Astrocyte-secreted glypican-4 drives APOE4-dependent tau hyperphosphorylation

Abstract

Tau protein aggregates are a major driver of neurodegeneration and behavioral impairments in tauopathies, including in Alzheimer's disease (AD). Apolipoprotein E4 (APOE4), the highest genetic risk factor for late-onset AD, has been shown to exacerbate tau hyperphosphorylation in mouse models. However, the exact mechanisms through which APOE4 induces tau hyperphosphorylation remains unknown. Here, we report that the astrocyte-secreted protein glypican-4 (GPC-4), which we identify as a binding partner of APOE4, drives tau hyperphosphorylation. We discovered that first, GPC-4 preferentially interacts with APOE4 in comparison to APOE2, considered to be a protective allele to AD, and second, that postmortem APOE4-carrying AD brains highly express GPC-4 in neurotoxic astrocytes. Furthermore, the astrocyte-secreted GPC-4 induced both tau accumulation and propagation in vitro. CRISPR/dCas9-mediated activation of GPC-4 in a tauopathy mouse model robustly induced tau hyperphosphorylation. In the absence of GPC4, APOE4-induced tau hyperphosphorylation was largely diminished using in vitro tau fluorescence resonance energy transfer-biosensor cells, in human-induced pluripotent stem cell-derived astrocytes and in an in vivo mouse model. We further show that APOE4-mediated surface trafficking of APOE receptor low-density lipoprotein receptor-related protein 1 through GPC-4 can be a gateway to tau spreading. Collectively, these data support that APOE4-induced tau hyperphosphorylation is directly mediated by GPC-4.

Keywords: APOE4; Alzheimer’s disease; astrocytes; glypican-4; tau pathology.

Fig. 1.

The brains of postmortem APOE4 AD patients accumulate more tau proteins and APOE4 enhances tau propagation/uptake. (A) Representative IHC images of postmortem PFC tissues from APOE2/3(control), APOE2/2 (AD), APOE3/3 (AD), and APOE4/4 (AD) individuals stained with AT8 and aceTau (Lys174) antibodies (n = 5 to 6) show the presence of Neurofibrillary tangles (arrow), neuropil threads (arrowhead), and neuritic plaque (asterisk). (Scale bar, 20 µm.) (B and C) Compared to APOE3/3 and APOE2/2, APOE4/4 carriers show the presence of more neurofibrillary tangle-containing neurons. (D) Neuronal monocultures from PS19 (Left), MAPT^KO (Center), and mixed neuronal culture of PS19*MAPT^KO (Right) showing the expression of tau (AT8), GFP, and uptake of tau proteins, respectively. PS19*MAPT^KO neurons were 3D-reconstructed using IMARIS software. (Scale bar, 20 µm.) (E) We treated the mixed neurons (PS19*MAPT^KO) with APOE2 or APOE4 particles isolated from human brains, and accessed tau spreading after 4 d. (Scale bar, 20 µm.) (F) Representative IHC staining of APOE treated neuronal culture shows that APOE4 treatments enhanced tau spreading whereas APOE2 significantly reduced APOE4-mediated tau spreading. (Scale bar, 20 µm.) n = 5, one-way ANOVA, *P < 0.05, ***P < 0.001 and ****P < 0.0001.

Fig. 2.

