Introduction
Tauopathies are a diverse group of neurodegenerative diseases, characterized by the presence of Tau aggregates composed of misfolded hyperphosphorylated Tau [
7,
41,
62]. In Tauopathies, symptoms correlate strongly with the regional distribution of Tau-aggregates in the brain. In Alzheimer’s Disease, the most common Tauopathy, Tau aggregate formation progresses in a stereotypic pattern, along functionally connected neuroanatomical pathways, used for staging disease progression [
2,
6,
34,
59]. In AD, Tau pathology starts in the entorhinal cortex (EC), progresses to the limbic regions such as subiculum, hippocampal cornu ammonis (CA) and amygdala (stages III and IV), and finally involves neocortical areas (stages V and IV), according to the Braak and Braak stages [
6,
59]. Defects in neuronal function and neuronal loss of the affected or connected neurons are believed to contribute to the disease symptoms by affecting functionally critical neuronal networks, while the mechanism remains poorly understood [
6,
15,
24,
58,
74].
Accumulating evidence indicates that Tau and related proteins linked to neurodegenerative proteinopathies display prion-like properties [
12‐
14,
19,
22,
25,
26,
28,
32,
35,
37,
72]. In vitro studies have demonstrated that extracellular misfolded Tau can be taken up by cultured cells, can seed aggregation of endogenous soluble Tau and can propagate a fibrillar, misfolded state between co-cultured cells [
20,
26,
32,
53]. Injections of brain lysates from neurofibrillary tangle (NFT) bearing transgenic mice into wild type Tau-expressing transgenic mice that do not develop NFTs, induce Tau pathology that spreads to brain regions remote from the injection site by connectivity rather than proximity [
1,
13]. This has been recapitulated with brain extracts from patients with different Tauopathies, mimicking distinct forms of Tau-aggregation—in a cell-type and region specific way-reminiscent for the respective Tauopathies [
5,
12]. Injection of pre-aggregated synthetic full length Tau and Tau fragments results in seeding and spreading of Tau-pathology in cells and in Tau transgenic mice, demonstrating that misfolded Tau per se, and not a different factor in the brain extracts, is sufficient to induce Tau-aggregation [
35]. Induction of Tau-aggregation by misfolded Tau has been demonstrated in non-transgenic mice [
40], albeit at later age and to a more limited extent. Indefinite and stable maintenance of unique conformations—“strains”—in vivo that link structure to patterns of pathology was recently demonstrated, further corroborating prion-like properties of misfolded Tau [
5,
53]. Although increasing dysfunction in neuronal networks is believed to explain the progression of behavioral disease symptoms, including memory loss [
22,
28,
53,
58,
59], it remains to be demonstrated whether this can be caused by prion-like spreading of Tau-pathology. This is not a trivial question, as previous reports have indicated that NFTs per se may not disturb neuronal function and could be an off-pathway disease side effect [
30,
38,
48,
52,
54,
61,
65,
66,
69].
In this work, we have addressed this question using in vitro, ex vivo and in vivo models with induction and spreading of Tau-pathology. Seeding of Tau pathology was shown to impair neuronal network function in primary neuronal cultures and in organotypic hippocampal slices. Furthermore, Tau-seeding caused prion-like spreading of Tau-aggregation through functionally connected neuronal networks and neuronal network dysfunction in TauP301S transgenic mice, leading to either cognitive or motoric deficits, depending on the initial site of Tau-seeding. Our data furthermore point to early pathological forms of Tau, including Tau oligomers, rather than somatic NFTs as culprits for the functional deficits.
Discussion
Accumulating evidence has demonstrated that Tau displays prion-like properties and that prion-like spreading of Tau-pathology occurs through connectivity rather than proximity [
1]. This process has been proposed to underlie the stereotyped progression along unique brain circuitries of Tau-pathology and symptoms in Tauopathies [
22,
28,
50,
53,
58,
74]. In this work, we demonstrated—for the first time—that templated Tau-misfolding induced by Tau-seeds through functionally connected neuro-anatomical pathways, impairs neuronal network function and associated behavioral outcomes, dependent on the initial site of seeding. More precisely, we demonstrated that small amounts of pre-aggregated synthetic Tau fragments, focally injected, without changing expression-level of Tau but only inducing Tau-misfolding, are sufficient to propagate Tau aggregation and impair neuronal network function and behavioral outcomes 6 months post-injection, dependent on the initial site of injection. These findings emphasize the potential relevance of this intensively studied process in the stereotypic progression of symptoms in Tauopathies. We have further extended our analysis to highlight early pathological forms of Tau, rather than fully mature fibrillar NFTs as causal culprits for the neuronal network dysfunction.
