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Abstract

Excitatory neurons are preferentially impaired in early Alzheimer’s disease but the pathways contributing to their relative vulnerability remain largely unknown. Here we report that pathological tau accumulation takes place predominantly in excitatory neurons compared to inhibitory neurons, not only in the entorhinal cortex, a brain region affected in early Alzheimer’s disease, but also in areas affected later by the disease. By analyzing RNA transcripts from single-nucleus RNA datasets, we identified a specific tau homeostasis signature of genes differentially expressed in excitatory compared to inhibitory neurons. One of the genes, BCL2-associated athanogene 3 (BAG3), a facilitator of autophagy, was identified as a hub, or master regulator, gene. We verified that reducing BAG3 levels in primary neurons exacerbated pathological tau accumulation, whereas BAG3 overexpression attenuated it. These results define a tau homeostasis signature that underlies the cellular and regional vulnerability of excitatory neurons to tau pathology.

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The data used to generate the results that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank M. Diamond (UT Southwestern Medical Center) for providing the Tau RD-P301S-YFP lentivirus and DS9 clone cell line; K. Zhang and B. Lake (UCSD) and A. Regev, N. Habib, I. Avraham-Davidi, and A. Basu (Broad Institute of MIT and Harvard) for sharing their single-nucleus RNA-seq datasets; L. Honig, J.P. Vonsattel, A. Teich, and E. Cortés (New York Brain Bank, Columbia University Medical Center), the NIH NeuroBrainBank at the University of Maryland Brain and Tissue Bank as well as T. Beach and G. Serrano (Banner Sun Health Research Institute Brain and Body Donation Program) for providing human de-identified brain tissue and for helpful discussions on brain regions and immunostaining techniques; P. Davies (The Feinstein Institute for Medical Research) for providing the MC1 and PHF1 tau antibodies; P. Dolan (Prothena) for providing the 12E8 tau antibody; C. Pröschel (University of Rochester Medical Center) for providing psPAX2 and VSVG; ACDBio for troubleshooting on RNAscope FISH; and W.H. Yu and C.L. Clelland for discussing the results. This work was funded by: NIH/NIA AG056673 (H.F.), Alzheimer’s Association AARF-17-505009 (H.F.), NIH/NINDS NS074874 (K.E.D.), NIH/NIA AG056151 (K.E.D) and by the BrightFocus Foundation, the Rainwater Foundation/Tau Consortium and the Cure Alzheimer’s Fund (K.E.D). The Banner Sun Health Research Institute Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimer’s Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinson’s Disease Consortium), and the Michael J. Fox Foundation for Parkinson’s Research.

Author information

Author notes

    • Paula V. M. Cauhy

    Present address: Federal University of Uberlândia, Uberlândia, Brazil

Affiliations

  1. Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, New York, NY, USA

    • Hongjun Fu
    • , Yoshikazu Nakano
    • , Nancy C. Hernandez Villegas
    • , Paula V. M. Cauhy
    • , Benjamin A. Lassus
    • , Shuo Chen
    • , Stephanie L. Fowler
    • , Helen Y. Figueroa
    • , Edward D. Huey
    •  & Karen E. Duff
  2. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA

    • Hongjun Fu
    •  & Karen E. Duff
  3. Department of Neuroscience, Chronic Brain Injury, Discovery Themes, The Ohio State University, Columbus, OH, USA

    • Hongjun Fu
  4. Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Cambridge, UK

    • Andrea Possenti
    • , Rosie Freer
    •  & Michele Vendruscolo
  5. Department of Anesthesiology, University of Rochester, Rochester, NY, USA

    • Maoping Tang
    •  & Gail V. W. Johnson
  6. Departments of Psychiatry and Neurology, Columbia University, New York, NY, USA

    • Edward D. Huey
  7. Division of Integrative Neuroscience, New York State Psychiatric Institute, New York, NY, USA

    • Karen E. Duff

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Contributions

K.E.D., H.F., and M.V. jointly designed and supervised the study, discussed the results, and wrote the paper. H.F. designed and performed experiments and analyzed the data. A.P., R.F., and M.V. analyzed single-nucleus RNA-seq datasets. A.P. discussed the results and wrote parts of the paper. Y.N., N.C.H.V., M.T., P.V.M.C., B.A.L., S.L.F., S.C., and H.Y.F. provided technical assistance. E.D.H. provided critical input into the paper. G.V.W.J. designed and supplied all the BAG3-related viruses. All authors discussed the results and contributed to the manuscript.

