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. 2024 Feb;20(2):1013-1025.
doi: 10.1002/alz.13493. Epub 2023 Oct 17.

The type-I interferon response potentiates seeded tau aggregation and exacerbates tau pathology

Sophie A I Sanford  1   2 , Lauren V C Miller  1   2 , Marina Vaysburd  3 , Sophie Keeling  1   2 , Benjamin J Tuck  1   2 , Jessica Clark  3 , Michal Neumann  3 , Victoria Syanda  3 , Leo C James  3 , William A McEwan  1   2
Affiliations

The type-I interferon response potentiates seeded tau aggregation and exacerbates tau pathology

Sophie A I Sanford et al. Alzheimers Dement. 2024 Feb.

Abstract

Introduction: Signatures of a type-I interferon (IFN-I) response are observed in the post mortem brain in Alzheimer's disease (AD) and other tauopathies. However, the effect of the IFN-I response on pathological tau accumulation remains unclear.

Methods: We examined the effects of IFN-I signaling in primary neural culture models of seeded tau aggregation and P301S-tau transgenic mouse models in the context of genetic deletion of the IFN-I receptor (IFNAR).

Results: Polyinosinic:polycytidylic acid (PolyI:C), a synthetic analog of viral nucleic acids, evoked a potent cytokine response that enhanced seeded aggregation of tau in an IFN-I-dependent manner. IFN-I-induced vulnerability could be pharmacologically prevented and was intrinsic to neurons. Aged P301S-tau mice lacking Ifnar1 had significantly reduced tau pathology compared to mice with intact IFN signaling.

Discussion: We identify a critical role for IFN-I in potentiating tau aggregation. IFN-I is therefore identified as a potential therapeutic target in AD and other tauopathies.

Highlights: Type-I IFN (IFN-I) promotes seeded tau aggregation in neural cultures. IFNAR inhibition prevents IFN-I driven sensitivity to tau aggregation. IFN-I driven vulnerability is intrinsic to neurons. Tau pathology is significantly reduced in aged P301S-tau mice lacking IFNAR.

Keywords: innate immunity; interferon; neuroinflammation; tau pathology; tauopathy.

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Conflict of interest statement

The authors report no competing interests. Author disclosures are available in the supporting information.

