Overexpression of TMEM100 in articular afferents induces secondary hyperalgesia in the hind paw but no knee joint pain. (A), Cartoon depicting the experimental approach (left), example image of a DRG cross section from a mouse that had received intraarticular AAV-PHP.S-TMEM100-Ires-dsRed (middle) and quantification of the total number of dsRed + neurons (right). Bars represent means ± SEM and values from individual DRGs are shown as black dots. (B), dsRed fluorescence in the tibial nerve distal to the knee (left) and the saphenous (right) nerve proximal to the knee which contains the medial articular nerve. (C), Comparison of the time courses of changes in stand time (left) and leg swing speed (right) of WT mice that intraarticularly received AAV-PHP.S-dsRed control virus (white circles) and AAV-PHP-S-TMEM100-Ires-dsRed (orange circles). Ratios at different time points were compared using multiple Mann-Whitney tests. (ns, P>0.05; *, P<0.05; N-numbers are provided in the graph legend). (D), Time courses of changes in mechanical paw withdrawal thresholds of AAV-PHP.S-dsRed (white circles) and AAV-PHP-S-TMEM100-Ires-dsRed (blue circles) injected WT mice (left) as well as comparison of the response rates at 14 dpi -i.e. % paw withdrawals in response to five successive stimulations with the indicated von Frey filaments (right). Mean paw withdrawal thresholds at different time points and response rates to different von Frey filaments were compared using Mann-Whitney test (***, P<0.001; number of tested animals is the same in both panels and is indicated in the graph legend). (E), Example traces of mechanicallyevoked action potentials recorded from cutaneous C-fiber nociceptors in the tibial nerve from control mice (top, AAV-PHP.S-dsRed) and from mice that overexpress TMEM100 in articular afferents (bottom, AAV-PHP.S-TMEM100-Ires-dsRed). (F), Comparison of the firing rates evoked by ramp-and-hold stimuli that exerted the indicated force to the receptive fields. Symbols represent means ± SEM numbers of action potentials, which were compared using multiple Mann-Whitney tests (*, P<0.05). (G), Comparison of the proportions of C-fiber nociceptors that respond to mechanical stimulation with the indicated von Frey filaments. The proportions were compared pairwise using the Chi-square test (ns, not significant; *, P<0.05). (H), Cartoon depicting the proposed mechanism underlying the induction of secondary hyperalgesia. Detailed statistical information and raw data is provided in supplemental Data S1.

Contexts in source publication

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... https://doi.org/10. 1101/2022 this end, we selectively overexpressed TMEM100 in knee joint afferents by intraarticular injection of an AAV-PHP.S-TMEM100-Ires-dsRed virus (30 µl, 1.5*10 11 vg; Figure 7A). Four days after intraarticular AAV-PHP.S-TMEM100-Ires-dsRed administration, we observed prominent dsRed fluorescence in a total of 339 ± 7 neurons in ipsilateral L3 and L4 DRG. ...

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... importantly, we observed numerous dsRed expressing nerve fibers in the saphenous nerve proximal to the knee, which includes the medial articular nerve that supplies the knee joint and in which silent nociceptors had first been described ( Schaible and Schmidt, 1988). Importantly, we observed hardly any dsRed + fibers in the tibial nerve distal to the knee, which contains cutaneous afferents that supply the plantar surface of the hind paw ( Figure 7B). Hence, intraarticularly administered AAV-PHP.S-TMEM100-Ires-dsRed causes selective overexpression of TMEM100 in knee joint but not in skin afferents. ...

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... intraarticularly administered AAV-PHP.S-TMEM100-Ires-dsRed causes selective overexpression of TMEM100 in knee joint but not in skin afferents. Interestingly, TMEM100-overexpressing mice exhibited normal gait, indicating that un-silencing of knee joint MIAs does not trigger knee joint pain ( Figure 7C). Strikingly, however, these mice developed profound mechanical hyperalgesia in the ipsilateral hind paw five days post AAV injection, which persisted until the end of the observation period (21 dpi; Figure 7D). ...

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... TMEM100-overexpressing mice exhibited normal gait, indicating that un-silencing of knee joint MIAs does not trigger knee joint pain ( Figure 7C). Strikingly, however, these mice developed profound mechanical hyperalgesia in the ipsilateral hind paw five days post AAV injection, which persisted until the end of the observation period (21 dpi; Figure 7D). Thus, the mechanical paw withdrawal thresholds decreased from 0.92 ± 0.066 g (-1 dpi) to 0.373 ± 0.035 g (14 dpi). ...