We demonstrated that templated Tau-misfolding by Tau-seeds affected neuronal network activity in primary neuronal cultures. Primary cortical neurons display synchronized calcium oscillations in culture, which were previously shown to be modulated by PTX, a GABA
A receptor antagonist [
44]. Exposure of cortical neurons to PTX renders calcium transients to exhibit increased amplitude, more regular frequency and higher synchronicity [
44]. A refractory period has previously been identified which may play a role in increased synchronization. Our data indicate a significant dysregulation of spontaneous and PTX-induced calcium oscillations, indicating that Tau-seeded Tau-misfolding affects neuronal network activity. Our findings suggest that Tau-seeding impairs GABA-ergic synaptic transmission, based on the similarity of the effect of PTX and Tau-seeds on spontaneous oscillations. As we further demonstrated that PTX elicited calcium transients are NMDA- and AMPA-dependent as shown previously [
44], their reduced amplitudes in Tau-seeded neuronal cultures indicate impaired glutamatergic, NMDA- and AMPA-dependent signaling. We further extended our analysis in models with conserved network architecture, i.e. to organotypic hippocampal slices [
21]. In organotypic cultures, we demonstrated impaired population spike amplitude measured in hippocampal CA1 region following stimulation of Schaffer collaterals. This basal synaptic transmission is predominantly generated by excitatory glutamatergic (NMDA/AMPA-mediated) neurotransmission, further corroborating data obtained in primary neuronal cultures. Furthermore, measurement of LTP revealed that synaptic plasticity is impaired in organotypic slices by Tau-seed induced Tau misfolding. Notably, NMDA- and AMPA-receptor functions are critically involved in hippocampal LTP in CA1. Our data were further corroborated in vivo. Electrophysiological analysis in acute hippocampal slices revealed that LTP was affected 6 months post-induction of Tau-pathology by Tau-seeding in vivo, although basic synaptic transmission was not. The latter may be explained by the fact that effects of Tau-aggregation can be more easily compensated in vivo than in vitro, or that the aggregation process occurred more rapidly in vitro, or that organotypic cultures are more sensitive than the intact hippocampus in vivo. It must be noted that electrophysiological analysis in acute hippocampal slices was performed in hippocampal CA1 region, while initial Tau-seeding was performed in entorhinal cortex, indicating that neuronal function remote from the initial seeding site was affected. We further demonstrated conserved presynaptic parameters including paired pulse ratio, synaptic fatigue and SV2 staining, pointing towards a postsynaptic mechanism. Together with the results obtained in primary neurons, indicating impaired NMDA/AMPA-dependent neuronal network function following Tau-seeding, our data are in line with a role of pathological Tau forms in postsynaptic dysfunction, including NMDA/AMPA dysfunction as previously shown [
8,
60,
70]. We further assessed behavioral effects, and demonstrated that cognition, measured in an object recognition task was impaired in bilaterally injected mice, 6 months post-injection. Together our data indicate that Tau-seed induced misfolding of Tau, leading to Tau-aggregation through connectivity, affects neuronal function, neuronal network activity and behavioral outcome, and hence could contribute to progression of the symptoms in Tauopathies.
