Several studies have demonstrated the capacity for seeding and spreading of tau-enriched fractions of brain homogenates from AD and other human and mouse tauopathies following intracerebral inoculation into transgenic mice bearing human tau or mutant human tau [8, 9, 11, 12, 16] and into WT mice [9, 13–16, 22–26].
In addition to neurons, deposits occur in glial cells, and the morphology of glial inclusions appears to mimic the glial aggregates of the corresponding human tauopathies in inoculated transgenic mice expressing human tau or mutant human tau [10–16]. However, our previous studies show differences between the phenotype of the original tauopathy and the resultant tauopathy following intracerebral inoculation of tau-enriched homogenates into WT mice [2–25]. In our experiments using WT mice, tau deposits in neurons resemble pre-tangles rather than tangles; oligodendrocytes contain tau deposits despite the tauopathy; and tau deposits are almost absent in astrocytes excepting first stages following tau ARTAG. Due to this uniform pattern following inoculation in WT mice, we speculated that host tau, in addition to inoculated tau, plays a role in the capacity for and pattern of seeding and spreading of tau .
Four tau genotypes used in the study
To assess this hypothesis, here we used four genotypes of mice carrying different forms of tau: adult WT mice expressing 4R murine tau, P301S transgenic mice expressing the human tau mutation P301S, hTau expressing mainly human 3Rtau and lesser amounts of 4Rtau in a KO murine tau background, and mtWT mice that do not express murine and human tau.
P301S transgenic mice at the age of 6 and 9/10 months show hyper-phosphorylated tau deposits in neurons with the appearance of pre-tangles in the cerebral cortex, amygdala, hippocampus, striatum, and thalamus; and neuropil threads in the cerebral cortex, and plexiform layers of the hippocampus. Tau deposits are positive with phospho-specific anti-tau antibodies AT8 (Ser202/Thr205), PHF1 (Ser396/404), Tau-P Ser422, Tau 100 (Tau-P Thr212/Ser214), and antibodies against epitopes within amino acids 312-322 (conformational antibody MC1). Truncated tau at the aspartic acid 421 (Tau C-3) is also expressed in a subset of neurons at the age of 9/10 months . Affected neurons in P301S transgenic mice also contain nitrated tau (Tau-N Tyr29). Although there is a positive 4Rtau background, neuronal tau deposits do not show 4Rtau immunoreactive enhancement.
Tau phosphorylation is accompanied by activation of p38, SAPK/JNK, and SRC kinases, as identified with the antibodies p38-P Thr180/182, SAPK/JNK-P Thr183/Thr185, and SRC-P Tyr416, respectively, but not by phosphorylation of GSK-3β Ser9. Affected neurons also show rare granular casein kinase δ immunoreactivity. However, markers of granulovacuolar degeneration linked to endoplasmic reticulum stress and autophagy (eIF2α-P Ser51, IRE1-P Ser 274, LC3 and LAMP1) are negative, in agreement with recent observations . In addition to phospho-tau, neuronal deposits are strongly immunoreactive with the MAP2-P Thr1620/1623 antibody. Astrocytes and oligodendrocytes barely contain abnormal tau deposits up to the age of 9/10 months in our series.
hTau transgenic mice express high levels of human 3Rtau and low levels of 4Rtau in western blots of brain homogenates. Phosphorylated tau deposition occurs in subpopulations of neurons in the cerebral cortex and hippocampus. Abnormal deposits are identified with the antibodies PHF1 and MC1, but tau deposits are not stained with AT8, tau-P Ser422, and Tau C-3 antibodies. This phenotype is similar to that described previously in hTau transgenic mice . Moreover, tau deposits in hTau do not contain Tau-N Tyr29. This contrasts with the presence of Tau-NTyr29 in NFT, dystrophic neurites, and neuropil threads in AD and NFTs in CBD and PSP  and P301S transgenic mice. There is no apparent activation of tau kinases p38, SAPK/JNK, GSK-3β Ser9, and SRC kinases co-localizing with abnormal tau deposits in hTau mice. Phosphorylated MAP2 is not deposited in neurons. Casein kinase δ immunoreactivity and markers of granulovacuolar degeneration linked to endoplasmic reticulum stress and autophagy are negative in hTau. Differences and similarities between P301S and hTau are shown in Table 2.
