Journal: bioRxiv

Article Title: Behavior- and circuit-specific cortico-striatal decoupling during the early development of Parkinson’s disease-like syndrome

doi: 10.1101/2024.08.13.607859

Figure Lengend Snippet: A . Schematic diagram showing the experimental design for analyzing cell type-specific cortico-striatal coupling. B . Immunofluorescence images showing the colocalization between DRD1 (red) and DIO-GCaMP (green) in D1R-Cre mice (top panels), and the colocalization between DRD2 (magenta) and DIO-GCaMP (green) in the D2R-Cre mice (bottom panels). Scale bar: 10μm. C, E, G . Z-scored average traces of M1 (red) and STR dMSN (light green) and iMSN (dark green) Ca 2+ activities temporally aligned to the initiation of contralateral turning (C), licking (E), and digging (G). D, F, H . Comparison of cell type-specific cortico-striatal coupling indices for turning (D), licking (F), and digging behavior (H, P = 0.0087). Mann-Whitney test. ** P <0.01.

Article Snippet: The drugs (all compounds from Merck), including D1R agonist (SKF81297; 1mg/kg), D2R agonist (quinpirole; 0.5mg/kg), L-DOPA (1mg/kg) together with benserazide (15mg/kg), were dissolved in 0.9% saline and filtered through a PES membrane filter unit (0.22μm, Millex).

Techniques: Immunofluorescence, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Behavior- and circuit-specific cortico-striatal decoupling during the early development of Parkinson’s disease-like syndrome

doi: 10.1101/2024.08.13.607859

Figure Lengend Snippet: A-E. Dual fiber photometry imaging of M1 and striatal dMSN in PFF- and PBS-injected mice at 2 Mpi (B-C), and 3-4 Mpi (D-E) during digging. (A) Scheme of virus injection and optic fiber implantation. Note the CreON-GCaMP virus was injected into the STR of D1R-Cre mice, while CreOFF-GCaMP virus was injected into the STR of D2R-Cre mice. Right panel, experiment timeline. (B, D) Z-scored average traces of M1 (red) and STR dMSN (aqua) Ca 2+ activities temporally aligned to the initiation of digging. Left, PBS; Right, PFF. (C, E) Quantitative analyses of the M1-dMSN coupling and plateau amplitude of dMSN Ca 2+ activities at 2 Mpi (C) and 3-4 Mpi (E). The hollow signal represents D1R-Cre mice; the cross signal represents D2-Cre mice. (C1, E1) Coupling indices (C1, P = 0.0043. E1, P = 0.0062). (C2, E2) Phase lags (E2, P = 0.0043. G2, P = 0.0062). (C3, E3) Plateau amplitude of dMSN Ca 2+ activities (E3, P = 0.0451). F-J. Dual fiber photometry imaging of M1 and striatal iMSN in PFF- and PBS-injected mice at 2 Mpi (G-H), and 3-4 Mpi (I-J) during digging. Right panel, experiment timeline. (F) Scheme of virus injection and optic fiber implantation. (G, I) Z-scored average traces of M1 (red) and striatal iMSN (dark green) Ca 2+ activities temporally aligned to the initiation of digging. Left, PBS; Right, PFF. (H, J) Quantitative analyses of the M1-iMSN coupling and plateau amplitude of iMSN Ca 2+ activities at 2 Mpi (H) and 3-4 Mpi (J). (H1, J1) Comparison of the coupling indices. (H2, J2) Comparison of the phase lags. (H3, J3) Comparison of the plateau amplitude of iMSN Ca 2+ activities. Mann-Whitney test. * P <0.05. ** P <0.01.

Article Snippet: The drugs (all compounds from Merck), including D1R agonist (SKF81297; 1mg/kg), D2R agonist (quinpirole; 0.5mg/kg), L-DOPA (1mg/kg) together with benserazide (15mg/kg), were dissolved in 0.9% saline and filtered through a PES membrane filter unit (0.22μm, Millex).

