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Young CSF restores oligodendrogenesis and memory in aged mice via Fgf17

Abstract

Recent understanding of how the systemic environment shapes the brain throughout life has led to numerous intervention strategies to slow brain ageing1,2,3. Cerebrospinal fluid (CSF) makes up the immediate environment of brain cells, providing them with nourishing compounds4,5. We discovered that infusing young CSF directly into aged brains improves memory function. Unbiased transcriptome analysis of the hippocampus identified oligodendrocytes to be most responsive to this rejuvenated CSF environment. We further showed that young CSF boosts oligodendrocyte progenitor cell (OPC) proliferation and differentiation in the aged hippocampus and in primary OPC cultures. Using SLAMseq to metabolically label nascent mRNA, we identified serum response factor (SRF), a transcription factor that drives actin cytoskeleton rearrangement, as a mediator of OPC proliferation following exposure to young CSF. With age, SRF expression decreases in hippocampal OPCs, and the pathway is induced by acute injection with young CSF. We screened for potential SRF activators in CSF and found that fibroblast growth factor 17 (Fgf17) infusion is sufficient to induce OPC proliferation and long-term memory consolidation in aged mice while Fgf17 blockade impairs cognition in young mice. These findings demonstrate the rejuvenating power of young CSF and identify Fgf17 as a key target to restore oligodendrocyte function in the ageing brain.

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Fig. 1: Young CSF improves memory consolidation and promotes OPC proliferation and differentiation.
Fig. 2: SRF is induced by young CSF and mediates CSF-induced OPC proliferation.
Fig. 3: SRF signalling is downregulated in hippocampal OPCs with ageing and induced by acute injection of young CSF.
Fig. 4: Fgf17 induces OPC proliferation and improves memory.

Data availability

All data are available in the main text or the Supplementary Information. Raw and processed sequencing data were deposited to NCBI’s Sequence Read Archive and Gene Expression Omnibus databases using accession code GSE198008.

Code availability

All analyses were carried out using freely available software packages. Custom code used to analyse the RNA-seq data and datasets generated and/or processed in the current study is available from the corresponding authors on request.

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Acknowledgements

We thank the members of the Wyss-Coray and Zuchero laboratories for feedback and support. Specifically, we thank H. Kantarci for advice on TEM tissue processing, imaging and analysis, H. Zhang and K. Dickey for laboratory management, and B. Carter for flow cytometry technical expertise. We thank V. Galata for graphical abstract design. We thank E. Mignot and J. Dalmau for providing human CSF samples. We also thank D. Jorgens and the staff at the University of California–Berkeley Electron Microscope Laboratory for EM sample preparation and data collection. This work was funded by the Department of Veterans Affairs (T.W.-C.), the National Institute on Aging (RF1-AG064897-02 to T.W.-C., T32AG000266 to M.S.H.), the NOMIS Foundation (T.W.-C.), the Nan Fung Life Sciences Aging Research Fund (T.W.-C.), the Glenn Foundation for Aging Research (T.W.-C.), the Big Idea Brain Rejuvenation Project and Interdisciplinary Scholar fellowship from the Wu Tsai Neurosciences Institute (T.W.-C. and T.I.), the Zuckerman STEM leadership fellowship and Tel Aviv University President Award for women postdoctoral scholars (T.I.), the National MS Society Harry Weaver Neuroscience Scholar Award (J.B.Z.), the McKnight Scholar Award (J.B.Z.), the Myra Reinhard Family Foundation and the National Institutes of Health (R01-NS119823 to J.B.Z.). H.Z. is a Wallenberg Scholar supported by grants from the Swedish Research Council (2018-02532), the European Research Council (681712), Swedish State Support for Clinical Research (ALFGBG-720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (201809-2016862) and the UK Dementia Research Institute at UCL.

