Journal: Pain

Article Title: Episodic pain in Fabry disease is mediated by a heat shock protein-TRPA1 axis

doi: 10.1097/j.pain.0000000000003814

Figure Lengend Snippet: Lumbar DRG neurons were isolated from Sprague-Dawley rats, cultured, and incubated overnight with the HSP70 inhibitor VER15508 (VER, 25 μM) or the HSP90 inhibitor 17-AAG (AAG, 0.5 μM). Neurons were then exposed to heat or sham treatment prior to calcium imaging (A). (B) Representative traces of AITC-induced calcium transients from sham ( top ) or heat ( bottom ) treated neurons. Incubation with VER increased the proportion of AITC-responsive neurons at baseline and following heat-treatment, whereas overnight incubation with AAG decreased the AITC responders regardless of heat or sham treatment (C). Neither VER nor AAG exposure sensitized the magnitude of AITC-induced calcium transients (D). Each dot represents the average of all neurons recorded per rat (n=3 rats, 50-90 neurons per rat). In a separate experiment, DRG neurons cultured from Sprague-Dawley rats were acutely incubated with VER or AAG for 30 minutes prior to calcium imaging (E). Representative traces are shown in (F). Acute incubation with either VER or AAG increased the proportion of AITC-responsive neurons (G) and the magnitude of AITC-evoked calcium transients (H) Each dot represents the average of all neurons per rat (n=4 rats, 50-90 neurons per rat). (C) Data were analyzed using 2-way ANOVA with repeated measures and exhibited a main effect of drug: p<0.05 along with a significant interaction between drug and heat treatment: p<0.05. (G-H) Data were analyzed using 1-way ANOVA with repeated measures and exhibited a main effect of drug: p<0.05. Tukey’s post hoc was performed for the effect of drug (C, G, and H) and the interaction between drug and heat treatment (C); * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.005.

Article Snippet: Between 1-2 hours after plating, cells were fed with either complete DRG media or complete DRG media supplemented with VER155008 (25 μM, MedChemExpress), 17-AAG (0.5 μM, MedChemExpress), or recombinant human HSP70 (5 μg/mL, R&D Systems, lot #DBPG0323101, Minneapolis, MN, USA).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Imaging

Journal: Pain

Article Title: Episodic pain in Fabry disease is mediated by a heat shock protein-TRPA1 axis

doi: 10.1097/j.pain.0000000000003814

Figure Lengend Snippet: DRG neurons cultured from wildtype or Fabry rats were treated cultured with 17-AAG or recombinant human HSP70 (rhHSP70) overnight. Neurons were exposed to heat (40°C for 30 minutes) before calcium imaging was performed (A). (B) Representative traces of AITC-induced calcium transients from sham ( top ) or heat ( bottom ) treated neurons exposed to AAG. Overnight AAG treatment reduced the proportion of both wildtype and Fabry neurons that responded to AITC at steady-state and after heat treatment (C) but did not significantly affect magnitude (D). Each dot represents the average of all neurons recorded per rat (n=4-5 rats, 50-90 neurons per rat). Incubation with rhHSP70 also reduced the proportion of Fabry neurons responding to AITC (F) and reduced the magnitude of AITC-evoked calcium transients (G). Representative traces are shown in panel E. Each dot represents the average of neurons recorded per rat (n=4 rats, 50-90 neurons per rat). H) Wildtype and Fabry rats were systemically injected (I.P.) with 17-AAG. 18 hours later, animals were exposed to heat or sham treatment in vivo and then tested for mechanical sensitivity. Heat exposure decreased the mechanical withdrawal thresholds (I) and the increased aversive responses to both pinprick (J) and dynamic brush (K) in vehicle-injected, but not in 17-AAG-injected Fabry rats (n=5 per treatment group). (C-G) Data were analyzed using a 3-Way ANOVA with repeated measures. In panels C and D, there was a main effect of genotype: p<0.005. In panels C, D, F, and G, there was a main effect of drug: p<0.005 (C-D, F), p<0.01 (G). In panel C, there was a significant interaction between genotype and drug: p<0.005. In panel D, there was a significant interaction genotype, drug, and heat treatment: p<0.01. Tukey’s post hoc comparisons are shown for the genotype and drug interaction in panel C and the genotype, drug, and heat treatment interaction in panel D, where * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.005. Data in panels I-K were analyzed by 2-way ANOVA with repeated measures and exhibited a significant main effect of group: p<0.005. Panel I also exhibited a significant interaction between group and time; Tukey’s post hoc comparisons are annotated with a red or blue * indicating difference to vehicle-treated wildtype: p<0.05, whereas *** indicates difference from vehicle-treated wildtype: p<0.005. For panels J and K, although there was a significant main effect of group, there was no significant interaction of time and genotype; here the Tukey’s post hoc comparison annotated with ### indicates a difference compared to vehicle-treated wildtype of p<0.005.

