Dissimilar accumulation of human β-amyloid and tau from enriched extracts in neuronal and endothelial cells

doi:10.21203/rs.3.rs-3343553/v1

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Dissimilar accumulation of human β-amyloid and tau from enriched extracts in neuronal and endothelial cells

https://doi.org/10.21203/rs.3.rs-3343553/v1

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BACKGROUND: While Aβ and Tau cellular distribution has been largely studied, the comparative internalization and subcellular accumulation of Tau and Aβ isolated from human brain extracts in endothelial and neuronal cells has not yet been unveiled. We have previously demonstrated that controlled enrichment of Aβ from human brain extracts constitutes a valuable tool to monitor cellular internalization in vitro and in vivo.

METHODS: Herein, we establish an alternative method to strongly enrich Aβ and Tau aggregates from human AD brains, which has allowed us to study and compare the cellular internalization, distribution and toxicity of both proteins within brain barrier endothelial (bEnd.3) and neuronal (Neuro2A) cells.

RESULTS: Our findings demonstrate the suitability of human enriched brain extracts to monitor the intracellular distribution of human Aβ and Tau, which, once internalized, show dissimilar sorting to different organelles within the cell and differential toxicity, exhibiting higher toxic effects on neuronal cells than on endothelial cells. While tau is strongly concentrated preferentially in mitochondria, Aβ is distributed predominantly within the endolysosomal system in endothelial cells, whereas the endoplasmic reticulum was its preferential location in neurons. Altogether, our findings display a picture of the cellular events that might occur in AD.

CONCLUSIONS: Monitoring the transit of internalized human Aβ and tau comparatively uncovers, for the first time, differences regarding their accumulation within the endolysosomal system, endoplasmic reticulum, mitochondria and autophagosomes, which might explain their differential cell-dependent toxicity. Overall, this study constitutes the first approach aimed at identifying the mechanism used by Aβ, tau and potential cofactors to exert cellular toxicity. Future studies must address the consequences of the accumulation of these proteins in different organelles and their relation to cell-to-cell transmission proposed for both proteins as part of the prion-like hypothesis of AD.

Alzheimer’s disease

amyloid-β

tau

prion-like hypothesis

internalization

subcellular compartment

propagation

seeding

The primary histopathologic hallmark of Alzheimer´s disease (AD) is the accumulation of neurofibrillary tangles (NFTs) and senile plaques, formed by the accumulation of tau oligomers and insoluble aggregates of amyloid-β (Aβ), respectively (1, 2). Several research groups have reported that injection of brain extracts from AD patients into Tg2567 mice (3, 4), APP23 mice (5), ALZ17 mice (6) or marmosets (7) results in the development of diffuse plaques far from the injection site and self-propagation and spreading of tau aggregates (reviewed in (8, 9)). Subsequent recent studies have demonstrated that intracerebral inoculation of synthetic tau fibrils and Aβ oligomers (10–13) in mice and primates, respectively, also mediates the transmission and progression of AD. These findings and the fact that both proteins in a pathological state share classic properties with prions support the prion-like hypothesis proposed for AD (reviewed in (6, 9, 14–18)). Notwithstanding this, it has been shown that native pathological tau aggregates derived from brain lysates exhibit 10-fold more seeding ability than equivalent aggregates generated in vitro (16) (reviewed in (18)), and it has been suggested that these enriched extracts must contain key protein partners or multiple distinct Aβ/tau prion strains capable of exhibiting self-propagation to initiate such a process (reviewed in (19, 20)).

This study aims to provide insights into the molecular mechanism of pathological tau and Aβ-mediated cellular spreading and propagation. Herein, we have obtained highly enriched Aβ and tau extracts from human AD brains that show robust internalization into neuronal and brain barrier microvascular endothelial cells. We have explored, for the first time and comparatively, similarities and differences between human tau and Aβ cellular internalization and distribution within different cellular organelles to identify potential hallmarks that could reveal information on cellular toxicity and major nucleation sites of both neurodegeneration components.