Author information

Authors and Affiliations

Authors

Contributions

T.I. and T.W.-C. conceptualized the study. T.I. performed all surgical procedures. A. Kaur, S.M., H.S. and T.I. performed and analysed histology and cell culture experiments. A. Kaur, L.Y., J.L. and T.I. designed and performed behaviour experiments. S.M. performed SLAMseq experiments, which were designed and analysed by F.K. and T.I. A.R.M. and T.I. performed nuclei sorting and RNA-seq experiments with guidance from N.L. and O.H. F.K. and T.I. analysed the datasets. M.A.G. isolated OPCs from mice with loxP-flanked Srf, and M.I. and M.S.H. assisted with cell culture experiments. A.C.Y., A.R.M. and T.I. preformed labelled CSF and Fgf17 experiments. S.R.S. assisted with CSF collection. R.P. and B.L. assisted with bioinformatic analysis. H.Z. provided human CSF samples. T.I. wrote the manuscript with input from all authors, T.I. and F.K. designed manuscript figures, and J.B.Z. and T.W.-C. edited the manuscript. A. Keller, J.B.Z. and T.W.-C. supervised the work.

Corresponding authors

Correspondence to Tal Iram or Tony Wyss-Coray.

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Competing interests

T.W.-C. and T.I. are co-inventors on a patent application related to the work published in this paper (STDU2-39617.101, S21-153-Methods and compositions for improved memory in the aging). H.Z. has served at scientific advisory boards and/or as a consultant for Abbvie, Alector, Annexon, Artery Therapeutics, AZTherapies, CogRx, Denali, Eisai, Nervgen, Novo Nordisk, Passage Bio, Pinteon Therapeutics, Red Abbey Labs, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics and Wave, has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure, Biogen and Roche, and is a cofounder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside the scope of the submitted work).

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Extended data figures and tables

Extended Data Fig. 1 Bulk RNAseq, infusion site details and overall overview of proliferating cells.

a, Relative proportions of cell types as predicted by deconvolution analysis of bulk RNAseq of aged mice infused with aCSF or YM-CSF (aCSF n = 8, YM-CSF n = 7). b, Predicted number of DEGs per cell type by deconvolution analysis of bulk RNAseq of aged mice infused with aCSF or YM-CSF (aCSF n = 8, YM-CSF n = 7). c, Effect size of the subset of oligodendrocyte genes in Fig. 1d 16 h following acute injection of YM-CSF or aged mouse CSF (AM-CSF) calculated over aCSF as control (n = 4; Wilcoxon rank sum test). d, Location of infusion site. Image source: Allen Institute, Mouse brain atlas (coronal). e, Location of analysis site. Image source: Allen Institute, Mouse brain atlas (coronal). f, Hippocampal slice of 10-month-old mice given an EdU pulse prior to surgery showing low baseline proliferation, and three pulses of BrdU at day 5 and 6 of infusion showing an overall increase in proliferating cells following YM-CSF infusion (n = 4 per group; repeated measures two-way ANOVA followed by Sidak’s post-hoc test; Means ± SEM). g, Representative images of EdU (red) and BrdU (green) cells in mice with no surgery or infused with aCSF or YM-CSF. Scale bar, 500 μm. h, RNAscope of Pdgfrα+EdU+ cells in hippocampus of 2-month-old (young) and 19-month-old (aged) mice (n = 3; two-sided t-test; mean ± s.e.m.). i, Representative images of analysis in panel h. Arrows pointing to Pdgfrα+EdU+ cells. Scale bar, 100 μm.

Source Data

Extended Data Fig. 2 Cortical Pdgfrα+EdU+ cells and identity of Pdgfrα EDU+ cells.

a, Hippocampal density of Pdgfrα+ EdU+ cells per mm2 (aCSF n = 7, YM-CSF n = 8; two-sided t-test; mean ± s.e.m.). b, Hippocampal density of Pdgfrα+ cells per mm2 (aCSF n = 7, YM-CSF n = 8; two-sided t-test; mean ± s.e.m.). c, Location of region of interest in the cortex. Scale bar, 100 μm. d, Percentage of Pdgfrα+ EdU+ / Pdgfrα+ cells showing very low proliferation rates of OPCs in the cortex (n = 6; two-sided t-test; mean ± s.e.m.). e, Cortical density of Pdgfrα+ EdU+ cells per mm2 (n = 6; two-sided t-test; mean ± s.e.m.). f, Cortical density of Pdgfrα+ cells per mm2 (n = 6; two-sided t-test; mean ± s.e.m.). g, Percentage of Pdgfrα+ EdU+ / EdU+ in the hippocampus of aged mice infused with YM-CSF (n = 3). h, Example of IBA+ EdU+ cells in the hippocampus (n = 3). Scale bar, 50 μm. Insert, 10 μm. i, Example of GFAP+ EdU+ cells in the hippocampus (n = 3). Scale bar, 50 μm. Insert, 10 μm.

