Introduction
Brains of Alzheimer’s disease patients are characterized by the presence of amyloid plaques and neurofibrillary tangles, respectively composed of Aβ peptides and hyperphosphorylated Tau, and both invariably associated with neuroinflammatory changes [
85,
86]. Genetic evidence, biomodelling data and biomarker analysis support an initiating role of accumulating Aβ in the pathogenetic process in EOFAD and sporadic AD patients [
39,
47,
48]. While accumulating Aβ presents as a key initiator, but difficult disease-modifying target, there is a growing focus on multi-targeted strategies aiming at including key pathogenetic targets downstream of Aβ, which include Tau [
11,
57] and its prion-like or templated propagation and neuroinflammatory changes [
40,
64,
80,
99] as key pathogenetic processes in AD.
An executive role of Tau in the pathogenetic process of AD and related Tauopathies is substantiated by (i) the existence of a family of neurodegenerative disorders all characterized by Tau-aggregation, by (ii) the identification of MAPT mutations autosomal dominantly linked to these Tauopathies, indicating that Tau dysfunction causes neurodegeneration, and by (iii) the strong correlation of progression of Tau pathology with progression of disease symptoms in AD—generally considered as a secondary Tauopathy [
9,
11,
85,
92]. Furthermore, (iv) prion-like or templated propagation of Tau pathology has been consistently and reproducibly shown in in vitro and in vivo models, highlighting a self-propagating effect of Tau pathology once initiated [
3,
12,
24‐
26,
30‐
33,
36,
37,
44,
49,
50,
56,
62,
77,
84,
89,
96,
97]. Prion-like propagation of Tau pathology thereby presents as a compelling mechanism for the progressive and characteristic development of Tau pathology, remarkably strong correlated with symptom progression in AD. Importantly, the presence of Tau seeds has been demonstrated not only in brains of Tau transgenic mice, but also in brains of patients [
26,
45,
52] using a sensitive Tau seeding detector assay. This not only highlights the relevance of this process in human AD brains [
75] but also emphasizes the interest to identify mechanisms to inhibit or target templated propagation of Tau pathology. Hence, the molecular and cellular processes involved in seeding and spreading of Tau pathology and their modulators present a topic of high interest, and represent highly interesting therapeutic targets.
Brains of AD patients and patients with Tauopathies are invariably characterized by neuroinflammatory changes, raising a keen interest in their contribution to the pathogenetic process. Neuroinflammation and innate immunity, in particular, are hence increasingly investigated for their translational potential in neurodegenerative disorders including AD [
40,
41,
74,
80]. Innate immunity and inflammasome are crucial in health and disease and, therefore, very intensively investigated [
40,
41,
59‐
61,
66,
80]. The inflammasomes are the sensors of the innate immune system and induce inflammation in response to infectious ‘attacks’ [
35] recognized by danger-associated signals [pathogen-associated molecular patterns (PAMPS) and danger-associated molecular patterns (DAMPS)] [
40,
41,
59‐
61,
66,
80]. Inflammasome becomes activated by a dual stimuli leading to the activation of pattern recognition receptors (PRRs), including NOD-like receptors or NLRs. The NLRP3 inflammasome can be activated by structurally diverse stimuli including ATP, imidazoquinoline derivatives, various crystals as well as by bacterial toxins and also Aβ peptides displaying an amyloid structure [
21,
51,
70,
72,
76]. NLRP3 activation induces heteromer formation or aggregation of ASC, leading to caspase-1 activation [
35], which subsequently can cleave pro-interleukin-1 beta (pro-IL-1β) to active IL-1β. NLRP3–ASC inflammasome activation induces an increase in cytokine and chemokine concentrations and microglial activation, while its activation can also induce microglial death through pyroptosis [
43]. Elegant work demonstrated that aggregated Aβ displaying amyloid structure [
38,
55] activates the NLRP3–ASC inflammasome, following endo-lysosomal uptake and damage [
38], resulting in the activation of caspase-1, subsequent cleavage of pro-IL-1β to IL-1β, and microglial activation [
38]. NLRP3 inflammasome assembly was subsequently demonstrated to actively contribute to amyloid pathology [
42,
95]. Heneka et al. elegantly demonstrated that inflammasome inhibition through NLRP3 or caspase-1 deficiency inhibits amyloid pathology in APP/PS1 transgenic mice [
42,
95], with alterations in microglial phenotypes and phagocytosis capacity as potential contributing mechanisms. These findings were further confirmed using pharmacological NLRP3 inhibitors [
16,
19]. Interestingly, ASC specks—fibrillar ASC aggregates formed upon inflammasome activation—have been shown to be released following inflammasome activation and to be taken up by ‘receptor’ microglial cells thereby contributing to the prion-like propagation of inflammasome activation [
10,
23] and microglial activation in a prion-like way. This is particularly interesting in the context of prion-like propagation in AD and related neurodegenerative disorders. Venegas et al. demonstrated that aggregation of ASC into prion-like ASC specks exacerbated amyloid pathology [
95], by a cross-seeding mechanism. While the relation between Aβ and inflammasome has been analyzed in exquisite detail, the relation between inflammasome and Tau and prion-like propagation of Tau pathology remains unexplored.
