Supplementary information:
Supplemental figures legend:
Fig. S1: ITPKB protein immunodetection in control neurons and astrocytes. (A)
Frontal cortex sections from a control human subject and from a 6-week-old C57BL/6 mouse
were labeled with an ITPKB antibody. The ITPKB antibody labeled human (upper panels)
and mouse (lower panels) neurons and astrocytes Scale bars: 50 µm. In 2 days postnatal
C57BL/6 mouse cortex primary cultures, the ITPKB protein was detected in (B) GFAPpositive astrocytes and in (C) MAP2-positive neurons. Scale bars: 50 µm. (D)
Immunodetection with an ITPKB antibody confirmed the presence of the protein in mouse
brain homogenate as well as in astrocytes and neurons from mouse cortex primary cultures.
ACTIN served as loading control.
Fig. S2: ITPKB protein immunodetection in dystrophic neurites surrounded
amyloid deposits in a human Alzheimer patient and 5XFAD transgenic mice.
Immunofluorescence studies on frontal cortex sections from an Alzheimer patient (A) and on
a brain section from a 6 month-old 5xFAD mice (B) with antibodies directed against APP
protein (red, left panel) and ITPKB (green, central panel): APP positive dystrophic neurites
are ITPKB positive (right panel). Scale bar: 50 µm. * illustrates the localization of the
amyloid deposits.
Fig. S3: ITPKB protein expression and Ins(1,3,4,5)P4 levels in Neuro-2a
APP(K695N + M596L) transfected cells. Neuro-2a APP(K695N + M596L) cells were transfected
with GFP, GFP-Itpkb or GFP-ItpkbD897N expression plasmids. (A) ITPKB protein levels were
determined by immunodetection with an ITPKB antibody. TUBULIN served as control
loading (left panel). A ~6 fold increased ITPKB protein expression is observed after
transfection with the GFP-Itpkb plasmid (right panel). ***P < 0.001 by Student’s t test. (B)
1
Ins(1,3,4,5)P4 levels were determined 12 hours after cell transfection. Inositol phosphate
levels were normalized to total protein levels and transfection efficiency, and expressed as
fold increase compared with GFP-transfected cells. Results are representative of 3
independent experiments. *P < 0.05 by Student’s t test.
Fig. S4: Cellular localization of GFP-ITPKB and hAPP proteins in Neuro-2a
APP(K695N + M596L) transfected cells. (A) Neuro-2a APP(K695N + M596L) cells were transfected
with GFP, GFP-Itpkb and GFP-Itpkb∆1-482 expression plasmids and processed for GFP and
hAPP proteins localization by immunofluorescence using GFP (green) and APP (red)
antibodies. GFP-ITPKB protein colocalized with APP in Neuro-2a APP cells, on the contrary
to GFP-ITPKB∆1-482 protein. Scale bars: 50 µm. (B) APP colocalized with GM130 (Golgi
matrix protein of 130 kDa), a Golgi apparatus marker in Neuro-2a APP cells. Cells were
processed for GM130 and APP proteins localization by immunofluorescence using GM130
(green) and APP (red) antibodies. Scale bars: 50 µm.
Fig. S5: Increased astrogliosis in 2Tg 5XFAD mice: Left panel: immunodetection of
GFAP with a GFAP antibody in brain homogenates from 6 month-old 5XFAD and 2Tg
5XFAD mice. TUBULIN served as loading control. Right panel: quantification of the GFAP
signal relative to the TUBULIN signal. A. U.: arbitrary units. Means ± SEM are shown, 7-8
mice per group. *P < 0.05 by Student’s t test.
Fig. S6: Expression of a catalytically-inactive ITPKB protein has no impact on
Alzheimer disease pathology in 5XFAD mice. (A) Immunodetection of HA-ITPKB (left
panels) and APP (right panels) proteins on brain sections from 6 month-old 5XFAD and 2Tg
Camk2a-tTA/HA-Itpkb(D743A + K745I) 5XFAD (or 2Tg mut 5XFAD) mice. Antibodies directed
against the HA tag and APP were used. Scale bars: 500 µm (left panels) and 1 mm (right
panels) (B) Immunodetection of astrocytes with a GFAP antibody on brain sections from 6
month-old 5XFAD and 2Tg mut 5XFAD mice. Insets show higher-magnification images.
