Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A and B) Quantitative Western-Blot analysis of WT and Msk1 IV KO cultured striatal neurons. (A) 1h BDNF (50 ng/mL) stimulation causes an increase in TrkB, ERK1/2 and MeCP2 S421 phosphorylation in WT cultured striatal neurons. ERK1/2 inhibition with U0126 prevents MeCP2 S421 phosphorylation. * P <0.05, ** P <0.01 (mean ± SEM; n=3; two tailed unpaired Student’s t test). (B) The absence of MSK1 prevents the BDNF-dependent MeCP2 S421 phosphorylation in cultured Msk1 IV KO striatal neurons, but not the phosphorylation of TrkB and ERK1/2. * P <0.05, ** P <0.01 (mean ± SEM; n=4; two tailed unpaired Student’s t test). (C) MSK1 and MeCP2 interact in the nucleus of HEK293-FT independently of MSK1 phosphorylation state. Lysates from HEK293-FT cells co-transfected with expression plasmids for MeCP2-HA and MSK1-myc were immunoprecipitated with anti-HA antibodies coupled to magnetic beads and western blots were performed to detect the interaction of MSK1 with MeCP2 before or after inducing MSK1 activation by PMA (200 nM; 1h). (D) Expression of MeCP2-regulated genes is also dependent of MSK1. qPCR analysis show downregulation of Gad1 , responsible of GABA production, Drd1 and Drd3 , which code for dopamine receptors, in the striatum of P60 Msk1 IV KO mice. However, transcript levels for the gene coding the GABA A receptor subunit gamma3 ( Gabrg3 ) and the dopamine receptor Drd2 are increased. * P <0.05, *** P <0.001 (mean ± SEM; n=3; two tailed unpaired Student’s t test).

Article Snippet: Antibodies and concentrations were as follow: rabbit monoclonal anti-MSK1 (C27B2; Cell Signaling) 1:1000, mouse monoclonal anti-β-actin (669-1-Ig; Proteintech) 1:20000, mouse monoclonal anti-phospho-pan-Trk (#9141; Cell Signaling) 1:1000, rabbit polyclonal anti-MeCP2 phospho S421 (ab254050; Abcam) 1:500, mouse monoclonal anti-pERK1/2 (#9101; Cell Signaling) 1:3000, mouse monoclonal anti-Myc (sc-41; SantaCruz) 1:200, mouse monoclonal anti-MeCP2 (Quimigen 10861-1-AP) 1:500; rabbit polyclonal anti-MSK1 phosho Thr581 (#9595; Cell Signaling).

Techniques: Western Blot, Cell Culture, Inhibition, Two Tailed Test, Transfection, Expressing, Immunoprecipitation, Magnetic Beads, Activation Assay

Journal: Aging and Disease

Article Title: Prelimbic Cortical Stimulation with L-methionine Enhances Cognition through Hippocampal DNA Methylation and Neuroplasticity Mechanisms

doi: 10.14336/AD.2022.0706

Figure Lengend Snippet: Protracted MET treatment and PrL DBS enhances neuroplasticity and hippocampal neurogenesis through the PKA-CaMKIIα-BDNF pathway. Representative Western blot images of neuroplasticity-associated proteins (A). Densitometric measurement by two-way ANOVA analysis of PSD95 (Treatment, p = 0.038; Stimulation, p = 0.01; Treatment x Stimulation, p = 0.01), SYP (p > 0.376), CREB (p > 0.369), pCREB (Treatment, p = 0.044) and pCREB/CREB ratio (Treatment, p = 0.074) showed enhanced neuroplasticity in MET and DBS animals (B). Representative Western blot images of proteins involved in memory neuroepigenetics (C). Two-way ANOVA revealed a significant stimulation effect (p = 0.041) for DNMT3a, insignificant treatment (p = 0.071) and significant stimulation (p = 0.041) effects for CaN, and significant treatment (p = 0.022) and insignificant stimulation (p = 0.068) effects for BDNF. No difference in the pMeCP2/MeCP2 ratio was observed among all groups (p > 0.205) (D). Representative Western blot images of proteins involved in the intermediate pathways of CREB phosphorylation (E). Two-way ANOVA showed no changes in pERK/ERK ratio (p > 0.499), significant changes in pPKA/PKA ratio (treatment, p = 0.048; stimulation, p = 0.046) and pCaMKIIα/CaMKIIα ratio (Treatment, p = 0.006; Stimulation, p = 0.004). (F). Scatter plot displaying significant correlations between CaN and pCaMKIIα/CaMKIIα ratio in SAL-DBS and MET-DBS animals, indicating that the inhibition of CaN by PrL DBS was strongly associated with the activation of CaMKIIα in the hippocampus. All groups: n=9. (G). Neurogenesis quantification by flow cytometry analysis for BrdU. Scatter plot displaying a selection of nuclei stained with DAPI, subsequent gating for BrdU, and removal of events doubly stained for GFAP and NeuN. Quantification of all groups revealed a significant main effect of stimulation (p = 0.012). More BrdU-positive events were found in SAL-DBS and MET-DBS animals compared to SAL-SHAM animals (H). Scatter plot displaying significant positive correlations between BrdU-positive cell count and BDNF protein expression in MET-DBS animals. SAL-SHAM: n=6; SAL-DBS: n=10; MET-SHAM: n=7; MET-DBS: n=10. (I). *, p<0.05; **, p<0.01; n.s., not significant. Data presented as mean ± s.e.m.