Human brain samples

Human tissue blocks were provided by National Network of Biobanks of Spain, namely, biobanks the Institut d’Investigacions Biomèdiques August Pi i Sunyer, Biobanco en Red de la Región de Murcia, Biobanco de Tejidos de la Fundación CIEN (registration numbers: B.0000575, B.0000859, B.0000741 respectively). Experimental procedures were approved by the Ethical Committee for Clinical Research of the Ciudad Real University Hospital (PID2019-108659RBI00). Ten human brain samples containing the olfactory cortex and amygdala were used (nine diagnosed AD cases and one case with no AD diagnosis; Suppl. Table 1).

Enrichment of tau and Aβ plaque-containing fractions from human brains

To obtain tau- and Aβ-enriched fractions, we used nine samples from human brains with AD (Stage VI; Suppl. Table 1). Approximately 0.1 g from each sample was homogenized in lysis buffer (20 mM Tris pH 7.4, 100 mM NaCl and 5 mM CaCl₂) supplemented with protease inhibitor. Homogenization was performed on a mechanical homogenizer on ice. The homogenates were incubated on ice with DNase I. Efficient enrichment of tau and Aβ plaques from the human brain was achieved using a protocol based on two centrifugation steps and one ultracentrifugation step using SDS (1.75%) followed by acetone precipitation. Four human samples from AD patients (AD; n = 4) were employed for the optimization of the protocol and the final optimized protocol was applied to one human brain sample with no evidence of AD pathology (non-AD; n = 1) and five samples from AD patients (AD; n = 5). An overview of the process is shown in Fig. 1A. The resulting precipitate was resuspended in PBS solution and stored at − 20°C.

Determination of the efficiency of tau and Aβ enrichment

Dot blot, Coomassie blue staining and western blot were performed to examine the content of tau and Aβ₁₋₄₂ in each fraction of the enrichment protocol. Dot blotting was carried out using 1 µl of each fraction generated in the enrichment procedure, which was set on nitrocellulose membranes. Nitrocellulose membranes (Bio-Rad) were dried overnight at room temperature and blocked with 5% skim milk powder and 5% BSA (VWR, Solon; Ohio, USA) in TTBS (200 mM Tris-HCl pH 8.8, 6 mM NaCl and 1% Tween 20) for 1 h at room temperature. Membranes were immunoblotted with tau (1:1000) and Aβ₁₋₄₂ (1:1000) antibodies. For Coomassie blue staining and western blotting, all protein extracts were quantified using a bicinchoninic acid assay (BCA; Sigma Aldrich). Ten micrograms of protein for each sample (all supernatants and pellet) were prepared using Laemmli buffer (1 M Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 10% β-mercaptoethanol and bromophenol blue) and diluted 4 times with deionized water and boiled for 5 min. Electrophoresis was performed on a 10% polyacrylamide gel with running buffer (25 mM Tris-HCl, 192 mM glycine, 1% SDS) at 160 V for 90 min. For Coomassie staining, gels were stained using Coomassie blue staining and destained with 15% acetic acid and 20% methanol, and for western blotting, proteins were transferred to a nitrocellulose membrane for 30 min at 0.25 mA in transfer buffer (25 mM Tris-HCl pH 8.8, 192 mM glycine and 20% methanol). Membranes were blocked in 5% skim milk in TTBS for 1 h at room temperature and then incubated with tau antibody (1:1000; 2.5% skim milk in TTBS) overnight at 4°C. After secondary antibody incubation, membranes were washed with TTBS and developed with Clarity Western ECL substrate according to the manufacturer’s instructions.

In vitro tau and Aβ _1–42 internalization into neuronal and endothelial cells

In vitro internalization of tau and Aβ₁₋₄₂ from enriched extracts was examined. Since endothelial dysfunction and neuronal loss constitute characteristic events of AD that are related to tau deposits and Aβ plaques, we employed the brain endothelial cell line bEnd.3 (ATCC CRL-2299) and the neuronal cell line Neuro2A (CCL-131).

Neuro2A and bEnd.3 cells were cultured in Minimum Essential Medium Eagle (MEM, Sigma Aldrich) and Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Aldrich), respectively, containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic at 37°C and 5% CO₂. They were seeded in 96-well Eppendorf Cell Imaging Plates and incubated with AD and non-AD extracts.

