What this is
- This research investigates the effects of () staining in the 5XFAD mouse model of Alzheimer's disease.
- 5XFAD mice are known for their amyloid-β (Aβ) plaques, but this study reveals that they also produce -positive aggregates.
- The findings suggest that complicates the accurate quantification of amyloid plaques in these mice, impacting drug evaluation.
Essence
- () staining in 5XFAD mice reveals aggregates alongside amyloid-β plaques, complicating plaque quantification and drug assessment.
Key takeaways
- 5XFAD mice exhibit -positive aggregates, indicating that does not exclusively bind to amyloid-β plaques. This finding challenges the reliability of for quantifying Aβ in this model.
- Administering an Aβ-dissociating drug, EPPS, resulted in a smaller reduction of plaques as measured by compared to anti-Aβ antibody staining. This discrepancy suggests that may misrepresent the efficacy of Aβ-targeting treatments.
Caveats
- The study focuses on a specific age range of 5XFAD mice, which may limit the generalizability of the findings to other ages. Further research is needed to confirm age-related changes in tau accumulation.
- The presence of -positive aggregates that are not Aβ may lead to misinterpretations in other studies using for plaque detection in different models.
Definitions
- Thioflavin S (ThS): A fluorescent dye that binds to β-sheet structures, used to visualize amyloid plaques and other protein aggregates.
- Phospho-tau (p-tau): A form of tau protein that is hyperphosphorylated, commonly associated with neurodegenerative diseases like Alzheimer's.
Simplified
Introduction
Alzheimer’s disease (AD) is defined by accumulation of amyloid-β (Aβ) plaques and neurofibrillary tau tangles in the brain, leading to neurodegeneration and cognitive dysfunction1. The need for deeper molecular understanding underlying AD pathogenesis and the discovery of effective therapeutics has led to the development of transgenic mouse models mimicking Aβ and tau pathologies. These neuropathological alterations in the brain are directly visualized by dyes, providing valuable information as an indicator of disease progression. As plaques and tangles share a common β-sheet-rich structure, both on the brain tissue are stained by β-sheet-binding dyes such as thioflavin S (ThS)2,3. Besides these AD-related proteins, ThS also binds to other β-sheet-containing deposits such as Lewy bodies of α-synuclein4,5.
5XFAD is a well-validated and widely used transgenic mouse model of AD that possesses a total of five mutations in amyloid precursor protein (APP) and presenilin (PSEN1) genes, which are involved in Aβ production. Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations in APP genes, and M146L and L286V mutations in PSEN1 genes contribute to the rapid development of Aβ deposits and progressive cognitive decline in 5XFAD mice. As the 5XFAD mouse model was originally generated to only develop amyloid pathology, single-staining with ThS is widely used for Aβ plaque detection6–11.
| Age (months) | Gender | Number of mice | Markers | Brain regions |
|---|---|---|---|---|
| 7–8 | Male | 3 | ThS/6E10 | Whole Cortex Hippocampus |
| ThS/AT8 | ||||
| ThS/α-Synuclein | ||||
| 5–6 | Female | 3 | ThS/6E10 | Whole Cortex Hippocampus |
| ThS/AT8 | ||||
| ThS/α-Synuclein | ||||
| 5–6 | Female | 3 | ThS/4G8 | Whole Cortex Hippocampus |
| 7.5 (including 6 weeks of drug administration) | Male | 5 (Vehicle) | ThS/6E10 | Whole Cortex Hippocampus |
| 4 (EPPS) | ThS/AT8 |
Results
ThS-positive and AT8-positive phospho-tau aggregates found in 5XFAD mouse brain
To confirm that yellow fluorescent signals in merged images were not due to the spectral overlap of ThS, we performed single-labeling with AT8 (Fig. 2c) or α-synuclein antibody (Fig. 3c). While AT8 single-staining showed a significant amount of tau aggregates in male and female 5XFAD mouse brains compared to age- and gender-matched wild type mouse brains, single-staining with α-synuclein antibody did not demonstrate visible fluorescence of α-synuclein. Therefore, we concluded that both male and female 5XFAD mice express aggregated phospho-tau (p-tau) that were detected by ThS.
