Journal: bioRxiv

Article Title: The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

doi: 10.1101/2021.07.06.451358

Figure Lengend Snippet: Differential gene expression (DE) for the interaction of Genotype and Treatment [(MUT_MA – MUT_SAL) – (WT_MA – WT_SAL)] was performed using limma and edgeR . Differential exon and intron usage analysis (alternatively spliced; AS) for interaction of Genotype and Treatment [(MUT_MA – MUT_SAL) – (WT_MA – WT_SAL)] was performed using Aspli . (A): Venn diagram comparing RNA-binding targets of hnRNP H with differential gene expression and differential exon or intron usage. The 19 DE genes and 469 AS genes were then compared with the hnRNP H targets that comprised the Genotype x Treatment interactions. Cacna2d2 is the only RNA-binding target of hnRNP H that was both a DE gene and an AS gene. Only the top overlapping and non-overlapping hnRNP H targets and AS genes are indicated. Out of the 19 DE genes, Cacna2d2, Elfn1 , Calb2 , and Zic1 are putative targets of Malat1 (predicted by TargetScan; ) while Camta1 and Pcdh8 are putative targets of Mir124a (predicted by LncRRIsearch; ). (B): Volcano plot of genes showing a Genotype x Treatment interaction in response to methamphetamine. The interaction is expressed as (MUT_MA – MUT_SAL) – (WT_MA – WT_SAL). Cacna2d2 is circled in red. The five overlapping hnRNP H targets and DE genes are circled in purple. (C): hnRNP H preferentially binds to the 3’UTR of Cacna2d2 . Visualization of reads by Integrative Genome Browser for the hnRNP CLIP peak at the 3’UTR of Cacna2d2 . Scale of the plot height is in counts per million (CPM). (D): A G-rich motif was detected at the hnRNP H CLIP peak within the 3’UTR of Cacna2d2 . De novo motif discovery of the binding site was performed in MEME . (E): Interaction plots showing hnRNP H binding to the 3’UTR of Cacna2d2 (left), differential usage of the 3’UTR (middle), and differential gene expression of Cacna2d2 (right) as a function of Genotype and Treatment. The increase in hnRNP H binding to the 3’UTR of Cacna2d2 was associated with decreased 3’UTR usage and increased gene expression of Cacna2d2 in Hnrnph1 mutants. Here, 3’UTR usage is defined as to the number of normalized reads mapped to the 3’UTR portion of Cacna2d2 . The interaction plots were generated from CLIP-seq and RNA-seq data showing average values with standard deviation of the means, with n = 3 per condition.

Article Snippet: Following the imaging, the membranes were blocked with 5% milk for 1 hand probed with anti-CACNA2D2 (1:1000; alomone, Cat#ACC-102) over night at 4°C followed by 1 hour probing with donkey anti-rabbit HRP (1:10,000; Jackson ImmunoResearch Labs Cat#711-035-152).

Techniques: Expressing, RNA Binding Assay, Binding Assay, Generated, RNA Sequencing Assay, Standard Deviation