As expected, WT mice do not have tauopathy, and mtWT mice do not express any type of tau using the same battery of anti-tau antibodies.
Inoculation of mice: variability of tau seeding and spreading depending on the host tau genotype.
We inoculated tau fibrils from the same AD case to randomize donor tau, and injected the same amount of the inocula unilaterally into the same region of the hippocampus in every case. Inoculations in the lateral ventricle, carried out to assess the capacity for seeding of tau fibrils in the CSF, resulted negative. This observation is in line with the failure to detect seeding of the CSF from AD using ultrasensitive tau biosensors .
Tau seeding and spreading does not occur in the absence of host tau, as no deposits develop in mtWt mice. This is particularly important as it indicates that host tau is essential for tau spreading in inoculated mice. Moreover, the latter experiment shows that the pathology of tau progression in inoculated mice is not due to the passive extension of the inocula.
The distribution of tau spreading following inoculation does not grossly differ in WT, P301S, and hTau mice inoculated at six months with a survival of three months, and in mice inoculated at three months and killed at the age of 9/10 months. Deposits of hyper-phosphorylated tau occur in neurons of the ipsilateral hippocampus, dentate gyrus, and cerebral cortex near the site of inoculation, and in threads in the corpus callosum and ipsilateral callosal radiation. The present results confirm pioneering observations demonstrating that the pattern of neuronal tau spread is largely determined by connectivity .
However, differences in the regional vulnerability to tau seeding and spreading depend on the genotype. The involvement of the CA1 region was higher in hTau mice when compared with P301 and, particularly, with WT mice. Tau deposits in the stratum radiatum and stratum oriens were more abundant in hTau in mice than in WT and P301S mice at the same survival time. In contrast, the contralateral corpus callosum and tau deposits in the fimbria were more marked in WT and P301S mice.
In addition to neurons, oligodendroglial cells in the fimbria, corpus callosum, and white matter contain hyper-phosphorylated tau, a feature not seen in AD. It is worth noting that oligodendrocytes in normal conditions produce and express tau protein [33, 34]. Abnormal tau deposition in oligodendrocytes occurs constantly in our paradigms following inoculation of tau from different tauopathies including AD, PART, AGD, PSP, ARTAG, and FTLD-P301L in WT mice [22–26]. Oligodendroglial participation is also observed following tau inoculation from CBD homogenates in humanized tau mice . These observations show that oligodendrocytes are principal participants in tau spreading following tau inoculation in mice expressing murine or human mutant tau. It is not clear whether tau spreading in oligodendrocytes occurs by continuity rather than connectivity. Differential vulnerability of oligodendrocytes is also dependent on the host tau genotype. Phospho-tau deposits in oligodendoglial cells are abundant in WT and P301S inoculated mice with AD-tau, but scanty if at all present in hTau transgenic mice. Since the involvement of oligodendrocytes differs in inoculated WT and P301S mice when compared with hTau mice, it may be inferred that oligodendrocyte vulnerability to identical tau inocula is also partially dependent on the host tau.
No evidence of unfolded protein response, autophagy, and apoptosis following tau inoculation
Abnormal deposits in neurons and glial cells are not stained with anti-eIF2α-P Ser51, IRE-P Ser274, LC3, and LAMP1 antibodies, suggesting that they do not present the biochemical markers of the unfolded protein response, and they aren’t either consistent with granulovacuolar degeneration [36, 37]. These observations are in contrast with previous studies stressing the activation of the unfolded protein response in pre-tangle neurons and the induction of granulovacuolar degeneration bodies by intracellular tau pathology [38, 39]. The discrepancies may originate in differences between human AD and AD-tau inoculation in mouse models, and in the methods employed, more precisely between tau seeding in cultured neurons versus tau seeding and spreading in vivo.
In the same line, although tau hyper-phosphorylation may produce oxidative stress and neuronal death by apoptosis , apoptosis is only seen in the hilus and dentate gyrus in P301S mice aged 9/10 months, but not in hTau mice. Additional apoptosis is not observed in inoculated WT, P301S, and hTau mice at three and six months after tau inoculation.