Article Snippet: Antibodies against PSD95 (1:1000, Cat# ab18258, Abcam), synaptophysin (1:1000, Cat# 5461, Cell Signaling Technology), CREB (1:1000, Cat# 9197, Cell Signaling Technology), pCREB (1:1000, Cat# 9198, Cell Signaling Technology), BDNF (1:1000, Cat# NB100-98682, Novus Biologicals), PKA C-α (1:1000, Cat# 4782, Cell Signaling Technology), pPKA C Thr197 (1:1000, Cat# 4781, Cell Signaling Technology), ERK1/2 (1:1000, Cat# 9102, Cell Signaling Technology), pERK1/2 T202/Y204 (1:1000, Cat# 9101, Cell Signaling Technology), CaMKIIα (1:1000, Cat# 3362, Cell Signaling Technology), pCaMKIIα Thr286 (1:1000, Cat# 12716, Cell Signaling Technology), MeCP2 (1:1000, Cat# NB600-1101, Novus Biologicals), pMeCP2 S421 (1:1000, Cat# NBP2-29524, Novus Biologicals), DNMT3a (1:1000, Cat# 3598, Cell Signaling Technology), Calcineurin A (1:1000, Cat# 2614, Cell Signaling Technology) and GAPDH (1:1000, Cat# 2118, Cell Signaling Technology) were used.

Techniques: Western Blot, Phospho-proteomics, Inhibition, Activation Assay, Flow Cytometry, Selection, Staining, Cell Counting, Expressing

Journal: Research

Article Title: ATP5O Hypo-crotonylation Caused by HDAC2 Hyper-Phosphorylation Is a Primary Detrimental Factor for Downregulated Phospholipid Metabolism under Chronic Stress

doi: 10.34133/2022/9834963

Figure Lengend Snippet: Upregulated HDAC2 phosphorylation is connected with downregulated ATP5O crotonylation in CS mice. (a, b) TMT-labeled phosphoproteomics showed that at the threshold of ≥1.2 or ≤ 0.833 , there are 117 downregulated and 60 upregulated phosphorylated proteins. (c) KEGG pathway showed that multiple pathways are lipid metabolism related. (d) We selected three deacetylases—HDAC2, KAT7, and CREBBP—to verify their relationship with ATP5O. Western blot showed that inhibition of HDAC2 phosphorylation at S424 with THP (CYGRKKRRQRRRSDSEDEGEGGRR) significantly decreased ATP5O, whereas inhibition of KAT7 phosphorylation at S102 with TKP (CYGRKKRRQRRRSSGSETEQVVDF) or CREBBP phosphorylation at S2361 and S2363 with TCP (CYGRKKRRQRRRQSQPPHSSPSPR) did not change ATP5O protein level at all. (e, f) We generated an ATP5O-S424 phosphorylation-specific antibody with a phosphorylated peptide SDS (phospho) EDEGEGGRR and verified the specificity through two ways. (e) Different amounts of non- and S424-phosphorylated HDAC2 peptide were loaded onto a dot blot PVDF membrane and detected by the HDAC2-S424 P antibody. Only phosphorylated HDAC2 peptide was detected in a dose-dependent manner. (f) When HDAC2-S424 P antibody was preblocked by the phosphorylated HDAC2 peptide, significantly less amount of endogenous ATP5O-K51 cr was detected. (g) Western blot showed that THP treatment significantly decreased HDAC2-S424 P (p-HDAC2) level and upregulated ATP5O-K51 cr (ATP5O cr ) level, whereas did not change KAT7-S102 P (p-KAT) level. Besides, THP treatment did not alter the level of HDAC1-S421 p at all, which shared the highest similarity with THP (Supp. Figure ). (h, i) By live imaging in Sf9 cells, when only ATP5O-WT-EGFP titer was added into sf9 culture media, as time progressed, green fluorescence rapidly increased; in contrast, when both ATP5O-EGFP and HDAC2-WT-TagRFP titers were added, as time went by, the red fluorescence (HDAC2) quickly increased, whereas the increment extent of green fluorescence (ATP5O) was significantly slowed down. (j) Coimmunoprecipitation and western blot showed that HDAC2 significantly interacted with ATP5O but rarely interacted with ATP5A1 or ATP5B. (k) GST-pulldown was carried out to verify the direct binding between ATP5O and HDAC2. GST-ATP5O and his-tagged HDAC2-TagRFP were cloned, expressed, and purified in E.coli (From left, the fourth and fifth lane, red asterisk-labeled) and sf9 cells (From left, the third lane, red asterisk-labeled), respectively. SDS-PAGE showed that GST-ATP5O could significantly pull down HDAC2-TagRFP (From left, the sixth lane, red asterisk-labeled). L-N. Western blot showed that when a fixed amount of ATP5O-WT and increasing amounts of HDAC2-WT were cotransfected into 293 T cells, ATP5O crotonylation level rapidly decreased (l, n); in contrast, when a fixed amount of ATP5O-WT and increasing amounts of HDAC2-S424A (a nonphosphorylable mutant) were cotransfected into 293 T cells, the decrement extent of ATP5O crotonylation level became much smaller (m, n). (o–s) When HDAC2-Flag and ATP5O-StrepII were cotransfected into 293 T cells at an increasing dosage, ATP5O crotonylation (o, p) and ATP levels both increased (q); accordingly, FAM126A and STAT5A levels also increased in a dose-dependent manner (r, s). (t–u) Western blot showed that compared with ATP5O-transfected 293 T cells, HDAC2 and ATP5O cotransfected cells had decreased ATP5O crotonylation, whereas inhibition of proteasome activity with MG132 significantly recovered ATP5O crotonylation level. (v, w) Western blot showed that in an in vitro ubiquitination assay with the ATP5O-E3 ubiquitin ligase complex immunoprecipitated from CS ovaries, the ATP5O ubiquitination level was significantly higher than the level in control ovaries. X. Model: ATP5O crotonylation is resistant to ubiquitination, whereas ATP5O-K51 cr decrotonylated by HDAC2 is susceptible to ubiquitination and protease degradation. Tubulin or actin was used as a loading control. * indicates p < 0.05 , *** indicates p < 0.001 , **** indicates p < 0.0001 .

