# Morphine Tolerance Gated through EZH2‐Mediated Suppression of Trpc5 in Spinal GABAergic Interneurons in Male Mice

## TL;DR

The study shows how repeated morphine use causes pain tolerance by reducing TRPC5 in spinal neurons, and suggests targeting TRPC5 or EZH2 could improve opioid effectiveness.

## Contribution

The novel finding is that morphine tolerance is caused by EZH2-mediated suppression of TRPC5 in spinal GABAergic interneurons, offering new therapeutic targets.

## Key findings

- Morphine tolerance is linked to TRPC5 downregulation in spinal GABAergic interneurons.
- TRPC5 activation or EZH2 inhibition reverses morphine tolerance in mice.
- EZH2 increases histone H3K27me3 at the Trpc5 promoter, suppressing its expression.

## Abstract

A major unresolved issue in managing severe pain is tolerance caused by repeated treatment of opioid analgesics. Here, it is demonstrated that tolerance‐inducing treatment with morphine results in the persistent downregulation of transient receptor potential canonical 5 (TRPC5), impairing the Ca2+ homeostasis in GABAergic interneurons of the spinal dorsal horn (SDH) and consequently reducing GABA release. Spinal activation of TRPC5 by riluzole (RLZ) or lentiviral‐mediated TRPC5 overexpression in GABAergic interneurons produces a long‐lasting enhancement of morphine's analgesic effect. In contrast, pharmacological inhibition of TRPC5 and mice lacking TRPC5 accelerates the development of morphine tolerance. Mechanistically, it is found that transcriptional suppression of Trpc5 results from enhancer of zeste homolog 2 (EZH2)‐mediated epigenetic modifications at the Trpc5 gene promoter. Morphine decreases the enrichment of RNA polymerase II at the Trpc5 promoter. Moreover, exposure to morphine increases EZH2 binding to the Trpc5 promoter, leading to the enrichment of histone H3 lysine‐27 trimethylation (H3K27me3). Pharmacological blockade of EZH2 by EPZ6438 or genetic silencing in GABAergic interneurons reverses morphine tolerance. Thus, it is proposed that the clinical translation of these findings may help reduce the suffering of individuals with intractable pain.

Chronic morphine suppresses Trpc5 in spinal GABAergic interneurons via EZH2‐mediated histone modification, reducing Ca2+ influx and GABA release. TRPC5 activation enhances morphine analgesia, while EZH2 inhibition restores efficacy and reverses tolerance. These results reveal TRPC5 and EZH2 as promising therapeutic targets to overcome opioid tolerance and improve pain management.