ThS stains Aβ plaques in 5XFAD mice brains and other protein aggregates as well. () The brains were double-stained by ThS and 6E10 anti-Aβ antibody. 7–8-month-old male (M2–M4) and 5–6-month-old female (F2–F4) 5XFAD mice were used. The brain of age-matched B6SJL wild type male (M1) or female mouse (F1) was also stained as a control. Hoechst 33342 was applied for nuclear counterstaining (scale bars = 400 µm, 150 µm). () The representative image of ThS- or 6E10-stained brains, which is indicated as yellow boxed images in () (scale bars = 150 µm). In merged images, yellow dots in white circles represent the double-staining of ThS and 6E10, whereas green dots marked by white arrows indicated 6E10-negative, ThS-positive aggregates. () ThS and 4G8 double-stained female 5XFAD brains at age of 5–6-month-old (F2–F4). The brain of age-matched B6SJL wild type female mouse (F1) was also stained as a control. Hoechst 33342 was used for nuclear counterstaining (scale bars = 400 µm). () Merged images of ThS and 4G8 double-staining (scale bars = 150 µm). () The representative image of ThS- or 4G8-stained brains, which is indicated as yellow boxed images in (). In merged images of cortical and hippocampal regions, yellow dots in white circles indicted the double-staining of ThS and 4G8, while green dots marked by white arrows represented 4G8-negative, ThS-positive aggregates (scale bars = 150 µm). wt, wild type; Tg, transgenic; ThS, thioflavin S; CTX, cortex; HP, hippocampus. a b a c d e d
P-tau aggregates in 5XFAD mice brains were stained by AT8 antibody and ThS. () The brains were double-stained by Thioflavin S and AT8 anti-phosphorylated tau antibody. We used male (M2-M4) and female (F2-F4) 5XFAD mice at 7–8 months and 5–6 months of age, respectively. The brain of age- and gender-matched B6SJL wild type male (M1 and F1) was also stained as a control. Hoechst 33342 was used for visualization of nuclei (Scale bars = 400 µm, 150 µm). () The representative image of ThS- or AT8-stained brains, which is indicated as yellow boxed images in () (scale bars = 150 µm). Yellow dots of overlapped fluorescence in white circles represent the aggregated p-tau double-stained by ThS and AT8, whereas green dots marked by white arrows indicate ThS-positive aggregates that were not p-tau aggregates. () Single-labeling with AT8 antibody showed 5XFAD mouse brains exhibit significant amount of aggregated p-tau compared to wild type mouse brains (Scale bars = 400 µm, 150 µm). wt, wild type; Tg, transgenic; ThS, thioflavin S; CTX, cortex; HP, hippocampus. a b a c
α-Synuclein aggregates were not detected in 5XFAD mice brains. () The brains were double-stained by Thioflavin S and AT8 anti-α-synuclein antibody. We used male (M2–M4) and female (F2–F4) 5XFAD mice at 7–8 months and 5–6 months of age, respectively. Age- and gender-matched B6SJL wild type male (M1 and F1) was used for a control (scale bars = 400 µm, 150 µm). () The representative image of ThS- or α-synuclein-stained brains, which is indicated as yellow boxed images in (). Several green dots of ThS fluorescence were observed but no overlapped signal was detected in double-staining by ThS and α-synuclein (scale bars = 150 µm). () Single-labeling with α-synuclein antibody displayed no remarkable α-synuclein aggregates in 5XFAD mouse brains compared to wild type mouse brains (scale bars = 400 µm, 150 µm). wt, wild type; Tg, transgenic; ThS, thioflavin S; α-syn, α-synuclein; CTX, cortex; HP, hippocampus. a b a c
Complicated interpretation of Aβ-targeting drug upon ThS brain staining
The different number of plaques were detected in 6E10- and ThS-stained 5XFAD mouse brains after the treatment of Aβ-disaggregating drug, EPPS. () The representative 6E10- and ThS-stained fluorescence half-brain images of the vehicle-treated wild type mouse, and vehicle- and EPPS-treated 5XFAD mice (scale bars = 2 mm). () Quantification of Aβ plaques in whole, hippocampal, and cortical regions of 6E10- or ThS-stained half-brains. The amount of remaining aggregates of EPPS group shown as a percentage in the bar graphs were normalized to the plaque numbers of vehicle group (100%). Significance was tested by unpaired two-tailed student’s-test (*P < 0.05, other comparisons were not significant). () Individual and merged brain images of the vehicle- and EPPS administered- groups stained with ThS and 6E10 or AT8 antibody (scale bars = 500 µm). wt, wild type; Tg, transgenic; veh, vehicle; ThS, thioflavin S; CTX, cortex; HP, hippocampus. a b c t
Discussion
Here, we report that single-staining with ThS for Aβ plaque measurements in the 5XFAD mouse brains can lead to incorrect interpretation because 5XFAD exhibits ThS-positive p-tau aggregates in their brains. We observed ThS-labeled insoluble protein aggregates that were not positive for anti-Aβ antibodies, 6E10 or 4G8, in the cortical and hippocampal regions of both male and female 5XFAD mice. Considering the possibility that the ThS + /anti-Aβ aggregates– could be composed of other aggregation-prone proteins, the brain sections of 5XFAD mice were stained with antibodies against phosphorylated tau and α-synuclein. Through double-staining with ThS and AT8, we deduced that the ThS + /anti-Aβ aggregates—were p-tau aggregates, ThS + /anti-hyperphosphorylated tau + . To test whether Aβ quantification with ThS in 5XFAD mouse brains interferes the evaluation of the efficacy of anti-Aβ drug, 5XFAD mice were orally administered an Aβ-dissociating agent, EPPS, and their brains were double-stained by ThS with 6E10 or AT8. We found that ThS-stained brains displayed a smaller extent of EPPS-induced plaque reduction than in the same brains stained with 6E10, demonstrating the need for extra attention in the use of ThS as an Aβ-detecting probe in 5XFAD mice.