2
Scale bars: 500 µm (50 µm for the higher-magnification images). (C) Immunodetection of
amyloid plaques with antibodies directed against Aβ40 or Aβ42 peptides in brain of 6 monthold 5XFAD and 2Tg mut 5XFAD mice. Scale bars: 500 µm. (D) Aβ40 and Aβ42 peptides
quantification by ELISA in insoluble formic acid fraction of brain homogenates from 6
month-old 5XFAD and 2Tg mut 5XFAD mice. Results are means ± SEM of 3-4 mice and
represent the Aβ40 and Aβ42 peptides concentrations. No Aβ40 or Aβ42 peptide was found in
C57BL/6 control mice (data not shown). (E) Immunodetection of hAPP and β-CTF in brain
homogenates from 6 month-old 2Tg mut 5XFAD and 5XFAD mice. TUBULIN served as
loading control. No significant difference was detected between 5XFAD and 2Tg mut
5XFAD mice for APP/TUBULIN (5XFAD mice: 1.47 ± 0.08 A. U.; 2Tg mut 5XFAD mice:
1.32 ± 0.06 A. U.) and β-CTF/APP (5XFAD mice: 0.42 ± 0.07; 2Tg mut 5XFAD mice: 0.41
± 0.08) ratios (P = 0.40 and P = 1.00, respectively; Mann Whitney test). (F) Immunodetection
of TAU proteins in brain homogenates from 6 month-old 2Tg mut-5XFAD and 5XFAD mice.
Antibodies directed against TAU, phosphoTAU (pTAU) Ser396/404, pTAU Ser202 and
pTAU Ser262 proteins were used. Total TAU protein and TUBULIN served as loading
control. No significant difference was detected between 5XFAD and 2Tg mut 5XFAD mice
for pTAU Ser396/404/TAU (5XFAD mice: 0.62 ± 0.20 A. U.; 2Tg mut 5XFAD mice: 0.48 ±
0.16 A. U.), pTAU Ser202/TAU (5XFAD mice: 0.92 ± 0.21 A. U.; 2Tg mut 5XFAD mice:
0.85 ± 0.23 A. U.) and pTAU Ser262/TAU (5XFAD mice: 0.25 ± 0.05 A. U.; 2Tg mut
5XFAD mice: 0.27 ± 0.06 A. U.) ratios (P = 1.00 for all differences, Mann Whitney test).
3
Supplementary Table 1: information on control subjects and Alzheimer
patients.
Case
Age at
number
death
Sex
Post-mortem
Braak
CERAD
interval (h)
stageing
Plaque
(NFT)
score
(years)
(amyloid)
Control subjects
Alzheimer patients
1
61
M
8
0
0
2
89
F
35
I
A
3
65
M
4,5
0
0
4
72
M
24
0
B
5
78
M
23
0
0
6
69
M
6
0
0
7
58
M
5.5
I-II
0
8
92
F
NA
0
0
9
60
F
28
0
0
1
NA
M
5
VI
C
2
81
F
8
VI
C
3
73
F
22
VI
C
4
70
F
21
VI
C
5
75
M
10
VI
C
6
91
F
5,5
VI
B
7
84
M
7
VI
C
8
89
F
7
VI
C
4
9
72
M
44
VI
C
10
76
M
10
VI
C
11
60
M
37
VI
C
12
78
F
24
VI
C
13
79
F
25
VI
C
14
83
F
23
VI
C
Post-mortem interval
Age at death (years)
(h)
Mean ± SEM
Sex F/M
Mean ± SEM
Control subjects
16.75 ± 4.27
71.56 ± 4.15
3/6
Alzheimer patients
17.75± 3.29a
77.77 ± 2.30b
8/6
NA : Non available ; NFT : neurofibrillary tangles.
a and b : mean post-mortem interval (a) and age (b) were not significantly different between
control subjects and Alzheimer patients (P = 0.86 and P = 0.17, respectively; Student’s t test).
5