Techniques: Imaging, Injection, Virus, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Behavior- and circuit-specific cortico-striatal decoupling during the early development of Parkinson’s disease-like syndrome

doi: 10.1101/2024.08.13.607859

Figure Lengend Snippet: A. Schematic diagram for pharmacological experiments. i.p., intraperitoneal administration. B-E. Dual fiber photometry imaging of M1 and STR in PFF- and PBS-injected mice at 4Mpi during digging, after ip. SAL (saline), L-dopa, SKF (SKF-81297, a D1R selective agonist), or Quin (quinpirole, a D2R selective agonist). (B) Z-scored average traces of M1 (red) and STR (green) temporally aligned, digging-associated Ca 2+ activities after administration of drugs. Left, PBS; Right, PFF. (C, D, E) Quantitative analyses of the digging-associated cortico-striatal coupling (C,D) and amplitude of striatal Ca 2+ activities (E) after administration of drugs. (C1) Comparison of the phase lags (SAL-PBS vs. SAL-PFF: Mann-Whitney test, P = 0.0152. PFF groups: SAL-PFF as control, F(1.588, 9.529) = 9.270, SAL-PFF vs. Ldopa-PFF, P =0.0156; SAL-PFF vs. SKF-PFF, P = 0.0328). (C2) Comparison of the phase lags, after saline or quinpirole administration. (D1) Comparison of the coupling indices (SAL-PBS vs. SAL-PFF: Mann-Whitney test, P = 0.0043). (D2) Comparison of the coupling indices, after saline or quinpirole administration. (E1) Comparison of the plateau amplitude of striatal activity (SAL-PBS vs. SAL-PFF: Mann-Whitney test, P = 0.0424. PFF groups: SAL-PFF as control, F(1.453, 8.719) = 6.152, SAL-PFF vs. SKF-PFF, P = 0.0459). (E2) Comparison of the plateau amplitude of striatal activity, after saline or quinpirole administration. F-G. Effect of dopaminergic drugs on digging behavior. (F) Comparison of digging duration (PFF groups: SAL-PFF as control, F(1.463, 7.317) = 19.97, SAL-PFF vs. Ldopa-PFF, P = 0.0018; SAL-PFF vs. SKF-PFF, P = 0.0388). (G) Comparison of digging frequency (PFF groups: SAL-PFF as control, F(2.120, 10.60) = 11.78, SAL-PFF vs. SKF-PFF, P = 0.0143; SAL-PFF vs. Quin-PFF, P = 0.0192). One-way ANOVA followed by Dunnett’s multiple comparisons test, unless otherwise noted. Wilcoxon matched-pairs test (C2, D2, E2). * P <0.05. ** P <0.01.

Article Snippet: The drugs (all compounds from Merck), including D1R agonist (SKF81297; 1mg/kg), D2R agonist (quinpirole; 0.5mg/kg), L-DOPA (1mg/kg) together with benserazide (15mg/kg), were dissolved in 0.9% saline and filtered through a PES membrane filter unit (0.22μm, Millex).

Techniques: Imaging, Injection, Saline, Comparison, MANN-WHITNEY, Control, Activity Assay

Journal: bioRxiv

Article Title: YTHDF1 mediates translational control by m6A mRNA methylation in adaptation to environmental challenges

doi: 10.1101/2024.08.07.607063

Figure Lengend Snippet: (A) A schematic showing the timeline to examine cocaine’s sensitization effect in the open field box. (B) A schematic showing the in vivo fiber photometry recording setup. (C, D) Cocaine-induced locomotor sensitization. (C) D1-Cre;Mettl14 f/+ (Ctrl, blue) and D1-Cre;Mettl14 f/f (KO, green) mice, n=7/genotype. (D) A2A-Cre;Mettl14 f/+ (Ctrl, blue) and A2A-Cre;Mettl14 f/f (KO, red) mice, n=7/genotype. Locomotor activity was recorded for 60 min after saline/cocaine injection. Total distance traveled was recorded. (E) Fiber photometry recordings from D1 striatal neurons. Left: representative Ca 2+ traces from D1-Cre;Mettl14 f/+ mice (Ctrl, blue) after saline and cocaine injection using fiber photometry. Right: representative Ca 2+ traces from D1-Cre;Mettl14 f/f mice (KO, green) after saline and cocaine injection using fiber photometry. (F) Left bar graph: Mean Ca 2+ activity of D1-Cre;Mettl14 f/+ mice (Ctrl, blue) and D1-Cre;Mettl14 f/f (KO, green) mice from 15 min fiber photometry recording after saline (S) and cocaine (C) injection. *: P=0.0163, paired T-test. ns: P=0.0702, paired T-test. **: P=0.0010, 2-way ANOVA. Right bar graph: Peak Ca 2+ transients level comparison. **: P=0.0029, paired T-test. Ns: P=0.1250, paired T-test. ****: P<0.0001, 2-way ANOVA, n=5. (G) Fiber photometry recordings from D2 striatal neurons. Left: representative Ca 2+ traces from A2A-Cre;Mettl14 f/+ mice (Ctrl, blue) after saline and cocaine injection using fiber photometry. Right: representative Ca 2+ traces from A2A-Cre;Mettl14 f/f mice (KO, red) after saline and cocaine injection using fiber photometry. (H) Left bar graph: Mean Ca 2+ activity of A2A-Cre;Mettl14 f/+ mice (Ctrl, blue) and A2A-Cre;Mettl14 f/f mice (KO, red) from 15 min fiber photometry recording after saline (S) and cocaine (C) injection. **: P=0.0020, paired T-test. Ns: P=0.0690, paired T-test. ***: P=0.0007, 2-way ANOVA. Right bar graph: Peak Ca 2+ transients level comparison. **: P=0.0011, paired T-test. *: P=0.0150, paired T-test. ***: P=0.0007, 2-way ANOVA, n=5. All data expressed as mean ± SEM. Overall, Mettl14 deficiency blunted the cellular responses in both D1 and D2 SPNs, but resulted in opposite behavioral outcomes observed in mice after cocaine treatment.