Figures

FIGURE 1
FIGURE 1
PolyI:C drives seeded tau aggregation in a type‐I IFN‐dependent manner. Mixed neural cultures were prepared from P301S‐tau transgenic mice. (A) Cytokines were measured in the supernatant of cultures treated repetitively with polyI:C (2.5 μg/mL) and compared to control untreated cultures; n = 3. Data presented are the log2 of cytokine concentration (pg/mL), which was normalized to the average of control concentrations. (B) Schematic of tau seeding assay in primary mixed neural cultures from P0/P1 mouse brain overexpressing human P301S‐tau. Recombinant tau assemblies were added at DIV7, and tau aggregation was monitored by immunofluorescence microscopy using antibody AT8 or pS422 at DIV14. (C) Tau monomer and assemblies (50 nM) were added to cultures and levels of AT8 and pS422 reactivity measured by immunofluorescence. (D, E) Seeded aggregation in Ifnar1 +/+ P301S‐tau and Ifnar1 −/− P301S‐tau cultures treated with/without polyI:C (2.5 μg/mL) and tau assemblies (50 nM); n = 5, N = 2 independent experiments. (F) Levels of tau aggregation in Ifnar1 +/+ P301S‐tau cultures treated with polyI:C (2.5 μg/mL) alone or tau assemblies (50 nM) alone; n = 5, N = 3 independent experiments. (G) Representative images of seeded tau aggregation in OHSCs from P301S‐tau mice treated with tau assemblies (‘Tau’) (300 nM) ± polyI:C (10 μg/mL) and stained with AT8. (H) Quantification of seeded tau aggregation measured by immunofluorescence; slices from N = 6 mice. n = wells/condition, each containing 30,000 plated cells. All error bars indicate mean ± SD, except for H, where median and interquartile range are presented. Scale bars = 100 μm. Significance calculated by two‐way ANOVA for E and Kruskal–Wallis test with Dunn's correction for F and H. ***p < 0.001; ****p < 0.0001; ns, not significant.
FIGURE 2
FIGURE 2
Type‐I interferons increase seeded tau aggregation. (A) Mixed neural cultures from Ifnar1 +/+ P301S‐tau or Ifnar1 −/− P301S‐tau transgenic mice were treated overnight with IFN‐α or IFN‐β and expression of interferon‐stimulated genes (ISGs) IFITM3, and STAT1 was measured by western blot. (B) Representative immunofluorescence images of STAT1 and MAP2 staining in cultures from Ifnar1 +/+ P301S‐tau mice following overnight incubation with IFN‐β. (C) Mixed neural cultures from Ifnar1 +/+ P301S‐tau or Ifnar1 −/− P301S‐tau mice were treated with tau assemblies (‘Tau’) (50 nM) with/without repetitive treatment of IFN‐α or IFN‐β (50U/mL). Seeded tau aggregation was measured by immunofluorescence staining and quantified in (D); n = 5, N = 3 independent experiments. (E) Seeded tau aggregation was quantified in Ifnar1 +/+ P301S‐tau cultures treated with IFN‐β (50 U/mL) alone or tau assemblies (50 nM) alone; n = 5, N = 3 independent experiments. (F) Tau assemblies were titrated onto Ifnar1 +/+ P301S‐tau cultures treated with IFNβ (50 U/mL) and seeded aggregation quantified; n = 5, N = 2 independent experiments. (G) Ifnar1 +/+ P301S‐tau cultures were treated with a type‐I IFN receptor blocking antibody (αIFNAR) or a non‐targeting IgG isotype control antibody, anti‐adenovirus 9C12 (1 h at 5 μg/mL or 1 μg/mL), before overnight treatment with IFN‐β (50 U/mL). Expression of IFITM3 and STAT1 was measured by western blot. (H) Seeded tau aggregation was quantified in Ifnar1 +/+ P301S‐tau cultures pretreated with IFN‐β and αIFNAR or 9C12 (1 h at 5 μg/mL or 1 μg/mL) and seeded with tau assemblies; n = 5, N = 2 independent experiments. (I) Representative images of seeded tau aggregation in OHSCs from P301S‐tau mice treated with tau assemblies (1000 nM) ± IFN‐α (50 U/mL) and stained with AT8. (J) Quantification of seeded tau aggregation measured by immunofluorescence; slices from N = 6 mice. Western blots representative of N = 3 independent experiments. n = wells/condition, each containing 30,000 plated cells. All error bars indicate mean ± SD, except for H, where median and interquartile range is presented. Scale bars = 100 μm. Significance calculated by two‐way ANOVA for D and Kruskal–Wallis test with Dunn's correction for E, H, and J and without Dunn's correction in F. *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant.
FIGURE 3
FIGURE 3
