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ApoE4 inhibition of VMAT2 in the locus coeruleus exacerbates Tau pathology in Alzheimer’s disease

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Abstract

ApoE4 enhances Tau neurotoxicity and promotes the early onset of AD. Pretangle Tau in the noradrenergic locus coeruleus (LC) is the earliest detectable AD-like pathology in the human brain. However, a direct relationship between ApoE4 and Tau in the LC has not been identified. Here we show that ApoE4 selectively binds to the vesicular monoamine transporter 2 (VMAT2) and inhibits neurotransmitter uptake. The exclusion of norepinephrine (NE) from synaptic vesicles leads to its oxidation into the toxic metabolite 3,4-dihydroxyphenyl glycolaldehyde (DOPEGAL), which subsequently activates cleavage of Tau at N368 by asparagine endopeptidase (AEP) and triggers LC neurodegeneration. Our data reveal that ApoE4 boosts Tau neurotoxicity via VMAT2 inhibition, reduces hippocampal volume, and induces cognitive dysfunction in an AEP- and Tau N368-dependent manner, while conversely ApoE3 binds Tau and protects it from cleavage. Thus, ApoE4 exacerbates Tau neurotoxicity by increasing VMAT2 vesicle leakage and facilitating AEP-mediated Tau proteolytic cleavage in the LC via DOPEGAL.

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Acknowledgements

We thank the Emory Goizueta Alzheimer’s Disease Research Center for postmortem human AD and healthy control samples. This study was supported in part by the Rodent Behavioral Core (RBC), Viral Vector Core, and HPLC Bioanalytical Core, which are subsidized by the Emory University School of Medicine and are part of the Emory Integrated Core Facilities.

Funding

This work was supported by the NIH (R01AG051538; RF1 AG061175 to KY and DW). Additional support was provided by the Emory Neuroscience NINDS Core Facilities (P30NS055077). Further support was provided by the Georgia Clinical and Translational Science Alliance of the National Institutes of Health under Award Number UL1TR002378.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 615 Michael St. Whitehead BLDG Room #141, Atlanta, GA, 30322, USA

    Seong Su Kang, Eun Hee Ahn, Xia Liu, Matthew Bryson & Keqiang Ye

  2. Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, USA

    Gary W. Miller

  3. Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, 30322, USA

    David Weinshenker

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  1. Seong Su Kang
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Supplementary Information

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401_2021_2315_MOESM1_ESM.tif

Supplementary Fig. 1. ApoE4 selectively interacts with VMAT2. a The binding between ApoE4 and VMAT2 was investigated in SH-SY5Y cells, which were co-transfected with GFP-APOE3, GFP-APOE4, and VMAT2 (3 more N number of Fig. 2A). Transfected cell lysates were immunoprecipitated with anti-GFP, and the precipitated proteins were analyzed by immunoblotting with anti-VMAT2. b ApoE TR mouse brain cell lysates were immunoprecipitated with anti-ApoE and the precipitated proteins were analyzed by immunoblotting with anti-VMAT2 (3 more N number of Fig. 2L). c The interaction between ApoE4 and VMAT2 was confirmed in LC of ApoE TR mice. The interacted proximity signals of anti-ApoE4 and anti-VMAT2 are shown in red, and the nuclei are shown in blue by Proximity ligation assay. Scale bar is 20 μm. (TIF 1539 KB)

401_2021_2315_MOESM2_ESM.tif

Supplementary Fig. 2. ApoE4 triggers AEP activation and Tau cleavage and phosphorylation. Primary neurons were cultured and infected with AAV-hTau, AAV-ApoE3 or ApoE4, and AAV-AEP or AEP C189S. a Western blot analysis showed that ApoE4 overexpression induced Tau phosphorylation and Tau N368 cleavage with AEP overexpression, but not inactive AEP C189S (2 more N number of Fig. 3A). b Western blot analysis showed that Tau and ApoE4-induced AEP activation, Tau phosphorylation, and Tau N368 cleavage were blocked by AEP inhibitor compound 11 (2 more N number of Fig. 3d). c Western blot analysis showed that non-cleavable Tau N255A/N368A suppressed the effects of Tau and ApoE4 on AEP activation, Tau phosphorylation, and Tau N368 cleavage (2 more N number of Fig. 3g). (TIF 1894 KB)