Article Snippet: To selectively delete Ythdf1 in D1 or D2 SPNs, we crossed Ythdf1 f/f mice to a D1 receptor promoter-driven Cre recombinase (D1-Cre) transgenic line (B6.FVB(Cg)-Tg(Drd1-cre)EY262Gsat/Mmucd, RRID: MMRRC-030989-UCD) or an adenosine 2A receptor promoter-driven Cre recombinase (A2A-Cre) transgenics line (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd, RRID: MMRRC_036158-UCD).

Techniques: In Vivo, Activity Assay, Saline, Injection, Comparison

Journal: bioRxiv

Article Title: YTHDF1 mediates translational control by m6A mRNA methylation in adaptation to environmental challenges

doi: 10.1101/2024.08.07.607063

Figure Lengend Snippet: (A) Cocaine-induced locomotor sensitization in D1-Cre;Ythdf1 f/+ mice (Ctrl, blue) and D1-Cre;Ythdf1 f/f mice (KO, cyan). Total distance traveled (cm) was recorded for 60 min after saline/cocaine injection. n=8. (B) Cocaine-induced locomotor sensitization in A2A-Cre;Ythdf1 f/+ mice (Ctrl, blue) and A2A- Cre;Ythdf1 f/f mice (KO, magenta). n=8. (C) The rotarod motor learning in D1-Cre;Ythdf1 f/+ mice (Ctrl, blue) and D1-Cre;Ythdf1 f/f mice (KO, cyan). Performance was recorded as latency to fall (s), n=5. (D) The rotarod motor learning in A2A-Cre;Ythdf1 f/+ mice (Ctrl, blue) and A2A-Cre;Ythdf1 f/f mice (KO, magenta). N=5. (E) The sensitization of haloperidol-induced catalepsy response in A2A-Cre;Ythdf114 f/+ mice (Ctrl, blue) and A2A-Cre;Ythdf1 f/f mice (KO, magenta). Catalepsy duration was recorded (s). ***: P=0.0003, 2-way ANOVA, n=7. All data expressed as mean ± SEM. Ythdf1 deletion resembles impairment caused by Mettl14 deletion in a cell type specific manner. YTHDF1 is potentially the main downstream reader protein that regulating translation in response to stimulation and during learning in the striatum.

Article Snippet: To selectively delete Ythdf1 in D1 or D2 SPNs, we crossed Ythdf1 f/f mice to a D1 receptor promoter-driven Cre recombinase (D1-Cre) transgenic line (B6.FVB(Cg)-Tg(Drd1-cre)EY262Gsat/Mmucd, RRID: MMRRC-030989-UCD) or an adenosine 2A receptor promoter-driven Cre recombinase (A2A-Cre) transgenics line (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd, RRID: MMRRC_036158-UCD).

Techniques: Saline, Injection

Journal: bioRxiv

Article Title: YTHDF1 mediates translational control by m6A mRNA methylation in adaptation to environmental challenges