APOE4 interacts with GPC-4 and APOE4 AD postmortem brains express more GPC-4 in neurotoxic astrocytes. (A) GPC-4 proteins were incubated with either APOE2 or APOE4 proteins at room temperature for 1 h, and then separated by a nondenaturing gel (without SDS). Western blot analysis with GPC-4 (Left) and APOE (Right) antibodies revealed that combinations of APOE4+GPC-4 were robustly shifted while some noticeable shifts were observed with APOE2+GPC-4. A red arrow (Left) indicates GPC-4 proteins were shifted when combined with APOE4, but not with APOE2. A red arrow (Right) indicates APOE4 proteins were shifted when combined with GPC-4, whereas APOE2 proteins were not shifted when combined with GPC-4. The samples treated with ME disturbed the GPC-4/APOE4 interactions. (B and C) Proteins isolated from APOE2/2 and APOE4/4 human brains were coimmunoprecipitated with APOE antibodies (n = 3) (B). The levels of immunoprecipitated GPC-4 proteins were normalized with corresponding APOE immunoreactive bands (C). It shows that the APOE/GPC-4 complex is significantly higher in APOE4/4 compared to APOE2/2. (D and E) Representative IHC staining of APOE2/3(control), APOE2/2(AD), APOE3/3 (AD) and APOE4/4 (AD) postmortem tissues (PFC) with GFAP and GPC-4 antibodies show that APOE4 carrying AD patients expressed significantly higher levels of GPC-4 in astrocytes compared to control and other APOE genotypes (E). n = 5 to 6. (Scale bars, 20 µm.) (F and G) Astrocytes from APOE4/4 AD brains were grouped into two categories based on the number of the branches at 15-μm radius (F). Group 1: fewer than 10 branches. Group 2: more than 10 branches. Astrocytes with more branches expressed significantly elevated levels of GPC-4 (G). (H) AD-associated astrocytic genes were selected from Habib et al. (43) and generated a heat map with subtypes of astrocytes from Grubman et al. (42). Disease-associated genes were enriched in subtypes 2 and 3, and GPC-4 was enriched in a subtype 3. (I–K) Western blot analysis from astrocyte culture shows that treatment with TNF-α and IL-1β significantly increased expression of GPC-4, and it also activated the NF-κB pathway. (L–N) Western blot analysis from astrocyte culture treated with NF-κB pathway blocker, IMD-0354, reversed TNF-α and IL-1β-induced expression of GPC-4. n = 4; one-way ANOVA or unpaired t test, **P < 0.01, ***P < 0.001, and ****P < 0.0001. O.D., optical density.

Fig. 3.

GPC-4 induces tau hyperphosphorylation in vitro. (A–D) Western blot analysis of proteins isolated from PS19 primary neuronal culture, which were treated with GPC-4 protein, shows that GPC-4 significantly enhanced pTau levels (C and D), whereas no changes were observed in total tau protein (B). (E and F) Representative IHC staining of GPC-4–treated neuronal culture with AT8 and MAP2 antibodies demonstrates that GPC-4 treatment enhanced pTau levels (E). (Scale bar, 20 µm.) (G) Schematic diagram shows that astrocytes (WT mice) were treated with control or GPC-4 shRNA and the resulting ACM was added to PS19 neuronal culture. (H–K) Addition of ACM to neuronal culture increased pTau (AT8 and PHF1) levels whereas total tau proteins were unaltered. On the other hand, addition of GPC-4–deprived ACM failed to induce tau phosphorylation in PS19 neurons. (L and M) Representative IHC staining of GPC-4 treated neuronal coculture (PS19*MAPT^KO mice) shows that GPC-4 treatment enhanced tau spreading/uptake. (Scale bar, 20 µm.) n = 4 to 5, two-way ANOVA or unpaired t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.

GPC-4 induces tau hyperphosphorylation in vivo. (A) Schematic diagram shows that donor cells express both GFP and hTau while synaptically connected neurons receive hTa. (B and C) We injected eGFP-2PA-hTauP301L AAV virus in the ipsilateral CA1 region and control/GPC-4 shRNA in the contralateral CA1 region (8-mo-old WT mice), and examined them after 4 wk. Representative IHC images human tau specific antibody Tau13 show that GPC-4 shRNA treatment significantly reduced tau spreading in the contralateral CA1 region. The levels of contralateral Tau13 were normalized by the levels of ipsilateral Tau13. (Scale bar, 20 µm.) (D and E) In order to induce the expression of GPC-4 proteins, we activated the GPC-4 gene by injecting GPC-4 CRISPR/dCas9 lentivirus activation systems in the cortex or hippocampus (4-mo-old WT mice). Representative IHC with GPC-4 and GFAP antibodies show that, following 1 wk of injection, GPC-4 CRISPR/dCas9 robustly induced GPC-4 expression compared to control lentiviral activation particles. (Scale bar, 20 µm.) (F and G). IHC images with the AT8 antibody show that GPC-4 induced significantly higher levels of pTau in the CA1 region of the hippocampus. (Scale bars, 20 µm.) (H and I). IHC images with AT8 antibody shows that GPC-4 induced significantly higher levels of pTau in the cortex. (Scale bar, 20 µm.) n = 4 to 5, unpaired t test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.