401_2021_2315_MOESM3_ESM.tif

Supplementary Fig. 3. ApoE3 but not ApoE4 selectively binds to Tau and protects it from AEP cleavage. SH-SY5Y cells were co-transfected with ApoE3/E4 and GST-Tau. a GST pull down and western blot analysis showed that ApoE3 but not ApoE4 bound to Tau and prevented AEP activation, Tau phosphorylation and Tau aggregation. b LDH assay indicating Tau and ApoE4 mediated cell death. c The activation of AEP was confirmed by the enzymatic assay. Data are shown as mean ± SEM. N=3 per group. *p<0.05, **p<0.01. HEK293 cells were co-transfected with GFP-ApoE3 and GST-Tau Fragments to investigate ApoE3’s binding domain on Tau. d GST pull down analysis showed that ApoE3 bound to Tau in the repeat motifs (fragment 256–368) including AEP cleavage sites. e Recombinant Tau (5 μg) was incubated with recombinant AEP (0.05 μg) and escalating doses of recombinant ApoE3/E4 (0.1, 0.5, and 1 μg). Western blot and densitometric quantification of cleaved Tau band showed that rTau cleavage by rAEP was attenuated by rApoE3. f HEK293 cells were co-transfected with ApoE3/ApoE4 and Tau. Western data and densitometric analysis of cleaved Tau band presented the protective effect of ApoE3 in Tau cleavage by AEP. Data are shown as mean ± SEM. N=3 per group. *p<0.05, **p<0.01. (TIF 1799 KB)

401_2021_2315_MOESM4_ESM.tif

Supplementary Fig. 4. AEP is required for mediating ApoE4-provoked AD pathologies in MAPT mice. AAV-ApoE3 or AAV-ApoE4 was injected into the hippocampus of MAPT/AEP WT or MAPT/AEP KO mice, and the pathologic events in the hippocampus for AD were measured by various analysis. a ELISA analysis showed that Aβ40 and Aβ42 in the hippocampus were not changed by ApoE4 in MAPT mice. b ELISA assay for IL-6, TNF-α, and IL-1β showed that inflammatory cytokines were increased in the hippocampus by ApoE4 in MAPT/AEP WT mice, but not MAPT/AEP KO mice. Data of ELISA assay in a and b are shown as mean ± SEM. N=6 per group. *p<0.05. c Representative images of Golgi staining demonstrating the reduction of spines in CA1 region of ApoE4-injected MAPT/AEP WT mice. Scale bar is 5 μm. d Quantification of dendritic spines in Golgi staining of CA1 region. Data are shown as mean ± SEM. N=6 per group. *p<0.05. e Representative images by electron microscopy demonstrating the reduction of synapses (red arrows) in CA1 region of ApoE4-injected MAPT/AEP WT mice. Scale bar is 2 μm. f Quantification of synapse number in electron microscopy images of CA1 region. Data are shown as mean ± SEM. N=6 per group. *p<0.05. (TIF 1341 KB)

401_2021_2315_MOESM5_ESM.tif

Supplementary Fig. 5. Tau is required for ApoE4-induced AEP activation and neuronal cell death. Primary neurons from MAPT or Tau–/– mice brains were infected with AAV-ApoE3 or AAV-ApoE4. a Western blot analysis showed that ApoE4 induced AEP activation, Tau aggregation, Tau phosphorylation, and Tau N368 cleavage in MAPT mice primary neurons. b LDH assay demonstrated that the cell death by ApoE4 was mediated in a Tau-dependent manner. c The activation of AEP was confirmed by enzymatic assay. d ELISA assay showed that the change of Aβ40 content by ApoE4 was not affected by Tau. Data of b–d are shown as mean ± SEM. N=3 per group. *p<0.05, **p<0.01. (TIF 576 KB)

