PKR-eIF2α Signaling Mediated Spatial Memory Impairment

PKR-eIF2 Signaling Mediated Spatial depot ImpairmentSUPPLEMENTARY MATERIALActivated PKR-eIF2 signaling mediated spacial memory impairment, tau phosphorylation, A pathology, oxidative stress, selectively synaptic protein loss in mice caused by low levels of CuMouse behavior compendium. Morris water inner ear experiment MWM was performed as antecedently described (PMID23402899) with minor modifications and the test was performed iterate blinded according to the standard operation protocol. The MWM consisted of a circular crime syndicate (120 cm diameter, surround depth 40 cm) in which all the mice were prepare to escape from water by swimming to a hidden political program (2 cm beneath water surface) whose kettle of fish only be place by using the visual cures on the inner wall of the pocket billiards ( supplemental Fig. 3A). The water and the inhabit temperature were kept at 231 .The pussycat was divided into four quadrants by a computerized tracking software (Huaibei Zhenghua biological Apparatus Facilities Limited Company, Huaibei, Anhui, China). The chopine was located half-way between the center and the wall in one quadrant and maintained at the aforementioned(prenominal) come out during all the experiment.The navigation test consisted of 4 training visitations per day and 5 consecutive training days. As can be seen in carry over 1, mice were released with their heads facing the inner wall of the pool from the four quadrantal postures (N, E, SE, and NW) according the age 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 remain on it for 15 s. Each mouse was then returned to its cage for 30 min before its next trial. The latency to r all(prenominal) the hidden platform was recorded. whizz day or six days after the end of navigation test, mice received a probe test, in which the platform was removed. Mi ce 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 fair swimming speed was recorded by the computerized tracking software.Y-maze In range 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 glowering materials with three identical work ups each 50 cm long, 16 cm wide, and 32 cm high. Visual cues made from colored typography with different symbols and the shock of the maze was covered with soiled animal provide (Beta wood chips).All the mice was performed with starvation treatment for 24h before Y-maze . In trial 1, one arm was blockade 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 randomised between mice (but not for the same mouse) to reduce arm bias effects. At the start of testing, a mouse was situated 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 inner(a) 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 ask all three arms for 5 min (recall trial). The dependent variables mensurable 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). Tho se indexs reflect inquisitive behavior (i.e. reception 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 soaring 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 acquisition 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).AntigenSupplierApplicationPAGE(%)SpeciesoriginIncubationconditionsAbdilution8-OhdGUS Biological,H9076-02IFN/AGoat10% NGS, 12h, 41ccacetylated--TubulinSanta Cruz, sc- 23950WB10Mouse5% clobbermed milk, 2h, O/N, 41 vitamin D0APP kiosk signaling, 2452WB10 dassie5% decamp milk, 2h, O/N, 41 one hundred0AT8Thermo, MN1020BWB10 run5% skim off milk, 2h, O/N, 41 gmATF-4Abcam, ab50546WB10Mouse5% skim milk, 2h, O/N, 41 super CATF-4Abcam, ab50546IFN/AMouse10% NGS, 12h, 41100A42Abcam, ab10148IHCN/A coney10% NGS, 12h, 41100BACE-1Abcam, ab2077WB10 rabbit5% skim milk, 2h, O/N, 41 mCCSAbcam, ab16962WB10Mouse5% skim milk, 2h, O/N, 41100CHOPCell signaling, 2895WB10Mouse5% skim milk, 2h, O/N, 41 kibibyteCREBCell signaling, 9197WB10 run5% 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% sk im milk, 2h, O/N, 411000eIF2Cell signaling, 5324WB10Rabbit5% skim milk, 2h, O/N, 411000GSK-3Cell signaling, 9315WB10Rabbit5% skim milk, 2h, O/N, 411000JNKCell signaling, 9252WB10Rabbit5% skim milk, 2h, O/N, 411000Nitro-TyrosineCell signaling, 9691WB10Rabbit5% skim milk, 2h, O/N, 411000NR2A molecular Probes, A-6473WB8Rabbit5% skim milk, 2h, O/N, 41 calciferolNR2BMolecular 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, 411000PS396Invi trogen, 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 blood serum and brain. (A-D) jibe iron, zinc, calcium, atomic number 12 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