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Nociceptive afferent signaling evoked by inflammation and nerve injury is mediated by the opening of ligand-gated and voltage-gated receptors or channels localized to cholesterol-rich lipid raft membrane domains. Dorsal root ganglion (DRG) nociceptors express high levels of toll-like receptor 4 (TLR4), which also localize to lipid rafts. Genetic deletion or pharmacologic blocking of TLR4 diminishes pain associated with chemotherapy-induced peripheral neuropathy (CIPN). In DRGs of mice with paclitaxel-induced CIPN, we analyzed DRG neuronal lipid rafts, expression of TLR4, activation of transient receptor potential cation channel subfamily V member 1 (TRPV1), and TLR4-TRPV1 interaction. Using proximity ligation assay, flow cytometry, and whole-mount DRG microscopy, we found that CIPN increased DRG neuronal lipid rafts and TLR4 expression. These effects were reversed by intrathecal injection of apolipoprotein A-I binding protein (AIBP), a protein that binds to TLR4 and specifically targets cholesterol depletion from TLR4-expressing cells. Chemotherapy-induced peripheral neuropathy increased TRPV1 phosphorylation, localization to neuronal lipid rafts, and proximity to TLR4. These effects were also reversed by AIBP treatment. Regulation of TRPV1-TLR4 interactions and their associated lipid rafts by AIBP covaried with the enduring reversal of mechanical allodynia otherwise observed in CIPN. In addition, AIBP reduced intracellular calcium in response to the TRPV1 agonist capsaicin, which was increased in DRG neurons from paclitaxel-treated mice and in the naïve mouse DRG neurons incubated in vitro with paclitaxel. Together, these results suggest that the assembly of nociceptive and inflammatory receptors in the environment of lipid rafts regulates nociceptive signaling in DRG neurons and that AIBP can control lipid raft-associated nociceptive processing.

Focal cortical dysplasia is a highly epileptogenic cortical malformation with few treatment options. Here, we generated human cortical organoids from patients with focal cortical dysplasia type II. Using this human model, we mimicked some focal cortical dysplasia hallmarks, such as impaired cell proliferation, the presence of dysmorphic neurons and balloon cells, and neuronal network hyperexcitability. Furthermore, we observed alterations in the adherens junctions zonula occludens-1 and partitioning defective 3, reduced polarization of the actin cytoskeleton, and fewer synaptic puncta. Focal cortical dysplasia cortical organoids showed downregulation of the small GTPase RHOA, a finding that was confirmed in brain tissue resected from these patients. Functionally, both spontaneous and optogenetically-evoked electrical activity revealed hyperexcitability and enhanced network connectivity in focal cortical dysplasia organoids. Taken together, our findings suggest a ventricular zone instability in tissue cohesion of neuroepithelial cells, leading to a maturational arrest of progenitors or newborn neurons, which may predispose to cellular and functional immaturity and compromise the formation of neural networks in focal cortical dysplasia.

Neuroinflammation is a major component in the transition to and perpetuation of neuropathic pain states. Spinal neuroinflammation involves activation of TLR4, localized to enlarged, cholesterol-enriched lipid rafts, designated here as inflammarafts. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia, leading to inflammaraft formation, induced tactile allodynia in naive mice. The apoA-I binding protein (AIBP) facilitated cholesterol depletion from inflammarafts and reversed neuropathic pain in a model of chemotherapy-induced peripheral neuropathy (CIPN) in wild-type mice, but AIBP failed to reverse allodynia in mice with ABCA1/ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. An AIBP mutant lacking the TLR4-binding domain did not bind microglia or reverse CIPN allodynia. The long-lasting therapeutic effect of a single AIBP dose in CIPN was associated with anti-inflammatory and cholesterol metabolism reprogramming and reduced accumulation of lipid droplets in microglia. These results suggest a cholesterol-driven mechanism of regulation of neuropathic pain by controlling the TLR4 inflammarafts and gene expression program in microglia and blocking the perpetuation of neuroinflammation.

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

Abstract: Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA. [ABSTRACT FROM AUTHOR]

Abstract: Rheumatoid arthritis is frequently associated with chronic pain that still remains difficult to treat. Targeting nerve growth factor (NGF) seems very effective to reduce pain in at least osteoarthritis and chronic low back pain but leads to some potential adverse events. Our aim was to better understand the involvement of the intracellular signalling pathways activated by NGF through its specific tyrosine kinase type A (TrkA) receptor in the pathophysiology of rheumatoid arthritis using the complete Freund adjuvant model in our knock-in TrkA/C mice. Our multimodal study demonstrated that knock-in TrkA/C mice exhibited a specific decrease of mechanical allodynia, weight-bearing deficit, peptidergic (CGRP+) and sympathetic (TH+) peripheral nerve sprouting in the joints, a reduction in osteoclast activity and bone resorption markers, and a decrease of CD68-positive cells in the joint with no apparent changes in joint inflammation compared with wild-type mice after arthritis. Finally, transcriptomic analysis shows several differences in dorsal root ganglion mRNA expression of putative mechanotransducers, such as acid-sensing ionic channel 3 and TWIK-related arachidonic acid activated K+ channel, as well as intracellular pathways, such as c-Jun, in the joint or dorsal root ganglia. These results suggest that TrkA-specific intracellular signalling pathways are specifically involved in mechanical hypersensitivity and bone alterations after arthritis using TrkA/C mice. [ABSTRACT FROM AUTHOR]

Repressing pain LATER: Opioids are the current standard of care for the treatment of chronic pain. However, they have severe side effects. Recent data have shown that loss-of-function mutations in the sodium channel NaV1.7 cause insensitivity to pain. Here, Moreno et al. developed an epigenetic strategy using CRISPR-dCas9 and zinc fingers called long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 (LATER) to repress NaV1.7. In vivo, LATER reduced hyperalgesia in multiple animal models and reversed chemotherapy-induced chronic pain in mice. The results suggest that LATER might be effective for treating chronic pain of multiple origins. Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)–dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states. [ABSTRACT FROM AUTHOR]