Journal: bioRxiv

Article Title: The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

doi: 10.1101/2021.07.06.451358

Figure Lengend Snippet: CLIP-seq analysis identified an increase in hnRNP H binding to the distal end of 3’UTR of Cacna2d2 in Hnrnph1 mutants that was associated with decreased usage of the 3’UTR, warranting further validation. (A): Three polyadenylation sites (pA-1, pA-2, and pA-3) are present within the 3’UTR of Cacna2d2 that distinguish isoforms containing 3’UTR of different lengths (UCSC genome browser). Primers were designed to detect difference in usage of the proximal and distal end of the 3’UTR of Cacna2d2 as well as exons 37-38 and exons 3-4. The schematic indicates the positions of these primers. SAL = saline; MA = methamphetamine; WT = wild-types; H1 MUT = Hnrnph1 mutants. (B): The left striata were harvested from mice in the same way as in CLIP-seq followed by RNA extraction, cDNA library generation with oligo-DT primers, and RT-qPCR to detect differential usage of the regions at the 3’UTR of Cacna2d2 and upstream of it. No differences were found at exons 3-4 [F(1,39) Genotype x Treatment = 0.650, p = 0.425], exons 37-38 [F(1,39) Genotype x Treatment = 0.900, p = 0.349], or at the proximal end of the 3’UTR [F(1,39) Genotype x Treatment = 0.198, p = 0.659]. Although no significant Genotype x Treatment interaction was detected for the usage of the distal end of the 3’UTR [F(1,39) Genotype x Treatment = 1.485, p = 0.230], there was a significant main effect of Treatment [F(1,39) Treatment = 10.772, p = 0.002]. In examining the effect of Treatment at each level of Genotype, a significant, simple main effect of Treatment was found in the Hnrnph1 mutants [F(1,40) Treatment = 9.368, p = 0.004] but not in the wild-types [F(1,40) Treatment = 2.993, p = 0.091]. Subsequent analysis revealed decreased methamphetamine-induced usage of the distal end of 3’UTR in Hnrnph1 mutants compared to wild-types [t(40) = −3.061, *p = 0.004], with no significant genotypic difference between saline groups [t(40) = −1.730, p = 0.091]. The normalized 2 −ΔΔCT values from three independent replicates are shown in the plots. (C): The right striata of the same mice from the RT-qPCR analysis were collected for Western blot analysis to assess differences in CACNA2D2 protein expression. Immunoblots from three separate replicates are shown on the left with quantification shown on the right. There was no significant genotypic difference in the level of CACNA2D2 protein in saline or methamphetamine treatment groups [F(1,40) Genotype x Treatment = 2.587, p = 0.117]. (D): Schematics showing the putative interaction between hnRNP H-mediated selection of polyadenylation site in Cacna2d2. We proposed that binding of hnRNP H to the pA-3 site within the 3’UTR of Cacna2d2 blocks the usage of pA-3 to produce transcripts with a shorter 3’UTR in Hnrnph1 mutants in response to methamphetamine, (shown on right). This observation was identified in alternative splicing analysis ( , middle panel) and subsequently validated in the RT-qPCR ( , rightmost panel).

Article Snippet: Following the imaging, the membranes were blocked with 5% milk for 1 hand probed with anti-CACNA2D2 (1:1000; alomone, Cat#ACC-102) over night at 4°C followed by 1 hour probing with donkey anti-rabbit HRP (1:10,000; Jackson ImmunoResearch Labs Cat#711-035-152).

Techniques: Binding Assay, RNA Extraction, cDNA Library Assay, Quantitative RT-PCR, Western Blot, Expressing, Selection

Journal: The Journal of Clinical Investigation

Article Title: Gabapentinoid treatment promotes corticospinal plasticity and regeneration following murine spinal cord injury

doi: 10.1172/JCI130391

Figure Lengend Snippet: (A) Schematic of retrograde labeling of corticospinal neurons. (B) Representative fluorescence images of corticospinal neurons identified by retrograde labeling after Fluoro-Gold injections into the cervical spinal cords of adult GFP-M mice. Sagittal sections of the mouse brain were immunostained with α2δ2 antibody (n = 4 independent replicates). Scale bar: 50 μm. (C) Immunoblot shows α2δ2 expression in the mouse sensory-motor cortex during postnatal development. Under reducing conditions, the α2δ2 antibody recognizes 2 bands at approximately 130 and 105 kDa. Tuj1 is used as loading control. (D) Quantification of C. Data normalized using loading control (linear trend test **P < 0.01, n = 3 biological replicates). (E) Representative fluorescence images of corticospinal neurons from mouse brains at different ages. Scale bar: 50 μm. (F) Quantification of E. Box plot (minimum to maximum) and line at median (1-way ANOVA followed by Dunnett post test *P < 0.05; **P < 0.01; P7 n = 5, P14 n = 3, and P28 n = 3 mice, 60–95 neurons per condition). (G) Raster plots show spontaneous firing within layer V of the sensory-motor cortex at different stages of brain development. (H) Quantification of G. Mean and SEM (linear trend test *P < 0.05; P7 n = 5, P14 n = 7, and P28 n = 8 mice). (I) Schematic representation of C5 SCI experimental model. (J) Representative fluorescence images of retrogradely labeled corticospinal neurons (yellow arrows) 7 days after C5 SCI. DPO, days after operation. Sagittal sections of the mouse brain (right hemisphere) were immunostained with α2δ2 antibody. Scale bar: 50 μm. (K) Quantification of J. Mean and SEM (unpaired 2-tailed Student’s t test **P < 0.01; sham n = 4 and SCI n = 4 mice, 229–302 neurons per condition).

