Accepted

Transient receptor potential vanilloid 1 (TRPV1), a nonselective, ligand-gated cation channel, responds to multiple noxious stimuli and is targeted by many kinases that influence its trafficking and activity. Studies on the internalization of TRPV1 have mainly focused on that induced by capsaicin or other agonists. Here, we report that constitutive internalization of TRPV1 occurred in a manner dependent on clathrin, dynamin, and adaptor protein complex 2 (AP2). The μ2 subunit of AP2 (AP2μ2) interacted directly with TRPV1 and was required for its constitutive internalization. Cyclin-dependent kinase 5 (CDK5) phosphorylated AP2μ2 at Ser, which reduced the interaction between TRPV1 and AP2μ2, leading to decreased TRPV1 internalization. Intrathecal delivery of a cell-penetrating fusion peptide corresponding to the Cdk5 phosphorylation site in AP2μ2, which competed with AP2μ2 for phosphorylation by Cdk5, increased the abundance of TRPV1 on the surface of dorsal root ganglion neurons and reduced complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia in rats. In addition to describing a mechanism of TRPV1 constitutive internalization and its inhibition by CDK5, these findings demonstrate that CDK5 promotes inflammatory thermal hyperalgesia by reducing TRPV1 internalization, providing previously unidentified insights into the search for drug targets to treat pain.

Spinal cord injury (SCI) usually causes a devastating lifelong disability for patients. After a traumatic lesion, disruption of the blood-spinal cord barrier induces the infiltration of macrophages into the lesion site and the activation of resident glial cells, which release cytokines and chemokines. These events result in a persistent inflammation, which has both detrimental and beneficial effects, but eventually limits functional recovery and contributes to the appearance of neuropathic pain. Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that regulate the expression of inflammatory genes by interacting with acetylated lysine residues. While BET inhibitors are a promising therapeutic strategy for cancer, little is known about their implication after SCI. Thus, the current study was aimed to investigate the anti-inflammatory role of BET inhibitors in this pathologic condition.

The cation channel TRPA1 transduces a myriad of noxious chemical stimuli into nociceptor electrical excitation and neuropeptide release, leading to pain and neurogenic inflammation. Despite emergent evidence that TRPA1 is regulated by the membrane environment, it remains unknown whether this channel localizes in membrane microdomains or whether it interacts with cholesterol. Using total internal reflection fluorescence microscopy and density gradient centrifugation we found that mouse TRPA1 localizes preferably into cholesterol-rich domains and functional experiments revealed that cholesterol depletion decreases channel sensitivity to chemical agonists. Moreover, we identified two structural motifs in transmembrane segments 2 and 4 involved in mTRPA1-cholesterol interactions that are necessary for normal agonist sensitivity and plasma membrane localization. We discuss the impact of such interactions on TRPA1 gating mechanisms, regulation by the lipid environment, and role of this channel in sensory membrane microdomains, all of which helps to understand the puzzling pharmacology and pathophysiology of this channel.

Endometriosis is characterized by the presence of ectopic endometrial cells outside the uterine cavity. Thyroid autoimmunity has been associated with endometriosis. This work investigated the potential pathophysiological link between endometriosis and thyroid disorders. Transcripts and proteins involved in thyroid metabolism are dysregulated in eutopic and ectopic endometrium of endometriotic patients, leading to resistance of ectopic endometrium to triiodothyronine (T3) action and local accumulation of thyroxine (T4). Thyroid-stimulating hormone (TSH) acts as a proliferative and prooxidative hormone on all endometria of endometriosis patients and controls, whereas T3 and T4 act to specifically increase ectopic endometrial cell proliferation and reactive oxygen species (ROS) production. Mouse studies confirmed the data gained in vitro since endometriotic implants were found to be bigger when thyroid hormones increased. A retrospective analysis of endometriosis patients with or without a thyroid disorder revealed an increased chronic pelvic pain and disease score in endometriotic patients with a thyroid disorder.

It has been suggested that interactions of neuronal nitric oxide synthase (nNOS) with postsynaptic density 95 (PSD95) play important roles in the development of chronic neuropathic pain. Here we examine the possible role of nNOS-PSD95 interactions in central sensitization as represented by phosphorylation of the NMDA receptor GluN1 subunit (pGluN1) in mice with chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the nNOS-PSD95 interactions inhibitor, IC87201 on post-operative days 0-3 significantly reduced the CCI-induced increase in total NO levels in the lumbar spinal cord dorsal horn. IC87201 administration on post-operative days 0-3 also attenuated the CCI-induced development of mechanical allodynia (MA) and PKC-dependent (Ser896) pGluN1. Sciatic nerve injury elicited a significant translocation of the PKC-ε isoform from the cytosol to the membrane fraction in the lumbar spinal cord dorsal horn on day 3 post-CCI surgery. Administration of IC87201 significantly inhibited this translocation of PKC-ε, while the expression of PKC-α and -ξ in the cytosol and membrane fractions was unaffected by sciatic nerve injury or injection of IC87201. Furthermore, administration of the PKC-ε inhibitor, εV1-2 on post-operative days 0-3 attenuated the CCI-induced development of MA and pGluN1. Collectively these results demonstrate that spinal nNOS-PSD95 interactions play an important role in PKC-dependent GluN1 phosphorylation via activation of the PKC-ε isoform, and ultimately contributes to the development of MA in peripheral neuropathy.

Immunohistochemical characterization of primary afferent fibers (intact or after nerve damage) is traditionally performed in thin sections from dorsal root ganglia (DRGs) or in teased fibers, as light scattering in whole-mounts compromises visualization. These procedures are time-consuming, require specific equipment and advanced experimental skills. Lipid-clearing techniques are increasing in popularity, but they have never been used for the peripheral nervous system. We established a modified, inexpensive clearing method based on lipid-removal protocols to make transparent peripheral nerve tissue (inCLARITY). We compared retrograde-labeling and free-floating immunostaining with cryo-sections. Confocal microscopy on whole-mount transparent DRGs showed neurons marked with retrograde tracers applied to experimental neuromas (Retrobeads, Fluoro-ruby, Fluoro-emerald, DiI, and Fluoro-gold). After immunostaining with calcitonin gene-related peptide (peptidergic) or isolectin IB4 (non-peptidergic), nociceptors were visualized. Immunostaining in transparent whole-mount nerves allows simultaneous evaluation of the axotomized branches containing the neuroma and neighboring intact branches as they can be mounted preserving their anatomical disposition and fiber integrity. The goal of our study was to optimize CLARITY for its application in peripheral nerve tissues. The protocol is compatible with the use of retrograde tracers and improves immunostaining outcomes when compared to classical cryo-sectioning, as lack of lipids maximizes antibody penetration within the tissue.