Phenotype- and species-specific skin proteomic signatures for incision-induced pain in humans and mice.

BACKGROUND: Acute pain after surgery is common and often leads to chronic post-surgical pain, but neither treatment nor prevention is currently sufficient. We hypothesised that specific protein networks (protein-protein interactions) are relevant for pain after surgery in humans and mice. METHODS: Standardised surgical incisions were performed in male human volunteers and male mice. Quantitative and qualitative sensory phenotyping were combined with unbiased quantitative mass spectrometry-based proteomics and protein network theory. The primary outcomes were skin protein signature changes in humans and phenotype-specific protein-protein interaction analysis 24 h after incision. Secondary outcomes were interspecies comparison of protein regulation as well as protein-protein interactions after incision and validation of selected proteins in human skin by immunofluorescence. RESULTS: Skin biopsies in 21 human volunteers revealed 119/1569 regulated proteins 24 h after incision. Protein-protein interaction analysis delineated remarkable differences between subjects with small (low responders, n=12) and large incision-related hyperalgesic areas (high responders, n=7), a phenotype most predictive of developing chronic post-surgical pain. Whereas low responders predominantly showed an anti-inflammatory protein signature, high responders exhibited signatures associated with a distinct proteolytic environment and persistent inflammation. Compared to humans, skin biopsies in mice habored even more regulated proteins (435/1871) 24 h after incision with limited overlap between species as assessed by proteome dynamics and PPI. Immunohistochemistry confirmed the expression of high priority candidates in human skin biopsies. CONCLUSIONS: Proteome profiling of human skin after incision revealed protein-protein interactions correlated with pain and hyperalgesia, which may be of potential significance for preventing chronic post-surgical pain. Importantly, protein-protein interactions were differentially modulated in mice compared to humans opening new avenues for successful translational research.

Proteome and Network Analysis Provides Novel Insights Into Developing and Established Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side-effect of cancer therapies. So far, the development of CIPN cannot be prevented, neither can established CIPN be reverted, often leading to the cessation of necessary chemotherapy. Thus, there is an urgent need to explore the mechanistic basis of CIPN to facilitate its treatment. Here we used an integrated approach of quantitative proteome profiling and network analysis in a clinically relevant rat model of paclitaxel-induced peripheral neuropathy. We analysed lumbar rat DRG at two critical time points: (1) day 7, right after cessation of paclitaxel treatment, but prior to neuropathy development (pre-CIPN); (2) 4 weeks after paclitaxel initiation, when neuropathy has developed (peak-CIPN). In this way we identified a differential protein signature, which shows how changes in the proteome correlate with the development and maintenance of CIPN, respectively. Extensive biological pathway and network analysis reveals that, at pre-CIPN, regulated proteins are prominently implicated in mitochondrial (dys)function, immune signalling, neuronal damage/regeneration, and neuronal transcription. Orthogonal validation in an independent rat cohort confirmed the increase of ß-catenin (CTNNB1) at pre-CIPN. More importantly, detailed analysis of protein networks associated with ß-catenin highlights translationally relevant and potentially druggable targets. Overall, this study demonstrates the enormous value of combining animal behaviour with proteome and network analysis to provide unprecedented insights into the molecular basis of CIPN. In line with emerging approaches of network medicine our results highlight new avenues for developing improved therapeutic options aimed at preventing and treating CIPN.

A proteome signature for acute incisional pain in dorsal root ganglia of mice.

ABSTRACT: After surgery, acute pain is still managed insufficiently and may lead to short-term and long-term complications including chronic postsurgical pain and an increased prescription of opioids. Thus, identifying new targets specifically implicated in postoperative pain is of utmost importance to develop effective and nonaddictive analgesics. Here, we used an integrated and multimethod workflow to reveal unprecedented insights into proteome dynamics in dorsal root ganglia (DRG) of mice after plantar incision (INC). Based on a detailed characterization of INC-associated pain-related behavior profiles, including a novel paradigm for nonevoked pain, we performed quantitative mass-spectrometry-based proteomics in DRG 1 day after INC. Our data revealed a hitherto unknown INC-regulated protein signature in DRG with changes in distinct proteins and cellular signaling pathways. In particular, we show the differential regulation of 44 protein candidates, many of which are annotated with pathways related to immune and inflammatory responses such as MAPK/extracellular signal-regulated kinases signaling. Subsequent orthogonal assays comprised multiplex Western blotting, bioinformatic protein network analysis, and immunolabeling in independent mouse cohorts to validate (1) the INC-induced regulation of immune/inflammatory pathways and (2) the high priority candidate Annexin A1. Taken together, our results propose novel potential targets in the context of incision and, therefore, represent a highly valuable resource for further mechanistic and translational studies of postoperative pain.

