half-life (curved line indicates confidence interval). o, Ex vivo autoradiography assessment of 64Cu-labelled TIMP2 localization in coronal or sagittal (right-most) brain sections from injected mice (top) with corresponding Nissl staining (middle) and 64Cu-labelled TIMP2/Nissl overlay to examine anatomical co-registration with radioactive signal (colour bar indicates radioactive signal from low (black) to high (white); n = 3 per group; ~20-month-old WT mice). p, Representative analytical high-performance liquid chromatography radioactivity chromatogram from mouse brain homogenates to assess stability of 64Cu-labelled TIMP2 24 h after injection. Dotted line corresponds to retention time of 11.5 min (n = 2 per group; ~20-month-old WT mice). q, Ultraviolet absorbance at 280 nm measured for TIMP2–DOTA before injection in vivo, exhibiting identical retention time as in m. r, Number of c-Fos+ cells in dentate gyrus from aged (21-month-old) WT mice treated seven times systemically every other day with vehicle (n = 8) or 50 μg kg−1 (intraperitoneal) recombinant TIMP2 (n = 7). s, t, Freezing levels for vehicle- or TIMP2-treated aged WT mice during baseline period (s) and during cued fear-conditioning task (t; n = 15 per group; eight intraperitoneal injections; 20-month-old). See Supplementary Fig. 1 for uncropped blot images. One-way ANOVA with Tukey’s post hoc test; two-way ANOVA with Bonferroni’s post hoc test for group × time comparisons; Student’s t-test for two-group comparisons. *P < 0.05, **P < 0.01, ****P < 0.0001; NS, not significant; mean ± s.e.m.

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Extended Data Figure 8 Effect of systemic TIMP2 treatment or whole-body TIMP2 deletion on dentate gyrus synaptic plasticity.

a, b, Representative dentate gyrus sections stained with doublecortin antibody (DCX, arrowheads; scale bar, 100 μm) in vehicle- or TIMP2-treated WT mice (a) and corresponding quantification (b) of total newborn neuron number in dentate gyrus (n = 15 mice per group; 20-month-old). c, Input–output relationship in dentate gyrus synapses from hippocampal slices taken from vehicle- or TIMP2-treated WT mice (n = 10 slices per group from n = 5 mice per group; eight intraperitoneal injections; 20-month-old), showing no difference in synaptic strength (basal synaptic transmission); mean ± s.d. d, e, Population spike amplitudes recorded in dentate gyrus granule cell layer after perforant path stimulation in slices from TIMP2 KO or WT mice (d) and quantification (e) of LTP maintenance phase (n = 10 slices per group from n = 5 mice per group; 2-month-old mice). f, g, Population spike amplitudes recorded in dentate gyrus granule cell layer after perforant path stimulation in TIMP2 KO slices incubated with the indicated ex vivo treatments (f) and quantification (g) of LTP maintenance phase (n = 8 slices (control IgG incubations) from n = 4 mice; n = 10 slices (anti-TIMP2 IgG incubations) from n = 5 mice; 2- to 3-month-old sex-matched mice); Student’s t-test for two-group comparisons; *P < 0.05; NS, not significant; mean ± s.e.m.

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Extended Data Figure 9 Systemic TIMP2 neutralization alters spatial memory but not activity or general health parameters.

a, b, Baseline freezing levels (a) and cued fear-conditioning freezing levels (b) in young WT mice treated with anti-TIMP2 IgG or control IgG (60 μg kg−1) for 2 weeks (n = 15 per group; 2-month-old). c, d, Serum metabolite measurements (c) and weekly weights (d) to assess general health and organ function in young WT mice treated for ~4 weeks with anti-TIMP2 IgG or control IgG. e, f, Proportion of trial time spent in centre (e) and velocity in centre (f) during open-field assessment of anxiety-like behaviour in the treated mice. gi, Velocity in zone outside the centre (g), total trial distance (h), and mean trial velocity (i; centre and outside) during open-field testing. jm, General activity (j), rearing activity (k), distance travelled (l), and mean trial velocity (m)—all monitored by SMARTCage beam-breaks in a home cage for the treated mice. n, o, Quantification of total newborn neuron number in dentate gyrus (n) with corresponding representative dentate gyrus sections stained with doublecortin antibody (o; DCX, arrowheads; scale bar, 100 μm) in control IgG- or anti-TIMP2 IgG-treated young mice; For do, n = 15 mice per group; 2.5-month-old; in c, one serum sample in each group was not submitted for metabolite testing owing to a high degree of haemolysis in these two samples; Student’s t-test; *P < 0.05; NS, not significant; mean ± s.e.m.

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Extended Data Figure 10 TIMP2 is critical for several cognitive benefits of cord plasma independent of activity behaviour.

a, Table of significant changes (ranked by effect size) in the relative level of plasma proteins in young (3-month-old) TIMP2 KO (n = 13) versus WT (n = 9) mice measured by customized protein microarray. The first two entries represent two different antibodies against TIMP2. b, Mean TIMP2 concentrations determined by ELISA (±s.d. for technical replicates) for cord plasma pre-depletion, or cord plasma after TIMP2 or control depletion. c, Silver-stained gel loaded with elution from beads used for TIMP2 (T2) or control (ctl) depletion. ‘#1’ and ‘#2’ reflect replicate plasma aliquots from the depletion process (see Supplementary Fig. 1 for uncropped gel image). d, e, Baseline freezing levels (d) and cued fear-conditioning freezing levels (e) in aged NSG mice (13.8 ± 0.1 months old) given eight intravenous injections of vehicle (n = 10), TIMP2-depleted cord plasma (n = 8), and IgG control-depleted cord plasma (n = 9). f, g, Proportion of trial time spent in centre (f) and velocity in centre (g) during open-field assessment of anxiety-like behaviour in the treated mice. hj, Velocity in zone outside the centre (h), total travel distance (i), and mean trial velocity (j; centre and outside) during open-field testing. km, General activity (k), distance travelled (l), and mean trial velocity (m) by SMARTCage beam-break monitoring for the treated mice. One-way ANOVA with Tukey’s post hoc test; Student’s t-test for WT versus TIMP2 KO comparisons with q < 0.15; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant; mean ± s.e.m.

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Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, the uncropped scans of the western blots, Ponceau S stains, and silver gel depicted in the main and Extended Data Figures and Supplementary Table 1, a list of human and mouse plasma protein microarray antibodies. (PDF 599 kb)

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Castellano, J., Mosher, K., Abbey, R. et al. Human umbilical cord plasma proteins revitalize hippocampal function in aged mice. Nature 544, 488–492 (2017). https://doi.org/10.1038/nature22067

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