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In Alzheimer’s disease, tau pathology is thought to spread via a prion-like manner along connected neuronal networks. For this to occur, the usually cytosolic tau protein must be secreted via an unconventional mechanism prior to uptake into the connected neuron. While secretion of healthy and pathological tau has been documented, it remains under-investigated whether this occurs via overlapping or distinct processes. Here, we established a sensitive bioluminescence-based assay to assess mechanisms underlying the secretion of pseudohyperphosphorylated and wild-type tau in cultured murine hippocampal neurons. We found that under basal conditions, both wild-type and mutant tau are secreted, with mutant tau being more robustly secreted. Pharmacological stimulation of neuronal activity led to a modest increase of wild-type and mutant tau secretion, whereas inhibition of activity had no effect. Interestingly, inhibition of heparin sulfate proteoglycan (HSPG) biosynthesis drastically decreased secretion of both wild-type and mutant tau without affecting cell viability. This shows that native and pathological tau share release mechanisms; both activity-dependent and non-activity-dependent secretion of tau is facilitated by HSPGs.

Amyloid-beta (Aβ) and tau protein are both involved in the pathogenesis of Alzheimer's disease. Aβ produces synaptic deficits in wild-type mice that are not seen in Mapt-/- mice, suggesting that tau protein is required for these effects of Aβ. However, whether some synapses are more selectively affected and what factors may determine synaptic vulnerability to Aβ are poorly understood. Here we first observed that burst timing-dependent long-term potentiation (b-LTP) in hippocampal CA3-CA1 synapses, which requires GluN2B subunit-containing NMDA receptors (NMDARs), was inhibited by human Aβ1-42 (hAβ) in wild-type (WT) mice, but not in tau-knockout (Mapt-/-) mice. We then tested whether NMDAR currents were affected by hAβ; we found that hAβ reduced the postsynaptic NMDAR current in WT mice but not in Mapt-/- mice, while the NMDAR current was reduced to a similar extent by the GluN2B-selective NMDAR antagonist Ro 25-6981. To further investigate a possible difference in GluN2B-containing NMDARs in Mapt-/- mice, we used optogenetics to compare NMDAR/AMPAR ratio of EPSCs in CA1 synapses with input from left vs right CA3. It was previously reported in WT mice that hippocampal synapses in CA1 that receive input from the left CA3 display a higher NMDAR charge transfer and a higher Ro-sensitivity than synapses in CA1 that receive input from the right CA3. Here we observed the same pattern in Mapt-/- mice, thus differential NMDAR subunit expression does not explain the difference in hAβ effect on LTP. Finally, we asked whether synapses with left vs right CA3 input are differentially affected by hAβ in WT mice. We found that NMDAR current in synapses with input from the left CA3 were reduced while synapses with input from the right CA3 were unaffected by acute hAβ exposure. These results suggest that hippocampal CA3-CA1 synapses with presynaptic axon originating in the left CA3 are selectively vulnerable to Aβ and that a genetic knock out of tau protein protects them from Aβ synaptotoxicity., Alzheimer's Research UK (ARUK)