Neuropathological and imaging studies have revealed microglial changes associated with Tau pathology in brains of primary Tauopathy patients and animal models recapitulating Tau pathology. It must be noted that neuroinflammatory changes are a prominent characteristic of Tauopathies per se, also in the absence of amyloid plaque pathology, highlighting the association between Tau pathology and microglial changes [
5,
18,
46,
64,
86,
90,
100]. This has been demonstrated in postmortem brain tissue [
5,
46,
90,
100] as well as using in vivo PET imaging in Tauopathy patients [
27,
28,
33]. Neuroinflammatory alterations associated with Tau pathology not only include altered chemokine and cytokine profiles and microglial activation, but also microglial degeneration [
5,
18,
46,
64,
86,
90,
100]. While detailed analysis of Tau-associated neuroinflammatory processes will yield insight into their association with different stages of the disease process, these changes are in line with a potential role for inflammasome activation by Tau. In vivo animal models have furthermore demonstrated an active role of microglial activation and IL-1β in the pathogenetic process of Tauopathies [
6,
101], and indicated microgliosis associated with early or soluble Tau aggregates and preceding the development of full-blown mature neurofibrillary tangles (NFTs) [
26,
45,
53,
78,
101]. As soluble Tau aggregates and Tau seeds display an “amyloid” or aggregated structure similar to Aβ [
13,
22,
34,
54,
55], we hypothesized that Tau seeds could activate the NLRP3–ASC inflammasome and thereby contribute to exogenously and non-exogenously seeded Tau pathology in mice in vivo. We here hence focus on the role of microglial activation, and particularly ASC-dependent inflammasome activation in the pathogenetic process of Tauopathies focusing on its effect on exogenously seeded and non-exogenously seeded Tau pathology.
In this work, we, therefore, analyzed whether Tau aggregates, which display similar amyloid structure as Aβ aggregates and prion-like properties, are capable of activating the NLRP3–ASC inflammasome. In addition, we analyzed whether inhibition of ASC-dependent inflammasome activation is capable of inhibiting exogenously seeded and non-exogenously seeded Tau pathology in Tau transgenic mice in vivo.
Materials and methods
Animals
In this project, transgenic mice overexpressing the human (1N4R) Tau protein harboring the P301S mutation driven by the mouse Prion promoter (PS19, denoted TPS; The Jackson Laboratory, Bar Harbor, US) backcrossed to C57BL/6J background were used [
87,
94,
101]. These mice exhibit a phenotype that mimics important aspects of Tauopathies. From the age of 11 months, filamentous Tau accumulates in the brain of these mice, and the mice subsequently develop a progressive neurodegenerative phenotype, characterized by clasping of the hind limbs, motoric problems, hippocampal atrophy, development of a hunchback and premature death [
87,
101]. Tau seeding at the age of 3 months induces strong Tau-seeded Tau pathology already 7 weeks post-injection, absent in the parental strain at that age. In addition, mice deficient for ASC (backcrossed to C57BL/6J background) (Charles River Laboratory, Brussels, Belgium) were used to investigate the role of ASC inflammasome in Tau pathology [
17,
69,
81]. Hemizygous TPS mice and hemizygous ASC knockout mice were crossed to obtain T +.ASC+/− , which were further crossed with ASC +/− mice to obtain T +.ASC−/− and T +.ASC +/+ littermates. Hemizygous ASC +/− mice were intercrossed to generate ASC −/− and ASC +/+ littermates for primary microglial cultures. All mice were genotyped by PCR analysis of tail biopsies. Animals were housed on a 12-h light/dark cycle in standard animal care facilities with access to food and water ad libitum. All animal experiments used in this study were performed in accordance with protocols approved by the institutional Ethics Committee for Animal Welfare.
