PKR-eIF2α Signaling Mediated Spatial Memory Impairment

PKR-eIF2 Signaling Mediated Spatial Memory ImpairmentSUPPLEMENTARY MATERIALActivated PKR-eIF2 signal mediated spatial memory impairment, tau phosphorylation, A pathology, oxidative stress, selectively synaptic protein loss in mice caused by low levels of Cu sneak behaviour analysis. Morris piddle maze test MWM was performed as antecedently described (PMID23402899) with minor modifications and the test was performed double blinded according to the standard operation protocol. The MWM consisted of a circular puss (120 cm diameter, wall depth 40 cm) in which all the mice were trained to escape from water by swimming to a hidden broadcast (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 agency temperature were kept at 231 .The pool was divided into quadruplet quadrants by a computerized tracking software (Huaibei Zhenghua Biologic Apparatus Facilities Limited Company, H uaibei, 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) (PMID17406317), 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 hang in on it for 15 s. separately mouse was then returned to its cage for 30 min before its next trial. The latency to reach the hidden platform was recorded. iodin 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 locati on of the platform. The swimming path, the fourth dimension 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 make 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 rivulet 2. The remaining two arms were designated as the appear 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 tribulation 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 make into each arm for each minute (Entry). Those indexs reflect inquisitive behavior (i.e. response to novelty) and spatial recognition m emory 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 undefendable 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).AntigenSupplierAppli cationPAGE(%)Species kickoffIncubationconditionsAbdilution8-OhdGUS Biological,H9076-02IFN/AGoat10% NGS, 12h, 41200acetylated--TubulinSanta Cruz, sc- 23950WB10Mouse5% drub milk, 2h, O/N, 41 calciferol0APPCell signaling, 2452WB10 das5% decamp milk, 2h, O/N, 41 meterAT8Thermo, MN1020BWB10 dassie5% skim milk, 2h, O/N, 41 gigabyteATF-4Abcam, ab50546WB10Mouse5% skim milk, 2h, O/N, 41 grand pianoATF-4Abcam, ab50546IFN/AMouse10% NGS, 12h, 41100A42Abcam, ab10148IHCN/ARabbit10% NGS, 12h, 41100BACE-1Abcam, ab2077WB10Rabbit5% skim milk, 2h, O/N, 411000CCSAbcam, ab16962WB10Mouse5% skim milk, 2h, O/N, 41100CHOPCell signaling, 2895WB10Mouse5% skim milk, 2h, O/N, 411000CREBCell signaling, 9197WB10Rabbit5% skim milk, 2h, O/N, 411000complexin-1/2Santa Cruz, sc-33603WB10Rabbit5% skim milk, 2h, O/N, 411000CpAbcam, ab48614WB10Rabbit5% skim milk, 2h, O/N, 411000DrebrinCell signaling, 12243WB10Rabbit5% skim milk, 2h, O/N, 411000eIF2Cell signaling, 5324WB10Rabbit5% skim milk, 2h, O/N, 411000GSK-3Cell sig naling, 9315WB10Rabbit5% skim milk, 2h, O/N, 411000JNKCell signaling, 9252WB10Rabbit5% skim milk, 2h, O/N, 411000Nitro-TyrosineCell signaling, 9691WB10Rabbit5% skim milk, 2h, O/N, 411000NR2AMolecular Probes, A-6473WB8Rabbit5% skim milk, 2h, O/N, 41 five hundredNR2BMolecular Probes, A-6474WB8Rabbit5% skim milk, 2h, O/N, 41500PKR (N-Term)GenWay Biotech, GWB-A4757EWB10Rabbit5% skim milk, 2h, O/N, 41500p-PKR (Thr 451)Invitrogen, 44668GWB10Rabbit5% skim milk, 2h, O/N, 41500p-eIF2 (Ser51)Cell signaling, 3398WB10Rabbit5% skim milk, 2h, O/N, 411000p-GSK-3 (Ser9)Cell signaling, 9336WB10Rabbit5% skim milk, 2h, O/N, 411000p-CREB (Ser133)Cell signaling, 9198WB10Rabbit5% skim milk, 2h, O/N, 411000p-JNKCell signaling, 4671WB10Rabbit5% skim milk, 2h, O/N, 411000p-PP2AEpitomics, 1155-1WB10Rabbit5% skim milk, 2h, O/N, 411000PP2A C subunitEpitomics, 1512-1WB10Rabbit5% skim milk, 2h, O/N, 411000PS396Invitrogen, 44752GWB10Rabbit5% skim milk, 2h, O/N, 411000PS404Invitrogen, 44-758GWB10Rabbit5% skim milk , 2h, O/N, 411000PSD-93Cell signaling, 9445WB10Rabbit5% skim milk, 2h, O/N, 411000PSD-95Cell signaling, 2507WB10Rabbit5% skim milk, 2h, O/N, 411000PSD-95Cell signaling, 2507IFN/ARabbit10% NGS, 12h, 41100synapsin 1Invitrogen, 51-5200WB10Rabbit5% skim milk, 2h, O/N, 411000sAPPCovance, SIG-39139WB10Rabbit5% skim milk, 2h, O/N, 411000sAPPCovance, SIG-39138WB10Rabbit5% skim milk, 2h, O/N, 411000Tau-1Chemicon, MAB3420WB10Mouse5% skim milk, 2h, O/N, 411000Tau-5Abcam, ab80579WB10Mouse5% skim milk, 2h, O/N, 41500-tubulinSanta Cruz, sc-58667WB8-10Mouse5% skim milk, 2h, O/N, RT11000-actinSanta Cruz, sc-47778WB10Mouse5% skim milk, 2h, O/N, RT11000N/A, not applicable NGS, normal-goat serum O/N, over-night RT, room temperature.SUPPLEMENTARY FIGURESSupplementary Figure 1. Content of Cu in Serum and brain. (A-D) Total iron, zinc, calcium, milligram content in the serum respectively (E-H) Total iron, zinc, calcium, magnesium content in the hippocampus respectively (I-L) Total iron, zinc, calcium, m agnesium content in the lens cortex respectively *P