To explore tau and Aβ uptake, cells were incubated with 1 µl of the extracts for 24, 48 and 72 hours and then fixed in 4% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 for 10 min and blocked in 5% BSA/PBS with 0.05% Tween for 1 h, all at room temperature. Then, the cells were incubated with rabbit anti-Aβ (1:500; 2454; Cell Signaling Technology) and mouse anti-tau (1:500; 4019; Cell Signaling Technology) overnight at 4°C, washed and incubated with Alexa Fluor® 488-conjugated anti-mouse antibody and Alexa Fluor® 647-conjugated anti-rabbit antibody (1:500; Thermo Fisher Scientific) for 2 h at room temperature. Finally, the cells were costained with 1 µg/ml Hoechst and ActinRed™ 555. Confocal images were collected on an LSM 800 microscope (Zeiss; Jena, Germany) using a 63× Plan-Apo/1.4-NA oil objective at room temperature. For presentation purposes, stacks were z-projected to maximum intensity, and the brightness was adjusted in ImageJ. The same adjustments were made across all images.

Cell survival after incorporation of enriched extracts

To evaluate cell survival and estimate the internalization efficiency of the extracts at different times, Neuro2A and bEnd.3 cells were incubated with non-AD and AD extracts for 24, 48 and 72 hours (as indicated in the previous section) and then imaged using a 4x objective Cytation Cell Imaging Reader (BioTek). The number of attached cells and the area occupied by cells were analyzed for each condition. For presentation purposes, AD samples were normalized to non-AD samples (controls).

Subcellular localization of tau and Aβ_1–42 enrichment extracts into neuronal and endothelial cells

To examine the subcellular distribution of each protein (tau and Aβ) after its cellular internalization, endothelial (bEnd.3) and neuronal (Neuro2A) cells were seeded in 96-well Eppendorf plates and incubated with AD extracts. After 48 hours, the cells were immunostained as previously described. The following primary antibodies were used: mouse Alexa Fluor® 594 anti-Aβ (1:200; 803018; Biolegend), mouse anti-tau (1:200; 4019; Cell Signaling Technology), rabbit anti-Aβ (1:200; 2454; Cell Signaling Technology), rabbit anti- tau (1:200; 46687S; Cell Signaling Technology), mouse anti-TOMM20 (1:200; ab56783; Abcam), rabbit anti-LAMP1 (1:200; ab62562; Abcam), rabbit anti-PDI (1:200; 3501; Cell Signaling Technology), mouse anti-LC3 (1:200; ALX-803-081-C100 LC3B; Enzo), rabbit anti-Rab5 (1:200; 3547; Cell Signaling Technology), and rabbit anti-Rab9 (1:200; 5118; Cell Signaling Technology). Cells were washed and incubated with the appropriate secondary antibodies (1:500; Thermo Fisher Scientific) for 2 h at room temperature. Secondary antibodies were conjugated with Alexa Fluor® 488 or 594. Cells were costained with 1 µg/ml Hoechst, and confocal images were collected on an LSM 800 microscope (Zeiss; Jena, Germany) using a 63× Plan-Apo/1.4-NA oil objective at room temperature. The acquisition software was Zeiss Zen 3.5. All analyses were performed in ImageJ 1.53 (National Institutes of Health, Bethesda, MD, USA).

Quantification and statistical analysis

Statistical analyses were carried out with GraphPad Prism 8.0.2 (San Diego, CA, USA), and two-tailed, unpaired Student’s t-tests were performed on experimental data from at least three individual experiments.

Efficient enrichment of insoluble tau and Aβ₁₋₄₂ from human brains

To perform a comparative internalization study of tau and Aβ in vitro, we first optimized the protocol previously generated (21) to increase the amount of purified tau, since the first protocol was designed for the specific enrichment of Aβ. After the development of several attempts to modify detergents and centrifugation speed, the method described in Fig. 1A was the most successful approach for concomitant enrichment of both proteins, as observed by the similar concentrations of tau (Fig. 1B) and Aβ₁₋₄₂ (Fig. 1C) in the last fraction (P3). Similar results were observed for five AD human brain extracts (Fig. 1B and C), demonstrating the reproducibility of the protocol, whereas the non-AD sample did not show either tau or Aβ immunostaining (Fig. 1B and C). Tau purification was also evaluated in all the fractions generated throughout the protocol by western blotting and showed similar trends for two human AD samples (Fig. 1D, top lane). Additionally, Coomassie staining revealed a significant reduction in concomitant protein contaminants (Fig. 1D, bottom lane) compared to the previous protocol (21). Tau enrichment was confirmed by comparing the low (almost negligible) total protein concentration in P3 (Fig. 1D, bottom lane) with tau expression in AD samples (Fig. 1D, top lane). Indeed, the calculation of the ratio tau/total protein indicated 2,18- and 3,54-fold enrichment of tau in AD1 and AD2, respectively.

