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Midkine attenuates amyloid-β fibril assembly and plaque formation

Nature Structural & Molecular Biology (2025)Cite this article

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Abstract

Proteomic profiling of Alzheimer disease (AD) brains has identified numerous understudied proteins, including midkine (MDK), that are highly upregulated and correlated with amyloid-β (Aβ) from the early disease stage but their roles in disease progression are not fully understood. Here, we present that MDK attenuates Aβ assembly and influences amyloid formation in the 5xFAD amyloidosis mouse model. MDK protein mitigates fibril formation of both Aβ40 and Aβ42 peptides according to thioflavin T fluorescence, circular dichroism, negative-stain electron microscopy and nuclear magnetic resonance analyses. Knockout of the Mdk gene in 5xFAD increased amyloid formation and microglial activation in the brain. Further comprehensive mass-spectrometry-based profiling of the whole proteome and detergent-insoluble proteome in these mouse models indicated significant accumulation of Aβ and Aβ-correlated proteins, along with microglial components. Thus, our structural and mouse model studies reveal a protective role of MDK in counteracting amyloid pathology in AD.

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Fig. 1: MDK is highly correlated and colocalized with Aβ in AD and in the FAD mouse model.
Fig. 2: Characterization of recombinant MDK proteins and their impact on Aβ fibrillation.
Fig. 3: MDK inhibits the fibril assembly of Aβ40 and Aβ42 peptides and associates with Aβ filaments from AD brain.
Fig. 4: MDK rescues NMR signals of Aβ peptides.
Fig. 5: Mdk gene KO in FAD mice results in Aβ accumulation, plaque increase and microglia activation.
Fig. 6: Brain tissue proteomics reveals that Mdk KO leads to the accumulation of Aβ and Aβ-correlated proteins, along with microglia activation in FAD.

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Data availability

The NMR chemical shift assignments are available from the BMRB under accession number 17795. Information on all human cases is provided in Supplementary Data 2. The MS proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE partner repository under dataset identifiers: PXD007985 (human whole-proteome dataset from previous publication16), PXD046539 and PXD061103 (whole proteome of AD mouse models) and PXD045746 and PXD061104 (detergent-insoluble proteome of the mice). Source data are provided with this paper.

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Acknowledgements

We thank I. Chen for critical readings and comments. We also thank the St. Jude Shared Resources and Core Facilities, including the Protein Technology Center, Biomolecular NMR Center, Cryo-EM and Tomography Center, Cell and Tissue Imaging Center, Animal Research Center, Center for Advanced Genome Engineering and Center for Proteomics and Metabolomics. This work was partially supported by National Institutes of Health grants R01AG053987, RF1AG064909, RF1AG068581, U19AG069701 and P30CA021765 and the American Lebanese Syrian Associated Charities foundation. The Banner Sun Health Research Institute Brain and Body Donation Program was supported by National Institutes of Health grants U24NS072026, P30AG019610 and P30AG072980, the Arizona Department of Health Services, the Arizona Biomedical Research Commission and the Michael J. Fox Foundation for Parkinson’s Research.

Author information

Author notes

  1. Shu Yang

    Present address: Department of Medical Research Center, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China

  2. These authors contributed equally: Masihuz Zaman, Shu Yang, Ya Huang.

Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Masihuz Zaman, Shu Yang, Ya Huang, Jay M. Yarbro, Yanhong Hao, Zhen Wang, Danting Liu, Kiara E. Harper, Hadeer Soliman, Alex Hemphill, Sarah Harvey, Ajay Singh Tanwar, Ravi Kalathur, Christy R. Grace, Martin Turk, Sagar Chittori, Yun Jiao, Zhiping Wu, Ping-Chung Chen & Junmin Peng

  2. Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Masihuz Zaman, Shu Yang, Ya Huang, Jay M. Yarbro, Yanhong Hao, Zhen Wang, Danting Liu, Kiara E. Harper, Hadeer Soliman, Alex Hemphill, Sarah Harvey, Valerie Stewart, Yun Jiao, Zhiping Wu, Ping-Chung Chen & Junmin Peng

  3. Center for Advanced Genome Engineering, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Shondra M. Pruett-Miller

  4. Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Shondra M. Pruett-Miller

  5. Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Anthony A. High & Xusheng Wang

  6. Banner Sun Health Research Institute, Sun City, AZ, USA

    Geidy E. Serrano & Thomas G. Beach

  7. Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Gang Yu

  8. Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA

    Yang Yang

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  1. Masihuz Zaman
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Contributions

J.P., P.-C.C., G.Y. and M.Z. conceptualized the project. M.Z., Y.Y, J.M.Y., Y.H., A.S.T., R.K., C.R.G., M.T. and S.C. engaged in the biophysical experiments. S.Y., Z. Wang, K.E.H., H.S., A.H., S.H., Y.J. and P.-C.C. performed the biological experiments. P.-C.C., S.Y., Y.J., S.M.P.-M. and V.S. generated and bred the mouse models. S.Y., Z. Wang, D.L., Y, H., Z. Wu, A.A.H. and X.W. contributed to the MS-based proteomics analysis. G.E.S. and T.G.B. characterized and provided the human brain samples. M.Z., Y.H., P.-C.C. and J.P. wrote the manuscript.

Corresponding authors

Correspondence to Yang Yang, Ping-Chung Chen or Junmin Peng.

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Extended data

Extended Data Fig. 1 Bottom-up MS analysis of recombinant MDK proteins and their effect on Aβ fibrillation.

a, Protein sequences of MDK proteins expressed in mammalian 293 or E. coli cells. The N-terminal His-tag was cleaved after expression by TEV protease. b, Workflow of bottom-up LC-MS/MS analysis to characterize the positions of disulfide (S-S) bonds. Proteins were subjected to IAA alkylation, trypsin digestion and LC-MS/MS analysis. Crosslinked peptides containing single or double S-S bonds were identified. c, Relative peak intensities of top five crosslinked peptides (n = 3 replicates). Data are shown as mean ± SEM. d-e, Effect of 293- or E. coli-expressed MDK (10 µM) on Aβ40/42 (5 µM) fibrillation kinetics in ThT fluorescence assays. Results represent the average of 3 replicates. Full statistical information is in Source Data Statistics.

Extended Data Fig. 2 Characterization of the effect of MDK on Aβ fibrillation.

a, Diagram of the secondary nucleation model illustrating Aβ assembly. The rate constants (k+ and k2) are defined with units, reflecting their dependency on protein concentration and time. b-c, Analysis of Aβ40 and Aβ42 elongation constants (k+) by fitting the ThT data. d, Effect of MDK (0-3 µM) on Aβ40 fibrillation kinetics (n = 3 replicates, averaged data shown). e, Effect of MDK (0-3 µM) on Aβ42 fibrillation kinetics (n = 3 replicates, averaged data shown). f, Time course CD spectra of Aβ40 alone (0-24 h). g, Time course CD spectra of Aβ40 with MDK (0-24 h). h, Time course CD spectra of Aβ42 alone (0-24 h). i, Time course CD spectra of Aβ42 with MDK (0-24 h). Full statistical information is in Source Data Statistics.

Extended Data Fig. 3 Characterization of ubiquitin protein by MS and its lack of effect on Aβ40/42 fibrillation.

a, Purified Ub protein on a stained SDS gel. b-c, Top-down mass spectrum and deconvoluted results of Ub protein showing different charge states. Similar results were obtained in two independent experiments. d, Bottom-up LC-MS/MS analysis of Ub, covering the full Ub sequence except two short tryptic peptides (in grey). e, ThT fluorescence assay measuring Aβ40 fibril formation at different Ub concentrations (n = 3 replicates, averaged data shown). f, CD spectroscopy of Aβ40 with or without Ub, with ellipticity reported in millidegrees (mdeg). g, Negative stain EM of Aβ40/Ub samples with a scale bar (100 nm). h, ThT fluorescence assay measuring Aβ42 fibril formation at different Ub concentrations (n = 3 replicates, averaged data shown). i. CD spectroscopy of Aβ42 with or without Ub. j, Negative stain EM of Aβ42/Ub samples with a scale bar (100 nm). Similar results were obtained in two independent experiments. Full statistical information is in Source Data Statistics.

Extended Data Fig. 4 MDK or ubiquitin cannot independently assemble into fibrils.

a, ThT assay of MDK alone at different concentrations (n = 3 replicates, averaged data shown). b, Time course CD spectra of MDK (0-24 h). c, Negative stain EM of the MDK samples, with a scale bar (100 nm). Similar results were obtained in two independent experiments. d, ThT assay of Ub alone at different concentrations (n = 3 replicates, averaged data shown). e, Time course CD spectra of Ub (0-24 h). f, Negative stain EM of the Ub samples, with a scale bar (100 nm). Similar results were obtained in two independent experiments. Full statistical information is in Source Data Statistics.

Extended Data Fig. 5 Structural prediction of Aβ-MDK interaction.

a, The alignment of human and mouse MDK protein sequences. MDK is a secreted protein containing a signal peptide for secretion (aa 1–22) and the main protein chain (aa 23–143). b, AlphaFold2-multimer predicted structures of the Aβ-MDK potential interacting interfaces. The confidence of the predicted 3D structures is evaluated by the predicted Local Distance Difference Test (pLDDT). Full statistical information is in Source Data Statistics.

