Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity

Abstract

In Alzheimer's disease and tauopathies, tau protein aggregates into neurofibrillary tangles that progressively spread to synaptically connected brain regions. A prion-like mechanism has been suggested: misfolded tau propagating through the brain seeds neurotoxic aggregation of soluble tau in recipient neurons. We use transgenic mice and viral tau expression to test the hypotheses that trans-synaptic tau propagation, aggregation, and toxicity rely on the presence of endogenous soluble tau. Surprisingly, mice expressing human P301Ltau in the entorhinal cortex showed equivalent tau propagation and accumulation in recipient neurons even in the absence of endogenous tau. We then tested whether the lack of endogenous tau protects against misfolded tau aggregation and toxicity, a second prion model paradigm for tau, using P301Ltau-overexpressing mice with severe tangle pathology and neurodegeneration. Crossed onto tau-null background, these mice had similar tangle numbers but were protected against neurotoxicity. Therefore, misfolded tau can propagate across neural systems without requisite templated misfolding, but the absence of endogenous tau markedly blunts toxicity. These results show that tau does not strictly classify as a prion protein.

Keywords: Alzheimer's disease; P301L tau; neurodegeneration; neurofibrillary tangles; prion‐like.

Figure 1. Trans‐synaptic propagation of human tau in ECrTgTau mice in the absence of endogenous mouse tau

1. 3D brain model, horizontal brain section illustrating transgenic human P301Ltau expression in the entorhinal cortex (green, EC) of the ECrTgTau mouse lines, and the propagation of transgenic tau to the dentate gyrus (DG). Tau composition in ECrTgTau and control mouse lines investigated.

2. Immunostained horizontal sections show the expression of human P301Ltau in EC neurons in the absence of endogenous mouse tau (ECrTgTau‐Mapt ^0/0). Fluorescence in situ hybridization of human tau mRNA combined with immunofluorescence labeling (immuno‐FISH) of human tau protein (huTau) verifies P301Ltau transgene expression in the EC. Scale bars, 50 μm.

3. Propagation of human tau protein to neurons in the DG (white arrowheads) in ECrTgTau‐Mapt ^0/0 mice. Close‐ups show DG neurons from three ECrTgTau‐Mapt ^0/0 mice (DG I‐III). Immuno‐FISH proofs the absence of human tau expression in DG neurons, which have huTau protein but no human tau mRNA. Scale bars, 50 μm.

4. Immunostained horizontal sections of ECrTgTau mice show the expression of human P301Ltau in EC neurons in the presence of endogenous mouse tau. Immuno‐FISH proofs the absence of human tau expression in these DG neurons. Scale bars, 50 μm.

5. Human P301Ltau propagation to DG neurons (white arrowheads) in the presence of endogenous mouse tau in ECrTgTau mice. Close‐ups show DG neurons from three ECrTgTau mice (DG I‐III). Scale bars, 50 μm.

6. Human (huTau, antibody Tau13) and total tau (hu+moTau, DAKO) levels in entorhinal cortex (EC) extracts from 18‐month‐old mice show equal human P301Ltau expression in ECrTgTau and ECrTgTau‐Mapt ^0/0 mice (Mean ± SEM, P = 0.201, n = 3 mice/group, one‐way ANOVA with Bonferroni correction).

7. The number of human tau‐positive cell bodies in the DG (Mean ± SEM, P = 0.58, n = 4 sections and 3 mice/group) and human tau in hippocampal (HPC) extracts (P = 0.14, n = 3 mice/group) were similar in ECrTgTau‐Mapt ^0/0 and ECrTgTau mice (two‐tailed Student's t‐test).

Source data are available online for this figure.