Tau- and Aβ-enriched fractions internalize into brain barrier microvascular endothelial and neuronal cells

To evaluate the in vitro incorporation of the extracts, bEnd.3 (Fig. 2left) and Neuro2A cells (Fig. 2right) were incubated with 1 µl of Non-AD and AD extracts for 24, 48 and 72 hours. Then, immunostaining of tau and Aβ₁₋₄₂ and labeling with Actin-Red™ were performed. As shown in Fig. 2(top lane), upon 24 h of incubation with the extracts, Aβ₁₋₄₂ and tau internalization was observed in both neuronal and endothelial cells. However, a low percentage of cells incorporated the extract, and a reduced abundance of tau and Aβ₁₋₄₂ internalized in those cells was observed. After 72 h of incubation, while tau and Aβ₁₋₄₂ uptake were substantially increased, preferential tau internalization was detected in both cell lines, and alterations in Neuro2A cell morphology were observed (Fig. 2, bottom lane). On the other hand, 48 h of incubation with the extracts allowed us to observe equivalent levels of Aβ₁₋₄₂ and tau within Neuro2A and bEnd.3 cells (Fig. 2, center lane), which appeared to be the most appropriate incubation time to perform an accurate quantification of their distribution while maintaining cell viability in both cell lines.

Irrespective of the incubation time with the extracts, confocal z-stacks led us to visualize the distribution of tau and Aβ₁₋₄₂ aggregates in vesicle-like structures (Fig. 2) scattered throughout the cytoplasm in both cell lines. Interestingly, although some colocalization was observed, different patterns of subcellular distribution of tau and Aβ₁₋₄₂ were found. As expected, no staining was detected in cells incubated with non-AD extracts. Extracts from four other AD patients were tested after 24, 48 and 72 h of incubation, and similar results were obtained in bEnd.3 cells (Suppl. Figure 1) and Neuro2A (Suppl. Figure 2) cells.

Brain barrier microvascular endothelial cells are more resistant than neuronal cells to tau and Aβ-enriched extracts

We also evaluated the potential negative effect of the extracts in bEnd.3 and Neuro2A cells after 24, 48 and 72 h of incubation (Fig. 3A). While bEnd.3 adhesion to the plate was unaltered regardless of the incubation time (Fig. 3A and B), a significant reduction of the cells adhered to the plate (Fig. 3B) and the area occupied by the cells were observed in Neuro2A cells after 48 and 72 h of incubation with the extracts when compared to cells incubated with Non-AD extracts (Fig. 3C). These results revealed a cell-dependent susceptibility to the extracts, which generated a higher cytotoxic effect in Neuro2A cells. Therefore, the following experiments were performed after 48 h of incubation with the extracts.

Different subcellular localization of tau and Aβ in endothelial and neuronal cells

We next explored and compared the specific subcellular distribution of tau and Aβ in neuronal and endothelial cells. To this end, cells were incubated with the extracts for 48 h and then immunostained against tau or Aβ and different cellular organelles, and the percentage of tau and Aβ localized in each organelle was calculated.

We first compared the distribution of tau and Aβ in mitochondria and ER through coimmunostaining with TOMM20 and PDI, respectively. Mitochondria appeared to be the preferential localization for tau since approximately 35% of tau incorporated into cells was found in this organelle in endothelial (Fig. 4A) and neuronal (Fig. 5A) cells. In both cell lines, tau localization into mitochondria was higher than Aβ, although these differences were statistically significant exclusively in endothelial cells.

On the other hand, while no differences were observed in ER distribution in endothelial cells (Fig. 4B), wherein tau and Aβ accumulated similarly, a significantly higher accumulation of Aβ in the ER was observed in neuronal cells. Indeed, Aβ localized preferentially in this organelle since approximately 40% of Aβ incorporated into neurons was found in the ER (Fig. 5B).