Source Data

Extended Data Fig. 3 Young CSF increases number of myelinated axons in the molecular layer.

a, Representative overview of 1mm diameter biopsy punch in the hippocampus. b, Representative overview of molecular layer (MoL, between dashed lines) before and after TEM imaging of three 10x10 montage squares (n=7). c, Representative montage of MoL of aged mouse infused with aCSF and YM-CSF (n = 7). Scale bar, 10 μm. d, Representative higher resolution image of aged mouse infused with aCSF and YM-CSF (n = 7). Scale bar, 1 μm. e, g-ratio analysis of myelinated axons in molecular layer. (n = 3 mice per group, aCSF n = 321 axons, YM-CSF n = 291 axons).

Source Data

Extended Data Fig. 4 Young CSF boosts OPC differentiation in vitro and validation of OPC culture purity.

a, Related to images in Fig 1o. Overview of MBP stain of OLs at day 4 of differentiation supplemented with 10% aCSF or YH-CSF (aCSF n = 3 coverslips, YH-CSF n = 2 coverslips). b, Quantification of MBP intensity of day 4 differentiated OLs. Scale bar, 200 μm. (aCSF n = 3 coverslips, YH-CSF n = 2 coverslips; two-sided t-test; mean ± s.e.m). c, Primary rat OPC cultures were supplemented with 10% aCSF or YH-CSF for 6 h and stained for NG2 (green), Olig2 (grey) and Acta2 (red). (n = 3 coverslips; Scale bar, 100 μm). d, Higher magnification of primary rat OPC cultures were supplemented with 10% aCSF or YH-CSF for 6 h and stained for NG2 (green), Olig2 (grey) and Acta2 (red). (n = 3 coverslips; Scale bar, 20 μm).

Source Data

Extended Data Fig. 5 SLAMseq QC and principal component analysis.

a, Overall conversion rates in all SLAMseq samples, showing an enrichment for T>C mutation rate (orange bar) which increases with longer incubation time (6 h). b–c, Distribution of T>C mutations across b, read position and c, 3’UTR position indicating an equal distribution of s4U incorporation along the positive strand. d–e, UMAP of aCSF and YH-CSF samples in both time points by all genes detected in the d, total and e, nascent mRNA counts. (young CSF 1 h n = 4, all the rest n = 5). f, Gene set enrichment analysis (GSEA) of 6hr genes sorted by log2FC showing an enrichment for SRF target genes by TRANSFAC75. g, Overall log2FC enrichment indicating upregulation of SRF target genes (TRANSFAC and curated list) and actin cytoskeleton genes in YH-CSF treated OPCs over aCSF. (SRF TRANSFAC (423 genes), validated SRF targets from literature (74 genes) and actin genes (212 genes); Wilcoxon rank sum test; box show the median and the 25–75th percentiles, and the whiskers indicate values up to 1.5-times the interquartile range).

Extended Data Fig. 6 YH-CSF induces actin cytoskeleton alterations in vitro.

a–b, Actin filament content measured by live imaging using SiR-actin (red) throughout 4hr of aCSF and YH-CSF exposure. Average SiR-actin a, intensity and b, area in rat OPC cultures exposed to aCSF or YH-CSF (n = 6 wells per condition; Means ± SEM). c, Representative images of experiment quantified in panel a and b. Scale bar 200 μm. d, OPC coverslips were treated with YH-CSF for 6 h and stained for phalloidin. Histogram of the percentage of OPC with the indicated number of growth cones per cell. YH-CSF treated cells show a shift towards more growth cones per cell (n = 3 coverslips per condition, total of 200 cells analyzed per condition; two-way ANOVA followed by Sidak’s post-hoc test; Means ± SEM). Scale bar 20 μm. e, mouse OPC primary cultures from SRF-fl/fl pups infected with CRE-GFP and CRE-GFP AAVs to induce recombination. Representative images of infected cells (green) 48 h after infection. Scale bar, 100 μm. f, Normalized SRF mRNA levels as measured by RT-PCR (n = 3 coverslips per condition; mean ± s.e.m.). g, Representative image of data presented in figure 2h. Scale bar, 20 μm. h, Quantification of GFP+ cells per image in SRF-WT and SRF-KO cells treated with 10% aCSF or YH-CSF. (n = 3; mean ± s.e.m.). i, Quantification of number of DAPI cells per image in SRF-WT and SRF-KO cells treated with 10% aCSF or YH-CSF. (n = 3; mean ± s.e.m.). Data in panels a-i were replicated in two independent experiments.