Myotubularin related protein-2 and its phospholipid substrate PIP2 control Piezo2-mediated mechanotransduction in peripheral sensory neurons.

Piezo2 ion channels are critical determinants of the sense of light touch in vertebrates. Yet, their regulation is only incompletely understood. We recently identified myotubularin related protein-2 (Mtmr2), a phosphoinositide (PI) phosphatase, in the native Piezo2 interactome of murine dorsal root ganglia (DRG). Here, we demonstrate that Mtmr2 attenuates Piezo2-mediated rapidly adapting mechanically activated (RA-MA) currents. Interestingly, heterologous Piezo1 and other known MA current subtypes in DRG appeared largely unaffected by Mtmr2. Experiments with catalytically inactive Mtmr2, pharmacological blockers of PI(3,5)P2 synthesis, and osmotic stress suggest that Mtmr2-dependent Piezo2 inhibition involves depletion of PI(3,5)P2. Further, we identified a PI(3,5)P2 binding region in Piezo2, but not Piezo1, that confers sensitivity to Mtmr2 as indicated by functional analysis of a domain-swapped Piezo2 mutant. Altogether, our results propose local PI(3,5)P2 modulation via Mtmr2 in the vicinity of Piezo2 as a novel mechanism to dynamically control Piezo2-dependent mechanotransduction in peripheral sensory neurons.

Optimization of Experimental Parameters in Data-Independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results.

Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Here, we present an implementation of data-independent acquisition using its parallel acquisition nature that surpasses the limitation of serial MS2 acquisition of data-dependent acquisition on a quadrupole ultra-high field Orbitrap mass spectrometer. In deep single shot data-independent acquisition, we identified and quantified 6,383 proteins in human cell lines using 2-or-more peptides/protein and over 7100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8121 when including the 382 proteins that were identified based on a single peptide sequence. Missing values for proteins were within 0.3 to 2.1% and median coefficients of variation of 4.7 to 6.2% among technical triplicates. In very complex mixtures, we could quantify 10,780 proteins and 12,192 proteins when including the 1412 proteins that were identified based on a single peptide sequence. Using this optimized DIA, we investigated large-protein networks before and after the critical period for whisker experience-induced synaptic strength in the murine somatosensory cortex 1-barrel field. This work shows that parallel mass spectrometry enables proteome profiling for discovery with high coverage, reproducibility, precision and scalability.

A novel mechanism for nicotinic potentiation of glutamatergic synapses.

Selective strengthening of specific glutamatergic synapses in the mammalian hippocampus is critical for encoding new memories. This is most commonly achieved by input-specific Hebbian-type plasticity involving glutamate-dependent coincident presynaptic and postsynaptic depolarization. Our results demonstrate a novel mechanism by which nicotinic signaling, independently of coincident fast glutamatergic transmission, increases synaptic strength in the hippocampus. Electrophysiological recordings from rat hippocampal neurons in culture revealed that 1-3 h of exposure to 1 µm nicotine, even with action potentials being blocked, produced increases in both the frequency and amplitude of miniature EPSCs. Possible mechanisms were analyzed both in mouse organotypic slice culture and in rat cell culture by inducing the cells to express super-ecliptic pHluorin-tagged GluA1-containing AMPA receptors, which fluoresce only on the cell surface. Pharmacological and genetic manipulation of the cells, in combination with fluorescence-recovery-after-photobleaching experiments, revealed that nicotine, acting through α7-containing nicotinic acetylcholine receptors on the postsynaptic neuron, induces the stabilization and accumulation of GluA1-containing AMPA receptors on dendritic spines. The process relies on intracellular calcium signaling, PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] interactions with members of the PSD-95 family, and lateral diffusion of the GluA1 receptors on the cell surface. These findings define a new avenue by which nicotinic signaling modulates synaptic mechanisms thought to subserve learning and memory.

PMCA2 via PSD-95 controls calcium signaling by α7-containing nicotinic acetylcholine receptors on aspiny interneurons.

Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain α7 nicotinic receptor subunits (α7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons, which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that α7-nAChRs are associated with plasma membrane calcium-ATPase pump isoform 2 (PMCA2). Analysis of α7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of α7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of α7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95 (postsynaptic density-95), which is associated with α7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of α7-nAChR clusters. This three-component combination of PMCA2, PSD-95, and α7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons.

Characterization of Eag1 channel lateral mobility in rat hippocampal cultures by single-particle-tracking with quantum dots.