Next, we examined the distribution of tau and Aβ along the endosomal pathway. Therefore, early and late endosomes were stained by using the endosomal markers Rab5 (plasma membrane and early endosomes) and Rab9 (late endosomes). Tau and Aβ similarly accumulated in early endosomes in neuronal cells (Fig. 5C), a significantly higher colocalization of Aβ with early endosomes was observed in endothelial cells when compared to tau (Fig. 4C), thereby suggesting different uptake mechanisms for tau and Aβ in both cell lines. Remarkably, while tau and Aβ were similarly incorporated into late endosomes in endothelial cells (Fig. 4D), the tau proportion in this cellular compartment was detected to a significantly higher extent in neuronal cells (Fig. 5D). Evaluation of tau and Aβ lysosomal distribution using the lysosomal marker LAMP1 showed that both proteins were equally distributed in lysosomes from endothelial (Fig. 4E) and neuronal cells (Fig. 5E).

We additionally explored tau and Aβ distribution in autophagosomes by analyzing their colocalization with the autophagy marker LC3. Interestingly, while lower levels of Aβ were found in autophagosomes, tau predominantly accumulated in these vesicles in endothelial cells (Fig. 4F), but no differences were observed in neuronal cells (Fig. 5F).

Since the presence of tau on the nucleus has been previously described, it is also interesting to mention that tau nuclear staining was observed in both cell lines (Suppl. Figure 3), while no Aβ was detected.

Taken together, our results suggest that the internalization and distribution of brain extracts differ among brain endothelial and neuronal cells, which could reflect different cellular targets and nucleation sites for tau and Aβ, thereby leading to specific mechanisms to induce cellular toxicity. While mitochondria could be the preferential localization for tau, early endosomes constitute the organelles wherein the lesser amount of tau was found in endothelial and neuronal cells. In contrast, Aβ accumulated similarly in mitochondria and the endolysosomal system in endothelial cells, whereas the ER was its preferential location in neurons.

The accumulation of Aβ plaques and tau-containing neurofibrillary tangles constitutes a hallmark of Alzheimer´s disease (1). Recent studies have demonstrated that aggregated tau (22) and Aβ (23), but not monomers (24–28), can be efficiently taken up by cells, and it has been postulated that this constitutes the first step toward a characteristic seeding process associated with prion-like proteins that involves pathology and neurodegeneration (reviewed in (29)). Emerging evidence suggests that tau and Aβ have synergistic effects and may reciprocally modulate each other, although it has also been proposed that they act independently and in the absence of specific interactions (reviewed in (29)). Therefore, identifying the internalization mechanisms and cellular targets of tau and Aβ may be critical to understanding how AD pathology initially spreads and leads to pathological progression.

Although the involvement of neurons and cerebral vasculature in AD pathogenesis is widely accepted, cellular mechanisms leading to the accumulation of tau and Aβ in endothelial and neuronal cells have not been clearly elucidated. This knowledge would be essential for the understanding of how these proteins mediate neurodegeneration. The current study describes a fast and robust protocol to obtain highly enriched Aβ and tau extracts from human AD brains that efficiently preserve their fibrillar structures and their ability to internalize in vitro, which allowed us to compare and contrast human Aβ- and tau-specific targets in neuronal and cerebrovascular endothelial cells that may resemble neurodegenerative processes.

Differential vulnerability of cell lines to tau/Aβ extracts

This study sought to define the impact of Aβ- and tau-enriched extracts on cellular toxicity in neuronal and brain microvascular endothelial cells. While Aβ- and tau-enriched extracts did not cause significant toxicity after 72 hours of incubation in endothelial cells, decreased adhesion of Neuro2A to the substrate was observed after 48 hours.

The comparative vulnerability of neuronal and endothelial cells in AD pathology has not been fully explored. Nonetheless, in agreement with our observations, Kandimalla et al. (30) described some differences in Aβ₄₀ internalization between endothelial and neuronal cells, which may provide essential clues to understand how particular cells differentially regulate Aβ peptides. Moreover, the uptake of tau partially determines the propagation of neurofibrillary tangles, and previous reports have shown that pathogenic tau is internalized by neurons through different mechanisms, such as macropinocytosis, micropinocytosis via tau binding to cell surface heparan sulfate proteoglycans (HSPG), phospholipid disruption or tunneling nanotubes (reviewed in (31)), while tau entry into primary endothelial cells HBEC is HSPG-dependent (32). Such differences in Aβ and tau internalization may provide evidence for mechanisms of pathogenic Aβ- and tau-induced neuronal and endothelial dysfunction and help explain the vulnerability of neurons to their toxicity.

