Journal: Brain and Behavior

Article Title: Enriched environment ameliorates postsurgery sleep deprivation‐induced cognitive impairments through the AMPA receptor GluA1 subunit

doi: 10.1002/brb3.2992

Figure Lengend Snippet: Exposure to enriched environment improved BDNF expression in the hippocampus of postsurgery sleep deprivation rats. The relative mRNA levels of Bdnf in the hippocampus were tested by RT‐qPCR (a). The relative protein levels of Bdnf in the hippocampus were assayed by Western blotting (b), and the expressions were normalized to β‐actin (c). n = 3 repeats for each group (8 hippocampus homogenates were mixed for each group). (d) Relative staining intensity of BDNF in CA3 region of rat hippocampus. Six rats in each group were used for staining. (e) The protein level of BDNF in the hippocampus was measured by ELISA. Six rats in each group were used. Data were shown with box plot. * p < .05, ** p < .01, *** p < .001. One‐way ANOVA followed Dunn's multiple comparisons test.

Article Snippet: After nonspecific blocking with 5% nonfat dry milk, the PVDF membranes were incubated with the primary antibodies against BDNF, β‐actin, synaptosomal (Syn) GluA1, Synapsin 1, GluA1, and phospho (p)‐Ser‐845 GluA1 (Thermo Fisher Scientific) at a 1:1000 dilution at 4°C overnight, and incubated with peroxidase‐conjugated secondary antibody (1:1000 dilution, at room temperature, 1 h) and developed with a ECL system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay

Journal: Brain and Behavior

Article Title: Enriched environment ameliorates postsurgery sleep deprivation‐induced cognitive impairments through the AMPA receptor GluA1 subunit

doi: 10.1002/brb3.2992

Figure Lengend Snippet: Exposure to enriched environment improved the level of synaptic GluA1 subunits in hippocampus of postsurgery sleep deprivation rats. (a) Synaptosomal (Syn) GluA1 proteins in the hippocampus were assayed with Western blotting. The relative expressions were normalized to Synapsin 1 (c). Phosphorylated GluA1 at Ser‐845 and total GluA1 proteins in the hippocampus were measured by Western blotting (b). The relative expressions were normalized to β‐actin (d and e). n = 3 repeats for each group (8 hippocampus homogenates were mixed for each group). Data were shown with box plot. ** p < .01, *** p < .001. One‐way ANOVA followed Dunn's multiple comparisons test.

Article Snippet: After nonspecific blocking with 5% nonfat dry milk, the PVDF membranes were incubated with the primary antibodies against BDNF, β‐actin, synaptosomal (Syn) GluA1, Synapsin 1, GluA1, and phospho (p)‐Ser‐845 GluA1 (Thermo Fisher Scientific) at a 1:1000 dilution at 4°C overnight, and incubated with peroxidase‐conjugated secondary antibody (1:1000 dilution, at room temperature, 1 h) and developed with a ECL system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

Techniques: Western Blot

Journal: The Journal of Neuroscience

Article Title: Transplantation of Astrocytic Mitochondria Modulates Neuronal Antioxidant Defense and Neuroplasticity and Promotes Functional Recovery after Intracerebral Hemorrhage