Article Snippet: Other primary antibodies: Mouse monoclonal anti-GAPDH (Cat#: 30201ES60; YEASEN, Shanghai, China); mouse monoclonal anti- β -Actin (Cat#: A5316-100; Sigma, MS, USA); Mouse monoclonal anti- β -Tubulin (Cat#: sc-5274; Santa Cruz, TX, USA); Mouse monoclonal anti-alpha Tubulin (Acetyl Lys40) (cat#: bsm-33235 M; Bioss, Beijing, China); Rabbit anti-Transferrin polyclonal antibody (Cat#: 17435-1-ap; Proteintech, Chicago, USA); Rabbit anti-ATP5O polyclonal antibody (Cat#: D126152; BBI Life science, Shanghai, China); Rabbit anti-ATP5A1 polyclonal antibody (Cat#: D154243; BBI Life science, Shanghai, China); Rabbit anti-ATPB polyclonal antibody (Cat#: A5286; Selleckchem, Shanghai, China); Mouse anti-COX4L1 monoclonal antibody (Cat#: D190618; BBI Life science, Shanghai, China); Anti-AKT (Ab-129) Rabbit polyclonal antibody (Cat#: D151616-0100; BBI Life science, Shanghai, China); Rabbit anti-Phosphpho AKT (Ser473, Cat# 4060, Cell Signaling Technology); Rabbit anti-Phosphpho RPS6 (Ser235/236, Cat#: D155178; BBI Life science, Shanghai, China); Rabbit anti-STAT5A polyclonal antibody (Cat#: D220085; BBI Life science, Shanghai, China); Rabbit anti-FAM126A polyclonal antibody (Cat#: bs-11554R; Bioss, Beijing, China); Rabbit anti-PTDSS1 polyclonal antibody (Cat#: BS-19583R; Bioss, Beijing, China); Rabbit anti-HDAC2 polyclonal antibody (Cat#: 12922-3-ap, Proteintech, Chicago, USA); Mouse monoclonal anti-strep II Tag (Cat#: YFMA0054, Yifeixue, Nanjing, China); Mouse monoclonal anti-flag Tag (Cat#: D190828, BBI Life science, Shanghai, China); Rabbit anti-HDAC1-S421 p polyclonal antibody (Cat#: AP59578, Abcepta, San Diego, CA, USA); Rabbit anti-Ubinuclein polyclonal antibody (Cat#: DF8873, Affinity Biosciences, Melbourne, Australia); Fibrinogen alpha chain (FGA) Rabbit pAb (Cat#: A1453, ABclonal, Wuhan, Hubei, China); Albumin Polyclonal antibody (Cat#: 16475-1-AP; Proteintech, Rosemont, IL, USA); AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, Cat#: 115-005-003, West Grove, PA, USA); Rabbit anti-ATP5O-K51 cr rabbit polyclonal antibody was made against ASK (crotonyl) EKKLDQVEKELLC by YuBo BioTech and purified by affinity purification; Rabbit anti-HDAC2-S424 p ,and anti-KAT7-S102 p polyclonal antibody was made against SDS(phospho)EDEGEGGRRC and CPLRQTRSSGS (phospho) ET by Zoonbio BioTech and purified by affinity purification.