Primary microglial cultures
All cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Primary microglial cultures were prepared from newborn (P0-2) mouse pups. The newborn mice were genotyped, euthanized by decapitation and dissected using standard procedures as described previously [
17]. Briefly, brains were dissected, and meninges removed in ice-cold sterile HBSS medium under a Primo Vert light microscope (Zeiss, Oberkochen, Germany). The isolated cortices were transferred into chilled Dulbecco’s modified Eagle’s medium (DMEM), with 1% penicillin–streptomycin (PS; Invitrogen, Carlsbad, USA) followed by mechanical trituration, and subsequently filtered through a 70-μm cell strainer and centrifuged at 300 g for 5 min at 4 °C. The cell pellet was resuspended in DMEM supplemented with 10% horse serum, 10% fetal calf serum and 1% PS (DMEM 10:10:1) at 37 °C and seeded on poly-
d-lysine-coated 175-cm
2 culture flasks. After incubation in a humidified 5% CO
2 at 37 °C for 10 days, the medium was replaced with fresh DMEM 10:10:1 supplemented with 1/3 colony stimulating factor 1 (CSF1) and incubated for 5 days at 37 °C and 5% CO
2. At day 15, the microglial cells were removed by shaking the culture flasks for 3 h at 230 rpm at 37 °C in an orbital shaker. The supernatant was filtered through a 70-μm cell strainer and centrifuged for 10 min at 300 g and 4 °C. The cell pellet was resuspended in 1 ml DMEM 10:10:1 and counted with trypan blue. Cells were plated on poly-
d-lysine-coated plates and coverslips and further used for experiments.
Generation of “Tau seeds”
Tau seeds were generated as previously described [
79,
87,
94]. Briefly, the human truncated 4R Tau, encompassing the four-repeat microtubule-binding domain with the P301L mutation and a Myc tag (K18-P301L; Q244-E372) was generated in
Escherichia coli. Tau fragments (monomeric Tau; 67 µM) were incubated in a 1:2 ratio with low molecular weight heparin (MP Biomedicals, Santa Ana, CA, USA) in 100 mM ammonium acetate buffer (pH 7) at 37 °C for 5 days. The fibrillization mixture was centrifuged (100,000 g for 1 h at 4 °C) and the resultant pellet resuspended in the same buffer without heparin to a final concentration of 333 µM, aliquoted and stored at − 80 °C (“Tau seeds”). Successful Tau fibrillization was confirmed by Thioflavin T (Sigma-Aldrich, St. Louis, MO, USA) assay and immunoblotting. For all experiments, Tau seeds were sonicated (eight pulses of 30% amplitude) before use.
Analysis of inflammatory activation in vitro
At the start of the experiment, fresh medium was added to the microglial culture. The cells were then primed with 1 μg/ml lipopolysaccharide (LPS) from Escherichia coli O26:B6 (Sigma-Aldrich) for 3 h at 37 °C and 5% CO2, washed with fresh medium and then treated with either 20 μM nigericin (Sigma-Aldrich) for 3 h or 5 μM of Tau seeds for 18 h, for each condition the medium was collected and the cells were fixed in 4% PFA in phosphate buffer saline (PBS). NLRP3 inhibitor MCC950 at 1 μM, or cathepsin B inhibitor CA-074 Me at 25 μM (both from Sigma-Aldrich), was added 15 min before treatment with either nigericin or Tau seeds. The various conditions were tested in both microglia cultures derived from ASC +/+ and ASC−/− mice minimally in three independent biological experiments.