Article Snippet: The following antibodies were used: mouse monoclonal anti-βIII tubulin (Tuj1) (801202, RRID:AB_10063408, BioLegend), rabbit polyclonal anti-βIII tubulin (T2200, RRID:AB_262133, Sigma-Aldrich), mouse monoclonal anti-NeuN (MAB377, RRID:AB_2298772, Millipore), Alexa Fluor 488 Phalloidin (A12379, RRID:AB_2315147, Invitrogen), rabbit polyclonal anti-α2δ2 (ACC-102, RRID:AB_11124467, Alomone Labs), rabbit monoclonal anti–c-Fos (9F6) (2250S, RRID:AB_2247211, Cell Signaling Technology), rabbit polyclonal anti-GFAP (Z0334, RRID:AB_10013382, Dako), rabbit polyclonal anti-mCherry (ab167453, RRID: AB_2571870, Abcam), rabbit monoclonal anti-PKC gamma (59090, RRID: AB_2799557, Cell Signaling Technology), chicken polyclonal anti-Homer1 (160006, RRID:AB_2631222, Synaptic Systems), guinea pig polyclonal anti-VGLUT1 (135304, RRID:AB_887878, Synaptic Systems), rabbit polyclonal anti–Caspase-3 (ab 13847, RRID AB_443014, Abcam),and chicken polyclonal anti-GFP (GFP-1020, RRID:AB_10000240, Aves Labs).

Techniques: Labeling, Fluorescence, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Gabapentinoid treatment promotes corticospinal plasticity and regeneration following murine spinal cord injury

doi: 10.1172/JCI130391

Figure Lengend Snippet: (A) Experimental scheme of B. (B) Representative fluorescence images of C7 spinal cord sections from mice with sham operation or unilateral PTX performed at P10. PKCγ staining is shown to confirm lesion completeness. Scale bar: 500 μm. D, dorsal; V, ventral. (C) Quantification of B. Mean and SEM (unpaired 2-tailed Student’s t test **P < 0.01; sham n = 6 and PTX n = 5 mice). (D) Coronal sections of the medullary pyramid showing BDA-labeled corticospinal axons. Scale bar: 50 μm. (E) Quantification of D (unpaired 2-tailed Student’s t test NS, not significant; sham n = 6 and PTX n = 5 mice). (F) Representative fluorescence images of corticospinal neurons 32 days after sham or PTX at P10. Sagittal sections of the mouse brain (right hemisphere) were immunostained with α2δ2 antibody. Scale bar: 25 μm. (G) Quantification of F (unpaired 2-tailed Student’s t test **P < 0.01; sham n = 6 and PTX n = 5 mice; 278–369 neurons per condition). (H) Representative fluorescence images of C7 spinal cord sections from mice with unilateral PTX performed at P10. The arrows indicate excitatory synaptic puncta along sprouting corticospinal axons. Inset, 3D reconstruction of the region in the main panel indicated by the yellow arrow. Scale bar: 100 μm. (I) Immunoblot showing α2δ2 expression in E17.5 cortical neurons cultured for 48 hours after electroporation with GFP plus either control (CTR) or Cacna2d2-expressing plasmids. Tuj1 is shown as a loading control (n = 3 independent replicates per condition). (J) Representative fluorescence images of E17.5 cortical neurons cultured for 48 hours after electroporation with GFP plus either CTR or Cacna2d2-expressing plasmids. DIV, day in vitro. Scale bar: 100 μm. (K) Quantification of J. Mean and SEM (unpaired 2-tailed Student’s t test ***P < 0.001; triplicate experiments; 134–144 neurons per condition). (L) Representative fluorescence images of E17.5 cortical neurons cultured for 48 hours in the presence of either vehicle (0.9% saline) or 250 μM GBP. Scale bar: 100 μm. (M) Quantification of L. Mean and SEM (unpaired 2-tailed Student’s t test ***P < 0.001; 4 independent experiments; 163–165 neurons per condition).