Techniques: Labeling, Western Blot, Inhibition, Generated, Dot Blot, Membrane, Imaging, Fluorescence, Binding Assay, Clone Assay, Purification, SDS Page, Mutagenesis, Transfection, Activity Assay, In Vitro, Ubiquitin Assay, Immunoprecipitation

Journal: Nature neuroscience

Article Title: Sustained effects of rapidly-acting antidepressants require BDNF-dependent MeCP2 phosphorylation

doi: 10.1038/s41593-021-00868-8

Figure Lengend Snippet: a,b, BDNF and pMeCP2 levels were measured in the hippocampus of C57BL/6J mice 30 min and 7 days after ketamine treatment. BDNF levels were increased at 30 min and returned to baseline at 7 days after treatment (30 min - P = 0.0189, Sal, Ket: n = 8, 7 mice, 7 days - P = 0.5872, Sal, Ket: n = 9, 9 mice). pMeCP2 levels were increased 7 days but not 30 min after injection (30 min - P = 0.7126, Sal, Ket: n = 8, 8 mice, 7 days - Sal, Ket: n = 9, 11 mice). c, In the FST, Mecp2 CTL and KI mice showed a significant reduction in time spent immobile 30 min after ketamine, compared to the respective saline-treated control groups (Genotype x Drug: P = 0.2875, Drug: P < 0.0001, CTL -Sal, CTL -Ket, KI-Sal, KI-Ket: n = 10, 10, 13, 12 mice). d, In the FST, Mecp2 CTL, but not KI, mice showed a significant reduction in time spent immobile 7 days after ketamine compared to the respective saline-treated control groups (Genotype x Drug: P = 0.0222, CTL-Sal, CTL-Ket, KI-Sal, KI-Ket: n = 9, 9, 9, 8 mice). e, Western blot analysis of hippocampi from Bdnf cKO and CTL mice 7 days after ketamine treatment. Ketamine increased pMeCP2 levels in the CTL but not in cKO mice (Genotype x Drug: P = 0.0465, CTL-Sal, CTL-Ket, cKO-Sal, cKO-Ket: n = 13, 13, 11, 12 mice). f, In the FST, Ketamine reduced time spent immobile in CTL mice but not in Bdnf cKO mice 7 days after injection (Genotype x Drug: P = 0.0456, CTL-Sal, CTL-Ket, cKO-Sal, cKO-Ket: n = 12, 12, 8, 8 mice). b: two-sided unpaired t-test. c-f: two-way ANOVA with Tukey’s multiple comparisons. Graphs represent mean ± S.E.M.; N.S.: not significant, *, P < 0.05, **, P < 0.01. Sal: saline, Ket: ketamine, CTL: littermate control. For detailed statistical information, see .

Article Snippet: After blocking the membranes for 1 hr at room temperature in blocking solution, blots were washed three times with 0.1% TBS-Tween and incubated overnight at 4°C with the following primary antibodies: MeCP2 (1:4,000, Thermo Fisher, PA1–888), phospho-MeCP2 (S421, 1:1,000, a generous gift from Dr. Anne West and ECM Biosciences, MP46111), GAPDH (1:100,000, Cell Signaling Technology, 2118), CREB (1:5,000, Cell Signaling Technology, 9197), phospho-CREB (Ser133, 1:5,000, Merck Millipore, 06–519), BDNF (1:1,500, Abcam, ab108319), CaMKIIα (1:5,000, Cell Signaling Technology, 3357S), CaMKIIβ (1:500,000, Abcam, ab34703), phospho-CaMKII (D21E4, 1:50,000, Cell Signaling Technology, 12716S).

Techniques: Injection, Saline, Control, Western Blot

Journal: Nature neuroscience

Article Title: Sustained effects of rapidly-acting antidepressants require BDNF-dependent MeCP2 phosphorylation

doi: 10.1038/s41593-021-00868-8

Figure Lengend Snippet: BDNF protein levels were measured 30 min after ketamine treatment in Mecp2 KI and CTL mice by Western blot analysis. BDNF levels were significantly increased by ketamine treatment in both the CTL and KI mice (two-way ANOVA with Tukey’s multiple comparisons, Genotype x Drug: F (1, 21) = 0.0157, P = 0.9016, Genotype: F (1, 21) = 0.0115, P = 0.9158, Drug: F (1, 21) = 31.93, P < 0.0001, CTL-Sal, CTL-Ket, KI-Sal, KI-Ket: n = 7, 7, 6, 5 mice). In Western blot analysis for pMeCP2, membrane above about 70 KDa was cropped and used for immunoblotting. The graph represents mean ± S.E.M., **, P < 0.01, Sal: saline, Ket: ketamine, CTL: littermate control. For detailed statistical information, see .

Article Snippet: After blocking the membranes for 1 hr at room temperature in blocking solution, blots were washed three times with 0.1% TBS-Tween and incubated overnight at 4°C with the following primary antibodies: MeCP2 (1:4,000, Thermo Fisher, PA1–888), phospho-MeCP2 (S421, 1:1,000, a generous gift from Dr. Anne West and ECM Biosciences, MP46111), GAPDH (1:100,000, Cell Signaling Technology, 2118), CREB (1:5,000, Cell Signaling Technology, 9197), phospho-CREB (Ser133, 1:5,000, Merck Millipore, 06–519), BDNF (1:1,500, Abcam, ab108319), CaMKIIα (1:5,000, Cell Signaling Technology, 3357S), CaMKIIβ (1:500,000, Abcam, ab34703), phospho-CaMKII (D21E4, 1:50,000, Cell Signaling Technology, 12716S).

Techniques: Western Blot, Membrane, Saline, Control

Journal: Nature neuroscience

Article Title: Sustained effects of rapidly-acting antidepressants require BDNF-dependent MeCP2 phosphorylation

doi: 10.1038/s41593-021-00868-8

Figure Lengend Snippet: a, The experimental design for measurement of ketamine-potentiation. Mice were treated with saline or ketamine 7 days later hippocampal slices were prepared and fEPSPs recorded in the CA1 region. b,c , Ketamine-potentiation was measured in hippocampal CA1 from C57BL/6J mice previously given either a saline (b) or ketamine (c) injection. Ketamine perfusion induced significant potentiation in both groups, and augmented potentiation was observed in the group previously treated with the ketamine (c: Ket+Ket, 140.7 ± 10.57%), compared to the group previously treated with saline (b: Sal+Ket, 116 ± 1.994%), (two-sided unpaired t-test, b - Sal+ACSF vs Sal+Ket: P < 0.0001, Sal+ACSF, Sal+Ket: n = 6, 11 slices, c - Ket+ACSF vs Ket+Ket: P = 0.0019, Ket+ACSF, Ket+Ket: n = 8, 10 slices, Sal+Ket in b vs Ket+Ket in c: P = 0.0266). d, e, Ketamine-potentiation was measured in the hippocampal CA1 from Mecp2 KI mice previously given either a saline (d) or ketamine injection (e). Ketamine-potentiation was intact in Mecp2 KI mice (d) but the augmented potentiation was not observed in the KI group previously treated with ketamine (e: Ket+Ket, 121.6 ± 6.139%), compared to the group previously treated with saline (d: Sal+Ket, 118.7 ± 4.103%), (two-sided unpaired t-test, d - Sal+ACSF vs Sal+Ket: P = 0.0017, Sal+ACSF, Sal+Ket: n = 7, 7 slices, e - Ket+ACSF vs Ket+Ket:, P = 0.0017, Ket+ACSF, Ket+Ket: n = 8, 9 slices, Sal+Ket in d vs Ket+Ket in e: P = 0.7249). Representative traces are from recorded results during the measurement of baseline and potentiation. Graphs represent mean ± S.E.M., **, P < 0.01, ***, P < 0.001. Ket: ketamine, Sal: saline, Sal+ACSF: ACSF perfusion onto hippocampal slices from saline-injected mice, Sal+Ket: ketamine perfusion onto hippocampal slices from saline-injected mice, Ket+ACSF: ACSF perfusion onto hippocampal slices from ketamine-injected mice, Ket+Ket: ketamine perfusion onto hippocampal slices from ketamine-injected mice. For detailed statistical information, see .

Article Snippet: After blocking the membranes for 1 hr at room temperature in blocking solution, blots were washed three times with 0.1% TBS-Tween and incubated overnight at 4°C with the following primary antibodies: MeCP2 (1:4,000, Thermo Fisher, PA1–888), phospho-MeCP2 (S421, 1:1,000, a generous gift from Dr. Anne West and ECM Biosciences, MP46111), GAPDH (1:100,000, Cell Signaling Technology, 2118), CREB (1:5,000, Cell Signaling Technology, 9197), phospho-CREB (Ser133, 1:5,000, Merck Millipore, 06–519), BDNF (1:1,500, Abcam, ab108319), CaMKIIα (1:5,000, Cell Signaling Technology, 3357S), CaMKIIβ (1:500,000, Abcam, ab34703), phospho-CaMKII (D21E4, 1:50,000, Cell Signaling Technology, 12716S).

Techniques: Saline, Injection

Journal: Nature neuroscience

Article Title: Sustained effects of rapidly-acting antidepressants require BDNF-dependent MeCP2 phosphorylation

doi: 10.1038/s41593-021-00868-8

Figure Lengend Snippet: a, Scopolamine decreased time spent immobile in the FST compared to the saline-treated group, both 8 hrs and 24 hrs after treatment (8 hr - Sal, SCA: n = 9, 9 mice, 24 hr - Sal, SCA: n = 8, 8 mice). b, Eight hours after scopolamine treatment, Bdnf mRNA levels were significantly increased (Sal, SCA: n = 7, 7 mice), and BDNF protein levels showed an increased tendency (Sal, SCA: n = 15, 15 mice). Both mRNA and protein levels returned to baseline by 24 hrs (Sal, SCA: n = 8, 8 mice). c, pMeCP2 levels were increased 24 hrs after scopolamine treatment in the hippocampus (8 hrs - Sal, SCA: n = 8, 8 mice, 24 hrs - Sal, SCA: n = 7, 8 mice). d, Scopolamine increased hippocampal pMeCP2 levels in the CTL mice but not in the Bdnf cKO mice 24 hrs after injection (CTL-Sal, CTL-SCA, cKO-Sal, cKO-SCA: n = 10, 12, 11, 11 mice). e, Time spent immobile was reduced in the CTL mice but not in the Bdnf cKO mice 8 hrs after scopolamine treatment (CTL-Sal, CTL-SCA, cKO-Sal, cKO-SCA: n = 16, 14, 15, 13 mice). f, Scopolamine decreased immobility time in the CTL mice but not in the B dnf cKO mice 8 hrs after treatment (CTL-Sal, CTL-SCA, cKO-Sal, cKO-SCA: n = 17, 18, 16, 14 mice). g, Scopoloamine reduced time spent immobile in both the CTL mice and the Mecp2 KI mice 8 hrs after treatment (CTL-Sal, CTL-SCA, KI-Sal, KI-SCA: n = 13, 16, 14, 14 mice). h, Scopolamine decreased immobility time in the CTL mice but not in the Mecp2 KI mice at 24 hrs after treatment (CTL-Sal, CTL-SCA, KI-Sal, KI-SCA: n = 9, 11, 9, 10 mice). a,d,e-h: two-way ANOVA with Tukey’s multiple comparisons. b,c: two-sided unpaired t-test or Welch’s correction. Graphs represent mean ± S.E.M., N.S.: not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. Sal: saline, SCA: scopolamine, CTL: littermate control. For detailed statistical information, see .

Article Snippet: After blocking the membranes for 1 hr at room temperature in blocking solution, blots were washed three times with 0.1% TBS-Tween and incubated overnight at 4°C with the following primary antibodies: MeCP2 (1:4,000, Thermo Fisher, PA1–888), phospho-MeCP2 (S421, 1:1,000, a generous gift from Dr. Anne West and ECM Biosciences, MP46111), GAPDH (1:100,000, Cell Signaling Technology, 2118), CREB (1:5,000, Cell Signaling Technology, 9197), phospho-CREB (Ser133, 1:5,000, Merck Millipore, 06–519), BDNF (1:1,500, Abcam, ab108319), CaMKIIα (1:5,000, Cell Signaling Technology, 3357S), CaMKIIβ (1:500,000, Abcam, ab34703), phospho-CaMKII (D21E4, 1:50,000, Cell Signaling Technology, 12716S).

Techniques: Saline, Injection, Control

Journal: Nature neuroscience

Article Title: Sustained effects of rapidly-acting antidepressants require BDNF-dependent MeCP2 phosphorylation

doi: 10.1038/s41593-021-00868-8

Figure Lengend Snippet: Bdnf mRNA levels were measured with quantitative real-time PCR in CTL and Mecp2 KI mice 8 hrs after scopolamine. Scopolamine treatment significantly increased Bdnf mRNA levels in the hippocampus of both the CTL and KI mice (two-way ANOVA with Tukey’s multiple comparisons, Genotype x Drug: F (1, 33) = 0.0467, P = 0.8302, Genotype: F (1, 33) = 0.3223, P = 0.5741, Drug: F (1, 33) = 19.22, P = 0.0001, CTL-Sal, CTL-SCA, KI-Sal, KI-SCA: n = 10, 9, 10, 8 mice). The Graph represents mean ± S.E.M., *, P < 0.05, Sal: saline, SCA: scopolamine, CTL: littermate control. For detailed statistical information, see .

Article Snippet: After blocking the membranes for 1 hr at room temperature in blocking solution, blots were washed three times with 0.1% TBS-Tween and incubated overnight at 4°C with the following primary antibodies: MeCP2 (1:4,000, Thermo Fisher, PA1–888), phospho-MeCP2 (S421, 1:1,000, a generous gift from Dr. Anne West and ECM Biosciences, MP46111), GAPDH (1:100,000, Cell Signaling Technology, 2118), CREB (1:5,000, Cell Signaling Technology, 9197), phospho-CREB (Ser133, 1:5,000, Merck Millipore, 06–519), BDNF (1:1,500, Abcam, ab108319), CaMKIIα (1:5,000, Cell Signaling Technology, 3357S), CaMKIIβ (1:500,000, Abcam, ab34703), phospho-CaMKII (D21E4, 1:50,000, Cell Signaling Technology, 12716S).

Techniques: Real-time Polymerase Chain Reaction, Saline, Control

Journal: Nature neuroscience

Article Title: Sustained effects of rapidly-acting antidepressants require BDNF-dependent MeCP2 phosphorylation

doi: 10.1038/s41593-021-00868-8

Figure Lengend Snippet: a-f, fEPSPs and PPRs were recorded in SC–CA1 synapses before and after perfusion of scopolamine or physostigmine onto hippocampal slices. Scopolamine application did not affect the fEPSP response (a) and PPRs (d) [All P > 0.05, n = 8 slices]. Physostigmine induced long-term depression for an hour (b, n = 7 slices) and increased PPRs at 20 to 100 msec interstimulus interval (e, n = 7 slices). Scopolamine treatment following physostigmine-induced long-term-depression rescued the depressed fEPSP (c, n = 11 slices) and normalized the increased PPRs at 30 to 100 msec interstimulus interval to control levels (f, n = 11 slices). g, PPRs were significantly decreased at 8 hrs and the reduction was still observed at 24 hrs at 20, 30, and 50 msec interstimulus interval [Sal, SCA (8 h), SCA (24 h): n = 20, 24, 20 slices]. h, Scopolamine decreased PPRs at 20, 30, 50, and 100 msec interstimulus intervals in slices from Mecp2 CTL mice 24hs after the injection. In hippocampal slices from Mecp2 KI mice, PPRs were decreased at 20, 30, and 50 msec interstimulus intervals 8 hr but not 24 hrs after scopolamine treatment [CTL-Sal, CTL-SCA (24 h), KI-Sal, KI-SCA (8 h), KI-SCA (24 h) : n = 20, 20, 17, 15, 14 slices ]. i, Scopolamine decreased PPRs in slices from Bdnf CTL mice 24 hrs after treatment. However, PPRs were not significantly changed in slices from Bdnf cKO mice at 8 or 24 hrs after scopolamine treatment [CTL-Sal, CTL-SCA (24 h), cKO-Sal, cKO-SCA (8 h), cKO-SCA (24 h): n = 31, 27, 21, 11, 26 slices]. Representative traces are from results recorded at 30 msec interstimulus interval in their respective experiments. a,b,d,e: two-sided paired t-test. c,f: One-way RM ANOVA with Tukey’s multiple comparison. g,h,i: One-Way ANOVA with Tukey’s multiple comparison. h,i: two-sided unpaired t-test, Mann-Whitney test, Welch’s correction. Graphs represent mean ± S.E.M., N.S.: not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. Sal: saline, SCA: scopolamine, CTL: littermate control. For detailed statistical information, see .

Article Snippet: After blocking the membranes for 1 hr at room temperature in blocking solution, blots were washed three times with 0.1% TBS-Tween and incubated overnight at 4°C with the following primary antibodies: MeCP2 (1:4,000, Thermo Fisher, PA1–888), phospho-MeCP2 (S421, 1:1,000, a generous gift from Dr. Anne West and ECM Biosciences, MP46111), GAPDH (1:100,000, Cell Signaling Technology, 2118), CREB (1:5,000, Cell Signaling Technology, 9197), phospho-CREB (Ser133, 1:5,000, Merck Millipore, 06–519), BDNF (1:1,500, Abcam, ab108319), CaMKIIα (1:5,000, Cell Signaling Technology, 3357S), CaMKIIβ (1:500,000, Abcam, ab34703), phospho-CaMKII (D21E4, 1:50,000, Cell Signaling Technology, 12716S).

Techniques: Control, Injection, Comparison, MANN-WHITNEY, Saline

Journal: Nature neuroscience

Article Title: Sustained effects of rapidly-acting antidepressants require BDNF-dependent MeCP2 phosphorylation

doi: 10.1038/s41593-021-00868-8

Figure Lengend Snippet: a, pMeCP2 levels were measured in the hippocampus of C57BL/6J mice 24 hrs after pirenzepine treatment (5mg/kg) with Western blot analysis. Pirenzepine significantly increased pMeCP2 levels but not pCREB levels (pMeCP2/MeCP - P = 0.0298, n = 7 mice per group, pCREB/CREB - P = 0.3604, Sal, PRZP: n = 8, 7 mice). b, To investigate whether the scopolamine-induced reduction of PPRs results from M1 antagonism, PPRs were measured in CA1 hippocampal slices from C57BL/6J mice treated with saline or pirenzepine 24 hrs before the hippocampal slice preparation. PPRs were significantly decreased in the pirenzepine-treated group at the 20 msec, 30 msec, 50 msec, and 100 msec interstimulus interval conditions (20, 30, 50, and 100 msec P < 0.05, Sal, PRZP: n = 20, 28 slices). c, To confirm whether the sustained reduction of PPRs by M1-receptor specific antagonism requires pMeCP2, PPRs were measured in CA1 hippocampal slices from Mecp2 KI or CTL mice 24 hrs after pirenzepine treatment. Pirenzepine-treated CTL group showed decreased PPRs compared to saline-treated CTL group, but the effects of pirenzepine were impaired in the Mecp2 KI mice [Genotype x Drug : 20 msec - P = 0.0049, 30 msec - P = 0.0069, 50 msec - P = 0.0083, CTL-Sal, CTL-PRZP, KI-Sal, KI-PRZP: n = 18, 18, 16, 18 slices]. Representative traces are from results recorded at 30 msec interstimulus interval in the respective groups. a,b: two-sided unpaired t-test or Mann-Whitney test, c: two-way ANOVA with Tukey’s multiple comparisons. Graphs represent mean ± S.E.M., N.S.: not significant, *, P < 0.05, **, P < 0.01, Sal: saline, PRZP: pirenzepine, CTL: littermate control. For detailed statistical information, see .

Article Snippet: After blocking the membranes for 1 hr at room temperature in blocking solution, blots were washed three times with 0.1% TBS-Tween and incubated overnight at 4°C with the following primary antibodies: MeCP2 (1:4,000, Thermo Fisher, PA1–888), phospho-MeCP2 (S421, 1:1,000, a generous gift from Dr. Anne West and ECM Biosciences, MP46111), GAPDH (1:100,000, Cell Signaling Technology, 2118), CREB (1:5,000, Cell Signaling Technology, 9197), phospho-CREB (Ser133, 1:5,000, Merck Millipore, 06–519), BDNF (1:1,500, Abcam, ab108319), CaMKIIα (1:5,000, Cell Signaling Technology, 3357S), CaMKIIβ (1:500,000, Abcam, ab34703), phospho-CaMKII (D21E4, 1:50,000, Cell Signaling Technology, 12716S).

Techniques: Western Blot, Saline, Slice Preparation, MANN-WHITNEY, Control

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Schematic (A) and photograph (B) of Raman spectrometry system, diagram of trapping and detection scheme (C), SERS signatures of the nanoparticle variants used (D) and example output of spectrometer during trapping (E).

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques:

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Diagrams of targeted magnetic nanoparticles (A), targeted SERS nanoparticles (B) and untargeted SERS nanoparticles (C). Chemical structures of PSMA targeted-, folate targeted- and untargeted-nanoparticles are presented in the insets.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques:

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Representative Flow Cytometry Analysis of CF 633-maleimide loading on S421 (A) and S440 (B) SERS NPs.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques: Flow Cytometry

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: FESEM images of all nanoparticle formulations: magnetic nanoparticles with (A) reactive thiols, (B) folate, and (C) PSMA inhibitor functionalizations; S421 SERS nanoparticles with (D) reactive thiols, (E) folate, and (F) PSMA inhibitor functionalizations; S440 SERS nanoparticles with (G) reactive thiols, and (H) methoxy group capped functionalizations. Representative close-up images with sizing information of a reactive thiol functionalized (I) magnetic, (J) S421 SERS, and (K) S440 SERS nanoparticle.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques:

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: EDX analysis of reactive thiol functionalized (A-E) magnetic nanoparticles and (F-J) S421 SERS nanoparticles.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques:

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Nanoparticle Tracking Analysis measurements of (A) S421 SERS, (B) S440 SERS and (C) magnetic nanoparticle species with either reactive thiol, methoxy group capped, folate or PSMA inhibitor functionalizations. n=3

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques:

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Statistical summary of hydrodynamic radii measurements of all nanoparticle formulations.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques:

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Statistical summary of hydrodynamic radii measurements of all nanoparticle formulations.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques:

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: UV-Vis absoprtion spectroscopy measurements of (A) S421 SERS, (B) S440 SERS and (C) magnetic nanoparticle species with either reactive thiol, methoxy group capped, folate or PSMA inhibitor functionalizations.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques: Spectroscopy

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Maximum intensity projection of fluorescence microscopy images of HeLa cells incubated with folate conjugated fluorescent magnetic nanoparticles(red) without(A) and with(B) blocking in excess folate. Counterstained with Alexa Fluor 488, WGA membrane stain(green) and Hoechst 33342 nuclear stain(blue).

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques: Fluorescence, Microscopy, Incubation, Blocking Assay, Staining

Journal: Biomedical Optics Express

Article Title: Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles

doi: 10.1364/BOE.410527

Figure Lengend Snippet: Maximum intensity projection of fluorescence microscopy images of LNCaP-PALM-GFP (A) and PC3-PALM-GFP (B) cells stained with anti-PSMA-Cy5 (red) as well as LNaP-PALM-GFP without (C) and with (D) blocking in excess free PSMA inhibitor and PC3-PALM-GFP (E) cells incubated with PSMA inhibitor conjugated fluorescent magnetic nanoparticles(red). Counterstained and Hoechst 33342 nuclear stain(blue) and endogenously expressing PALM-GFP(green) on the membrane.

Article Snippet: For detection of trapping events, two variants of thiolated 120-nm Surface-Enhanced Raman scattering nanoparticles (S440 and S421, Oxonica Materials) were utilized.

Techniques: Fluorescence, Microscopy, Staining, Blocking Assay, Incubation, Expressing