Hope Center Member Publications: June 25, 2023

Neuron-associated macrophage proliferation in the sensory ganglia is associated with peripheral nerve injury-induced neuropathic pain involving CX3CR1 signaling
(2023) eLife, 12, art. no. e78515, . 

Guimarães, R.M.^a b , Aníbal-Silva, C.E.^a b , Davoli-Ferreira, M.^a b c , Gomes, F.I.F.^a , Mendes, A.^a , Cavallini, M.C.M.^a b , Fonseca, M.M.^a d , Damasceno, S.^a , Andrade, L.P.^a b , Colonna, M.^e , Rivat, C.^f , Cunha, F.Q.^a , Alves-Filho, J.C.^a , Cunha, T.M.^a

^a Center for Research in Inflammatory Diseases (CRID), Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Ribeirão Preto, Brazil
^b Graduate Program in Basic and Applied Immunology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Ribeirão Preto, Brazil
^c Department of Physiology and Pharmacology, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Canada
^d Department of Anesthesiology, Pain Mechanisms Laboratory, Wake Forest University School of Medicine, Winston-Salem, United States
^e Department of Pathology and Immunology, Washington University School of Medicine in Saint Louis, Saint Louis, MO, United States
^f Univ Montpellier, Montpellier, France

Abstract
Resident macrophages are distributed across all tissues and are highly heterogeneous due to adaptation to different tissue-specific environments. The resident macrophages of the sensory ganglia (sensory neuron-associated macrophages, sNAMs) are in close contact with the cell body of primary sensory neurons and might play physiological and pathophysiological roles. After peripheral nerve injury, there is an increase in the population of macrophage in the sensory ganglia, which have been implicated in different conditions, including neuropathic pain development. However, it is still under debate whether macrophage accumulation in the sensory ganglia after peripheral nerve injury is due to the local proliferation of resident macrophages or a result of blood monocyte infiltration. Here, we confirmed that the number of macrophages increased in the sensory ganglia after the spared nerve injury (SNI) model in mice. Using different approaches, we found that the increase in the number of macrophages in the sensory ganglia after SNI is a consequence of the proliferation of resident CX3CR1+ macrophages, which participate in the development of neuropathic pain, but not due to infiltration of peripheral blood monocytes. These proliferating macrophages are the source of pro-inflammatory cytokines such as TNF and IL-1b. In addition, we found that CX3CR1 signaling is involved in the sNAMs proliferation and neuropathic pain development after peripheral nerve injury. In summary, these results indicated that peripheral nerve injury leads to sNAMs proliferation in the sensory ganglia in a CX3CR1-dependent manner accounting for neuropathic pain development. In conclusion, sNAMs proliferation could be modulated to change pathophysiological conditions such as chronic neuropathic pain. © 2023, eLife Sciences Publications Ltd. All rights reserved.

Funding details
Washington University in St. LouisWUSTL
Agency for Science, Technology and ResearchA*STAR
Fundação de Amparo à Pesquisa do Estado de São PauloFAPESP2013/08216-2
Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorCAPES

Document Type: Article
Publication Stage: Final
Source: Scopus

Kappa opioid receptor agonists produce sexually dimorphic and prolactin-dependent hyperalgesic priming
(2023) Pain, 164 (6), pp. E263-E273. 

Kopruszinski, C.M.^a , Watanabe, M.^a b , Martinez, A.L.^a b , De Souza, L.H.M.^a b , Dodick, D.W.^c , Moutal, A.^a b , Neugebauer, V.^d , Porreca, F.^a b , Navratilova, E.^a b

^a Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, United States
^b Department of Pharmacology and Physiology, Saint Louis University, School of Medicine, St. Louis, MO, United States
^c Department of Neurology, Mayo Clinic, Phoenix, AZ, United States
^d Department of Pharmacology and Neuroscience, Garrison Institute on Aging, Texas Tech University, Health Sciences Center, Lubbock, TX, United States

