Pathogenic PS1 phosphorylation at Ser367
- Massachusetts General Hospital, Harvard Medical School, United States
- University of Ulm, Germany
Figures
Figure 1 with 1 supplement
Ca2+ influx-triggered PS1 conformational change and increased phosphorylation.
(A) FLIM analysis of PS1 conformation. Pseudo-colored FLIM images of primary neurons treated with vehicle control or 50 mM KCl for 5 min. The neurons were stained with the antibodies to PS1 NT …
Figure 1—figure supplement 1
Glutamate treatment induces PS1 phosphorylation.
Primary neurons treated with 5 mM glutamate or with vehicle control for 2 min, were immunoprecipitated with mouse and rabbit anti-phosphoserine antibodies mixture, followed by the immunoblotting …
Figure 2 with 2 supplements
Phosphorylation mimicking mutations at S365-S366-S367 result in the PS1 conformational shift.
(A) FLIM analysis of the PS1 conformation in 7 W cells transfected with WT or phosphorylation-mimicking mutant G-PS1-R. The FRET efficiency between GFP and RFP in phospho-mutants is normalized to …
Figure 2—figure supplement 1
Calcium imaging and validation of the phosphorylation at the three domains-driven PS1 conformational change by the G-PS1-R FRET reporter probe.
This figure is related to the data displayed in Table 1. (A) Indo-1/Ca2+ imaging shows changes in intracellular Ca2+ levels of the 7 W cells transfected with FLAG-WT PS1, following 15 min treatment …
Figure 2—figure supplement 2
Schematic image of the three domains in PS1 involved in Ca2+-triggered pathogenic ‘closed’ conformation.
This figure is related to the data displayed in Table 1. Schematic image of PS1 molecule and the three domains involved in Ca2+-triggered PS1 pathogenic ‘closed’ conformation (blue squares). …
Figure 3 with 1 supplement
PKA activity is involved in the Ca2+-driven PS1 conformational change.
(A) Spectral FRET analysis of the PS1 conformation in 7 W cells transfected with WT G-PS1-R and pre-treated with 1 µM KT5720 (KT, PKA inhibitor, 16 hr), 20 µM SP600125 (SP, JNK inhibitor, 4 hr), 100 …
Figure 3—figure supplement 1
PKA is involved in the Ca2+-triggered PS1 pathogenic ‘closed’ conformation.
(A) FLIM analysis of the PS1 conformation in 7 W cells. The cells were pre-treated with 30 µM H-89 or 1 µM KT5720 for 16 hr, followed by the treatment with vehicle control (−) or 5 µM A23187 (+) for …
Figure 4 with 4 supplements
Ca2+ triggers the PS1/γ-secretase pathogenic conformation via PS1 phosphorylation in vivo.
(A) Schematic representation of the pAAV8-hSyn1-WT G-PS1-R construct. (B) Two-photon image of the WT G-PS1-R expression in the somatosensory cortex of WT mouse. Laser at 775 nm wavelength was used …
Figure 4—figure supplement 1
Establishment of the two-photon spectral FRET settings for monitoring PS1 conformation.
(A) 7 W cells expressing G-PS1-R were excited by different wavelength laser from 750 nm to 975 nm in 25 nm steps. The top panel shows representative images of the emission intensities in green and …
Figure 4—figure supplement 2
YC3.6-based Ca2+ imaging in vivo after KCl application.
300 mM KCl or vehicle (PBS) was topically applied under the cranial window to mice expressing YC3.6. The YFP/CFP ratio was measured in neurons of the somatosensory cortex in vivo (n = 18–22 cells, …
Figure 4—figure supplement 3
Spectral FRRT assay of PS1 conformation in vivo.
Additional time traces and pseudo-coloured images of the neurons in vivo showing changes in PS1 conformation. A scale bar indicates 10 µm.
Figure 4—figure supplement 4
Spectral FRET assay for monitoring endogenous PS1 conformation in mouse brain sections.