Voltage-gated ion channels are main players involved in fast synaptic events. However, only slow intracellular mechanisms have so far been described for controlling their localization as real-time visualization of endogenous voltage-gated channels at high temporal and spatial resolution has not been achieved yet. Using a specific extracellular antibody and quantum dots we reveal and characterize lateral mobility as a faster mechanism to dynamically control the number of endogenous ether-a-go-go (Eag)1 ion channels inside synapses. We visualize Eag1 entering and leaving synapses by lateral diffusion in the plasma membrane of rat hippocampal neurons. Mathematical analysis of their trajectories revealed how the motion of Eag1 gets restricted when the channels diffuse into the synapse, suggesting molecular interactions between Eag1 and synaptic components. In contrast, Eag1 channels switch to Brownian movement when they exit synapses and diffuse into extrasynaptic membranes. Furthermore, we demonstrate that the mobility of Eag1 channels is specifically regulated inside synapses by actin filaments, microtubules and electrical activity. In summary, using single-particle-tracking techniques with quantum dots nanocrystals, our study shows for the first time the lateral diffusion of an endogenous voltage-gated ion channel in neurons. The location-dependent constraints imposed by cytoskeletal elements together with the regulatory role of electrical activity strongly suggest a pivotal role for the mobility of voltage-gated ion channels in synaptic activity.

Participation of HERG channel cytoplasmic structures on regulation by the G protein-coupled TRH receptor.

Human ether-a-go-go-related gene (HERG) channels heterologously expressed in Xenopus oocytes are regulated by the activation of G protein-coupled hormone receptors that, like the thyrotropin-releasing hormone (TRH) receptor, activate phospholipase C. Previous work with serially deleted HERG mutants suggested that residues 326-345 located in the proximal domain of the channels amino terminus might be required for the hormonal modulation of HERG activation. Generation of new channel mutants deleted in this region further point to the amino acid sequence between residues 326 and 332 as a possible determinant of the TRH effects, but individual or combined single-point mutations in this sequence demonstrate that maintenance of its consensus sites for phosphorylation and/or interaction with regulatory components is not important for the modulatory response(s). The TRH-induced effects also remained unaltered when a basic amino acid cluster located between residues 362 and 366 is eliminated. Additionally, no effect of TRH was observed in channels carrying single-point mutations at the beginning of the intracellular loop linking transmembrane domains S4 and S5. Our results indicate that a correct structural arrangement of the amino terminal domains is essential for the hormone-induced modifications of HERG activation. They also suggest that the hormonal regulatory action is transmitted to the transmembrane channel core through interactions between the cytoplasmic domains and the initial portion of the S4-S5 linker.

Monoclonal antibody blockade of the human Eag1 potassium channel function exerts antitumor activity.

The potassium channel ether à go-go has been directly linked to cellular proliferation and transformation, although its physiologic role(s) are as of yet unknown. The specific blockade of human Eag1 (hEag1) may not only allow the dissection of the role of the channel in distinct physiologic processes, but because of the implication of hEag1 in tumor biology, it may also offer an opportunity for the treatment of cancer. However, members of the potassium channel superfamily are structurally very similar to one another, and it has been notoriously difficult to obtain specific blockers for any given channel. Here, we describe and validate the first rational design of a monoclonal antibody that selectively inhibits a potassium current in intact cells. Specifically blocking hEag1 function using this antibody inhibits tumor cell growth both in vitro and in vivo. Our data provide a proof of concept that enables the generation of functional antagonistic monoclonal antibodies against ion channels with therapeutic potential. The particular antibody described here, as well as the technique developed to make additional functional antibodies to Eag1, makes it possible to evaluate the potential of the channel as a target for cancer therapy.

Different relevance of inactivation and F468 residue in the mechanisms of hEag1 channel blockage by astemizole, imipramine and dofetilide.

The relevance of a point mutation at the C-terminal end of the S6 helix (F468) and the introduction of C-type inactivation in the blockage of hEag1 channels by astemizole, imipramine and dofetilide was tested. C-type inactivation decreased block by astemizole and dofetilide but not imipramine, suggesting different binding sites in the channel. F468C mutation increased IC(50) for astemizole and imipramine but in contrast to HERG channels, only slightly for dofetilide. Together with measurements on recovery of blocking, our observations indicate that the mechanism of hEag1 blockage by each of these drugs is different, and suggest relevant structural differences between hEag1 and HERG channels.

Specificity of TRH receptor coupling to G-proteins for regulation of ERG K+ channels in GH3 rat anterior pituitary cells.