Figure EV1. Genotype confirmation of ECrTgTau(‐Mapt ^0/0) mice by qPRC

qPCR using RNA extracted from fresh frozen brain tissue of 18‐month‐old tau knockout mice (Mapt ^0/0), ECrTgTau, and ECrTgTau crossed to Mapt ^0/0 mice (ECrTgTau‐Mapt ^0/0, n = 4 mice/group) was transcribed into cDNA using primers recognizing human tau transgene (P301L tau, top) or genomic mouse tau (MapT, bottom). GAPDH mRNA was co‐transcribed as amplification control. ECrTgTau‐Mapt ^0/0 and ECrTgTau mice showed human tau expression, and Mapt ^0/0 and ECrTgTau‐Mapt ^0/0 mice lacked mouse tau expression.

Figure EV2. DG neurons do not express but have human tau in ECrTgTau(‐Mapt ^0/0 ) mice

1. Representative images of the fluorescence in situ hybridization of transgenic human tau mRNA combined with immunofluorescence labeling of human tau protein (Tau13 antibody) shows human tau protein (green) in neuronal cell bodies in the EC and the DG (white arrowheads), but human tau mRNA only in EC neurons both in ECrTgTau‐Mapt ^0/0 and ECrTgTau mice. n = 3 sections/mouse and 3 mice/group. Scale bars, 50 μm.

2. Human tau (huTau, Tau13 antibody) and total tau (hu+moTau, DAKO antibody) levels in hippocampal extracts from 18‐month‐old mice are similar in ECrTgTau and ECrTgTau‐Mapt ^0/0 mice. Mean ± SEM, n = 3 mice/group, two‐tailed Student's t‐test. ns, non‐significant.

Source data are available online for this figure.

Figure EV3. Propagation of full‐length tau to neurons not glia in the DG of ECrTgTau(‐Mapt ^0/0 ) mice

1. A

   Horizontal ECrTgTau‐Mapt ^0/0 brain section co‐immunolabeled with Tau13 (mouse antibody recognizing the N‐terminal end of human not mouse tau; epitope: aa20–35; red) and DAKO (polyclonal rabbit antibody recognizing the C‐terminal half of all mouse and human tau; epitope: multiple sites in aa243–441; green). Human tau in cell bodies in both EC and DG neurons (white arrowheads) was recognized by both antibodies against the N‐terminus and the C‐terminal half, suggesting the trans‐synaptic propagation of full‐length tau. Scale bars, 50 μm.

2. B, C

   Co‐immunostaining of human tau with GAD67 and Parvalbumin suggest the propagation of tau to a few GABAergic interneurons (white arrowheads) in the DG of ECrTgTau‐Mapt ^0/0 (B) and ECrTgTau (C) mice. Astrocytes (GFAP) and microglia (Iba1) did not have human tau in either mouse line. n = 4 sections/mouse, 3 mice/group. Scale bars, 50 μm.

Figure 2. P301Ltau propagation after viral expression in the entorhinal cortex

1. Adeno‐associated virus (AAV) construct designed for expression of eGFP and human P301Ltau as individual proteins, separated by the self‐cleaving 2a peptide, under the CBA promoter (AAV8 CBA‐eGFP‐2a‐huTauP301L). AAV‐transduced “donor neurons” express eGFP and huTauP301L, and tau “recipient neurons” are identified after immunostaining for human tau as huTau⁺ but GFP ⁻ neurons.

2. Primary cortical neuron cultures that were transduced with AAV eGFP‐2a‐P301Ltau at 7 DIV, and fixed and immunostained for GFP and human tau (Tau13 antibody) at 14 DIV, show tau donor (GFP ⁺, huTau⁺; ˜10% neurons) and a small number of tau recipient neurons (GFP ⁻, huTau⁺; ˜1% neurons). Western blot of whole cell lysates verified efficient cleavage (˜95%) of eGFP and P301Ltau by the 2a peptide (n = 3).

3. Eight weeks after AAV injection into right EC of aged Mapt ^0/0 mice (n = 3), immunostained brain sections showed that huTauP301L (red) propagated to a few DG “recipient neurons” (white arrowheads). Scale bar, 50 μm.

4. Unilateral AAV‐mediated human P301L tau expression in the EC and DG of age‐matched WT mice (n = 3). Representative images of brain sections show donor neurons in the injected EC and DG, and a few tau recipient neurons (white arrowheads) adjacent to the AAV injection site. Scale bar, 50 μm.