Article Snippet: The following antibodies were used: mouse monoclonal anti-βIII tubulin (Tuj1) (801202, RRID:AB_10063408, BioLegend), rabbit polyclonal anti-βIII tubulin (T2200, RRID:AB_262133, Sigma-Aldrich), mouse monoclonal anti-NeuN (MAB377, RRID:AB_2298772, Millipore), Alexa Fluor 488 Phalloidin (A12379, RRID:AB_2315147, Invitrogen), rabbit polyclonal anti-α2δ2 (ACC-102, RRID:AB_11124467, Alomone Labs), rabbit monoclonal anti–c-Fos (9F6) (2250S, RRID:AB_2247211, Cell Signaling Technology), rabbit polyclonal anti-GFAP (Z0334, RRID:AB_10013382, Dako), rabbit polyclonal anti-mCherry (ab167453, RRID: AB_2571870, Abcam), rabbit monoclonal anti-PKC gamma (59090, RRID: AB_2799557, Cell Signaling Technology), chicken polyclonal anti-Homer1 (160006, RRID:AB_2631222, Synaptic Systems), guinea pig polyclonal anti-VGLUT1 (135304, RRID:AB_887878, Synaptic Systems), rabbit polyclonal anti–Caspase-3 (ab 13847, RRID AB_443014, Abcam),and chicken polyclonal anti-GFP (GFP-1020, RRID:AB_10000240, Aves Labs).

Techniques: Fluorescence, Staining, Labeling, Western Blot, Expressing, Cell Culture, Electroporation, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: Gabapentinoid treatment promotes corticospinal plasticity and regeneration following murine spinal cord injury

doi: 10.1172/JCI130391

Figure Lengend Snippet: (A) Experimental scheme of B. (B) Representative fluorescence images of C7 spinal cord sections from adult mice with unilateral PTX performed at 8 weeks of age. PKCγ staining is shown to confirm lesion completeness. The arrows indicate sprouting corticospinal axons (bottom panels). Scale bar: 500 μm. D, dorsal; V, ventral. (C) Quantification of B. Mean and SEM (unpaired 2-tailed Student’s t test *P < 0.05; vehicle n = 6 and GBP n = 6 mice). (D) Coronal sections of the medullary pyramid showing BDA-labeled corticospinal axons. Scale bar: 50 μm. (E) Quantification of D (unpaired 2-tailed Student’s t test NS, not significant; vehicle n = 6 and GBP n = 6 mice). (F) α2δ2 expression in corticospinal neurons 4 weeks after PTX. Sagittal sections of the mouse brain (left and right hemispheres) were immunostained with α2δ2 antibody. Mean and SEM (1-way ANOVA followed by Dunnett post test *P < 0.05; **P < 0.01; vehicle n = 6 and GBP n = 6 mice, 349–396 neurons per condition). (G) Representative fluorescence images of C7 spinal cord sections from mice with unilateral PTX performed in adulthood treated with GBP. The arrows indicate excitatory synaptic puncta along sprouting corticospinal axons. Inset, 3D reconstruction of the region in the main panel indicated by the yellow arrow. Scale bar: 100 μm.