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Supplementary Fig. 6. Tau is required for ApoE4-triggered AD pathologies and LC neuronal loss. AAV-ApoE3 or AAV-ApoE4 was injected into the hippocampus of Tau–/– or MAPT mice, and pathologic events in the hippocampus for AD were assessed. a ELISA analysis showed that Aβ40 and Aβ42 in the hippocampus were not changed by ApoE4 in MAPT mice. b ELISA assay for IL-6, TNF-α, and IL-1β showed that inflammatory cytokines were increased in the hippocampus by ApoE4 in MAPT mice but not Tau–/– mice. Data of ELISA assay in a and b are shown as mean ± SEM. N=6 per group. *p<0.05. c Representative images of Golgi staining demonstrating the reduction of spines in CA1 region of ApoE4-injected MAPT mice. Scale bar is 5 μm. d Quantification of the dendritic spines in Golgi staining of CA1 region. Data are shown as mean ± SEM. N=6 per group. *p<0.05. e Representative images by electron microscopy demonstrating the reduction of synapses (red arrows) in CA1 region of ApoE4-injected MAPT mice. Scale bar is 2 μm. f Quantification of synapse number in electron microscopy images of CA1 region. Data are shown as mean ± SEM. N=6 per group. *p<0.05. g Stereological cell counting of TH+ cells in the LC region of Fig. 5d showed LC neurodegeneration mediated by ApoE4 injection into the hippocampus of MAPT mice. h Quantification of Tau N368+ cells and T22+ cells in the LC of Fig. 5d to show the retrograde spread of Tau pathologies. Data are shown as mean ± SEM. N=6 per group. *p<0.05. (TIF 1424 KB)

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Supplementary Fig. 7. ApoE4 inhibits VMAT2 and increases DOPEGAL and triggers cognitive impairments in MAPT mice. AAV-ApoE3/ApoE4 and Lenti-Sh-VMAT2 were injected into the LC of MAPT or Tau–/– mice, and then mice were assessed for Tau pathology and memory dysfunction 6 months later. a Quantification of ApoE+ cells and VMAT2+ cells in the LC of Fig. 6A to verify virus infection. b Representative images of T22 (red) and Thioflavin S immunofluorescence co-staining verifying Tau aggregation and fibrillization. Scale bar is 100 μm. c Quantification of Thioflavin S/T22+ cells in the LC shows Tau fibrillization is mediated by ApoE4 overexpression and VMAT2 deficiency. d MAO-A enzymatic assay in the LC for AAV-ApoE3/ApoE4 and Lenti-Sh-VMAT2-injected MAPT or Tau–/– mice. e Measurement of DOPEGAL in the LC following ApoE4 overexpression and VMAT2 deficiency via HPLC analysis. Behavioral tests for Morris water maze (f–h) and fear conditioning test (i and j) demonstrating memory dysfunctions mediated by ApoE4 overexpression and VMAT2 deficiency in MAPT mice. Distance travelled to the platform (f), area under the curve for total distance travelled (g), and percent time spent in the quadrant (h) previously containing the platform during the probe trial in the Morris water maze. Percent time spent freezing during the cued fear (i) and contextual fear (j) tests following fear conditioning. All data were analyzed using two-way ANOVA and shown as mean ± SEM. N=6 per group. * <0.05, ** <0.01 (TIF 2458 KB)

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Supplementary Fig. 8. VMAT2 knockout elicits DOPEGAL production, AEP activation, and LC neuronal loss in an age-dependent manner. LC neurodegeneration and DOPEGAL concentrations in VMAT2 LO mice were examined by immunofluorescence staining, enzymatic analysis, and HPLC assay. a Representative images of staining in LC sections of VMAT2 LO mice by TH (red), VMAT2 (green), and DAPI (blue). Scale bar is 100 μm. b Quantification of TH + cells representing the slight but significant reduction of LC neurons at 12 months of age in VMAT2-LO mice. c and d Enzymatic assays in LC of age-dependent VMAT2 LO mice for AEP activity (c) and MAO-A activity (d). e Measurement of age-dependent increase of DOPEGAL in the LC of VMAT2-LO mice via HPLC analysis. Data of b–e were analyzed using two-way ANOVA and shown as mean ± SEM, N = 3 per group. *p<0.05, **p < 0.01. f Western blot analysis showed the change of endogenous protein expression for Tau and TH in LC tissue of VMAT2 LO mice. (TIF 1940 KB)

Supplementary Fig. 9. The full western bolts used in Figures are presented (TIF 4367 KB)

Supplementary Fig. 10. The full western bolts used in Supplementary Figures are presented (TIF 4103 KB)

Supplementary Fig. 11. The full western bolts used in Supplementary Figures are presented (TIF 3969 KB)

Supplementary Table 1. Information of postmortem human patients for LC sections (TIF 342 KB)

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Kang, S.S., Ahn, E.H., Liu, X. et al. ApoE4 inhibition of VMAT2 in the locus coeruleus exacerbates Tau pathology in Alzheimer’s disease. Acta Neuropathol 142, 139–158 (2021). https://doi.org/10.1007/s00401-021-02315-1

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  • DOI: https://doi.org/10.1007/s00401-021-02315-1

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