Distribution of tau and Aβ after cellular internalization

While several studies have reported that pathological tau and Aβ aggregates can colocalize in neurons in human postmortem tissue and 3xTg mice (33), our observations argue against a major physical interaction of tau and Aβ at the subcellular level in both neuronal and endothelial cells. In support of our data, a recent quantitative array tomography study of human postmortem tissue, as well as APP/PS1-rTg21221 mice, described that only rare synapses were positive for both tau and Aβ staining (34). When we explored the specific subcellular distribution of both proteins, summarized in Fig. 6, we observed that they trafficked along the endosomal–lysosomal system, which has already been shown for both tau (35–37) and Aβ (38, 39). Nonetheless, it is interesting to mention that contradictory findings in different cell models regarding the mechanisms for tau internalization have been shown. For instance, tau aggregates are taken up into human induced-pluripotent stem cell-derived neurons via dynamin-mediated and actin polymerization-dependent pathways (35), short tau fibrils and tau aggregates enter mouse hippocampal, cortical primary neurons (40), and human embryonic kidney cells (41) via micropinocytosis, and tau oligomers are internalized by primary cortical neurons by HSPG-mediated endocytosis (37). Notwithstanding this, a recent study has reported that oligomeric tau traffics along the endosomal–lysosomal system in the early phase of translocation to induce autophagy–lysosomal pathway alterations in the latter phase (37). In our study, we observed that upon 48 h of incubation with the extracts, tau was primarily concentrated in late endosomes in neuronal cells, whereas autophagosomes were the preferential localization in endothelial cells. Therefore, these results could reflect different tau internalization kinetics in Neuro2A and bEnd.3 cells.

The previously described ability of tau (42, 43) and Aβ (reviewed in (44)) to leave the normal transit to lysosomes and spread within the cell was also supported by our study, since both human proteins were also found in the ER, autophagosomes and mitochondria.

Additionally, although the main function of tau is associated with microtubule binding, we also identified tau immunoreactivity inside the nucleus in a reduced number of cells (Supplemental Fig. 3). This nuclear distribution has been shown in different reports (45–47) wherein it is proposed that tau could be involved in nucleolar transcription, nuclear transport and stress response (48, 49). Indeed, it has been reported that pathological tau impairs nuclear import and export by directly interacting with nucleoporins of the nuclear pore complex and affecting their structural and functional integrity, which could contribute to tau-induced neurotoxicity in AD (50). To our knowledge, this is the first study that identifies exogenous internalized human tau within the nucleus and demonstrates the suitability of this experimental approach as a tool to investigate intracellular events of both proteins.

The endolysosomal system and ER constitute major targets for Aβ

The internalization and processing of Aβ and tau is a complex process that involves different cellular mechanisms and receptors, and it is also influenced by the aggregation state of the proteins (51–54). After entering the cell by endocytosis, Aβ is dissociated in the early endosome, transported to late endosomes and, ultimately, lysosomes, where it is degraded or excreted from the cell (reviewed in (38). There is clear evidence that the endolysosomal system is damaged in AD (reviewed in (55)). Abnormally enlarged early endosomes in AD brains and Aβ colocalization with these endosomes have been observed in early stages of the disease (56, 57), thus suggesting that endocytic compartments constitute one likely site for the production of pathogenic Aβ (57). Since macropinocytosis seems to be one of the most relevant pathways in Aβ internalization, especially for its oligomeric form (54), this could explain the greater accumulation of Aβ in the early endosome observed in our study, thus reflecting a higher efficiency in Aβ internalization compared to tau.

Interestingly, our observations suggest that in cells with neuronal origin Aβ accumulated preferentially in the ER. Emerging evidence shows that Aβ peptide accumulation triggers neuronal ER stress in both murine models and human AD (58–60). Therefore, it is plausible to speculate that constitutive ER stress would explain the higher toxicity of the extracts, thus leading to neuronal cell death.

