Transcriptome analysis reveals novel pain targets enriched in TRPV1(+) neurons.

<p>(A) qPCR quantification of TRPV1 mRNA levels in samples hybridized on microarrays (n = 4, p<0.001, One-way ANOVA). (B) Relative expression levels of TRPV1 mRNA determined by microarray hybridizations (n = 4, p<0.001, One-way ANOVA with Bonferroni's multiple comparisons test). (C) Correlation matrix of the expression level of all genes (normalized fluorescence intensities) detected using micorarrays. Clustering of overall gene expression is visible between RTX and capsaicin treated samples. (D) qPCR quantification of TRPV1 mRNA levels in samples used for RNA-Seq (n = 3, p<0.001, paired two-tailed t-test). (E) Relative expression levels of TRPV1 mRNA determined by RNA-Seq (n = 3, p<0.001, One-way ANOVA). (F) Overview of the number of target transcripts identified by microarray hybridizations and RNA-Seq. Raw fluorescence intensities of microarrays were background corrected, log2 transformed, normalized, and filtered for expressed genes (Illumina detection p-value <0.01 in at least one of the samples, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115731#s4" target="_blank">Material and Methods</a> sections for details). Sequencing was performed using a 5500xl SOLiD System resulting in 664 million reads in total (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115731#s4" target="_blank">Material and Methods</a> sections for details). The reads were filtered to remove ribosomal RNA, tRNAs, and vector sequences. The remaining reads mapped to 16590 genes of the reference genome (rn5). Read counts were transformed to RPKM values (Reads per kilo base per million), normalized, and filtered to remove weakly expressed transcripts (RPKM>0.1). P-values of differentially expressed genes identified with both methods were adjusted for multiple testing with Benjamini and Hochberg's method, adjusted p-values <0.05 were considered significant.</p

Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

<div><p>Normal and painful stimuli are detected by specialized subgroups of peripheral sensory neurons. The understanding of the functional differences of each neuronal subgroup would be strongly enhanced by knowledge of the respective subgroup transcriptome. The separation of the subgroup of interest, however, has proven challenging as they can hardly be enriched. Instead of enriching, we now rapidly eliminated the subgroup of neurons expressing the heat-gated cation channel TRPV1 from dissociated rat sensory ganglia. Elimination was accomplished by brief treatment with TRPV1 agonists followed by the removal of compromised TRPV1(+) neurons using density centrifugation. By differential microarray and sequencing (RNA-Seq) based expression profiling we compared the transcriptome of all cells within sensory ganglia versus the same cells lacking TRPV1 expressing neurons, which revealed 240 differentially expressed genes (adj. p<0.05, fold-change>1.5). Corroborating the specificity of the approach, many of these genes have been reported to be involved in noxious heat or pain sensitization. Beyond the expected enrichment of ion channels, we found the TRPV1 transcriptome to be enriched for GPCRs and other signaling proteins involved in adenosine, calcium, and phosphatidylinositol signaling. Quantitative population analysis using a recent High Content Screening (HCS) microscopy approach identified substantial heterogeneity of expressed target proteins even within TRPV1-positive neurons. Signaling components defined distinct further subgroups within the population of TRPV1-positive neurons. Analysis of one such signaling system showed that the pain sensitizing prostaglandin PGD₂ activates DP1 receptors expressed predominantly on TRPV1(+) neurons. In contrast, we found the PGD₂ producing prostaglandin D synthase to be expressed exclusively in myelinated large-diameter neurons lacking TRPV1, which suggests a novel paracrine neuron-neuron communication. Thus, subgroup analysis based on the elimination rather than enrichment of the subgroup of interest revealed proteins that define subclasses of TRPV1-positive neurons and suggests a novel paracrine circuit.</p></div

Validation of the transcriptome data by single cell based quantitative High Content Screening (HCS) microscopy focusing on selected signaling-relevant proteins.

<p>(A) Triple staining of the neuronal marker UCHL1 and two different TRPV1 antibodies derived from rabbit and goat, respectively, to facilitate the analysis of various targets. The staining intensities obtained with both TRPV1 antibodies correlated significantly (Spearmans ρ = 0.96, p<2.2e-16). (B-E, G) Co-labeling of TRPV1 and CART (B), Nos1 (C), KChIP1 (D), KChIP2 (E), and CaMKIIα (G). Plots of respective controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115731#pone.0115731.s001" target="_blank">S1 Fig</a>. (F) Average fluorescence intensities of TRPV1 and the indicated targets in TRPV1-negative (grey) and -positive (black) neurons. Signal intensities of all analyzed targets were significantly higher within the TRPV1(+) population (n = 3 with>3000 analyzed neurons per experiment, paired two-tailed t-tests).</p

PGD₂ is a paracrine mediator synthesized in myelinated large-diameter neurons that acts on TRPV1(+) neurons.