(A) Immunohistochemical detection of the phosphorylated CREB in mouse brain injected with 100 mM 8-Bromo-cAMP (right hemisphere) or vehicle (left hemisphere). The brain sections were immunostained …
Figure 5 with 2 supplements
Phosphorylated PS1 level is increased in AD brains.
Western blot analysis of PS1 phosphorylated at S310 in 20 control brains and 23 AD brains. The level of PS1 S310 phosphorylation (phospho-PS1/total PS1) in AD brains is normalized to that in the …
Figure 5—figure supplement 1
Validation of the PKA-mediated PS1 phosphorylation at S310.
(A) Confocal microscope images of the 7 W cells immunostained with anti-phosphorylated PS1 at S310 (green) or anti-FLAG antibodies (red). 7 W cells transfected with FLAG-WT PS1 or FLAG-S310A PS1 …
Figure 5—figure supplement 2
Correlation between the relative phosphorylation level of PS1 and age, or PMI.
Correlation analysis of phosphorylation of PS1 at S310 with age in control and AD brains (A), or with post mortem interval (PMI) (B). Spearman's nonparametric correlation analysis.
Figure 6 with 1 supplement
Mechanism of the Ca2+-triggered PS1 pathogenic conformational change.
The schematic image of the molecular events involved in the Ca2+-triggered pathogenic ‘closed’ conformational change and increase of the Aβ42/40 ratio. The elevated Ca2+ levels induce PKA …
Figure 6—figure supplement 1
Model of the PKA-mediated PS1 phosphorylation.
(A) Model. PKA first phosphorylates domain 2, followed by changing local conformation around domain 1. PKA then phosphorylates domain 1, which subsequently leads to local rearrangement around domain …
Tables
Table 1
FLIM analysis of the PS1 NT-CT proximity in phosphorylation-inhibited PS1 mutants.
Construct | *Relative FRET efficiency (%) | p value | ||
|---|---|---|---|---|
DMSO | A23187 | ‡vs WT (in DMSO) | §DMSO vs A23187 | |
PS1 Wild type (WT) | 100 ± 8.9 (n = 18) | 134.7 ± 7.5 (n = 25) | - | †p<0.05 |
PS1 S28A | 106.9 ± 7.0 (n = 19) | 143.4 ± 8.6 (n = 19) | n.s. | †p<0.05 |
PS1 T74A | 96.5 ± 13.9 (n = 14) | 98.5 ± 8.3 (n = 22) | n.s. | n.s. |
PS1 S310A | 88.8 ± 10.2 (n = 17) | 95.1 ± 8.6 (n = 24) | n.s. | n.s. |
PS1 S313A | 92.7 ± 8.4 (n = 15) | 108.0 ± 8.8 (n = 21) | n.s. | n.s. |
PS1 S310A/S313A | 84.2 ± 6.7 (n = 26) | 87.8 ± 9.0 (n = 17) | n.s. | n.s. |
PS1 S319A/T320A | 106.2 ± 8.1 (n = 20) | 131.8 ± 8.1 (n = 20) | n.s. | †p<0.05 |
PS1 S324A | 93.8 ± 6.2 (n = 19) | 129.9 ± 8.4 (n = 15) | n.s. | †p<0.05 |
PS1 S337A | 99.9 ± 8.8 (n = 13) | 132.4 ± 10.9 (n = 16) | n.s. | †p<0.05 |
PS1 S346A | 100.1 ± 8.9 (n = 18) | 135.0 ± 5.2 (n = 19) | n.s. | †p<0.05 |
PS1 S353A | 89.6 ± 7.4 (n = 18) | 129.8 ± 8.3 (n = 23) | n.s. | †p<0.05 |
PS1 T354A | 74.4 ± 5.6 (n = 14) | 113.4 ± 7.7 (n = 23) | n.s. | †p<0.05 |
PS1 S357A | 91.2 ± 7.9 (n = 18) | 119.8 ± 9.2 (n = 23) | n.s. | †p<0.05 |
PS1 S353A/S357A | 98.9 ± 9.1 (n = 14) | 125.9 ± 7.1 (n = 13) | n.s. | †p<0.05 |
PS1 S365A | 102.3 ± 6.4 (n = 19) | 87.3 ± 9.9 (n = 21) | n.s. | n.s. |
PS1 S366A | 102.8 ± 6.7 (n = 16) | 101.6 ± 12.6 (n = 16) | n.s. | n.s. |
PS1 S367A | 99.7 ± 6.1 (n = 10) | 99.1 ± 11.4 (n = 15) | n.s. | n.s. |
PS1 S365A/S367A | 104.1 ± 6.7 (n = 19) | 102.8 ± 8.4 (n = 19) | n.s. | n.s. |
PS1 S366A/S367A | 102.7 ± 11.2 (n = 10) | 108.2 ± 9.9 (n = 19) | n.s. | n.s. |
-
*The FRET efficiency in DMSO-treated cells expressing WT PS1 is set as 100%, and relative FRET efficiency in phosphorylation-inhibited mutants of PS1 is shown. Mean ± SEM, Student’s t-test, n: cell number, †: p<0.05, n.s.: not significant.