Tau preferentially localizes into mitochondria

Aβ and tau have been found in mitochondria in postmortem AD brains (61–63). Since mitochondria are not classically associated with endosomal pathways, tau and Aβ may gain access to mitochondria by alternative transport from the cytosol.

It has been hypothesized that oligomeric Aβ has the ability to permeabilize cellular membranes and lipid bilayers, thereby entering organelles such as mitochondria (44, 64). Recent findings, however, point to a specific uptake mechanism for Aβ by mitochondria rather than simply being adsorbed to the external surface of the mitochondria (62, 65). In this model, Aβ is transported into mitochondria mainly via translocases of the TOM import machinery (62, 66), as has been shown for synthetic Aβ ₁₋₄₀ and Aβ ₁₋₄₂ (62, 66).

On the other hand, in our study, tau predominantly accumulated within mitochondria. Previous reports link tau to impaired mitochondrial functions (67, 68) and suggest that mitochondrial tau may play a role in cellular dysfunction associated with AD. Indeed, it has been recently reported that tau localizes at submitochondrial compartments, the outer mitochondrial membrane and within the inner mitochondrial space (69). Toxic cytosolic entities of tau target mitochondria by associating with cardiolipin-rich membrane domains, triggering membrane poration and ultimately leading to compromised mitochondrial structural integrity (70). To be released into the cytosol and bind to mitochondria, internalized tau aggregates escape the endosomal pathway by rupturing the endosome membrane (42, 71).

It is also interesting to mention that, as an alternative possibility for Aβ and tau to gain access to the mitochondria, ER-to mitochondrial transfer might occur (69, 72).

Taken together, our findings demonstrate the suitability of human enriched brain extracts to monitor the intracellular distribution of human Aβ and tau. This study broadens the cell-dependent impact of tau and Aβ and shows that, in addition to mediating cellular internalization, human brain-extracted pathogenic tau and Aβ exhibit different toxicities and specific nucleation sites to propagate pathology in neuronal and brain microvascular endothelial cells, thereby contributing to cellular dysfunction in AD and displaying a picture of the cellular events that might occur in AD. Future studies must address the consequences of the accumulation of these proteins in different organelles and their relation to cell-to-cell transmission as part of the prion-like hypothesis of AD.

Controlled enrichment of Aβ and tau-enriched extracts from the human brain tissue constitutes a valuable tool to monitor cellular internalization in vitro.

Pathogenic tau and Aβ exhibit different toxicities and cellular-specific targets in neuronal and brain microvascular endothelial cells.

Alzheimer´s disease

Aβ

Amyloid-β

FBS

Fetal bovine serum

HSPG

Heparan sulfate proteoglycans

NFTs

Neurofibrillary tangles

Ethics approval and consent to participate

Experimental procedures were approved by the Ethical Committee for Clinical Research of the Ciudad Real University Hospital (PID2019-108659RBI00).

Consent for publication

All authors read and approved the final manuscript.

Availability of data and materials

The datasets generated during this study are included in this article and its supplementary information files.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was funded by the Spanish Ministry of Science and Innovation (grant no. PID2019-108659RB-I00 to AMM), the Ministry of Economy, Industry and Competitiveness (grant number SAF201679311-R, to MDP) and the Autonomous Government of Castilla-La Mancha/ERDF (grant no. 26 SBPLY/21/180501/000093 to AMM and IUB) and Consejería de Educación de la Junta de Comunidades de Castilla-La Mancha (grant number SBPLY/19/180501/000245, to YRR).

Authors´ contributions

Y. R-R and CM. P-P performed cell biology experiments, data curation and formal bioimaging analyses; L. S-R; A. F-C; D. S‒S; I. U-B; J. F-R performed cell biology experiments Y. R-R, CM P-P, M. D-P, A. M-M and JR. P designed and supervised elements of the study; A. M-M., M. D-P and Y. R-R obtained the funding; A. M-M and JR. supervised the entire project; Y. R-R, CM. P-P and JR. P wrote the manuscript with help from all authors.

Acknowledgements

The authors are indebted to donors, and IDIBAPS, BIOBANKMUR and BTCIEN integrated into the National Network of Biobanks of Spain (registration numbers: B.0000575, B.0000859, B.0000741 respectively). Figure 6 was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. We are grateful to UCAI (Unidad Científico Técnica de Apoyo a la Investigación, Ciudad Real) for technical assistance.