<p>(A) Dose-dependent induction of RII phosphorylation in sensory neurons after 1 min stimulation with PGD₂ (EC₅₀ = 377 nM, n = 3,>2000 neurons/condition; one-way ANOVA with Bonferroni's multiple comparisons test). (B) PGD₂ did not induce pRII in non-neuronal cells of the same cultures shown in A. (C) Time course of RII phosphorylation indicating long-lasting effects of PGD₂ (10 µM) on sensory neurons. (D) Stimulation with PGD₂ also results in phosphorylation of the ERK1/2 measured in the same cultures shown in D. (E) Representative experiment demonstrating that induction of RII phosphorylation is enhanced in TRPV1(+) neurons (total of 3664 neurons). Plots of respective controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115731#pone.0115731.s002" target="_blank">S2 Fig</a>. (F) Fold changes of pRII intensities in TRPV1(−) (grey bars) and TRPV1(+) (black bars) neurons after 1 min stimulation with 10 µM PGD₂ (n = 3,>2000 neurons/condition, one-way ANOVA with Bonferroni's multiple comparisons test). (G) Co-labeling of TRPV1 and PTGDS revealing that PTGDS is expressed in neurons lacking TRPV1 (total of 9951 neurons, also refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115731#pone.0115731.s002" target="_blank">S2 Fig</a>. for control plots). (H) Co-labeling of NF200 and PTGDS showing that PTGDS(+) neurons express NF200 (total of 12966 neurons, also refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115731#pone.0115731.s002" target="_blank">S2 Fig</a>. for control plots).(I) Size distribution of PTGDS(+) (green), NF200(+) (red), and all sensory neurons (black) indicating that PTGDS(+) neurons are larger than other neurons. (J) Suggested pathway of interneuronal communication between subgroups of sensory neurons. Large-diameter mechanosensitive neurons express PTGDS resulting in the production of PGD₂, which activates DP1 receptors present on nociceptive neurons expressing TRPV1.</p

Validation of transcriptome data.

<p>(A) Correlation of fold-changes obtained by microarray hybridizations and RNA-Seq (Spearmans ρ = 0.66, p<0.0001). (B) qPCR validation of 14 transcripts identified as differentially expressed by microarray hybridizations or RNA-Seq. The depletion of TRPV1(+) neurons was performed with capsaicin (1 and 10 µM) for 30 min. The reduction of all target transcripts is dose-dependent.</p

Agonist-treatment enables the selective removal of TRPV1(+) neurons.

<p>(A) Immunolabeling of the neuronal marker UCHL1 and TRPV1 in frozen DRG sections and cultured sensory neurons from rat. TRPV1 is selectively expressed in a subpopulation of sensory neurons. (B) Representative view field showing the automated image analysis to quantify TRPV1 expression in cultured sensory neurons. Green encircled objects represent sensory neurons marked by UCHL1. (C) Distribution of TRPV1 immunofluorescence intensities in frozen DRG sections (red line) and cultured sensory neurons (blue line). The scattered line indicates the threshold used to determine the number of TRPV1(+) neurons. (D) Work flow to remove TRPV1(+) neurons from freshly isolated sensory neurons. (E) Immunoblot showing the reduction of TRPV1 protein following depletion of TRPV1(+) neurons with 10 µM capsaicin for 30–120 min. (F) Time-dependent reduction of TRPV1 mRNA after removal of TRPV1(+) neurons with 10 µM capsaicin or 100 nM RTX determined by qPCR. (G) Dose-dependent reduction of TRPV1 mRNA following depletion of TRPV1(+) neurons with RTX for 30 min determined by qPCR. (H) Agonist-treated neurons were cultured overnight, immunostained for TRPV1, and analyzed by quantitative microscopy. Both agonists effectively reduced the number of TRPV1(+) neurons. Scattered lines indicate the threshold used to determine the number of TRPV1(+) neurons. (I) Quantification of TRPV1(+) neurons following treatment with 10 µM Cap or 100 nm RTX, respectively (n = 3, p<0.01, one-way ANOVA with Bonferroni's multiple comparisons test).</p

Expression of BOD1 and PLK1 in human tissues.

<p>(A) BOD1-specific quantitative RT-PCR experiments were carried out in triplicates, using RNA from the indicated tissues. All splice variants (indicated by the respective exon combinations) were investigated. Error bars represent the SEM. (B) Expression levels i.e. reads per kilobase of transcript per million reads mapped (RPKM), corresponding to BOD1 (NM_138369.2) and PLK1 (NM_005030.5) obtained by RNA-Sequencing of commercially available RNA-samples from different brain tissues, induced pluripotent stem cells (IPSC) and human embryonic stem cells (hES).</p

Functional consequences of the absence of BOD1 in patient-derived fibroblasts.