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... accordance with the behavioral outcome of TMEM100 over-expression in articular afferents, single-unit action potential recordings from a tibial nerve-glabrous skin preparation showed that cutaneous C-fiber nociceptors fire about twice as many action potentials in response to a given stimulus and have significantly reduced mechanical activation thresholds in mice that overexpress TMEM100 in articular afferents compared to mice that had received a control virus ( Figure 7E-G). Similar to mice that received intraarticular CFA, the mechanical activation thresholds of cutaneous Aδ-fiber nociceptors were slightly reduced and the action potential firing rate in response to suprathreshold stimuli was significantly increased ( Figure S4). ...

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... summary, our data shows that TMEM100 overexpression-induced un-silencing of mechanically insensitive articular afferents is sufficient to trigger mechanical hyperalgesia in remote skin regions ( Figure 7D). Together with the observation that TMEM100 is specifically up-regulated in MIAs during CFA-induced inflammation and that knock-out of TMEM100 exclusively abolishes long-lasting secondary hyperalgesia, these results suggest that sensory input from unsilenced MIAs is the . ...

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... https://doi.org/10. 1101/2022 main trigger for the induction of secondary hyperalgesia ( Figure 7H). While we have not examined the well-established contribution of central sensitization, our data reveals a previously unrecognized contribution of peripheral sensitization to secondary hyperalgesia. ...

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... we show that blocking the un-silencing of articular MIAs by knocking out TMEM100 prevents the development of long-lasting secondary hyperalgesia in remote skin regions, but does not alter pain at the actual site of CFAinduced inflammation (Figure 4). Moreover, TMEM100 overexpression-induced unsilencing of articular MIAs in the absence of inflammation or injury, induces secondary hyperalgesia but not pain hypersensitivity in the knee joint ( Figure 7C and D). Finally, our skin-nerve recordings demonstrate that secondary hyperalgesia, in addition to the previously described central sensitization, is partly driven by peripheral sensitization of cutaneous C-fiber nociceptors (Figure 6 and 7E-G). ...

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... however, seems unlikely, considering that TMEM100 expression is exclusively upregulated in MIAs in CFA-induced monoarthritis ( Figure 3C) and that mice lacking TMEM100 only show functional deficits in MIAs but not in other articular nociceptors (Figure 3 and 5). It should further be noted, that we observed off-target expression of TMEM100 in a few fibers in the tibial nerve distal to the knee, which supplies the plantar surface of the hind paw ( Figure 7B). Cutaneous C-fiber nociceptors that detect von Frey stimuli express MRGPRD ( Cavanaugh et al., 2009) and require PIEZO2 and TRPA1 for normal mechanosensitivity ( Garrison and Stucky, 2014;Murthy et al., 2018), but do not express TRPV1 (Cavanaugh et al., 2009;Zeisel et al., 2018). ...

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... have not explicitly tested if un-silencing of MIAs also triggers central sensitization, which is thought to be the major cause of secondary hyperalgesia. Yet, considering that the AAV-PHP.S-TMEM100 induced reduction of paw withdrawal thresholds ( Figure 7D) was larger than the reduction of the mechanical activation thresholds of individual cutaneous C-and Aδ-fiber nociceptors ( Figure 7G and Figure . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...

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... have not explicitly tested if un-silencing of MIAs also triggers central sensitization, which is thought to be the major cause of secondary hyperalgesia. Yet, considering that the AAV-PHP.S-TMEM100 induced reduction of paw withdrawal thresholds ( Figure 7D) was larger than the reduction of the mechanical activation thresholds of individual cutaneous C-and Aδ-fiber nociceptors ( Figure 7G and Figure . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...

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... our data support a mechanistic model of secondary hyperalgesia in which inflammation-induced upregulation of TMEM100 un-silences MIAs, which subsequently triggers central sensitization and -via a yet unknown central mechanism -peripheral sensitization of cutaneous nociceptors, which eventually leads to secondary hyperalgesia in skin regions remote from the site of inflammation ( Figure 7H). By demonstrating that primary and secondary hyperalgesia are triggered by separate subclasses of primary sensory afferents and considering that MIAs constitute almost fifty percent of all nociceptors in viscera and deep somatic tissues, our study provides an invaluable framework for future studies that aim at deciphering the contribution of different afferent subtypes to other clinically relevant forms of pain and to develop new strategies for preventing the chronification of pain after injury and inflammation. ...

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... /2022 Supplemental Figure S4, Sensitization of cutaneous Aδ-fiber nociceptors by TMEM100 overexpression in articular afferents, related to Figure 7 (A), Example traces of mechanically-evoked action potentials recorded from cutaneous Aδ-fiber nociceptors in the tibial nerve from control mice (top, AAV-PHP.S-dsRed) and from mice that overexpress TMEM100 in articular afferents (bottom, AAV-PHP.S-TMEM100-Ires-dsRed). (B), Comparison of the firing rates evoked by ramp-and-hold stimuli that exerted the indicated force to the receptive fields. Symbols represent means ± SEM numbers of action potentials, which were compared using multiple Mann-Whitney tests (ns, not significant; *, P<0.05; **, P<0.01). ...