Mouse IL-1β ELISA
For measuring IL-1β concentrations, the Mouse IL-1β Ready-SET-GO! ELISA kit (eBioscience, San Diego, US) was used according to the manufacturer’s protocol. Briefly, a 96-well plate was coated overnight with anti-mouse IL-1β capture antibody, washed three times for 1 min each with PBS, 0.05% Tween 20, followed by blocking with 1 × ELISPOT diluent for 1 h at room temperature (RT), after which 100 μl of sample was applied per well. The standard curve [eight samples (from 1000 pg/ml to 8 μg/ml) provided in the kit] was included in duplicate in the analysis, as well as two blank controls. After incubation overnight at 4 °C, the anti-mouse IL-1β detection antibody was added for 1 h at RT, followed by incubation with avidin–HRP solution for 30 min at RT. Tetramethylbenzidine solution was used as a substrate and 1 M H3PO4 was used as a stop solution. The absorbances were read at 450 nm with a BioRad iMark microplate absorbance reader (BioRad, Hercules, US). The results were processed using GraphPad Prism software.
Tau seeding experiments in vivo
To analyze the effect of Tau seeding on Tau pathology and the role of microglial inflammation, we performed injection of Tau seeds in TPS mice. The mice were deeply anesthetized by intraperitoneal injection of Ketamine/Xylazine mixture (Ketalar/Rompun) and placed in the stereotaxic apparatus (Kopf Instruments). Stereotactic injections of pre-aggregated Tau (Tau seeds) were performed as described previously [
87]. Briefly, sonicated Tau seeds (5 µl; 333 µM) were injected using a 10-μl Hamilton syringe in the frontal cortex (A/P, + 2.0; L, + 1.4; D/V, − 1.0; relative to bregma) at a rate of 1 μl/min. After injection, the needle was kept in place for additional 5 min before gentle withdrawal. The injected mice were sacrificed at the indicated time post-injection for immunohistochemical analysis.
Pharmacological inhibition by osmotic mini pump in Tau mice in vivo
To analyze the effect of the inflammasome inhibitor MCC950 (Sigma-Aldrich) on Tau-seeded Tau pathology, 3-month-old TPS mice were unilaterally injected into the right brain hemisphere with 5 µl (333 µM) Tau seeds (A/P, − 4.8; L, − 3.0; D/V, − 3.7; relative to bregma), as described above. In addition, an Alzet mini-osmotic pump (model 2006; Alzet, Cupertino, CA, US) attached via a catheter (20–25 mm) to a brain infusion cannula (brain infusion kit III; Alzet, Cupertino, CA, US) was implanted in a subcutaneous pocket towards the left hind limb as previously described [
20]. The brain infusion cannula was slowly inserted through the skull into the right lateral ventricle (A/P, − 0.5; L, − 1.1; relative to bregma) and attached to the skull with two drops of adhesive (Loctite 454). The skin was sutured with a running horizontal mattress suture using 3-0 braided silk suture thread. The mice were monitored until complete recovery and were housed individually for 24 h. Thereafter the mice were monitored daily during the first week post-surgery and three times per week for 6 weeks. The filling of the mini-osmotic pumps and the preparation of the brain infusion assembly was performed according to the manufacturer’s instructions. The fully assembled mini-osmotic pumps were placed into a sterile 50-ml conical tube with 0.9% NaCl for priming at 37 °C for 60 h prior to implantation. The mini-osmotic pumps were filled with two different concentrations of MCC950 (to obtain a final concentration of 0.1 µM and of 0.5 µM) in sterile PBS. Calculations were based on a release rate of 0.15 µl/h from the mini-osmotic pumps into the total brain volume to obtain a final concentration of 0.1 µM and 0.5 µM of MCC950 in the brain (calculated for an estimated brain volume of 400 mm
3). In the control mice, PBS was administered by mini-osmotic pump for 7 weeks. The treated mice were sacrificed 7 weeks post-injection.