IFN‐I vulnerability is intrinsic to neurons. (A) Fluorescence microscope images of mixed neural cultures from Ifnar1 +/+ P301S‐tau transgenic mice (P0/P1) treated with L‐leucine methyl ester (LME, 15 mM for 4 h) revealing reduction of Iba1 positive cell populations. (B) The Iba1+ area fraction was quantified in untreated (NTC) and LME‐treated cultures; n = 5, N = 3 independent experiments. (C) Seeded tau aggregation was quantified in untreated or LME‐treated Ifnar1 +/+ P301S‐tau cultures, seeded with tau assemblies (50 nM) ± IFN‐β (50 U/mL); n = 5, N = 3 independent experiments. (D) Cultures from Ifnar1 +/+ P301S‐tau mice treated with DMSO or PLX3397 (PLX, 2 μM) revealing reduction of Iba1‐positive cell populations. (E) The Iba1+ area fraction was quantified in untreated and DMSO or PLX3397‐treated cultures; n = 5, N = 3 independent experiments. (F) Seeded tau aggregation was quantified in untreated or PLX3397‐treated Ifnar1 +/+ P301S‐tau cultures, seeded with tau assemblies (25 nM) ± IFN‐β (50 U/mL); n = 5, N = 3 independent experiments. (G) Cortical/hippocampal neuronal cultures were prepared from Ifnar1 +/+ P301S‐tau mice at E15.5 using a papain‐based protocol. Addition of cytosine arabinoside (AraC, 1 μM) at DIV0/5/7 revealed a complete ablation of GFAP+ cell populations and an absence of Iba1+ cells. (H) The GFAP+ area fraction was quantified in untreated and AraC‐treated cultures; n = 5, N = 3 independent experiments. (I) Representative images of NeuN staining at DIV12. (J) The number of NeuN+ nuclei was quantified by image analysis and values are presented normalized to the DAPI count; n = 10 from n = 2 independent plates. (K) Representative images and (L) quantification of seeded tau aggregation in AraC treated Ifnar1 +/+ P301S‐tau neuronal cultures, seeded with tau assemblies (20 nM) on DIV6 and treated with/without IFN‐β (25 U/mL), with fixation at DIV12; n = 10, n = 2 independent plates. (M) Tau entry assay in primary mixed neural cultures from Ifnar1 +/+ P301S‐tau mice. Cultures were infected with hSyn::‐eGFP‐P2A‐LgBiT‐NLS and treated overnight at DIV6 with polyI:C (2.5 μg/mL), IFN‐α, IFN‐β (both 50 U/mL), or methyl‐β‐cyclodextrin (MBCD), (2 mM) for 2 h on DIV7. Cytosolic entry of tau was quantified by luminescence intensity 1 h after addition of tau‐HiBiT assemblies to the medium. Cytosolic entry was normalized to the number of viable cells per well; n = 3, N = 3 independent experiments. n = wells/condition, each containing 30,000 plated cells. All error bars indicate mean ± SD. Scale bars = 100 μm. Significance calculated by Mann–Whitney test for B, H and J and Kruskal–Wallis test with Dunn's correction for C, E, F, L and M. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.
FIGURE 4
FIGURE 4
Genetic depletion of Ifnar1 reduces tau pathology in vivo. (A) Cortical cytokine profile of wildtype (n = 6 M), Ifnar1 +/+ P301S‐tau (n = 3 M, n = 4 F), Ifnar1 −/− P301S‐tau (n = 4 M, n = 3 F) mice at 22 weeks of age, measured by Luminex 48‐plex assay. Data presented are log2 of cytokine concentration (pg/mL), which was normalized to average of WT controls for each cytokine. Gray = not detected. (B) Replot of selected cytokines CXCL10 and CCL7 observed to be significantly different between Ifnar1 +/+ and Ifnar1 −/− P301S‐tau animals. Subanalysis of male‐only mice showed the same effects. (C) Representative images and (D) quantification of Iba1 staining in cerebral cortex of Ifnar1 +/+ P301S‐tau and Ifnar1 −/− P301S‐tau mice. Points represent n = 3/4 sections/mouse from Ifnar1 +/+ P301S‐tau (n = 3 M, n = 3 F), Ifnar1 −/− P301S‐tau (n = 3 M, n = 3 F) mice. (E) Representative images and (F) quantification of GFAP staining in hippocampus of Ifnar1 +/+ P301S‐tau and Ifnar1 −/− P301S‐tau mice. Points represent n = 3 or 4 sections/mouse from Ifnar1 +/+ P301S‐tau (n = 2 M, n = 2 F), Ifnar1 −/− P301S‐tau (n = 2 M, n = 2 F) mice. (G) Representative images and (H) quantification of AT8 staining in 22‐week‐old Ifnar1 +/+ P301S‐tau and Ifnar1 −/− P301S‐tau brain sections. Points represent average of n = 3 sections/mouse (at least n = 6 mice per group, n = 3 M and n = 3 F in each group). (I) Quantification of seeded tau aggregation in HEK293 cells expressing P301S tau‐venus, treated with spinal cord homogenate from Ifnar1 +/+ P301S‐tau, Ifnar1 −/− P301S‐tau at 14 weeks (n = 6 mice per group, n = 3 M and n = 3 F in each group) and 22 weeks (n = 7 mice per group). Points represent n = 3 technical replicates per animal. Scale bar = 100 μm for C, E and 1000 μm for G. Significance calculated by one‐way ANOVA for B, Welch's t‐test for D, F, and I, and Mann–Whitney test for H. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Mean and SD are presented.
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