doi: 10.1523/JNEUROSCI.2222-21.2022

Figure Lengend Snippet: Astrocytic Mt upregulates the expression of synaptic plasticity-related genes and promotes neurite outgrowth under ICH-like injury. A, mRNA expression analysis for synaptic plasticity-related genes by qRT-PCR in cultured neurons (3.0 × 105 neurons/well) subjected to ICH-like injury (I) in vitro (RBC lysates, 1.0 × 107 RBCs/well) for 48 h versus untreated control (C). B–D, Cultured neurons were pretreated with ACM or mdACM for 24 h, followed by exposure to ICH-like injury in vitro (RBC lysates, 1.0 × 107 RBCs/well) for 48 h. A, The significant loss of synapsin 1, synaptophysin, PSD-95, and Mn-SOD mRNA was calculated using two-tailed unpaired t test (n = 6 per group), synapsin 1, **p < 0.01 (p = 0.0028, C vs I), t value (t = 3.944); synaptophysin, *p < 0.05 (p = 0.0355, C vs I), t value (t = 2.43); PSD-95, **p < 0.01 (p = 0.0068, C vs I), t value (t = 3.4); Mn-SOD, **p < 0.0001 (C vs I), t value (t = 8.995). B, Values represent fold change of synapsin 1 mRNA levels compared with VEH plus ICH-like injury group. The significance in mRNA levels changes was assessed by one-way ANOVA/Fisher's LSD test (n = 9 per group), **p < 0.01 (p = 0.0003, VEH plus ICH-like injury vs ACM plus ICH-like injury), t value (t = 4.196); **p < 0.01 (p = 0.0098, ACM plus ICH-like injury vs mdACM plus ICH-like injury), t value (t = 2.807). C, Values represent fold change of synaptophysin mRNA levels compared with VEH plus ICH-like injury group. The significance in mRNA levels changes was assessed by one-way ANOVA/Fisher's LSD test (n = 13 per group), **p < 0.01 (p = 0.0039, VEH plus ICH-like injury versus ACM plus ICH-like injury), t value (t = 3.084), NS; Non Significant. D, Values represent fold change of PSD-95 mRNA levels compared with VEH plus ICH-like injury group. The significance in mRNA levels changes was assessed by one-way ANOVA/Fisher's LSD test (n = 12 per group), **p < 0.01 (p = 0.0003, VEH plus ICH-like injury vs ACM plus ICH-like injury), t value (t = 3.99); *p < 0.01 (p = 0.0099, ACM plus ICH-like injury vs mdACM plus ICH-like injury), t value (t = 2.737). E, Neurite outgrowth quantification in rat cortical neurons (5.0 × 104 neurons/well) pretreated with ACM or mdACM, followed by exposure to ICH-like injury in vitro (RBC lysates, 1.7 × 106 RBCs/well) for 96 h. Eight individual neurites in culture from each treatment group (3 independent replicates for each treatment condition) were manually traced, and the length of each neurite was measured using ImageJ software. The significance in neurite length changes was assessed by one-way ANOVA/Fisher's LSD test (3 different colors represent 1 of 3 independent experiments-biological replicates, n = 3). Data are shown as mean ± SEM. The mean was calculated by averaging three values for each experiment; **p < 0.01 (p < 0.0001, VEH vs VEH plus ICH-like injury), t value (t = 8.413); **p < 0.01 (p = 0.0018, VEH plus ICH-like injury vs ACM plus ICH-like injury), t value (t = 4.574); **p < 0.01 (p = 0.0004, ACM plus ICH-like injury vs mdACM plus ICH-like injury), t value (t = 5.87). F, Representative Western blot images and quantitating bar graphs showing synapsin 1/2 and PSD-95 protein levels in cultured neurons pretreated with ACM or mdACM for 24 h, followed by exposure to ICH-like injury in vitro for 48 h. The significant changes in synapsin 1/2/β-actin and PSD-95/β-actin protein levels were assessed by one-way ANOVA/Fisher's LSD test (n = 9 in synapsin 1/2 and 7 in PSD-95), **p < 0.01 (p = 0.0005, VEH plus ICH-like injury vs ACM plus ICH-like injury in synapsin 1/2/β-actin), t value (t = 4.023); *p < 0.05 (p = 0.0121, VEH plus ICH-like injury vs ACM plus ICH-like injury in PSD-95/β-actin), t value (t = 2.789). G, Representative confocal microscopy images of cultured neurons pretreated with ACM for 24 h, followed by exposure to ICH-like injury in vitro for 48 h. Synapsin 1 and 2 are shown in red. Neurons were stained with MAP2 (green). Nuclei were counterstained with DAPI (blue). Scale bars: 20 µm. Data are shown as mean ± SEM. Synap1/2, Synapsin 1/2. Synapsin 1/2 is increased in perihematoma area of the brain after ICH in mice receiving astrocytic Mt in vivo (Extended Data Fig. 6-1).

Article Snippet: After washing with PBS, cells were incubated with primary antibodies against Synapsin 1/2 (1:200; catalog #106003, Synaptic Systems) and MAP-2 (1:200; catalog #MA5-12826, Invitrogen) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, In Vitro, Two Tailed Test, Software, Western Blot, Confocal Microscopy, Staining, In Vivo