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Extended Data Fig. 7 Bulk RNAseq of hippocampal OPC and OL nuclei from young and aged mice.

a, Gating strategy for sorting of hippocampal OPC and OL nuclei. b, Heatmap of expression OPC and OL specific genes across young and aged OPC and OL samples (aged OL n = 3, rest n = 4). c, Volcano plot showing OL genes up and downregulated with age (n = 4; p. adjusted value by Wald test in DESeq2). d, Pathways enriched (red) or depleted (blue) in hippocampal OLs with age (unweighted Kolmogorov-Smirnow test).

Extended Data Fig. 8 Bulk RNAseq of hippocampal OPC and OL nuclei from aged mice following acute injection and Srf levels in neurons.

a, Box plot of effect size of Srf targets (TRANSFAC database) in hippocampal OLs from aged vs. young, YM-CSF vs. aCSF at 1 h and 6 h timepoints (n = 4; genes pre-filtered by p < 0.05 cutoff; Wilcoxon rank sum test, box show the median and the 25–75th percentiles, and the whiskers indicate values up to 1.5-times the interquartile range). b, Pathways enriched (red) or depleted (blue) in hippocampal OPCs 1hr following injection of aCSF or YM-CSF (n = 4; p. adjusted value by Wald test in DESeq2). c, Volcano plot showing OPC genes up and down regulated 1hr following CSF injection (n = 4; p. adjusted value by Wald test in DESeq2). d, Volcano plot showing OPC genes up and down regulated 6hr following CSF injection (n = 4; p. adjusted value by Wald test in DESeq2). e, Neuronal Srf intensity in CA1 in young and aged mice. (n = 3; two-sided t-test; mean ± s.e.m). f, Representative image of panel e. Scale bar, 70 μm. g, Neuronal Srf intensity in CA1 in aged mice following YM-CSF infusion. (n = 4; two-sided t-test; mean ± s.e.m). h, Representative image of panel g. Scale bar, 70 μm.

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Extended Data Fig. 9 Fgf8 induces OPC proliferation and Fgf17 induces SRF reporter activation mediated by actin dynamics and Fgfr3.

a, Dose-dependent activation of SRE-GFP reporter by increasing concentrations of Fgf8 and representative images of the experiment at 15.5 h. Scale bar, 400 μm. (n = 3; similar control as in Fig. 4c; one-way ANOVA followed by Sidak’s post-hoc test; mean ± s.e.m.). b, Percentage of BRDU+/DAPI primary rat OPCs treated with 10, 20, 40 ng/ml Fgf8. (n = 4; one-way ANOVA followed by Tukey’s post-hoc test; mean ± s.e.m.). c, Quantification of OPC proliferating cells (Pdgfrα+EDU+ / Pdgfrα+ cells) in the CA1 region of the hippocampus of 20-month-old mice following a week of aCSF or Fgf8 infusion. (aCSF n = 8 similar control as in Fig. 4l, Fgf8 n = 4; two-sided t-test; mean ± s.e.m.). d, SRE-GFP activation with 200 ng/ml Fgf17 following 30 min pre-treatment with Jasplakinolide (Jasp, 125 or 250 nM) or Latrunculin A (LatA, 250 or 500 nM). (n = 3; Two-way ANOVA with Tukey’s multiple comparisons test; mean ± s.e.m.). e, SRE-GFP activation with 200 ng/ml Fgf17 following 30 min pre-treatment with blocking antibodies for FgfR1, FgfR2, FgfR3 (all 50 μg/ml) or FgfR3 alone (n = 3; One-way ANOVA with Sidak’s multiple comparisons test; mean ± s.e.m.). f, Example of Pdgfrα+ Fgfr3+ cells in the hippocampus of young mice. (n = 3). Scale bar, 5 μm.

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Extended Data Fig. 10 Fgf17 is predominantly expressed in the brain by a subset of neurons and is downregulated with age.

a, Fgf17 is predominantly expressed in the brain based on the human protein atlas. b, Fgf17 is expressed by cortical glutamatergic neurons in the young adult mouse (Allen brain atlas). c, Sub-clustering of mouse cortical layer 4/5 neurons indicates expression by a subset of cortical neurons (Allen brain atlas). d, Gene set enrichment analysis of genes mostly correlated with Fgf17 in layer 4/5 neurons (Allen brain atlas). e, Fgf17 is expressed by cortical glutamatergic and GABAergic neurons in the human cortex (Allen brain atlas). f, Representative image of analysis in panel g. Scale bar, 100 μm. g, Fgf17 mRNA expression in cortical neurons drops dramatically in aged mice. (n = 3; two-way student t-test; mean ± s.e.m.). h, Fgf17 protein expression in cortical and hippocampal neurons drops dramatically in aged mice. (n = 3; mean ± s.e.m.). i, Representative images of analysis in panel h and Fig. 4f. Scale bar, 20 μm.

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Extended Data Fig. 11 Perfusion of labeled YH-CSF and mouse Fgf17 to the brain parenchyma and working model.

a, Deposition of labeled Fgf17 on ventricular walls 3 h post ICV acute injection (n = 3). Scale bar, 300 μm. b, Deposition of labeled YH-CSF on lateral ventricle walls 2 h post ICV acute injection (n = 3). Scale bar, 100 μm. c, Labeled Fgf17 in perivascular spaces in the molecular layer of the hippocampus (n = 3). Scale bar, 50 μm. d, Labeled YH-CSF in perivascular spaces in the molecular layer of the hippocampus (n = 3). Scale bar, 20 μm. e, YH-CSF in the perivascular space in between the vessel (green) and astrocyte endfeet (white; n = 3). Scale bar, 20 μm. f, Orthogonal slice of YH-CSF (magenta) in perivascular space, in between the vessel (green) and astrocyte endfeet (white; n = 3). Scale bar, 20 μm. g, Working model. OPC proliferation and differentiation (termed oligodendrogenesis) slow down with age40,41,42,43. Re-exposure of the aged brain to young CSF or the brain-specific growth factor Fgf1745, boost hippocampal oligodendorgenesis, concomitant with improvement in long term memory recall.

Supplementary information

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Supplementary Table 1

Normalized gene counts and summary statistics of bulk RNA-seq of aged hippocampi infused with aCSF or YM-CSF for 6 d (linked to Fig. 1).

Supplementary Table 2

Normalized gene counts and summary statistics of hippocampal OPC nuclei 16 h after acute ICV injection with aCSF, YM-CSF or AM-CSF (linked to Extended Data Fig. 1c).

Supplementary Table 3

SLAMseq gene counts and statistics: nascent (TC) and total mRNA counts for rat OPCs treated with aCSF or YM-CSF for 1 or 6 h (linked to Fig. 2).

Supplementary Table 4

Gene lists used in the study. a, b, SRF targets according to the human TRANSFAC database (aligned to rat and mouse). c, Curated list of SRF targets from the literature. d, GO term actin cytoskeleton (rat).

Supplementary Table 5

Normalized gene counts and summary statistics for sorted OPC and OL nuclei from young and aged hippocampi (linked to Fig. 3).

Supplementary Table 6

Normalized gene counts and summary statistics for aged hippocampal OPC and OL nuclei 1 or 6 h after acute ICV injection with aCSF or YM-CSF (linked to Fig. 3).

Supplementary Table 7

SRF targets present in CSF proteomic datasets. a, Proteins tested in the SRE reporter assay. b, SRF targets in CSF datasets that were not tested in the SRE reporter assay.

Supplementary Video 1

OPCs growing under differentiation conditions with 10% aCSF. OPCs were treated with 10% aCSF under differentiation conditions (with T3) for 4 d and imaged in the IncuCyte every 2 h for 4 d. Scale bar, 200 μm.

Supplementary Video 2

OPCs growing under differentiation conditions with 10% YM-CSF. OPCs were treated with 10% YH-CSF under differentiation conditions (with T3) for 4 d and imaged in the IncuCyte every 2 h for 4 d. Scale bar, 200 μm.

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Iram, T., Kern, F., Kaur, A. et al. Young CSF restores oligodendrogenesis and memory in aged mice via Fgf17. Nature 605, 509–515 (2022). https://doi.org/10.1038/s41586-022-04722-0

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