-
‡p-value is shown for the comparison between WT PS1 and phosphorylation-inhibited mutants of PS1 in DMSO-treated conditions, or §for the comparison between DMSO-treated and A23187 (5 µM for 15 min)-treated cells expressing the same PS1 construct.
Table 2
List of the human brain samples used in the study.
Case | Age | PMI | Sex | Braak | Cerad |
|---|---|---|---|---|---|
Control 1 | 60 | 15 | F | ||
Control 2 | 73 | 20 | F | ||
Control 3 | 88 | 20 | F | II | |
Control 4 | 91 | 8 | F | I | A |
Control 5 | 63 | 16 | M | ||
Control 6 | 85 | 8 | M | I | |
Control 7 | 87 | 48 | M | I | |
Control 8 | 91 | 19 | F | II | A |
Control 9 | 86 | 10 | M | ||
Control 10 | 94 | 17 | M | I | |
Control 11 | 54 | 6 | M | ||
Control 12 | 58 | 18 | F | ||
Control 13 | 88 | 16 | F | II | |
Control 14 | 60 | 14 | M | ||
Control 15 | 92 | unknown | M | II | |
Control 16 | 68 | 27 | M | I | |
Control 17 | 76 | 48 | F | I | possibly A |
Control 18 | 92 | 12 | M | II | |
Control 19 | 85 | unknown | M | II | |
Control 20 | 92 | 23 | M | II | A |
Control average (Mean ± SEM) | 79.15 ± 3.0 | 19.16 ± 2.6 | (F:M = 8:12) | ||
AD 1 | 58 | 12 | F | VI | C |
AD 2 | 73 | 18 | F | V | C |
AD 3 | 84 | 24 | F | VI | C |
AD 4 | 85 | 5 | F | VI | C |
AD 5 | 60 | 24 | M | VI | C |
AD 6 | 78 | 18 | F | VI | C |
AD 7 | 85 | 24 | M | VI | C |
AD 8 | 86 | 20 | M | VI | C |
AD 9 | 87 | 4 | F | VI | C |
AD 10 | 69 | 5 | M | VI | C |
AD 11 | 94 | 12 | F | VI | C |
AD 12 | 86 | 12 | M | VI | B |
AD 13 | 89 | 18 | F | VI | B |
AD 14 | 71 | 16 | F | VI | C |
AD 15 | 70 | 17 | M | VI | C |
AD 16 | 96 | 18 | M | VI | C |
AD 17 | 91 | 6 | M | VI | C |
AD 18 | 83 | 14 | F | VI | C |
AD 19 | 87 | 12 | F | IV | B |
AD 20 | 87 | 13 | M | VI | C |
AD 21 | 73 | 14 | M | VI | C |
AD 22 | 78 | 8 | M | VI | C |
AD 23 | 79 | 8 | M | VI | C |
AD average (Mean ± SEM) | 80.39 ± 2.1 | 14 ± 1.3 | (F:M = 11:12) |