GPC-4 drives APOE4-mediated tau hyperphosphorylation. (A and B) Cultured neurons (WT) were treated with ACM alone, ACM with APOE4, and GPC-4–deprived (shRNA treated) ACM with APOE4. After 24 h, neurons were incubated with purified human tau proteins for 1 h. Representative IHC images of APOE4+ACM treated neuronal culture shows that APOE4 treatment enhanced tau uptake, but in the absence of GPC-4 (GPC-4 shRNA-treated ACM) APOE4-induced tau uptake was significantly reduced (B). Magnification of insets in (A): 60×. (Scale bar, 20 µm.) (C and D) Tau FRET-biosensor cells were used to monitor seeding activity of AD-tau proteins in the presence of APOE4 and GPC-4. We incubated Tau FRET-biosensor cells with 0.6 μg/mL tau proteins and 1 μg/mL APOE4 with/without GPC-4 shRNA for 2 d, and then counted the number of tau inclusion containing cells/area. Compared to tau alone, tau with APOE4 induced significantly higher numbers of tau inclusions in Tau FRET biosensor cells. Interestingly, APOE4-mediated tau inclusions were reduced in the presence of GPC-4 shRNA. n = 8 to 10. (Scale bar, 20 µm.) (E and F) We injected APOE2 or APOE4 particles isolated from corresponding human brains, in the absence or presence of GPC-4 shRNA. Following 3 wk of injections, no tau phosphorylation (AT8) was detected with APOE2. APOE4 robustly induced tau phosphorylation, but APOE4 failed to induce tau phosphorylation in the absence of GPC-4. (Scale bar, 20 µm.) (G and H) IHC staining with MC1 antibodies show that APOE4-mediated tau pathology was diminished in the presence of GPC-4 shRNA. (Scale bar, 20 µm.) (I–M) Human APOE4 iPSCs were differentiated into astrocytes, and the ACM was collected after 3 d of shRNA treatments. The PS19 neurons were treated with ACM for 4 d. Western blot analysis shows that ACM significantly increased AT8 and PHF1 levels, and the ACM-induced tau pathologies were reduced in the absence of GPC-4. n = 4 to 5, two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.

GPC-4 regulates trafficking of APOE receptor LRP1. Using a Pierce Cell Surface Protein Isolation Kit, the total surface proteins were isolated from primary neuronal culture (WT mice). (A–C) Western blot analysis from primary neuronal culture treated with APOE (s) shows that addition of APOE4 significantly enhanced the surface LRP1 (S.LRP1) (C). There were no changes in total LRP1 levels (T.LRP1) (B). (D–F) Western blot analysis from primary neuronal culture treated with APOE4 or APOE4 with exocytosis inhibitor, Exo-1, suggest that active exocytosis is required for APOE4-mediated surface trafficking of LRP1. (G) Coimmunoprecipitation of human postmortem brain protein samples with LRP1 antibodies and subsequent Western blotting revealed that LRP1 and GPC-4 are in the same complex. (H–J) Western blot analysis from primary neuronal culture treated with GPC-4 protein shows that GPC-4 significantly enhanced trafficking of surface LRP1 levels (J), whereas total LRP1 levels were not affected (I). (K–M) Western blot analysis from neuronal culture treated with ACM alone (control), APOE4+ACM, and with APOE4+GPC-4 shRNA treated ACM show that APOE4-induced surface expression of LRP1 is likely mediated through GPC-4. The GPC-4 shRNA treatment reduced the surface expression of LRP1 in the presence of APOE4 (J). (N and O) Representative IHC images with AT8 antibodies show that GPC-4-induced tau phosphorylation was reduced in the absence of LRP1. (Scale bar, 20 µm.) n = 4 to 5, one-way ANOVA or unpaired t test, **P < 0.01, ***P < 0.001, and ****P < 0.0001.