Journal: Nature

Article Title: Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

doi: 10.1038/nature22067

Figure Lengend Snippet: a, Heat maps from unsupervised hierarchical cluster analysis of human (n = 15 cord, 19 young, 16 elderly subjects; |d-score| ≥ 1.30) or mouse (n = 8 mice per group; 3, 6, 12, 18, and 24 months of age; |d-score| ≥ 1.3) plasma protein changes analysed by significance analysis of microarray (SAM) for quantitative ageing correlations. Relative Z-scored values indicated in graded yellow (high) or blue (low). b, Number of c-Fos-positive cells in dentate gyrus from aged (16-month-old) WT mice treated four times every other day with 50 μg kg−1 (intraperitoneal) recombinant GAS6, CSF2, or TIMP2 or vehicle (n = 6 mice per group). c, ELISA-based plasma TIMP2 measurements from subjects in a. d, e, Representative TIMP2 immunoblot from equal volumes of 7- to 8-week-old or 20-month-old WT mouse plasma with corresponding Ponceau S stain (d) and quantification (e; n = 8 per group); see Supplementary Fig. 1 for uncropped image. f, Quantification of TIMP2+ cells with NeuN+ nuclei in dentate gyrus (hilus) by confocal microscopy of WT mice at indicated ages (n = 8, 8, 6, 8, 9 mice per group (left to right)). g, h, Brain uptake of 64Cu-labelled bovine serum albumin (BSA) (n = 4 mice per time point (n = 3 at 24 h)) and TIMP2 (n = 5 mice per time point) in WT mice (21-month-old) euthanized at indicated time points after injected (intraperitoneal) tracer dose (ID; g) and as a proportion of tracer remaining in blood (h). i, c-Fos-positive cell counts within dentate gyrus from WT mice in normal housing (NH) or enriched housing (EH) injected four times with vehicle or TIMP2 (n = 8, 8, 8, 7 per group (left to right); 20-month-old; *versus vehicle/normal housing; ‡versus vehicle/enriched housing; † versus TIMP2/normal housing). One-way ANOVA with Tukey’s post hoc test (Dunnett’s in b); two-way ANOVA with Bonferroni’s post hoc test for group × time comparisons; Student’s t-test for two-group comparisons; one, two, or four symbols denote P < 0.05, 0.01, 0.0001, respectively; mean ± s.e.m. Mouse schematic adapted from ref. 16.

Article Snippet: In young WT mice that had been injected with 60 μg kg −1 anti-TIMP2 antibody (R&D Systems, AF971; dose based on the in vitro ND 50 (50% neutralizing dose from the manufacturer) or goat control IgG (R&D Systems), novel location recognition was performed as previously described 43 .

Techniques: Clinical Proteomics, Microarray, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Confocal Microscopy, Injection

Journal: Nature

Article Title: Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

doi: 10.1038/nature22067

Figure Lengend Snippet: 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.

Article Snippet: In young WT mice that had been injected with 60 μg kg −1 anti-TIMP2 antibody (R&D Systems, AF971; dose based on the in vitro ND 50 (50% neutralizing dose from the manufacturer) or goat control IgG (R&D Systems), novel location recognition was performed as previously described 43 .

Techniques: Clinical Proteomics, Western Blot, Staining, Control, Confocal Microscopy, Injection, Ex Vivo, Autoradiography, High Performance Liquid Chromatography, Radioactivity, In Vivo, Recombinant

Journal: Nature

Article Title: Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

doi: 10.1038/nature22067

Figure Lengend Snippet: a–d, Barnes maze escape latency for all trial days (a), fourth-day acquisition rate (b), freezing levels during contextual fear-conditioning (c), and nesting behaviour within 24 h of providing intact nestlet (d) for aged WT mice given eight systemic vehicle or TIMP2 injections every other day (n = 15 per group; 20-month-old). e, f, Population spike amplitudes recorded in dentate gyrus granule cell layer following perforant path stimulation (e) and maintenance-phase LTP quantification (f; n = 10 slices per group from n = 5 mice per group; eight intravenous injections of vehicle or TIMP2; 20-month-old). g, h, PSA recorded in dentate gyrus granule cell layer following perforant path stimulation in slices incubated with indicated ex vivo treatments (g) and maintenance-phase LTP quantification (h; n = 10 slices per group from n = 7, 4, 5 WT mice per group (left to right); 2-month-old mice). i, Discrimination for novel object displacement on day 2 for young WT mice treated every other day for 4 weeks with anti-TIMP2 IgG or control IgG (n = 15 per group; 2.5-month-old). j–l, Latency to escape hole in Barnes maze (j), day 4 acquisition rate (k), and contextual fear-conditioning freezing levels (l) in aged NSG mice (13.8 ± 0.1 months old) given eight intravenous injections of vehicle (n = 10), TIMP2-depleted cord plasma (n = 8), or IgG control-depleted cord plasma (n = 9) (in j, * and † indicate control-depleted cord plasma versus either vehicle or TIMP2-depleted cord plasma groups, respectively). One-way ANOVA with Tukey’s post hoc test for h, k, l; two-way repeated-measures ANOVA with Bonferroni’s post hoc test for a, j; Student’s t-test for two-group comparisons; χ2 analysis for d; two-way ANOVA with Tukey’s post hoc test for i; *P < 0.05, #P = 0.05, **P < 0.01, † † † or ***P < 0.001, ****P < 0.0001; mean ± s.e.m.

Article Snippet: In young WT mice that had been injected with 60 μg kg −1 anti-TIMP2 antibody (R&D Systems, AF971; dose based on the in vitro ND 50 (50% neutralizing dose from the manufacturer) or goat control IgG (R&D Systems), novel location recognition was performed as previously described 43 .

Techniques: Incubation, Ex Vivo, Control, Clinical Proteomics

Journal: Nature

Article Title: Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

doi: 10.1038/nature22067

Figure Lengend Snippet: 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.

Article Snippet: In young WT mice that had been injected with 60 μg kg −1 anti-TIMP2 antibody (R&D Systems, AF971; dose based on the in vitro ND 50 (50% neutralizing dose from the manufacturer) or goat control IgG (R&D Systems), novel location recognition was performed as previously described 43 .

Techniques: Staining, Transmission Assay, Incubation, Ex Vivo, Control

Journal: Nature

Article Title: Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

doi: 10.1038/nature22067

Figure Lengend Snippet: 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. g–i, Velocity in zone outside the centre (g), total trial distance (h), and mean trial velocity (i; centre and outside) during open-field testing. j–m, 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 d–o, 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.

Article Snippet: In young WT mice that had been injected with 60 μg kg −1 anti-TIMP2 antibody (R&D Systems, AF971; dose based on the in vitro ND 50 (50% neutralizing dose from the manufacturer) or goat control IgG (R&D Systems), novel location recognition was performed as previously described 43 .

Techniques: Neutralization, Activity Assay, Control, Staining

Journal: Nature

Article Title: Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

doi: 10.1038/nature22067

Figure Lengend Snippet: 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. h–j, Velocity in zone outside the centre (h), total travel distance (i), and mean trial velocity (j; centre and outside) during open-field testing. k–m, 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.

Article Snippet: In young WT mice that had been injected with 60 μg kg −1 anti-TIMP2 antibody (R&D Systems, AF971; dose based on the in vitro ND 50 (50% neutralizing dose from the manufacturer) or goat control IgG (R&D Systems), novel location recognition was performed as previously described 43 .

Techniques: Clinical Proteomics, Activity Assay, Microarray, Enzyme-linked Immunosorbent Assay, Control, Staining