PKR-eIF2α Signaling Mediated Spatial Memory Impairment

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28th Nov 2017 Chemistry Reference this

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SUPPLEMENTARY MATERIAL

Activated PKR-eIF2α signaling mediated spatial memory impairment, tau phosphorylation, Aβ pathology, oxidative stress, selectively synaptic protein loss in mice caused by low levels of Cu

Mouse behavior analysis. Morris water maze test: MWM was performed as previously described (PMID:23402899) with minor modifications and the test was performed double blinded according to the standard operation protocol. The MWM consisted of a circular pool (120 cm diameter, wall depth 40 cm) in which all the mice were trained to escape from water by swimming to a hidden platform (2 cm beneath water surface) whose location only be identified by using the visual cures on the inner wall of the pool (Supplementary Fig. 3A). The water and the room temperature were kept at 23±1 ℃.

The pool was divided into four quadrants by a computerized tracking software (Huaibei Zhenghua Biologic Apparatus Facilities Limited Company, Huaibei, Anhui, China). The platform was located half-way between the center and the wall in one quadrant and maintained at the same position during all the experiment.

The navigation test consisted of 4 training trials per day and 5 consecutive training days. As can be seen in Table 1, mice were released with their heads facing the inner wall of the pool from the four quadrantal locations (N, E, SE, and NW) according the sequence as previous report (Supplementary Fig. 3B) (PMID:17406317), and not allowed to swim and search for the platform more than 60 s, after which they were guided to the platform and allowed to remain on it for 15 s. Each mouse was then returned to its cage for 30 min before its next trial. The latency to reach the hidden platform was recorded.

One day or six days after the end of navigation test, mice received a probe test, in which the platform was removed. Mice were released from the NE location and allowed to a 120 s swim to find the previous location of the platform. The swimming path, the time spent in each quadrant, the distance traveled each quadrant, the probe time, the platform crossing number, the total distance traveled, and the average swimming speed was recorded by the computerized tracking software.

Y-maze: In order to study the PKR role in exploratory behavior and spatial memory, we performed the Y-maze in the PKR+/+Tg+/- and PKR+/-Tg+/- mice as described previously (PMID: 8986335, 1393562). Response to novelty was tested in a Y-maze, adopting a two-trial procedure in this test. The apparatus was equipped with black materials with three identical arms each 50 cm long, 16 cm wide, and 32 cm high. Visual cues made from colored paper with different symbols and the floor of the maze was covered with soiled animal bedding (Beta wood chips).

All the mice was performed with starvation treatment for 24h before Y-maze. In trial 1, one arm was blocked with black Plexiglas and referred to as the “novel” arm in Trial 2. The remaining two arms were designated as the ‘start’ arm and “other” arm respectively. Three arms were randomized between mice (but not for the same mouse) to reduce arm bias effects. At the start of testing, a mouse was placed in the start arm and was allowed to explore the start and other arms for 10 min (acquisition trial). At the end of Trial 1, the mouse was returned to its home cage and the bedding inside the maze was mixed to reduce the possibility of using odors as a cue. After an intertrial interval (ITI) of 1 h, the mouse was placed in the same start arm as in Trial 1. The previously blocked arm was opened in Trial 2 and the mouse was allowed to investigate all three arms for 5 min (recall trial). The dependent variables measured in Trial 2 were: (1) the amount of time spent in each arm for each minute; (2) the number of entries made into each arm for each minute (Entry). Those indexs reflect inquisitive behavior (i.e. response to novelty) and spatial recognition memory of the previously unvisited arm.

Step-down test: This test was used to measure inhibitory avoidance and short-term memory, according to the previously described method (PMID: 24678498). The apparatus comprised a plastic chamber (12x12x18cm) with an elevated rubber platform (4.8×4.8×4.5cm) placed on the left side wall. The floor was made of caliber stainless steel bars (0.1cm in length) placed in parallel, 0.5cm apart. On the first training day, mice were exposed to a 5-min learning course, if the animals stepped down from the platform, they were exposed to an electric foot shock (36V, AC). After 24h, latency was reassessed and recorded as the learning grade (latency), which was taken as a measure of memory retention. Each acquisition trial was performed 5min in the PKR+/+Tg+/- and PKR+/-Tg+/- mice.

Supplementary Table 1. Primary antibodies used for protein immunodetection in western blot analysis (WB), immunohistochemistry (IHC) and immunofluorescence (IF).

