PKR-eIF2α Signaling Mediated Spatial Memory Impairment

PKR-eIF2ÃŽ ± Signaling Mediated Spatial Memory Impairment SUPPLEMENTARY MATERIAL Activated PKR-eIF2ÃŽ ± signaling mediated spatial memory impairment, tau phosphorylation, AÃŽ ² 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 à ¢Ã¢â‚¬Å¾Ã†â€™. 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 â€Å"novel† arm in Trial 2. The remaining two arms were designated as the ‘start’ arm and â€Å"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 (4.84.84.5cm) 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, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:200 acetylated-ÃŽ ±-Tubulin Santa Cruz, sc- 23950 WB 10 Mouse 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:5000 APP Cell signaling, #2452 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 AT8 Thermo, MN1020B WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 ATF-4 Abcam, ab50546 WB 10 Mouse 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 ATF-4 Abcam, ab50546 IF N/A Mouse 10% NGS, 12h, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:100 AÃŽ ²42 Abcam, ab10148 IHC N/A Rabbit 10% NGS, 12h, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:100 BACE-1 Abcam, ab2077 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 CCS Abcam, ab16962 WB 10 Mouse 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:100 CHOP Cell signaling, #2895 WB 10 Mouse 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 CREB Cell signaling, #9197 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 complexin-1/2 Santa Cruz, sc-33603 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 Cp Abcam, ab48614 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 Drebrin Cell signaling, #12243 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 eIF2ÃŽ ± Cell signaling, #5324 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 GSK-3ÃŽ ² Cell signaling, #9315 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 JNK Cell signaling, ##9252 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 Nitro-Tyrosine Cell signaling, #9691 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 NR2A Molecular Probes, A-6473 WB 8 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:500 NR2B Molecular Probes, A-6474 WB 8 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:500 PKR (N-Term) GenWay Biotech, GWB-A4757E WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:500 p-PKR (Thr 451) Invitrogen, 44668G WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:500 p-eIF2ÃŽ ± (Ser51) Cell signaling, #3398 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 p-GSK-3ÃŽ ² (Ser9) Cell signaling, #9336 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 p-CREB (Ser133) Cell signaling, #9198 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 p-JNK Cell signaling, #4671 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 p-PP2A Epitomics, 1155-1 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 PP2A C subunit Epitomics, 1512-1 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 PS396 Invitrogen, 44752G WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 PS404 Invitrogen, 44-758G WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 PSD-93 Cell signaling, #9445 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 PSD-95 Cell signaling, #2507 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 PSD-95 Cell signaling, #2507 IF N/A Rabbit 10% NGS, 12h, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:100 synapsin 1 Invitrogen, 51-5200 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 sAPPÃŽ ± Covance, SIG-39139 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 sAPPÃŽ ² Covance, SIG-39138 WB 10 Rabbit 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 Tau-1 Chemicon, MAB3420 WB 10 Mouse 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:1000 Tau-5 Abcam, ab80579 WB 10 Mouse 5% skim milk, 2h, O/N, 4à ¢Ã¢â‚¬Å¾Ã†â€™ 1:500 ÃŽ ±-tubulin Santa Cruz, sc-58667 WB 8-10 Mouse 5% skim milk, 2h, O/N, RT 1:1000 ÃŽ ²-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