5. In the contralateral hemisphere of the same brain section as in (D), some tau recipient neurons (white arrowheads) were also present in the (non‐injected) axonal projection areas in the contralateral EC (GFP‐filled terminal ends). Scale bar, 100 μm.

Figure 3. Tau phosphorylation, misfolding, and gliosis in ECrTgTau(‐Mapt ^0/0) mice

1. A

   Brain sections from 18‐month‐old ECrTgTau‐Mapt ^0/0 and ECrTgTau mice were co‐immuolabeled for human tau and misfolded tau (Alz50). Misfolded tau was only found in EC and DG neurons (white arrowheads) of ECrTgTau, but not ECrTgTau‐Mapt ^0/0 animals (n = 4 sections/mouse, 3 mice/group). Scale bars, 50 μm.

2. B, C

   Immunofluorescence labeling and stereological counting of microglia in entorhinal cortex (B) and astrocytes in hippocampus (C) indicated early signs of neurodegeneration in ECrTgTau mice. The significantly increased number of Iba1‐positive microglia in the EC layer II/III of ECrTgtau mice (compared to WT) was partially rescued in ECrTgTau‐Mapt ^0/0 mice (non‐significant). The number of GFAP‐positive astrocytes was similar across all genotypes (non‐significant). Mean ± SEM, n = 4 sections per mouse, 3 mice/group, one‐way ANOVA with Bonferroni correction. Scale bars, 100 μm.

Figure EV4. Increased phospho‐tau and axonal changes in ECrTgTau mice

1. Stereological counting of DAPI nuclei in EC layer II/III suggested no obvious neuronal loss in ECrTgTau(‐Mapt ^0/0) mice at 18 months of age.

2. Western blotting for pre‐synaptic marker synapsin‐1 (Syn‐1) showed similar levels, indicating no major synapse loss in ECrTgTau(‐Mapt ^0/0) mice at 18 months of age.

Data information: Mean ± SEM, n = 4 sections/mouse, 3 mice/group, one‐way ANOVA with Bonferroni correction. ns, not significant. Source data are available online for this figure.&!#6;

Figure 4. Tau knockout rescues P301Ltau‐induced atrophy and neurodegeneration

1. Human, mouse, and total tau protein levels in cortical TBS‐extracts of rTg4510, rTg4510‐Mapt ^0/0, and control mice: The amount of human tau (Tau13 antibody) was comparable in rTg4510 and rTg4510‐Mapt ^0/0, moTau (Tau/5) was comparable in WT and rTg4510, and total tau levels (hu+moTau, DAKO antibody) were (expected) highest in rTg4510 mice. n = 3 mice/group, non‐significant.

2. Whole brain weights of 9‐month‐old animals revealed pronounced brain matter loss in rTg4510 compared to WT mice (weight loss > 16%), which was rescued in rTg4510‐Mapt ^0/0 mice to > 96%. n = 5 mice/group.

3. Cortical thickness measured adjacent to HPC, from CTX surface to corpus callosum, was decreased in rTg4510 mice by ˜25% compared to WT mice. rTg4510‐Mapt ^0/0 showed no CTX thinning compared to Mapt ^0/0 or WT mice. n = 3 mice/group.

4. The number of neurons (NeuN⁺ cells) in the cortex of rTg4510 mice was significantly reduced to ˜67% compared to both WT and rTg4510‐Mapt ^0/0. n = 3 mice/group.

5. The volume of hippocampal region CA1, with CT the most affected regions in rTg4510 mice, was significantly reduced in rTg4510 by ˜70% volume; rTg4510‐Mapt ^0/0 had significantly larger CA1 volume left (reduced by only ˜40%). n = 3 mice/group.

6. rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia (GFAP ⁺, red) and microglia (Iba1⁺, white) in the CTX compared to WT mice. Both astro‐ and microgliosis were reduced by ˜50% in rTg4510‐Mapt ^0/0 mice., n = 3 sections/mouse and 5 mice per/group. Scale bars, 100 μm.

Data information: Mean ± SEM. Two‐tailed Student's t‐test and one‐way ANOVA with Bonferroni for multiple comparison. ns, not significant.Source data are available online for this figure.

Figure EV5. Reduced neurodegeneration in 12‐month‐old rTg4510‐Mapt ^0/0 mice

1. Whole brain weights of 12‐month‐old animals show severe brain matter loss in rTg4510 compared to WT mice (weight loss > 23%), which was partially rescued in rTg4510‐Mapt ^0/0 mice.

2. Cortical thickness, measured from CTX surface to corpus callosum, decreased in rTg4510 mice at 12 months by ˜50% compared to WT, and rTg4510‐Mapt ^0/0 showed CTX thinning of ˜30% at 12 months.

3. The number of cortical neurons (NeuN⁺ cells) in 12‐month‐old rTg4510 mice was significantly reduced (˜63% of WT). The number of neurons in rTg4510‐Mapt ^0/0 slightly decreased to ˜88% of Mapt ^0/0 mice (ns).

Data information: Mean ± SEM, n = 3 mice/group, one‐way ANOVA with Bonferroni for multiple comparison. ns, not significant.

Figure EV6. Elevated ER stress in the presence of mouse tau in rTg4510 mice

Western blot analysis of extracts from 9‐month‐old mice revealed equally high ubiquitin levels in rTg4510 and rTg4510‐Mapt ^0/0 mice compared to controls; P301ltau aggregates appear to expose a similar challenge to the proteasome both in the presence and in the absence of mouse tau. Levels of the endoplasmatic reticulum (ER) stress marker CHOP seemed elevated only in rTg4510 mice, suggesting less ER stress and subsequent triggering of apoptotic pathways in the absence of mouse tau. Mean ± SEM, n = 3 mice/group, one‐way ANOVA with Bonferroni. ns, not significant.Source data are available online for this figure.

Figure 5. Reduced P301Ltau and NFT neurotoxicity in the absence of endogenous tau

1. Cortical extracts from rTg4510‐Mapt ^0/0 brains had significantly less phospho‐tau (CP13, PHF1, 12E8) than rTg4510 extracts. Compared to WT mice, both transgenic tau lines had high levels of phospho‐tau. n = 3 mice/group.

2. Representative images of gallyas silver‐stained aggregated tau in cortices from 12‐month‐old mice unravel stunning differences in the degree of tau pathology in rTg4510 compared to rTg4510‐Mapt ^0/0 mice. n = 3 mice/group.

3. Higher magnification images of silver (12‐month‐old) and thioflavine‐S (9‐month‐old)‐stained cortices show mature tangles (white arrowheads in Thio‐S stain) in rTg4510 and rTg4510‐Mapt ^0/0 mice; enhanced pathological changes such as neuritic tau accumulation and neuropil vacuolation around NFTs are found only in rTg4510 mice. Stereological counting revealed similar numbers of cortical NFTs between rTg4510 and rTg4510‐Mapt ^0/0 mice at 9 and 12 months of age. Because of the pronounced neuronal death only in rTg4510 mice, the percentage of tangle‐bearing neurons was ˜1.6‐ to 1.8‐fold higher in 9‐ and 12‐month‐old rTg4510 mice. n = 3 sections/mouse, 3 mice/group.

4. Immuno‐FISH for huTau mRNA (green) and phospho‐tau (PHF1, red) shows obvious differences in the distribution of neurons in cortex layer II/III: 9‐month‐old rTg4510 mice had more neurons filled with NFT‐like phosphorylated tau (PHF1⁺, red), and rTg4510‐Mapt ^0/0 mice had significantly more huTau mRNA‐positive neurons (FISH ⁺). rTg4510‐Mapt ^0/0 mice had also more neurons still expressing both PHF1 and huTau mRNA (PHF1⁺ FISH ⁺), suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510‐Mapt ^0/0 mice. n = 3 sections per mouse, 3 mice/group.

5. Immuno‐FISH showing EC neurons having both misfolded somatic tau (Alz50, red) and human tau mRNA (green; white arrowheads) in rTg4510‐Mapt ^0/0 but rarely in rTg4510 mice. n = 3 sections, 2 mice/group.

Data information: Mean ± SEM. Two‐tailed Student's t‐test and one‐way ANOVA with Bonferroni for multiple comparison. ns, not significant. (B–D) Scale bars, 100 μm. (E) Scale bar, 50 μm.Source data are available online for this figure.

Figure EV7. Lack of mouse tau largely delays onset of NFT‐induced neurodegeneration

1. A, B

   Average ratios of (A) tangle number:neuron loss (=tangles/([number of neurons in WT or Mapt ^0/0 controls]‐[number neurons in rTg4510 or rTg4510‐Mapt ^0/0]) and (B) average rations of tangle number:cortex thinning (=tangles/([CTX thickness of WT or Mapt ^0/0 controls]‐[CTX thickness of rTg4510 or rTg4510‐Mapt ^0/0]) at 9 and 12 months of age highlight the improved neuronal survival at given tangle load in rTg4510‐Mapt ^0/0 mice. Comparing the effect of NFTs on neuronal loss (A) and cortex thinning (B) between 9‐ and 12‐month‐old animals discovers a delayed onset of pathological changes—in the context of tangles—in tau‐null animals. n = 3 mice/group.

2. C

   Representative images of an immuno‐FISH for human tau protein (TauY9 antibody, pink) and human tau mRNA (green) on brain sections of 9‐month‐old rTg4510(‐Mapt ^0/0) mice. Human tau transgene expression appears diminished in the outer CTX layers in rTg4510 but not rTg4510‐MapT ^0/0 animals (low magnification images). In rTg4510‐MapT ^0/0 animals, more EC neurons still express human tau mRNA while having human tau protein accumulated in the somata (white arrowheads) compared to rTg4510 mice; the neurotoxicity caused by P301Ltau expression, missorting into the soma, and aggregation seems to be reduced in the absence of mouse tau. n = 4 sections/mouse, 2 mice/group. Scale bars, 100 μm.

Figure 6. Differences in tau oligomers and reduced seeding activity in rTg4510‐Mapt ^0/0 mice

1. Extraction of cortices revealed similar human tau (Tau13) in TBS‐extracts (not significant) but significantly more human tau in Triton X‐100 (TX‐100) extracts of rTg4510‐Mapt ^0/0 compared to rTg4510 mice. Mean ± SEM, n = 3 mice/group, two‐tailed Student's t‐test, ns, non‐significant.

2. Native gel electrophoresis of cortical TBS‐extracts showed small differences in HMW (oligomeric) human tau between rTg4510 and rTg4510‐Mapt ^0/0 brains. Western blot lanes were averaged across ˜2/3 of the width (black rectangular and arrow in Tau13 blot). The mean ± SEM (n = 3 mice/group) of these averages was plotted as longitudinal lane profiles. Differences in HMW tau are indicated by red and black arrowheads.

3. TBS‐brain extracts were applied to a HEK293 cell tau aggregation seeding assay (Holmes et al, 2014; Sanders et al, 2014), in which TauRDP301S‐CFP and TauRDP301S‐YFP are co‐expressed intracellularly. The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer (FRET) activity between co‐aggregated CFP and YFP‐tags and correlates with the tau aggregation seeding activity of the applied brain extracts. After 24 h, cells treated with extract (0.5 and 1.0 μg total protein per 96 well) from 9‐month‐old rTg4510 had significantly more intracellular YFP‐positive (white arrowheads) aggregates compared to rTg4510‐Mapt ^0/0 extracts; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510‐Mapt ^0/0. WT and Mapt ^0/0 extracts never showed seeding activity. Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510‐Mapt ^0/0 mice. Two‐tailed Student's t‐test, mean ± SEM, n = 3 replicates. ns, not significant. Insets, 100 μm. Scale bars, 50 μm.