Journal: The Journal of Neuroscience

Article Title: Transplantation of Astrocytic Mitochondria Modulates Neuronal Antioxidant Defense and Neuroplasticity and Promotes Functional Recovery after Intracerebral Hemorrhage

doi: 10.1523/JNEUROSCI.2222-21.2022

Figure Lengend Snippet: Mt-encoded peptide HN promotes antioxidative mechanisms, upregulates synaptogenesis-related genes, and stimulates neuronal growth under ICH-like stress. A, Representative Western blot image and quantitating bar graph showing phosphorylation of STAT3 at Y705 in cultured neurons pretreated with 100 ng/ml recombinant HN protein for 24 h. The significant changes in p-STAT3/β-actin protein levels were assessed by two-tailed unpaired t test (n = 7 per group), **p < 0.01 (p < 0.0001, CON vs HN), t value (t = 7.561). CON, Control. B, Representative Western blot image and quantitating bar graph showing Mn-SOD protein levels in cultured neurons pretreated with 100 ng/ml recombinant HN protein for 24 h, followed by exposure to ICH-like injury for 48 h. The significant changes in Mn-SOD/β-actin protein levels were assessed by one-way ANOVA/Fisher's LSD test (n = 9 per group), **p < 0.01 (p < 0.0001, CON vs ICH-like injury), t value (t = 4.993); **p < 0.01 (p = 0.0050, ICH-like injury vs HN plus ICH-like injury), t value (t = 3.092). C, ROS generation in cultured neurons pretreated with 100 ng/ml recombinant HN protein for 24 h, followed by exposure to ICH-like injury for 12 h. The significant changes in ROS generation were assessed by one-way ANOVA/Fisher's LSD test (n = 16 per group), **p < 0.01 (p < 0.0001, CON vs ICH-like injury), t value (t = 4.535); *p < 0.05 (p = 0.0140, ICH-like injury vs HN plus ICH-like injury), t value (t = 2.556). D, Neurite outgrowth quantification in cultured neurons (5.0 × 104 neurons/well) pretreated with 100 ng/ml recombinant HN protein for 24 h, followed by exposure to ICH-like injury (RBC lysates, 1.7 × 106 RBCs/well) for 96 h. The six individual neurites in culture from each treatment group (3 independent replicates for each treatment condition) were manually traced, and the length of each neurite was measured using ImageJ software. The significance in the neurite length changes was assessed by one-way ANOVA/Fisher's LSD test (3 different colors represent 1 of 3 independent experiments-biological replicates, n = 3). Data are shown as mean ± SEM. The mean was calculated by averaging three values for each experiment; **p < 0.01 (p = 0.0023, CON vs ICH-like injury), t value (t = 5.073); *p < 0.05 (p = 0.0146, ICH-like injury vs HN plus ICH-like injury), t value (t = 3.394). E, Representative Western blot images and quantitating bar graphs showing synapsin 1/2 and PSD-95 protein levels in neurons pretreated with 100 ng/ml recombinant HN for 24 h, followed by ICH-like injury for 48 h. The significant changes in synapsin 1/2/β-actin and PSD-95/β-actin protein levels were assessed by one-way ANOVA/Fisher's LSD test (n = 10 in synapsin 1/2 and 9 in PSD-95), **p < 0.01 (p = 0.0087, CON vs ICH-like injury in synapsin 1/2/β-actin), t value (t = 2.828); **p < 0.01 (p = 0.0058, ICH-like injury vs HN plus ICH-like injury in synapsin 1/2/β-actin), t value (t = 2.996); **p < 0.01 (p < 0.0001, CON vs ICH-like injury in PSD-95/β-actin), t value (t = 5.361); **p < 0.01 (p < 0.0001, ICH-like injury vs HN plus ICH-like injury in PSD-95/β-actin), t value (t = 5.031). Data are shown as mean ± SEM.

Article Snippet: After washing with PBS, cells were incubated with primary antibodies against Synapsin 1/2 (1:200; catalog #106003, Synaptic Systems) and MAP-2 (1:200; catalog #MA5-12826, Invitrogen) overnight at 4°C.

Techniques: Western Blot, Cell Culture, Recombinant, Two Tailed Test, Software

Journal: Cell reports

Article Title: Neuronal VCP loss of function recapitulates FTLD-TDP pathology

doi: 10.1016/j.celrep.2021.109399

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were stained in 10% NGS/0.3% Triton X-100 in PBS overnight at room temperature with primary antibodies against synapsin 1/2 (1:1000, Synaptic Systems Cat# 106004 Lot# 2-27; RRID: AB_1106784) and PSD-95 (1:200, Invitrogen Cat# 51-6900; RRID: AB_2533914).

Techniques: Ubiquitin Proteomics, Transduction, Recombinant, Sample Prep, In Situ, Sequencing, RNA Sequencing, Knock-In, Software