Competing interests

K.E.D. is on the board of directors and SAB of Ceracuity LLC. All other authors declare no competing interests.

Corresponding authors

Correspondence to Hongjun Fu or Michele Vendruscolo or Karen E. Duff.

Integrated supplementary information

  1. Supplementary Figure 1 Tau pathology co-localizes with EX, but not with IN neurons in secondary affected regions in EC-tau mice.

    (a-d) Representative images of MC1+ tau staining co-localized with TBR1+ and SATB2+ EX neurons, but not PVALB+, SST+ or CALB2+ IN neurons, in the PRH (a) and NC (b) of EC-tau mice at 22 months (n = 6 animals, 2 sections each animal), and in the PRH (c) and NC (d) of EC-tau mice at 30+ months (n = 5-6 animals, 2 sections each animal). Scale bar, 20 µm.

  2. Supplementary Figure 2 Tau pathology co-localizes with EX, but not with IN neurons or glial cells in human AD brain.

    (a) Representative images of MC1+ tau staining co-localized with TBR1+ and SATB2+ EX neurons, but not PVALB+, SST+ or CALB2+ IN neurons in the prefrontal cortex (BA9) of human AD brain at Braak stage V-VI. (b, c) Representative images of AT8+ tau staining did not co-localize with IBA-1+ microglia (b) or GFAP+ astrocytes (c) in the EC of human AD brain at different Braak stages. Scale bars, 20 µm (a) and 10 µm (b and c). Three independent experiments were repeated with similar results.

  3. Supplementary Figure 3 Tau pathology detected by specific phospho-tau antibodies co-localizes with EX, but not IN, neurons in EC-tau mice and in human AD brain.

    (a) Representative images of phospho-tau staining (AT8+, PHF1+, pS422+) co-localized with SATB2+ EX neurons, but not GAD1+ IN neurons in the MEC of EC-Tau mice at 22 months. (b) Representative images of phospho-tau staining (AT8+, PHF1+, AT100+) co-localized with SATB2+ EX neurons, but not GAD1+ IN neurons in the BA9 of human AD at Braak stage V-VI. Scale bar, 20 µm. Three independent experiments were repeated with similar results.

  4. Supplementary Figure 4 Null model for seven different subproteomes between EX and IN neurons.

    The random distribution of the mean values (orange histogram) computed on each sample, the normal fit (solid orange line), and the observed value (dashed red vertical line) are reported in each panel (see Methods for details). Next to each observed value, the probability computed with the cumulative distribution obtained from the fit and evaluated at the observed mean is reported. (a-g) Null models from the SNS dataset; (h-n) Null models from the DroNc-Seq dataset. Here nsns and ndrnc represent the sample sizes corresponding to SNS and DroNc-seq datasets, respectively. (a, h) EX markers:): nsns=ndrnc=2; (b, i) tau: nsns=ndrnc=1; c, j) promoters (tau aggregation promoters): nsns=ndrnc=6; (d, k) MS (metastable subproteome): nsns=162, ndrnc=179; (e, l) tangles (tangle co-aggregators): nsns=57, ndrnc=68; f, m) protectors (tau aggregation protectors): nsns=ndrnc=6; (g, n) IN markers: nsns=ndrnc=3. A p-value is computed as the probability to have a value more extreme than the observed one (one-tailed).

  5. Supplementary Figure 5 Null model for five different subproteomes in EX neurons between early- and late-affected brain regions.

    The statistical significance of the results is studied by creating a null model for each subproteome under scrutiny (see Supplementary Figure 4). (a-e) Null models from the SNS dataset (BA21+22+10+41 vs BA17); (f-j) Null models from the DroNc-Seq dataset (HP vs PFC). (a, f) protectors; (b, g) promoters; (c, h) MS; (d, i) tangles; (e, j) tau. A p-value is computed as the probability to have a value more extreme than the observed one (one-tailed). Sample sizes are the same as Supplementary Figure 4.

  6. Supplementary Figure 6 Comparison of the differential expression of the subproteomes for the two independent single-nucleus RNA-seq datasets.