Prion like induction of Tau-pathology—a process which raised considerable scientific interest—has been elegantly demonstrated and analyzed in exquisite detail for different aspects [
1,
12‐
14,
19,
20,
22,
26,
28,
32,
35,
37,
40,
53,
72]. The functional repercussions of this process remained however, to be analyzed. This question is important to evaluate its relevance in the pathogenetic process of Tauopathies. In AD, Tau-pathology is known to progress according to stereotypical and predictable patterns reflected in its incorporation in criteria for the neuropathological diagnosis of AD [
6,
34]. The first neurons to be affected are in entorhinal cortex (EC) (stage I), the neurons that give rise to the perforant pathway, the major projection connecting cerebral cortex with the hippocampus [
23,
33]. Next NFTs spread to the CA1 region of the hippocampus and accumulate in limbic structures such as the subiculum (stage II–III) and the amygdala, thalamus, and claustrum (stage IV). Finally, NFTs spread to isocortical areas (stage V–VI). Symptoms in AD are strongly correlated with this characteristic appearance of NFTs. In this work, we mimic spreading of Tau-pathology following seeding in entorhinal cortex of TauP301S mice, similar as observed in AD. Tau pathology starts first in the entorhinal cortex, subsequently in CA1, the subiculum and CA3-4 and later in the DG, and finally in neocortical areas (stages V and IV), according to the Braak and Braak stages. Noteworthy, minor discrepancy between spreading of Tau-pathology in AD [EC, subsequently CA1/subiculum/CA3-4 and later dentate gyrus (DG)] and functional connectivity (EC, subsequently DG/CA3-4 and then CA1/subiculum) may be ascribed to additional factors determining selective vulnerability or protection in these regions, which appear to be recapitulated in our model, as the AD pattern was mimicked following seeding in EC. It must be noted however that in contrast to AD and sporadic Tauopathies, characterized by aggregation of wild type Tau, we used in the current work recipient Tau and inducer Tau-seeds mutated at Proline 301, as this combination results in the highest Tau-aggregation efficiency. Previous work also indicated that mutation of Proline 301, rather than its substitution by Serine or Leucine, is determining aggregation efficiency [
27]. However, wild type Tau acting as recipient, in combination with either wild-type or mutant Tau seeds as inducer has been demonstrated to be capable of prion-like Tau seeding, albeit much less efficient. The potential of wild type Tau to undergo prion-like Tau seeding allows a (cautious) extrapolation of our data in the context of AD or sporadic Tauopathies, characterized by aggregation of wild type Tau. Hence, neuronal network dysfunction and associated behavioral dysfunctions induced by prion-like seeding and spreading of Tau-pathology along functionally connected circuitries could relate to symptomatic progression in AD and related Tauopathies.
Besides in AD patients, spreading of Tau-aggregation according to a characteristic pattern along functionally connected brain circuitries is also observed in argyrophilic grain disease, a different Tauopathy. Other Tauopathies are very heterogeneous between individuals with identical mutations, including for patients with Tau mutated at Proline 301 (P301L) [
4]. This heterogeneity hampers the delineation of a specific progression pattern. However, symptoms progressively affect certain behaviors driven by a particular brain circuitry. We here demonstrate for the first time that the initial site of Tau-seeding determines the behavioral outcomes, resulting in different behavioral outcomes depending on the initiation site. Initiation of prion-like seeding in transgenic mice expressing mutant TauP301S, in entorhinal cortex resulted in spreading of Tau-pathology along functionally connected circuits, resulting in impaired LTP in hippocampal CA1 neurons. Cognition measured in an object recognition task was demonstrated to be impaired following Tau-seeded Tau-aggregation. In contrast, prion-like seeding in basal ganglia resulted in spreading to brain regions involved in motor control, thereby resulting in motor impairments, 6 months after Tau-seeding. Motoric impairments were not observed following injection of Tau-seeds in EC. These findings are reminiscent of the heterogeneity of symptoms in TauP301 patients, where the initial site of Tau seeding by environmental, genetic or accidental factors, may determine the symptomatic outcome (cognitive symptoms or Parkinsonism).