As the 5XFAD mouse model was originally designed for the investigation of APP- and Aβ-related pathology, this model is not expected to develop significant tauopathy in the brain. Subsequently, tau aggregates were reported to be absent in this model when using a phosphorylated tau antibody recognizing phosphorylation at Ser199/2028,16. However, recent studies revealed that 5XFAD mice display a gradual increase of total tau level in cerebrospinal fluid17 as well as significant aggregated p-tau stained by AT8 recognizing phosphorylation at Ser202 and Thr20518,19. Furthermore, accumulating reports indicate that a pretangle state, in which tau is phosphorylated and accumulated20, was observed in Aβ-overexpressing transgenic mouse models including 5XFAD mice21–25. Consistent with these results, we observed ThS-positive tau phenotypes in the brains of both male and female aged 5XFAD mice by double-staining with ThS and AT8. As the tau hyperphosphorylation plays a critical role as a trigger for the development of neurofibrillary tangles, further investigation is needed to demonstrate the exact mechanisms on the alterations of endogenous tau in pretangle state of Aβ-overexpressing mouse models. The further analyses are also required to investigate the phosphorylation of the other pathologic tau epitopes in the brain of 5XFAD mice, except for Ser202 and Thr205 that were detected by AT8 antibody. Additionally, an in-depth study on whether these p-tau aggregates were neurofibrillary tau tangles or phospho-tau within dystrophic neurites surrounding Aβ plaques is also warranted.
To conclude, due to the presence of p-tau aggregates in the brain of 5XFAD mice, the use of ThS in Aβ quantification in this model requires caution since binding property of ThS against both Aβ and tau aggregates can lead inaccurate results. As these findings were only observed in the specific age of mice, further studies are needed to confirm age-dependent accumulation of tau in the brain of both male and female 5XFAD mice. Furthermore, the presence of phosphorylated tau at other epitopes also needs to be investigated.
Materials and methods
Animals
For histochemical analysis, male 5XFAD mice (strain name; B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V) 6799Vas/Mmjax) from 7 to 8 months of age (n = 3) and female from 5 to 6 months of age (n = 6) were obtained from Jackson Laboratory (USA). Age- and gender-matched wild type mice (n = 1, each) were used as controls. All mice were bred and provided constant temperature, humidity, and 12:12 h light–dark cycle in the animal facility of Yonsei University. Mice were given ad libitum access to water and food. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications) as well as the ARRIVE guidelines and approved by the Animal Institutional Animal Care and Use Committee of Yonsei University (Korea, IACUC-202003–1038-01).
Drug administration
A dose of 100 mg/kg of EPPS (Sigma-Aldrich, USA), a previously reported Aβ-dissociating drug candidate, was freely administered to 6-month-old male 5XFAD mice (n = 4) via drinking water for six weeks. After the administration, all mice were deeply anesthetized by intraperitoneal injection of 4% avertin (2,2,2-Tribromoethanol, Sigma-Aldrich, USA) and sacrificed by cervical dislocation to obtain brain tissue samples and compare the number of labeled plaques by 6E10 antibody and ThS. Age- and gender-matched 5XFAD transgenic mice (n = 5) and B6/SJL wild type mice (n = 4) were also sacrificed as controls.
Histochemistry
All mice were deeply anesthetized by intraperitoneal injection of 4% avertin for the brain extraction. After perfusion with 0.9% NaCl, mice were sacrificed by cervical dislocation and extracted brains were fixed in 4% paraformaldehyde (Biosesang, Korea). After 24 h of fixation, brains were immersed in 30% sucrose for 2 days. For immunofluorescence staining, 35 µm-thick frozen sections were incubated with 6E10 (Biolegend, USA, Catalog# SIG-39320, 1:200), 4G8 (Biolegend, USA, Catalog# SIG-39220, 1:200), AT8 (Invitrogen, USA, Catalog# MN1020, 1:200), or α-synuclein (BD Transduction Laboratories, USA, Catalog# 610786, 1:250) antibody diluted in 5% horse serum (Gibco, USA), then with Alexa555-conjugated secondary antibody (1:200 in PBS). Each stained section was incubated in 500 µM of Thioflavin S (ThS, Sigma-Aldrich, USA) dissolved in 50% ethanol for 7 min for ThS double-staining. Hoechst 33342 (10 µg/mL, Sigma-Aldrich, USA) was used to observe nuclear morphology. All images were taken using a fluorescence microscope (Leica DM2500, Germany). The number and area of plaques detected by ThS and 6E10 were quantified using Image J software.
Statistical analysis
Graphs were obtained by GraphPad Prism 7 and statistical analyses were performed using unpaired two-tailed Student’s t-test. All data were represented as mean ± SEM.
Supplementary information
Supplementary Information.