Antigen

Supplier

Application

PAGE

(%)

Species

origin

Incubation

conditions

Ab

dilution

8-OhdG

US Biological,

H9076-02

IF

N/A

Goat

10% NGS, 12h, 4℃

1:200

acetylated-α-Tubulin

Santa Cruz, sc- 23950

WB

10

Mouse

5% skim milk, 2h, O/N, 4℃

1:5000

APP

Cell signaling, #2452

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

AT8

Thermo, MN1020B

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

ATF-4

Abcam, ab50546

WB

10

Mouse

5% skim milk, 2h, O/N, 4℃

1:1000

ATF-4

Abcam, ab50546

IF

N/A

Mouse

10% NGS, 12h, 4℃

1:100

42

Abcam, ab10148

IHC

N/A

Rabbit

10% NGS, 12h, 4℃

1:100

BACE-1

Abcam, ab2077

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

CCS

Abcam, ab16962

WB

10

Mouse

5% skim milk, 2h, O/N, 4℃

1:100

CHOP

Cell signaling, #2895

WB

10

Mouse

5% skim milk, 2h, O/N, 4℃

1:1000

CREB

Cell signaling, #9197

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

complexin-1/2

Santa Cruz, sc-33603

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

Cp

Abcam, ab48614

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

Drebrin

Cell signaling, #12243

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

eIF2α

Cell signaling, #5324

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

GSK-3β

Cell signaling, #9315

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

JNK

Cell signaling, ##9252

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

Nitro-Tyrosine

Cell signaling, #9691

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

NR2A

Molecular Probes, A-6473

WB

8

Rabbit

5% skim milk, 2h, O/N, 4℃

1:500

NR2B

Molecular Probes, A-6474

WB

8

Rabbit

5% skim milk, 2h, O/N, 4℃

1:500

PKR (N-Term)

GenWay Biotech, GWB-A4757E

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:500

p-PKR (Thr 451)

Invitrogen, 44668G

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:500

p-eIF2α (Ser51)

Cell signaling, #3398

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

p-GSK-3β (Ser9)

Cell signaling, #9336

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

p-CREB (Ser133)

Cell signaling, #9198

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

p-JNK

Cell signaling, #4671

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

p-PP2A

Epitomics, 1155-1

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

PP2A C subunit

Epitomics, 1512-1

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

PS396

Invitrogen, 44752G

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

PS404

Invitrogen, 44-758G

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

PSD-93

Cell signaling, #9445

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

PSD-95

Cell signaling, #2507

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

PSD-95

Cell signaling, #2507

IF

N/A

Rabbit

10% NGS, 12h, 4℃

1:100

synapsin 1

Invitrogen, 51-5200

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

sAPPα

Covance, SIG-39139

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

sAPPβ

Covance, SIG-39138

WB

10

Rabbit

5% skim milk, 2h, O/N, 4℃

1:1000

Tau-1

Chemicon, MAB3420

WB

10

Mouse

5% skim milk, 2h, O/N, 4℃

1:1000

Tau-5

Abcam, ab80579

WB

10

Mouse

5% skim milk, 2h, O/N, 4℃

1:500

α-tubulin

Santa Cruz, sc-58667

WB

8-10

Mouse

5% skim milk, 2h, O/N, RT

1:1000

β-actin

Santa Cruz, sc-47778

WB

10

Mouse

5% skim milk, 2h, O/N, RT

1:1000

N/A, not applicable; NGS, normal-goat serum; O/N, over-night; RT, room temperature.

SUPPLEMENTARY FIGURES

Supplementary Figure 1. Content of Cu in Serum and brain. (A-D) Total iron, zinc, calcium, magnesium content in the serum respectively; (E-H) Total iron, zinc, calcium, magnesium content in the hippocampus respectively; (I-L) Total iron, zinc, calcium, magnesium content in the cortex respectively; *P<0.05, **P<0.001, ***P<0.01 vs. the control (n=5 for each group).

Supplementary Figure 2. The level of nitrotyrosine in hippocampus and 8-OhdG in CA1 region in the hippocampus. (A) The brain sections containing hippocampal CA1 region were stained with anti-8-OHdG antibody to detect the level of oxidative stress. Representative images were selected from the copper-treated mice and the control mice. Scale bar=100 µm.

Supplementary Figure 3. Low level of copper exposure impaired spatial memory abilities of mice. (A) High contrast visual cues was used in the Morris water maze. (B) Morris water maze spatial (hidden platform) start positions during learning and memory detection phage. (C) The representative swimming path was recorded, (D) average speed and (E) total distance were measured duiring short-term memory detection after Cu exposure (n=15 for each group). (F) The representative swimming path was recorded, (G) average speed and (H) total distance were measured during long-term memory detection after Cu exposure (n=15 for each group).

Supplementary Figure 4. PKR+/+3Tg+/- mice show shory-term and long-term memory impairment compared with PKR+/-3Tg+/- mice. In the Morris water maze, (A) the representative swimming path was recorded, (B) crossing number, (C) total distance and (D) percentage of time spent were measured in correct quadrant during shory-term memory detection in the PKR+/+3Tg+/- mice; (E) The representative swimming path, (F) average speed and (G) total distance were measured during long-term memory detection in the PKR+/+3Tg+/- mice. (H) In the Y-maze, the representative path was recored (n=9 for each group).

Supplementary Figure 5. The effects of chronic copper exposure on apoptosis in CA1 region of hippocampus. (A) TUNEL staining was performed to reveal the change of apoptosis of neuronal cells in hippocampal CA1 region. Scale bar=100 µm.

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