The identity of the G-protein coupling thyrotropin-releasing hormone (TRH) receptors to rat ether-à-go-go related gene (r-ERG) K+ channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH(3) cells and dominant-negative variants (Galpha-QL/DN) of G-protein alpha subunits. Expression of dominant-negative Galpha(q/11) that minimizes the TRH-induced Ca2+ signal had no effect on r-ERG current inhibition elicited by the hormone. In contrast, the introduction of dominant-negative variants of Galpha13 and the small G-protein Rho caused a significant loss of the inhibitory effect of TRH on r-ERG. A strong reduction of this TRH effect was also obtained in cells expressing either dominant-negative Galpha(s) or transducin alpha subunits, an agent known to sequester free G-protein betagamma dimers. As a further indication of specificity of the dominant-negative effects, only the dominant-negative variants of Galpha13 and Rho (but not Galpha(s)-QL/DN or Galpha(t)) were able to reduce the TRH-induced shifts of human ERG (HERG) activation voltage dependence in HEK293 cells permanently expressing HERG channels and TRH receptors. Our results demonstrate that whereas the TRH receptor uses a G(q/11) protein for transducing the Ca2+ signal during the initial response to TRH, this G-protein is not involved in the TRH-induced inhibition of endogenous r-ERG currents in pituitary cells. They also identify G(s) (or a G(s)-like protein) and G13 as important contributors to the hormonal effect in these cells and suggest that betagamma dimers released from these proteins may participate in modulation of ERG currents triggered by TRH.

Role of BK potassium channels shaping action potentials and the associated [Ca(2+)](i) oscillations in GH(3) rat anterior pituitary cells.

Measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch clamped GH(3) cells to determine the contribution of large-conductance calcium-activated K(+) (BK) channels to action potential repolarization and size of the associated Ca(2+) oscillations. By examining the dependence of action potential (AP) duration on extracellular Ca(2+) levels in the presence and the absence of the specific BK channel blocker paxilline, it is observed that plateau-like action potentials are associated to low densities of paxilline-sensitive currents. Extracellular Ca(2+) increases or paxilline additions are not able to largely modify action potential duration in cells showing a reduced expression of BK currents. Furthermore, specific blockade of these currents with paxilline systematically elongates AP duration, but only under conditions in which short APs and/or prominent BK currents recorded under voltage-clamp mode are present in the same cells. Our data indicate that in GH(3) cells, BK channels act primarily ending the action potential and suggest that by contributing to fine-tuning cellular electrical properties and hence intracellular Ca(2+) variations, BK channels may play an important role on time- and cell-dependent modulation of physiological outputs in adenohypophyseal cells.

Relevance of the proximal domain in the amino-terminus of HERG channels for regulation by a phospholipase C-coupled hormone receptor.

We used Xenopus oocytes co-expressing thyrotropin-releasing hormone (TRH) receptors and human ether-a-go-go-related gene (HERG) K+ channel variants carrying different amino-terminal modifications to check the relevance of the proximal domain for hormonal regulation of the channel. Deletion of the whole proximal domain (Delta 138-373) eliminates TRH-induced modifications in activation and deactivation parameters. TRH effects on activation are also suppressed with channels lacking the second half of the proximal domain or only residues 326-373. However, normal responses to TRH are obtained with Delta 346-373 channels. Thus, whereas residues 326-345 are required for the hormonal modulation of HERG activation, different proximal domain sequences contribute to set HERG gating characteristics and its regulation by TRH.

Protein kinase C is necessary for recovery from the thyrotropin-releasing hormone-induced r-ERG current reduction in GH3 rat anterior pituitary cells.

The biochemical cascade linking activation of phospholipase C-coupled thyrotropin-releasing hormone (TRH) receptors to rat ERG (r-ERG) channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH3 cells and pharmacological inhibitors. To check the recent suggestion that Rho kinase is involved in the TRH-induced r-ERG current suppression, the hormonal effects were studied in cells pretreated with the Rho kinase inhibitors Y-27632 and HA-1077. The TRH-induced r-ERG inhibition was not significantly modified in the presence of the inhibitors. Surprisingly, the hormonal effects became irreversible in the presence of HA-1077 but not in the presence of the more potent Rho kinase inhibitor Y-27632. Further experiments indicated that the effect of HA-1077 correlated with its ability to inhibit protein kinase C (PKC). The hormonal effects also became irreversible in cells in which PKC activity was selectively impaired with GF109203X, Gö6976 or long-term incubation with phorbol esters. Furthermore, the reversal of the effects of TRH, but not its ability to suppress r-ERG currents, was blocked if diacylglycerol generation was prevented by blocking phospholipase C activity with U-73122. Our results suggest that a pathway involving an as yet unidentified protein kinase is the main cause of r-ERG inhibition in perforated-patch clamped GH3 cells. Furthermore, they demonstrate that although not necessary to trigger the ERG current reductions induced by TRH, an intracellular signal cascade involving phosphatidylinositol-4,5-bisphosphate hydrolysis by phospholipase C, activation of an alpha/betaII conventional PKC and one or more dephosphorylation steps catalysed by protein phosphatase 2A, mediates recovery of ERG currents following TRH withdrawal.