Immunohistochemical and immunocytological analysis
Immunohistological analysis was performed as described previously [
79,
87,
94]. Briefly, the brains were dissected, after 2 min transcardiac perfusion with ice-cold PBS (Sigma-Aldrich, St. Louis, USA) and fixed for 24 h in 4% PFA–PBS at 4 °C. Free-floating sagittal sections (40 μm) were generated with a vibrating HM650 V microtome (Thermo Fisher Scientific, Waltham, MA, USA) and were preserved in PBS–sodium azide 0.1%. The sagittal brain sections were first washed twice in PBS and then three times in PBS + 0.1% Triton X-100 (PBST). Permeabilization of the tissue was performed using PBST + methanol (1:1) for 10 min, and subsequently blocked with PBST + 5% milk. Anti-Tau P-S202/T205 (AT8, 1:100; Thermo Fisher Scientific was used as a marker for Tau pathology and anti-Iba1 (Iba1, 1:500; Wako-Chemical GmbH, DE) was used as marker for microglial cells. The primary antibodies were incubated for 2 h at room temperature (RT) or overnight at 4 °C, followed by appropriate Alexa-coupled secondary antibody (1:500) in PBST + 5% milk for 1 h at RT. The slices were finally washed three times with PBST then twice with PBS and finally mounted with Fluoroprep mounting medium (BioMérieux, Marcy-l’Etoile, France). Staining with Thioflavin S (ThioS; Sigma-Aldrich), a specific β-sheet strand intercalant, and Gallyas silver (all chemicals from Sigma-Aldrich) staining were performed on vibratome sections as previously described [
87] and were used to demonstrate the presence of NFTs in brain sections. For ThioS staining the brain slices were mounted on 3% gelatin-coated glass slides, washed twice in PBS and incubated for 5 min in 0.3% KMnO
4. Subsequently, the slides were washed in a solution of 1% K
2S
2O
5/1% oxalic acid until the brown color was removed, followed by a solution of 1% NaBH
4 (prepared 2 h before use) for 20 s. Then the brain sections were incubated with 0.05% ThioS in 50% ethanol for 8 min, followed by two changes of 80% ethanol for 10 s each and three washes with large volumes of demineralized water. Slides were then placed in a high-concentrated phosphate buffer overnight in dark at 4 °C. For silver staining the free-floating brain sections were washed in demineralized water and placed for 5 min in 5% periodic acid solution, then washed twice in demineralized water and treated for 1 min with an alkaline silver iodide solution (1 M NaOH, 0.6 M KI, 0.035% silver nitrate). Subsequently, the brain slices were washed twice for 5 min with 0.5% acetic acid solution and placed in developer solution (combining solutions A—0.5% sodium carbonate:B—0.025 M NH
4NO
3, 0.012 M AgNO
3, 0.0035 M tungstosilicic acid:C—0.025 M NH
4NO
3, 0.012 M AgNO
3, 0.0035 M tungstosilicic acid, 0.28% formaldehyde in a 10:3:7 ratio) for 5 min. Then the brain slices were rinsed twice in 0.5% acetic acid, washed with demineralized water and placed in 0.1% gold chloride solution for 5 min followed by a 1% sodium thiosulphate solution for 5 min and a final wash in water. All chemicals used were from Sigma-Aldrich. Immunocytochemistry on primary microglial cells was performed similarly as above, using the lysosomal marker anti-LAMP-1 antibody (clone H4A3, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-c-Myc antibody (1:50; Sigma-Aldrich) for Tau seeds. Fixation of the cells was performed with 4% PFA in PBS for 10 min at RT. Image acquisition was performed with a Leica DM450B fluorescence microscope (Leica, Wetzlar, DE), EVOS FL Auto Imaging System (Thermo Fisher Scientific) and standard light microscope. Image analysis was performed with Image J (National Institutes of Health) blinded to the genotype and/or treatment of the mice. Briefly, the fluorescent TIFF images were converted to 16-bit images and then thresholded using the Image J Default method to allow quantification of the stained area without detection of the background staining; the threshold was then applied to all sections. For quantification of the silver staining area, the 16-bit images were inverted prior to the same thresholding procedure as above. The amount of staining was measured as the percentage of area occupied by positive signal within the brain region (frontal cortex, hippocampal CA1). The results were statistically processed with GraphPad Prism 7.04 software (GraphPad, San Diego, CA, USA).
Statistical analysis
The number of samples or animals is specified in the caption for each experiment. Results are expressed as the mean ± SEM. Statistical analysis was performed using Mann–Whitney t test and one-way ANOVA or two-way ANOVA with Dunnett’s/Tukey’s multiple comparison post hoc test (specified in the accompanying figure legend). All analyses were performed using GraphPad Prism software (GraphPad Software Inc). Statistical significance was defined as P < 0.05.