The clearcut demonstration of impaired neuronal network function and behavior by prion-like seeding raises questions about the causal culprit, in terms of pathological forms of Tau. The combination of our functional analysis with biochemical and immunological analysis points to early pathological forms of Tau, including pathological hyperphosphorylated and misfolded Tau and Tau oligomers rather than fully mature NFTs as potential pathological culprits. Notably, strong somatic AT8 aggregation was only observed in 5–10 % of neurons following Tau-seeding in primary neurons. Furthermore, the presence of fully mature fibrils was below detection limit using electron microscopy and ThioS staining was not observed in PNC. It must be noted that Tau-seeding in primary neurons in this work was less robust than previously published [
27], probably due to lower concentration of Tau-seeds used. We have however, demonstrated the abundant presence of “early” AT8-positive forms of Tau detected as diffuse staining and punctated synaptic staining in the current model. This staining was present throughout the primary neuronal culture and robustly induced following Tau-seeding. Furthermore, oligomeric forms of Tau stained with T22 or with AT8 were increased following Tau-seeding in all models. Although an approximately, twofold increase may seem rather low, the spatio-temporal characteristics (e.g. at the synapse) of this increase may underlay its pronounced effect on neuronal function. Taken together our data indicate that either a few neurons displaying strong somatic AT8 staining are responsible for impaired neuronal network activity, or most parsimoniously, that early forms of aggregated Tau or “pre-tangle stage” pathological forms of Tau, are responsible for neuronal network dysfunction. This is further supported by their abundant induction in brains and in organotypic cultures following Tau-seeding. Taken together our data suggest that early pathological forms of Tau rather than fully mature NFTs may represent the causal culprits. Final proof can only be delivered by selective elimination of different pathological forms of Tau and analysis of the functional repercussions. Our observations fit however with previous observations using elegant approaches to determine the repercussions of Tau-pathology, and more particularly of NFTs on neuronal function in vivo [
18,
30,
38,
48,
52,
54,
61,
65,
66,
69]. This demonstrated either no effects of NFTs in neurons on calcium or Arc responses [
38,
52]. Furthermore, using inducible expression of Tau or pro-aggregant Tau, reversibility of synaptic deficits despite the continued presence of NFTs was demonstrated, indicating association of synaptic defects with the aggregation process and early pathological Tau forms, rather than with the full-blown NFTs [
3,
30,
54,
61,
65,
66,
69]. These data are in line with clear demonstration of detrimental effects of Tau-oligomers on neuronal function and behavior, as demonstrated following acute injection of oligomeric Tau and immunization with anti-Tau oligomeric antibodies [
10,
11,
39,
40]. Our findings are furthermore in line with previous reports demonstrating that Tau overexpression or Tau-alterations affect neuronal function or function of connected neurons [
29,
31,
42,
47,
48,
56,
57,
63,
73]. It must be noted that in the current work, Tau-expression is not affected, but only misfolding of Tau is induced by templated seeding by focal administration of small amounts of seeds. We demonstrate that this templated Tau-misfolding is sufficient to propagate neuronal network dysfunction and behavioral outcomes, which correlate with the presence of early pathological forms of Tau, including Tau oligomers, rather than with the presence of fully mature Tau fibrils.
In summary, our data indicate for the first time and unequivocally, using in vitro, ex vivo and in vivo approaches that (i) prion-like Tau-seed induced Tau-aggregation with NFT formation—a mechanism under intensive investigation—causes synaptic and neuronal network dysfunction, resulting in behavioral impairments. Our data thereby indicate that prion-like spreading of Tau-pathology may contribute to progression of disease symptoms in Tauopathies by affecting intrinsic functional critical networks. Furthermore, our data indicate unequivocally that (ii) behavioral outcomes following seeding in Tau P301 transgenic mice, are determined by the initial site of Tau seeding. Hence, motoric problems were demonstrated following Tau-seeding in basal ganglia (substantia nigra), while absent following injection in entorhinal cortex, resulting in cognitive defects. These findings are reminiscent of the heterogeneity of clinical symptoms in familial cases with P301 mutations, in which initial seed formation by environmental, genetic or accidental factors, may determine the behavioral outcomes. Finally, (iii) our results support a role of early pathological forms of Tau, including oligomeric Tau rather than fully mature NFTs as pathogenic culprits of prion-like induced spreading of neuronal dysfunction. Our findings provide a basis to further identify the molecular mechanisms involved in Tau-seed induced synaptic dysfunction and provide a model to develop novel therapeutic strategies targeting Tau-seed induced neuronal dysfunction, and to analyze repercussions on neuronal function induced by different Tau-“strains”.