Additional file 1. Fig. S1. Automated analysis and semi-quantitative scales used for analysis. A and B) Original images and images after analysis with quantification. Analysis performed as specified in Materials and methods. A) 3 sample images with low, middle and high density of diffuse small tau-PLA labelled structures were processed with ImageJ for the automated measurement of diffuse signal, insets show final counts. B) Samples from the 4 different immunolabellings were processed with ImageJ for the automated measurement of large labelled lesions, insets show final counts. C) Semi-quantitative scale used for semi-quantitative analysis of all brain regions. Fig. S2. Detection of tau–tau interactions in HEK 293 cells. Representative fields of view of the tau-PLA puncta quantification of Fig. 2B. Tau-PLA puncta are in red. Nuclei were identified by DAPI staining (blue). Scale bar: 10 μm. Fig. S3. In situ specificity of tau-PLA in mouse brain tissue. Top: Representative images from 6 months-old P301S transgenic, C57BL/6 wild-type and MAPT KO mice (N = 6 per genotype), stained with Tau-PLA and AT8. Brain histological analysis revealed an absence of tau-PLA signal in MAPT KO mouse brain tissue. C57BL/6 control mice showed almost negligible load of tau-PLA, whereas this signal appeared to be quite strong in the P301S mice. Tau-PLA revealed prominent tau multimerization in the CA1 region of hippocampus and striatum, anatomical areas negative for AT8. Bottom: Representative images from P301S transgenic animals at the age of 3 (N = 2), 6 (N = 6), and 9 (N = 2) months old. This analysis shows an age-dependent accumulation of tau-PLA signal. Scale bar 100 μm. Fig. S4. Tau-PLA detects endogenous human tau–tau interaction in situ. A) Tau-PLA recognized tau pathology in the CA4/dentate gyrus of Alzheimer’s disease (Braak V) as compared to Braak 0 controls. B) Tau-PLA was performed in samples of different Braak stages in the presence or absence of ligase. The absence of ligase in the PLA reaction resulted in the ablation of all PLA signal, indicating the staining is dependent on the ligation of two conjugates. C) 6xHisTag IHC and 6xHisTag-PLA performed in samples of Braak stage 0 and V. No signal was detected after performing immunohistochemistry with 6xHisTag antibody and 6xHisTag-PLA. Scale bar 50 μm. Fig. S5. A proportion of diffuse tau complexes locates to astrocytes and microglia. Representative co-immunofluorescence images (Braak IV) of indicated markers and tau-PLA. Arrowheads indicate astrocyte/microglial cells in top and bottom panels, respectively. Fig S6. Tau-PLA revealed that cases in Braak 0 group presented heterogenicity. A) Quantification of tau-PLA labelled small diffuse complexes in the different hippocampal regions, revealing the distribution of the population in Braak 0 group. ONE-WAY ANOVA (Tukey). AV; average. B) Representative images of EC stained with tau-PLA that illustrate the heterogenicity that characterize the Braak 0 group. Scale bar 50 μm. Fig. S7. Quantification of lesions in samples labelled with tau5 IHC. Samples immunolabelled with tau5 IHC were used for automated analysis. All groups were compared to control (Braak 0) through a ONE-WAY ANOVA (Dunnet). N = 7/7/7/4/6/6/6. AV; average. IHC: immunohistochemistry. Fig. S8. Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex – CA4. A) Representative images of selected hippocampal regions are shown here. FFPE sections of posterior hippocampus at the level of lateral geniculate nucleus from Braak 0 to III were stained with tau-PLA and AT180-, AT8-, MC1-immunohistochemistry. Minimal PLA and immunohistochemistry signal is seen in Braak 0. Tau-PLA reveals abundant pathology in Braak stages I to III, while AT180-immunohistochemistry shows a modest increase in signal in Braak stages I to III. AT8- and MC1-immunohistochemistry is absent or relatively low at initial stages. Scale bar 50 μm. B) Quantification of tau-PLA, AT8-, MC1-, and AT8- IHC labelled diffuse/thread pathology. All groups in each Braak stage were compared to tau-PLA through a ONE-WAY ANOVA (Dunnet). N = 11/12/12/9/7/8/8. AV; average. IHC: immunohistochemistry. Fig. S9. Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex – Entorhinal Cortex. A) Representative images of selected hippocampal regions are shown here. FFPE sections of posterior hippocampus at the level of lateral geniculate nucleus from Braak 0 to III were stained with tau-PLA and AT180-, AT8-, MC1-immunohistochemistry. Minimal PLA and immunohistochemistry signal is seen in Braak 0. Tau-PLA reveals abundant pathology in Braak stages I to III, while AT180-immunohistochemistry shows a modest increase in signal in Braak stages I to III. AT8- and MC1-immunohistochemistry is absent or relatively low at initial stages, with signal appearing in Braak III. Scale bar 50 μm. B) Quantification tau-PLA, AT8-, MC1-, and AT8- IHC labelled diffuse pathology. All groups in each Braak stage were compared to tau-PLA through a ONE-WAY ANOVA (Dunnet). N = 11/12/12/9/7/8/8. AV; average. IHC: immunohistochemistry. Fig. S10. Early tau multimerization detection, prior to detection of tau hyperphosphorylation and misfolding across hippocampal regions and temporal isocortex – CA1. A) Representative images of selected hippocampal regions are shown here. FFPE sections of posterior hippocampus at the level of lateral geniculate nucleus from Braak 0 to III were stained with tau-PLA and AT180-, AT8-, MC1-immunohistochemistry. Minimal PLA and immunohistochemistry signal is seen in Braak 0. Tau-PLA reveals abundant pathology in Braak stages I to III, while AT180-immunohistochemistry shows a modest increase in signal in Braak stages I to III. AT8- and MC1-immunohistochemistry is absent or relatively low at initial stages, with signal appearing in Braak III. Scale bar 50 μm. B) Quantification tau-PLA, AT8-, MC1-, and AT8- IHC labelled diffuse pathology. All groups in each Braak stage were compared to tau-PLA through a ONE-WAY ANOVA (Dunnet). N = 11/12/12/9/7/8/8. AV; average. IHC: immunohistochemistry. Fig. S11. Quantification of diffuse pathology/threads and lesions labelled by AT180- and MC1- IHC in hippocampal regions and temporal isocortex. Quantification of diffuse pathology/threads and lesions labelled by AT180- and MC1- IHC in hippocampal regions and temporal isocortex. All groups were compared to control (Braak 0) through a ONE-WAY ANOVA (Dunnet). N = 11/12/12/9. AV; average. IHC: immunohistochemistry. Fig. S12. Quantification of lesions in samples labelled with 4G8 IHC. Clinically processed samples immunolabelled with 4G8 were used for automated analysis. All groups were compared to control (Braak 0) through a ONE-WAY ANOVA (Dunnet). N = 7/6/6/4/5/5/4. AV; average. IHC: immunohistochemistry. Table S1. Summary of patient information. AV; average. Table S2. Additional information of brain samples. PMI: post-mortem interval, CERAD: consortium to establish a registry for Alzheimer’s disease, MCI: mild cognitive impairment.

Although many disease models exist for neurodegenerative disease, the translation of basic research findings to clinic is very limited. Studies using freshly resected human brain tissue, commonly discarded from neurosurgical procedures, should complement on-going work using stem cell-derived human neurons and glia thus increasing the likelihood of success in clinical trials. Areas covered: Herein, the authors discuss key issues in the lack of translation from basic research to clinic. They also review the evidence that human neurons, both freshly resected brain tissue and stem cell-derived neurons, such as induced pluripotent stem cells (iPSCs), can be used for analysis of physiological and molecular mechanisms in health and disease. Furthermore, the authors compare and contrast studies using live human brain tissue and studies using induced human stem cell-derived neuron models. Using an example from the area of neurodegeneration, the authors suggest that replicating elements of research findings from animals and stem cell models in resected human brain tissue would strengthen our understanding of disease mechanisms and the therapeutic strategies and aid translation. Expert opinion: The use of human brain tissue alongside iPSC-derived neural models can validate molecular mechanisms identified in rodent disease models and strengthen their relevance to humans. If drug target engagement and mechanism of cellular action can be validated in human brain tissue, this will increase the success rate in clinical research. The combined use of resected human brain tissue, alongside iPSC-derived neural models, could be considered a standard step in pre-clinical research and help to bridge the gap to clinical trials.