Abstract
Repeated stress produces hyperalgesic priming in preclinical models, but underlying mechanisms remain uncertain. As stress engages kappa opioid receptors (KORs), we hypothesized that repeated administration of KOR agonists might mimic, in part, stress-induced hyperalgesic priming. The potential contribution of circulating prolactin (PRL) and dysregulation of the expression of PRL receptor (PRLR) isoforms in sensory neurons after KOR agonist administration was also investigated. Mice received 3 daily doses of U-69593 or nalfurafine as a “first-hit” stimulus followed by assessment of periorbital tactile allodynia. Sixteen days after the first KOR agonist administration, animals received a subthreshold dose of inhalational umbellulone, a TRPA1 agonist, as the second-hit stimulus and periorbital allodynia was assessed. Cabergoline, a dopamine D2 receptor agonist, was used to inhibit circulating PRL in additional cohorts. Prolactin receptor isoforms were quantified in the V1 region of the trigeminal ganglion after repeated doses of U-69593. In both sexes, KOR agonists increased circulating PRL and produced allodynia that resolved within 14 days. Hyperalgesic priming, revealed by umbellulone-induced allodynia in animals previously treated with the KOR agonists, also occurred in both sexes. However, repeated U-69593 downregulated the PRLR long isoform in trigeminal neurons only in female mice. Umbellulone-induced allodynia was prevented by cabergoline co-treatment during priming with KOR agonists in female, but not male, mice. Hyperalgesic priming therefore occurs in both sexes after either biased or nonbiased KOR agonists. However, a PRL/PRLR-dependence is observed only in female nociceptors possibly contributing to pain in stress-related pain disorders in females. © 2023 Lippincott Williams and Wilkins. All rights reserved.

Author Keywords
Functional pain syndrome;  Hyperalgesic priming;  Kappa opioid receptors;  Prolactin;  Stress

Funding details
National Institutes of HealthNIHR01NS120395
National Institutes of HealthNIH

Document Type: Article
Publication Stage: Final
Source: Scopus

AIBP regulates TRPV1 activation in chemotherapy-induced peripheral neuropathy by controlling lipid raft dynamics and proximity to TLR4 in dorsal root ganglion neurons
(2023) Pain, 164 (6), pp. E274-E285. 

Navia-Pelaez, J.M.^a , Borges Paes Lemes, J.^b , Gonzalez, L.^a , Delay, L.^b , Dos Santos Aggum Capettini, L.^a , Lu, J.W.^a , Goncalves Dos Santos, G.^b , Gregus, A.M.^c , Dougherty, P.M.^d , Yaksh, T.L.^b , Miller, Y.I.^a

^a Departments of Medicine, United States
^b Anesthesiology, University of California, San Diego, CA, United States
^c School of Neuroscience, Virginia Polytechnic, State University, Blacksburg, VA, United States
^d Departments of Anesthesia and Pain Medicine, University of Texas, MD Anderson Cancer Center, Houston, TX, United States

Abstract
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. © 2023 Lippincott Williams and Wilkins. All rights reserved.

Author Keywords
AIBP;  Allodynia;  Capsaicin;  Chemotherapy-induced peripheral neuropathy;  Dorsal root ganglia;  Lipid rafts;  Nociceptor;  Pain;  TLR4;  TRPV1

Funding details
National Institute of Neurological Disorders and StrokeNINDSNS102432, NS104769
National Institute of Neurological Disorders and StrokeNINDS
National Institute of Arthritis and Musculoskeletal and Skin DiseasesNIAMSAR075241
National Institute of Arthritis and Musculoskeletal and Skin DiseasesNIAMS
U.S. Department of Veterans AffairsVAI01BX004848, IBX005224, NS047101
U.S. Department of Veterans AffairsVA

Document Type: Article
Publication Stage: Final
Source: Scopus

Probing the conformational dynamics of thiol-isomerases using non-canonical amino acids and single-molecule FRET
(2023) Methods, 214, pp. 8-17. 

Ponzar, N., Pozzi, N.

Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, United States

Abstract
Disulfide bonds drive protein correct folding, prevent protein aggregation, and stabilize three-dimensional structures of proteins and their assemblies. Dysregulation of this activity leads to several disorders, including cancer, neurodegeneration, and thrombosis. A family of 20+ enzymes, called thiol-isomerases (TIs), oversee this process in the endoplasmic reticulum of human cells to ensure efficacy and accuracy. While the biophysical and biochemical properties of cysteine residues are well-defined, our structural knowledge of how TIs select, interact and process their substrates remains poorly understood. How TIs structurally and functionally respond to changes in redox environment and other post-translational modifications remain unclear, too. We recently developed a workflow for site-specific incorporation of non-canonical amino acids into protein disulfide isomerase (PDI), the prototypical member of TIs. Combined with click chemistry, this strategy enabled us to perform single-molecule biophysical studies of PDI under various solution conditions. This paper details protocols and discusses challenges in performing these experiments. We expect this approach, combined with other emerging technologies in single-molecule biophysics and structural biology, to facilitate the exploration of the mechanisms by which TIs carry out their fascinating but poorly understood roles in humans, especially in the context of thrombosis. © 2023 Elsevier Inc.

Author Keywords
Protein disulfide isomerase;  Protein dynamics;  Single-molecule biophysics;  Single-molecule FRET;  Structure–function;  Thiol isomerases;  Thrombosis

Funding details
National Heart, Lung, and Blood InstituteNHLBI

Document Type: Article
Publication Stage: Final
Source: Scopus