Article Snippet: The following antibodies were used: mouse monoclonal anti-βIII tubulin (Tuj1) (801202, RRID:AB_10063408, BioLegend), rabbit polyclonal anti-βIII tubulin (T2200, RRID:AB_262133, Sigma-Aldrich), mouse monoclonal anti-NeuN (MAB377, RRID:AB_2298772, Millipore), Alexa Fluor 488 Phalloidin (A12379, RRID:AB_2315147, Invitrogen), rabbit polyclonal anti-α2δ2 (ACC-102, RRID:AB_11124467, Alomone Labs), rabbit monoclonal anti–c-Fos (9F6) (2250S, RRID:AB_2247211, Cell Signaling Technology), rabbit polyclonal anti-GFAP (Z0334, RRID:AB_10013382, Dako), rabbit polyclonal anti-mCherry (ab167453, RRID: AB_2571870, Abcam), rabbit monoclonal anti-PKC gamma (59090, RRID: AB_2799557, Cell Signaling Technology), chicken polyclonal anti-Homer1 (160006, RRID:AB_2631222, Synaptic Systems), guinea pig polyclonal anti-VGLUT1 (135304, RRID:AB_887878, Synaptic Systems), rabbit polyclonal anti–Caspase-3 (ab 13847, RRID AB_443014, Abcam),and chicken polyclonal anti-GFP (GFP-1020, RRID:AB_10000240, Aves Labs).

Techniques: Fluorescence, Staining, Labeling, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca 2+ channels

doi: 10.1073/pnas.1702991114

Figure Lengend Snippet: Antibodies used in the current study with corresponding dilutions and origin

Article Snippet: A list of antibodies used in the current study with corresponding dilutions and origin are presented in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody Dilution Active zone proteins RBP-1 (RIM-binding protein-1) 316003 (SySy) 1:1,000 RBP-2 (RIM-binding protein-2) 316103 (SySy) 1:1,000 RBP-2 (RIM-binding protein-2) 4193 (custom) 1:1,000 RIM 1 (RIM1 central domains) R809 (custom) 1:2,000 RIM 1/2 (1α/2α N terminus + rabphillin) U1565 (custom) 1:2,000 Liprin α3 4396 (custom) 1:5,000 ELKS 1/2aB 4790 (custom) 1:500 CASK 75–000 (Neuromab) 1:1,000 Mint1 P932 (custom) 1;1000 Veli1,2,3 T813 (custom) 1:1,000 Munc13-1 126103 (SySy) 1:1,000 Ribeye Maxeiner et al. ( 32 ) 1:1,000 Ca 2+ channels Ca 2+ v1.2-α1C voltage-gated channel ACC-003 (Alomone) 1:200 Ca 2+ v1.3-α1D voltage-gated channel ACC-005 (Alomone) 1:200 Ca 2+ v1.4-α1D voltage-gated channel Gift from Frank Schmitz 1:1,000 Ca 2+ v-α2δ1 voltage-gated channel ACC-015 (Alomone) 1:500 Ca 2+ v-α2δ2 voltage-gated channel ACC-102 (Alomone) 1:500 Ca 2+ v-α2δ3 voltage-gated channel ACC-103 (Alomone) 1:500 Ca 2+ v-α2δ4 voltage-gated channel ACC-104 (Alomone) 1:500 Ca 2+ β1 voltage-gated channel ACC-106 (Alomone) 1:250 Ca 2+ β2 voltage-gated channel ACC-105 (Alomone) 1:250 Ca 2+ β3 voltage-gated channel ACC-108 (Alomone) 1:250 Ca 2+ β4 voltage-gated channel 75–054 (Neuromab) 1:250 Exocytosis proteins Syntaxin 1 HPC-1 (custom) 1:500 SNAP 25 71.1 (custom) 1:2,000 Synaptobrevin 2 69.1 (custom) 1:2,500 Synaptotagmin 1 41.1 (custom) 1:1,000 Loading control TUJ1 T2200 (Sigma) 1:5,000 Open in a separate window Antibodies used in the current study with corresponding dilutions and origin

Techniques: