Background & Aims

Chronic primary pain conditions (CPPCs), such as fibromyalgia and vestibulodynia, affect millions of patients worldwide [1]. The origin of CPPCs is linked to low activity of the catechol-O-methyltransferase (COMT) enzyme, high levels of environmental stress, and corresponding increases in circulating levels of catecholamines that drive nociception and pain via activation of peripheral beta-3 adrenergic receptor (ADRB3) [2]. However, the downstream consequences of ADRB3 activation in this context remain understudied. Here, we sought to identify pain-relevant microRNAs (miRNAs) downstream of ADRB3 activation and determine their functional effects in humans with CPPCs and in ‘high catecholamine’ rodent model of primary pain (PP). After identifying the downregulation of a single miRNA (miR-133a-3p) across species and linking its expression to white adipose tissue (WAT), we next tested the hypothesis of overexpressing miR-133a-3p in WAT to reverse mechanical sensitivity in our rodent model.

Methods

The PP model is 3 days of swim stress then i.p. injection of COMT-inhibitor OR486 if acute (<6 hours after injection) or s.c. osmotic minipump delivery if sustained time point (14 days). We used RNA from plasma samples and quantified expression with DESeq2 [3]. We performed RT-qPCR [4] to measure miR-133a-3p in plasma of 2 clinical cohorts (fibromyalgia and vestibulodynia), in central and peripheral sites in the PP model, as well as in primary cell culture (WAT, dorsal root ganglion (DRG)) after treatment with ADRB3 agonists. We delivered rAAV8-mAP2.2.-miR-133a-3p-eGFP or nonsense control virus to mice on Day 7 of the model and tracked mechanical sensitivity until Day 28. We measured the von Frey 50% paw withdrawal method for allodynia and hyperalgesia tests [5,6]. Additional experiments studying the effects of miR-133a-3p on predicted target mRNA gene regulation and nociception used in vitro luciferase assays [7] and calcium imaging [8].

Results

RNA sequencing shows that OR486 treatment decreased levels of miR-133a-3p, which was prevented by ADRB3 antagonism. Downregulation in plasma was replicated in patients with fibromyalgia, vestibulodynia, and both time points of the PP mouse model. Downregulation occurs in WAT at both times, but only at the 14 day time point in male mice in the spinal cord. We stimulated ADRB3 in primary adipocytes and found a significant reduction in miR-133a-3p after treatment, with no changes in DRG culture in either sex. We also delivered miR-133a-3p by AAV with an adipose specific promotor on Day 7 of our model and found that mechanical sensitivity is reversed in both sexes starting on Day 21 in line with the expression of AAV. Additionally, we supported the relationship of miR-133a-3p with its strongest predicted binding partners via luciferase assay and observed the ability of a miR-133a-3p mimic to block sensory neurons’ typical response to a capsaicin challenge via in vitro calcium imaging.

Conclusions

These data provide the first link between miR-133a-3p and multiple pain conditions linked to heightened catecholamine tone across species and support the use of peripherally-targeted miR-133a-3p overexpression strategies for the treatment of CPPCs. We discovered the unique role of ADRB3 on adipose in controlling the expression of miR-133a-3p and observe sex differences in expression patterns that highlight the need for studying sex as a variable in pain conditions. Future work will explore the sex-specific mRNA target networks impacted by this differential expression, as well as examine the cell type source(s) of miRNA expression. Additionally, we will isolate exosomes and measure miR-133a-3p across conditions to test the hypothesis that adipose-neuron crosstalk occurs through exosome signaling between these tissue sites.

References

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[2] Ciszek BP, O’Buckley SC, Nackley AG. Persistent Catechol-O-methyltransferase-dependent Pain Is Initiated by Peripheral beta-Adrenergic Receptors. Anesthesiology 2016;124(5):1122-1135.

[3] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15(12):550.

[4] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-408.

[5] Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53(1):55-63.

[6] Zhang X, Kanter K, Chen J, Kim S, Wang Y, Adeyemi C, O’Buckley SC, Nackley AG. Low catechol-O-methyltransferase and stress potentiate functional pain and depressive behavior, especially in female mice. Pain 2020;161(2):446-458.

[7] Jin Y, Chen Z, Liu X, Zhou X. Evaluating the microRNA targeting sites by luciferase reporter gene assay. Methods Mol Biol 2013;936:117-127.

[8] Wang Y, Scarneo SA, Kim SH, Zhang X, Chen J, Yang KW, Hughes P, Haystead T, Nackley AG. Expression of ectopic heat shock protein 90 in male and female primary afferent nociceptors regulates inflammatory pain. Pain 2022;163(6):1091-1101.

[9] Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16(2):109-110.

[10] Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, Clark A, Cuthill IC, Dirnagl U, Emerson M, Garner P, Holgate ST, Howells DW, Karp NA, Lazic SE, Lidster K, MacCallum CJ, Macleod M, Pearl EJ, Petersen OH, Rawle F, Reynolds P, Rooney K, Sena ES, Silberberg SD, Steckler T, Wurbel H. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. BMJ Open Sci 2020;4(1):e100115.

Presenting Author

Nathaniel Hernandez

Poster Authors

Nathaniel Hernandez

BSc

Duke University

Lead Author

E-mail

Jiegen Chen

PhD

Duke University

Lead Author

E-mail

Xin Zhang

MD PhD

Duke University

Lead Author

E-mail

Yaomin Wang

PhD

Duke University

Lead Author

E-mail

Yiling Qian

MD PhD

Duke University

Lead Author

E-mail

Xianglong Gao

BS

Duke University

Lead Author

E-mail

Brittney Ciszek

DDS PhD

White Cap Institute

Lead Author

E-mail

Matt Kanke

PhD

Cornell University

Lead Author

E-mail

Mohamad Karaky

McGill University

Lead Author

E-mail

Carolina Beraldo Meloto

PhD

McGill University

Lead Author

E-mail

Francesca Montagna

McGill University

Lead Author

E-mail

Marguerita Klein

BS

Duke University

Lead Author

E-mail

Praveen Sethupathy

PhD

Cornell University

Lead Author

E-mail

Luda Diatchenko

McGill University

Lead Author

E-mail

Topics

  • Treatment/Management: Pharmacology: Monoclonal Antibodies, Biologics, and Biosimilars