<p>(A) Flow cytometric analysis of cell cycle profile in WT and <i>BOD1</i>^-/- primary fibroblasts electroporated with control or <i>BOD1</i> siRNA. Error bars represent standard deviation. (B) Immunoblotting of BOD1 and tubulin from cell lysates simultaneously electroporated with samples analysed in (A). (C) Representative DIC timelapse imaging of primary fibroblast cells undergoing mitosis. Nuclear Envelope Breakdown (NEB) and Anaphase Onset (AO) are indicated. (D) Cumulative timing of NEB to AO timing in Primary Fibroblast cell lines. Error bars represent standard deviation. P<0.001 for <i>BOD1</i>^-/- cells to each WT sample. Insufficient data collected for <i>BOD1</i>^-/- cells to determine statistical significance. (E) Immunofluorescence localization of PP2A-B56 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (blue), centromeres (detected with ACA) (green), anti-PP2A-B56α (red). (F) Mean B56α levels at kinetochores of WT and <i>BOD1</i>^-/- Primary fibroblasts (P<0.001). Error bars represent SEM. (G) Immunofluorescence localization of PLK1 in WT and <i>BOD1</i>^-/- Primary fibroblasts. DAPI (white), anti-PLK1 (green), ACA (blue). (H-J) Mean PLK1 levels at kinetochores and centrosomes of WT and <i>BOD1</i>^-/- Primary fibroblasts, respectively (P<0.001 in each instance). Error bars represent SEM. (K) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous WT and <i>BOD1</i>^-/- primary fibroblasts. (L) Immunoblotting of PLK1, BOD1 and tubulin in asynchronous and Monastrol arrested WT and <i>BOD1</i>^-/- Primary fibroblasts. (M) Immunofluorescence localization of Bod1 in WT Primary Fibroblasts. DAPI (white), ACA (blue), anti-Plk1 (red), anti-Bod1 (green). Scale = 5 μm. Inset shows a single bioriented kinetochore pair. (N) Immunoblotting of PP2A-B56δ, PLK1 and tubulin in WT primary fibroblast electroporated with indicated combinations of CTR, B56-pool or <i>BOD1</i> siRNA. Rescue of WT primary fibroblasts after siRNA depletion of Bod1 with plasmids expressing GFP fused to either siRNA resistant WT Bod1 or Bod1^T95E. (O) Mitotic profile of WT primary fibroblasts and <i>BOD1</i>^-/- fibroblasts 1 hr after release from RO 3306 into the indicated concentrations of BI 2536. Results show average of three independent experiments. A minimum of 100 mitotic cells counted per condition per experiment. Error bars represent SEM.</p

Neuronal knockdown of Drosophila Bod1 affects learning and synapse development.

<p>(A-B') Knockdown of <i>Drosophila</i> Bod1 using the postmitotic, pan-neuronal promoter elav-Gal4 and three inducible RNAi lines affects non-associative learning in the light-off jump habituation paradigm. Jump responses were induced by repeated light-off pulses for 100 trials with a 1s inter-trial interval. Bod1 knockdown flies of genotypes (A) UAS-Bod1^vdrc105166/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+, plotted as red squares, (B) UAS-Bod1^vdrc27445/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+, plotted as blue squares, and (C) 2xGMR-wIR/+; UAS-Bod1^HMS00720/elav-Gal4, UAS-Dicer2, plotted as green squares, failed to habituate, i.e. to efficiently reduce their jump response upon repeated stimulation. The genetic background controls, generated by crossing the driver line to the respective genetic background of the RNAi line, are shown as grey circles (2xGMR-wIR/+; elav-Gal4, UAS-Dicer2/+). (A', B', C’) Quantification of average jump responses revealed that all three mutant genotypes habituated significantly slower (*** p<0,001). Red bar in (A') Bod1^vdrc105166, TTC = 49.75, n = 143 versus controls: TTC = 20.88, n = 134. Blue bar in (B'): Bod1^vdrc27445, TTC = 61.92, n = 93 versus controls TTC = 28.93, n = 87. Green bar in (C)’ Bod1^HMS00720, TTC = 10.03, n = 70 versus controls TTC = 5.51, n = 68. (D) Knockdown of <i>Drosophila</i> Bod1 using the elav-Gal4 promoter and RNAi lines Bod1^vdrc27445 and Bod1^vdrc105166 consistently affects synaptic branching at the <i>Drosophila</i> Neuromuscular Junction (see text). L3 muscle 4 synapses were labelled with anti-dlg1 and quantified using an in house-developed macro. A Bod1^vdrc27445 (UAS-Bod1^vdrc27445/2xGMR-wIR; elav-Gal4, UAS-Dicer2/+) and control (2xGMR-wIR/+; elav-Gal4, UAS-Dicer2/+) synapse is shown. Top panel in red: dlg1 labelling; bottom panels show the macro-annotated, quantified synapse). Asterisks highlight the increased number of synaptic branching points at the mutant synaptic terminal.</p

Presynaptic localisation of BOD1 in murine corticoneuronal cells.

<p>Representative indirect immunofluorescence confocal image (LSM510) of mouse cortical neurons transfected at day 7 after preparation with 0.1μg BOD1-GFP for 7hrs. Arrows indicate co-localization of BOD1-GFP (in green) with the (pre)synaptic marker anti-Bassoon (red). Insets are magnifications of the boxed area. The range indicator (RI) shows that the images are not overexposed.</p