Post-translational modifications of recruited tau in inoculated mice depend on the tau genotype of the host
Tau inoculation triggers an active process of post-translational modifications in vulnerable cells which is partially dependent on the host genotype. This is manifested by the appearance of new sites of tau phosphorylation and nitration in affected neurons in inoculated mice compared with the corresponding sites under baseline conditions in the different genotypes. Tau deposits in inoculated WT, P301S, and hTau are stained with antibodies AT8 and Tau-P Ser422 whereas no such deposit types occur in WT and hTau transgenic mice under normal conditions. This observation suggests that AD tau has the capacity to induce post-translational modifications in the host tau which are reminiscent of AD tau. Activation of p38 and SAPK-JNK co-localizing tau deposits in inoculated WT, P301S, and hTau mice further points to the active phosphorylation of retrieved tau from the host following tau inoculation, as already reported in WT mice inoculated with sarkosyl-insoluble brain fractions from various human tauopathies [22–26]. However, the characteristics of tau deposits in inoculated mice differ among genotypes. The morphology and composition of neuronal tau deposits are similar in inoculated WT and P301S mice, and they are reminiscent of pre-tangles. In contrast, neuronal tau aggregates in inoculated hTau mice resemble neurofibrillary tangles. This striking difference is manifested using a wide variety of specific antibodies including 3Rtau, 4Rtau, AT8, PHF1, tau-PSer422, tau MC1, and MAP2-P.
Moreover, abnormal tau protein aggregates in inoculated hTau mice, but not in WT and P301S mice, contain tau-N Tyr29, and co-localize with cytoplasmic granules immunoreactive with CK1-δ, CLK1, GSK-3β-P Ser9, and PKAα/β-P Tyr197 antibodies.
Modulation of tau transcription in the host following identical inoculum is host genotype-dependent
Tau deposits in inoculated mice with AD fibrils are composed of 4Rtau and 3Rtau. Murine 4Rtau and human mutant 4Rtau predominate in WT and P301S mice, respectively; and human 3Rtau predominates in hTau transgenic mice. Yet, the amount of 3Rtau and 4Rtau isoforms produced in inoculated mice varies from one genotype to another. However, deposits of 3Rtau prevail in neuronal deposits in inoculated WT and P301S transgenic mice, whereas deposits of 4Rtau prevail in hTau transgenic mice. Not only is 3Rtau expressed de novo in mice with predominance of 4Rtau, but the composition of de novo tau deposits in the inoculated mice is the reverse of that expressed in the corresponding non-inoculated mice.
Curiously, modifications in the expression of tau isoforms have been observed in oligodendrocytes following middle cerebral occlusion in rats and mice. Some peri-infarct oligodendrocytes shift from 4Rtau expression to 3Rtau isoform immunoreactivity thus suggesting exon 10 splicing plasticity under determinate conditions .
Several factors may contribute to differences between the different genotypes. One of them is species differences of tau .
A shift from foetal to adult tau isoform expression occurs in mice and humans . Exon 10 splicing rests on the activity of various SR proteins and diverse RNA-interacting and RNA/DNA-binding proteins . SR protein kinase (SRPK) and cdc-like kinase (CLK/Sty) phosphorylate SR proteins and control their functions [45–47]. Other kinases, such as cAMP-dependent protein kinase (PKA), serine/threonine-specific protein kinase AKT1, glycogen synthase kinase-3β (GSK-3β), have the additional capacity to phosphorylate selected SR proteins [48–51].
The present observations in hTau mice inoculated with AD-tau show the activation by phosphorylation at specific sites of PKA, AKT1, GSK-3β, and CLK1 in neurons, mainly in the CA1 region, containing higher expression of 4Rtau than 3Rtau. Although these kinases might be involved in altered tau transcription in inoculated hTau mice, this scenario does not apply to inoculated WT and P301S mice.
Several tau fractions in AD have differential prion-like activities . Following this line of thinking, the diversity of tau strains may contribute to clinical diversity in AD [52, 53]. As a working hypothesis, it may be postulated that variable composition of 3Rtau and 4Rtau isoforms is expressed in different brain regions and in different neurons in a particular individual. Baseline differences in the composition of tau would modulate the vulnerability of specific neuronal and glial populations to the presence of pathological tau strains. The present findings may have implications in human tauopathies. Individual neurons and glial cells may have differences in tau composition. Therefore, tau variability, in addition to other factors, would contribute to individual regional and cell vulnerability not only in the context of different tauopathies but also in the phenotypic variability between individuals affected by the same tauopathy.