Extended Data Fig. 6 Ubiquitin, used as a control, does not prevent the loss of NMR signals in Aβ peptides.

a, 1H-15N HSQC spectra of Aβ40 (10 μM) with or without Ub (10 μM) in 50 mM Tris buffer (pH 7.5). Spectra were collected before incubation (left panel) and after 48 h incubation (right two panels). b, Relative cross-peak intensities for each residue, excluding D1 and H14. Intensities were normalized to a maximum value of 1. c-d, NMR analysis of Aβ42 under similar conditions as Aβ40, but with 24 h incubation.

Extended Data Fig. 7 MDK expression in KO mice, cell-type specificity, and effects on Aβ pathology.

a, Predicted mouse MDK protein sequences in WT and CRISPR-mediated KO mice. A 23 bp deletion in Mdk gene disrupts the open reading frame in KO mice, potentially generating a truncated protein with distinct C-terminus (blue). Peptides detected by MS in brain proteomic analysis are marked with lines. The two shared peptides (aa 30-47 and aa 31-47, red line) allow quantification of MDK full length proteins and the tentative N-terminal truncation. b, Quantification of shared peptides in brain lysates (FAD n = 5, FAD/KO n = 7), normalized to the highest value (100). The absence of signals in FAD/KO mice suggests that the tentative N-terminal truncation was not expressed at a detectable level. c, Uniform Manifold Approximation and Projection plots showing expression of Mdk, Gfap (astrocyte marker) and Pdgfra (oligodendrocyte progenitor cell marker) in mouse brain, from single-cell RNA sequencing of more than 1.2 million cells in the whole cortex and hippocampus cells using the 10x Genomics Chromium platform44. Expression levels are indicated by color intensity (red). Mdk is highly expressed in astrocyte (377_Astro; trimmed mean (25-75%) Log2(CPM + 1) = 4.13), and oligodendrocyte (366_Oligo; trimmed mean value = 0.72). d, ELISA analysis of Aβ40/Aβ42 ratio in the Sarkosyl-soluble and -insoluble cortical fractions (n = 10 per group). e, Quantification of X34-positive amyloid plaque density in the cortices of male (FAD n = 5; FAD/KO n = 5) and female (FAD n = 5, FAD/KO n = 6) mice. f, Quantification of X34-positive amyloid plaque area in the same mice as in e; value normalized to the FAD mean (set to 1). g, Example images of X34 staining showing amyloid plaque quantification in the cortices of FAD and FAD/KO mice. Cortices were outlined by the white lines. Experiments were independently repeated three times. Scale bar, 500 µm. b, d-f. Results were analyzed using a two-tailed unpaired Student’s t-test when equal variances were confirmed, or Welch’s t-test when unequal variances were identified based on preliminary variance testing. Data are shown as mean ± SEM. Full statistical information is available in Source Data.

Extended Data Fig. 8 Proteomic comparison of four genotypes derived from FAD and Mdk KO mice.

a, Boxplot of whole proteome data showing equal loading across samples, with WT, KO, FAD and FAD/KO represented in purple, blue, orange, and green, respectively. b, PCA of whole proteome data using DEPs, showing genotype-specific separation. c, Representative volcano plot showing DEPs between FAD and FAD/KO mice, with FDR values from limma analysis and log2 ratios converted to z values. d, Selected DEPs in four whole proteome clusters (WPC), with each protein represented by a colored box after log2 conversion and Z score analysis. e, Boxplot of insoluble proteome data. f, PCA of insoluble proteome data using DEPs. g, Volcano plot comparing the insoluble proteome of FAD and FAD/KO mice. h, Heatmap of selected DEPs shared between the insoluble and whole proteomes. i, Full-length APP protein diagram showing the position of a representative non-Aβ tryptic peptide. j, Quantification of the non-Aβ peptide levels in whole proteome across four genotypes (WT n = 8, KO n = 8, FAD n = 15, FAD/KO n = 17), with statistical significance determined by a two-tailed Student’s t-test. Data are shown as mean ± SEM.

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Source data

Source Data Figures and Extended Data Figures

Uncropped western blots for Figs. 1b and 5c.

Source Data Figures and Extended Data Figures

Statistical source data for Figs. 1a,b, 2e,f, 3a–c,f, 4c–f and 5b,d–f and Extended Data Figs. 1c–e, 2d–h,i, 3e,i, 4a,b,d,e, 5b,d and 7b,d–f.

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Zaman, M., Yang, S., Huang, Y. et al. Midkine attenuates amyloid-β fibril assembly and plaque formation. Nat Struct Mol Biol (2025). https://doi.org/10.1038/s41594-025-01657-8

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