Article Snippet: Between 1-2 hours after plating, cells were fed with either complete DRG media or complete DRG media supplemented with VER155008 (25 μM, MedChemExpress), 17-AAG (0.5 μM, MedChemExpress), or recombinant human HSP70 (5 μg/mL, R&D Systems, lot #DBPG0323101, Minneapolis, MN, USA).

Techniques: Cell Culture, Recombinant, Imaging, Incubation, Injection, In Vivo, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 inhibition suppresses nerve growth factor signaling and nociception in pain models

doi: 10.1172/JCI183873

Figure Lengend Snippet: ( A ) Immunofluorescence detection of TrkA and NRP1 in mouse DRG. TrkA was largely intracellular (arrows), whereas NRP1 was localized to the plasma membrane (arrowheads). Scale bar: 50 μm. ( B ) RNAScope detection of Ntrk1 (TrkA) and Nrp1 (NRP1) mRNA in mouse DRG neurons identified by NeuN immunofluorescence. Arrowheads indicate neurons coexpressing Ntrk1 and Nrp1 . Scale bar: 50 μm. ( C ) Immunofluorescence detection of CGRP and RNAScope detection of Nrp1 mRNA in mouse DRG. Arrows indicate neurons coexpressing CGRP and Nrp1 . Scale bar: 20 μm. ( D ) Immunofluorescence detection of NRP1 and GS in mouse DRG. Arrows indicate satellite glial cells expressing NRP1. Scale bar: 50 μm. ( E ) RNAScope detection of NTRK1 and NRP1 mRNA in human DRG. Arrowheads indicate neurons coexpressing NTRK1 and NRP1 . Scale bar: 500 μm. ( F ) Immunofluorescence of P2X3 and CGRP and RNAScope detection of NRP1 mRNA in human DRG. Arrowheads indicate neurons coexpressing CGRP and NRP1 . Arrows indicate neurons expressing P2X3 but not NRP1 . Scale bar: 50 μm. *Denotes fluorescence in human neurons due to lipofuscin. Nuclei shown in blue. ( G ) Percentage of mouse DRG neurons expressing Ntrk1 or CGRP that coexpress Nrp1 . ( H ) Percentage of human DRG neurons expressing NTRK1 , CGRP, or P2X3 that coexpress NRP1 . A – F show representative images from n = 4–5 mice and n = 3 humans. G and H show hybridized positive neurons (%) from n = 3–4 mice and n = 3 humans.

Article Snippet: Human DRG suspension cells were from AnaBios Corp. and were studied within 96 hours.

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, RNAscope, Expressing, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Neuropilin-1 inhibition suppresses nerve growth factor signaling and nociception in pain models

doi: 10.1172/JCI183873

Figure Lengend Snippet: ( A – C ) RNAScope localization of Gipc1 mRNA in mouse DRG ( A ) and of NTRK1 and GIPC1 mRNA in human DRG ( B ). Arrows indicate mRNA expression within the same cell. Representative images, n = 5 mice and n = 3 humans. Scale bars: 500 μm. ( C ) Percentage of human DRG neurons expressing NTRK1 or GIPC1 that coexpress GIPC1 or NRP1 . Hybridized positive neurons (%) from n = 3 humans. ( D ) Effect of GIPC1 siRNA on BRET measurements of TrkA levels at the plasma membrane of HEK293T cells under basal conditions and after coexpression with NRP1. ( E and F ) Effect of 30 minutes preincubation of GIPC1 antagonist (300 μM CR1023 or inactive control, Ctrl) or myosin VI inhibitor (50 μM 2,4,6-triiodophenol, TIP) on NGF-induced TrkA-Rluc8 trafficking from a marker of the plasma membrane (RGFP-CAAX) in CAD cells. ( G ) Effect of GIPC1 siRNA on NGF-induced downstream ERK transcription in CAD cells. Data from 5–6 independent experiments with triplicate wells. ( H and I ) Sample traces of action potential firing in mouse DRG neurons evoked by injecting a 1-second ramp pulse from 0 to 250 pA ( G ), with the number of evoked action potentials ( H ). n = 7–10 cells. ( J – N ) NGF-induced pain. Effects of GIPC1 or Ctrl siRNA (i.t.) on NGF-induced (50 ng/10 μl, i.pl.) mechanical allodynia ( K and L ) and thermal hyperalgesia ( M and N ) in the ipsilateral paw. ( L and N ) AUC of time courses. ( O – S ) CFA-induced pain. Effects of GIPC1 or Ctrl siRNA (i.t.) on CFA-induced (i.pl.) mechanical allodynia ( P and Q ) and thermal hyperalgesia ( R and S ). ( Q and S ) AUC of time courses. n = 6–8 mice per group. B, basal. Data are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( D and F ) One-way ANOVA, Sídák’s multiple comparisons. ( I ) Tukey’s multiple comparison. ( K , M , P , and R ) Two-way ANOVA, Sídák’s multiple comparisons. ( L , N , Q , and S ) Unpaired t test.

Article Snippet: Human DRG suspension cells were from AnaBios Corp. and were studied within 96 hours.

Techniques: RNAscope, Expressing, Clinical Proteomics, Membrane, Control, Marker, Comparison