doi: 10.1101/2024.08.07.607063

Figure Lengend Snippet: (A) Representative images of de novo protein synthesis measured by HPG incorporation in the striatal neurons from wild type and Ythdf1 KO P1 mice. Three experimental conditions were compared: HPG+CHX group as negative control, HPG group as baseline condition and HPG+SKF (dopamine D1 receptor agonist) group to test the response after cAMP elevation. Blue: DAPI, red: MAP2, green: HPG tagged newly synthesized protein. Scale bar, 20um (B) Quantification of the HPG expression intensity in CHX, HPG and SKF group in wild type (Ctrl) and Ythdf1 KO striatal neurons. Genotype main effect, p<0.0001, genotype x time interaction, p<0.0001, 2-way ANOVA. HPG vs. SKF treatment: ****: P<0.0001(ctrl), ns: P=0.8390 (KO), paired t-test. Each group contained 3 replicates. (C) Gene ontology (GO) analysis of the upregulated YTHDF1 transcripts after cocaine treatment. (D) Venn diagram depicting the number of YTHDF1 targets after saline and cocaine treatment. (E) UHPLC-MS/MS analysis of m 6 A level in the striatum after saline and cocaine treatment. Striatal neurons with Ythdf1 deficiency have a higher baseline de novo protein synthesis rate but are incapable of responding to stimulations. At the molecular level, boosting dopamine release by cocaine drastically increased YTHDF1 binding to many mRNA targets in the striatum.

Article Snippet: To selectively delete Ythdf1 in D1 or D2 SPNs, we crossed Ythdf1 f/f mice to a D1 receptor promoter-driven Cre recombinase (D1-Cre) transgenic line (B6.FVB(Cg)-Tg(Drd1-cre)EY262Gsat/Mmucd, RRID: MMRRC-030989-UCD) or an adenosine 2A receptor promoter-driven Cre recombinase (A2A-Cre) transgenics line (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd, RRID: MMRRC_036158-UCD).

Techniques: Negative Control, Synthesized, Expressing, Saline, Tandem Mass Spectroscopy, Binding Assay

Journal: bioRxiv

Article Title: Beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy

doi: 10.1101/2024.07.31.605623

Figure Lengend Snippet: ( A ) Initial velocity of optimal Src peptide (AEEEIYGEFEAKKKK) phosphorylation by Src (V0) alone or in the presence of βarr1 WT or βarr1 mutants. V2Rpp was added to each reaction. Individual data, mean ±SD of five independent experiments is shown. Statistical differences were determined by one-way ANOVA and Dunnett’s multiple comparison test. ( B ) Effect of βarr1 mutations in SH3 binding sites on Src activity downstream of β2V2. β2V2, Src and βarr1 were transiently expressed in HEK-293 βarr1/βarr2 dKO cells. 48 hours after transfection with 16-hour starvation, cells were stimulated with 10 uM of BI-167107 for 10 minutes at 37 °C and lysed. Left panel: representative Western blots. Right panel: densitometry analysis of Src phosphorylation normalized to Src phosphorylations in control cells (no βarr1 transfected). Statistical differences were determined by one-way ANOVA and Dunnett’s multiple comparison test (n=6). ( C ) Effect of βarr1 mutations in SH3 binding sites on endogenous Src activity downstream of D1R. D1R and βarr1 were transiently expressed in HEK-293 βarr1/βarr2 dKO cells. 48 hours after transfection with 16-hour starvation, cells were stimulated with 10 uM of dopamine for 5 minutes at 37 °C and lysed. Left panel: representative Western blots. Right panel: densitometry analysis of Src phosphorylation normalized to phospho-Src in unstimulated cells. Statistical differences were determined by one-way ANOVA and Dunnett’s multiple comparison test (n=4). βarr1 mutants numbering is the same as in (B). ( D ) Structural superposition of Src–βarr1 (SH3, magenta; βarr1, green) with the β1V2R–βarr1 complex (β1V2R, slate; PDB: 6TKO), cartoon representation. The clashing region is indicated with the red arrow. ( E ) SH3 binding interferes with the core coupling of βarr1 to phosphorylated V2R in membranes. Top left panel: representative experiment of βarr1 finger loop bimane fluorescence. Top right panel: peak fluorescence of βarr1 finger loop bimane; individual data, mean ±SD of six independent experiments is shown. Statistical differences were determined by one-way ANOVA and Dunnett’s multiple comparison test (* - P=0.0174, **** - P<0.0001). Bottom panel: schematic of SH3-mediated disruption of βarr1 core coupling to V2R.

Article Snippet: HEK-293 βarr1/βarr2 dKO cells ( ) were co-transfected with receptor (FLAG-D1R or chimeric FLAG-β2V2), Src (only for chimeric FLAG-β2V2) and wild-type or mutant βarr1 with a C-terminal HA tag with a 1:5 DNA:FuGENE®6 (Promega) ratio according to the manufacturer’s instructions.

Techniques: Comparison, Binding Assay, Activity Assay, Transfection, Western Blot, Control, Fluorescence, Disruption