Authors information

Yoana Rabanal-Ruiz, Email: [email protected]

Cristina M. Pedrero-Prieto, Email: [email protected]

Lucía Rodríguez Sánchez, Email: [email protected]

Alicia Flores-Cuadrado, Email: [email protected]

Daniel Saiz-Sánchez, Email: [email protected]

Javier Frontiñán-Rubio, Email: [email protected]

Isabel Úbeda-Bañón, Email: [email protected]

Mario Durán-Prado, Email: [email protected]

Alino Martínez-Marcos, Email: [email protected]

Juan R. Peinado, Email: [email protected]

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No competing interests reported.

Allsupplemental010923.pdf
Supplemental table 1. Data of patients used in the present study. AD, Alzheimer´s disease; non-AD, non-Alzheimer´s disease; M, Male; F, Female; y, years. *Human brain tissue used for the optimization of the enrichment protocol. Supplemental figure 1. Internalization of βA and tau from enriched extracts in endothelial cells. Immunofluorescence against tau (green, yellow arrow) and Aβ_1–42 (deep red, red arrow) in ActinRed™ 555-labeled endothelial cells (bEnd.3) after 24, 48 and 72 h of incubation with the enriched extracts. Images show representative cells after incubation with extracts from four different brains affected with AD (AD2-AD5). Z-stacks are shown on the top and right sides of each image. Scale bars, 10 μm. Supplemental figure 2. Internalization of βA and tau from enriched extracts in neuronal cells. Immunofluorescence against tau (green, yellow arrow) and Aβ_1–42 (deep red, red arrow) in ActinRed™ 555-labeled neuronal cells (Neuro2A) after 24, 48 and 72 h of incubation with the enriched extracts. Images show representative cells after incubation with extracts from four different brains affected with AD (AD2-AD5). Z-stacks are shown on the top and right sides of each image. Scale bars, 10 μm. Supplemental figure 3. Nuclear and cytosolic location of tau and Aβ in endothelial and neuronal cells. bEnd.3 and Neuro2A cells were incubated with the enriched extracts for 48 h and then immunostained against tau and DAPI. Scale bars, 10 µm.

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Dissimilar accumulation of human β-amyloid and tau from enriched extracts in neuronal and endothelial cells

Status:

Version 1

Abstract

Figures

Background

Methods

Human brain samples

Enrichment of tau and Aβ plaque-containing fractions from human brains

Determination of the efficiency of tau and Aβ enrichment

Cell survival after incorporation of enriched extracts

Subcellular localization of tau and Aβ_1–42 enrichment extracts into neuronal and endothelial cells

Quantification and statistical analysis

Results

Efficient enrichment of insoluble tau and Aβ₁₋₄₂ from human brains

Tau- and Aβ-enriched fractions internalize into brain barrier microvascular endothelial and neuronal cells

Different subcellular localization of tau and Aβ in endothelial and neuronal cells

Discussion

Differential vulnerability of cell lines to tau/Aβ extracts

Distribution of tau and Aβ after cellular internalization

The endolysosomal system and ER constitute major targets for Aβ

Tau preferentially localizes into mitochondria

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Dissimilar accumulation of human β-amyloid and tau from enriched extracts in neuronal and endothelial cells

Status:

Version 1

Abstract

Figures

Background

Methods

Human brain samples

Enrichment of tau and Aβ plaque-containing fractions from human brains

Determination of the efficiency of tau and Aβ enrichment

Cell survival after incorporation of enriched extracts

Subcellular localization of tau and Aβ1–42 enrichment extracts into neuronal and endothelial cells

Quantification and statistical analysis

Results

Efficient enrichment of insoluble tau and Aβ1−42 from human brains

Tau- and Aβ-enriched fractions internalize into brain barrier microvascular endothelial and neuronal cells

Different subcellular localization of tau and Aβ in endothelial and neuronal cells

Discussion

Differential vulnerability of cell lines to tau/Aβ extracts

Distribution of tau and Aβ after cellular internalization

The endolysosomal system and ER constitute major targets for Aβ

Tau preferentially localizes into mitochondria

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Subcellular localization of tau and Aβ_1–42 enrichment extracts into neuronal and endothelial cells

Efficient enrichment of insoluble tau and Aβ₁₋₄₂ from human brains