    Each point in the scatterplot indicates a Δ score in the DroNc-Seq dataset (x-axis) and the SNS dataset (y-axis) shown in Fig. 3. Data are presented as mean ± SEM. Sample sizes are the same as Supplementary Figure 4.

  7. Supplementary Figure 7 BAG3 protein is differentially regulated in neurons in human non-AD and AD cases.

    (a) Representative immunofluorescent images of the co-staining of IN neuronal marker (GAD1, purple), pan neuronal marker (NeuN, green), and BAG3 (red) in the BA9 of human AD (Braak stage V/VI) brain. The white arrow represents the IN neurons (GAD1+/NeuN+), while the white arrowhead indicates the putative EX neurons (GAD1-/NeuN+). The letter “g” stands for glial cells. The nuclei (blue) were counterstained with Hoechest3342. Three independent experiments were repeated with similar results. Scale bar, 20 µm. (b) Comparison of the mean intensity of BAG3 in individual neurons in the BA9 regions (n = 3 human brains, 20 GAD1-/NeuN+ and 20 GAD1+/NeuN+ neurons from each case). Data are presented as mean ± SEM. The statistical significance was assessed by the two-tailed nonparametric Mann-Whitney test. *** P < 0.0001 vs non-AD and/or AD EX neurons (The Mann-Whitney U is 1551, 714, 531, and 130, respectively).

  8. Supplementary Figure 8 Null model for seven different subproteomes between microglia and EX or IN neurons.

    The statistical significance of the results is studied by creating a null model for each subproteome under scrutiny (see Supplementary Figure 4). (a, c, e, g) Null models for MG (microglia) vs EX (excitatory) neurons; (b, d, f, h) Null models for MG (microglia) vs IN (inhibitory) neurons. (a, b) tau; (c, d) promoters; (e, f) tangles; (g, h) protectors. A p-value is computed as the probability to have a value more extreme than the observed one (one-tailed). Sample sizes are the same as Supplementary Figure 4.

  9. Supplementary Figure 9 Null model for seven different subproteomes between astrocytes and EX or IN neurons.

    The statistical significance of the results is studied by creating a null model for each subproteome under scrutiny (see Supplementary Figure 4). (a, c, e, g) Null models for ASC (astrocytes) vs EX (excitatory) neurons; (b, d, f, h) Null models for ASC (astrocytes) vs IN (inhibitory) neurons. (a, b) tau; (c, d) promoters; (e, f) tangles; (g, h) protectors. A p-value is computed as the probability to have a value more extreme than the observed one (one-tailed). Sample sizes are the same as Supplementary Figure 4.

  10. Supplementary Figure 10 Null model for seven different subproteomes between oligodendrocytes and EX or IN neurons.

    The statistical significance of the results is studied by creating a null model for each subproteome under scrutiny (see Supplementary Figure 4). (a, c, e, g) Null models for ODC (oligodendrocytes) vs EX (excitatory) neurons; (b, d, f, h) Null models for ODC (oligodendrocytes) vs IN (inhibitory) neurons. (a, b) tau; (c, d) promoters; (e, f) tangles; (g, h) protectors. A p-value is computed as the probability to have a value more extreme than the observed one (one-tailed). Sample sizes are the same as Supplementary Figure 4.

  11. Supplementary Figure 11 Full length of the western blot image.

    Original western blot images of Fig. 7a in primary cortical neurons transduced with lentivirus expressing scrambled BAG3 or shBAG3, or overexpressing BAG3 (OE) as described in Methods. BAG3 is the target protein detected by primary rabbit anti-BAG3 antibody. GAPDH is a housekeeping protein used as the loading control.

Supplementary information

  1. Supplementary Figures 1–11

    Supplementary Figures 1–11

  2. Reporting Summary

  3. Supplementary Table 1

    Single-nucleus RNA-seq datasets analysis in neurons.

  4. Supplementary Table 2

    Single-nucleus RNA-seq datasets analysis in glia.

  5. Supplementary Table 3

    Weighted gene co-expression network analysis of the subproteomes relevant to tau homeostasis.

  6. Supplementary Table 4

    Supplementary Table 4.

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https://doi.org/10.1038/s41593-018-0298-7