Mycolactone-mediated neurite degeneration and functional effects in cultured human and rat DRG neurons

Abstract

BackgroundMycolactone is a polyketide toxin secreted by the mycobacterium Mycobacterium ulcerans, responsible for the extensive hypoalgesic skin lesions characteristic of patients with Buruli ulcer. A recent pre-clinical study proposed that mycolactone may produce analgesia via activation of the angiotensin II type 2 receptor (AT2R). In contrast, AT2R antagonist EMA401 has shown analgesic efficacy in animal models and clinical trials for neuropathic pain. We therefore investigated the morphological and functional effects of mycolactone in cultured human and rat dorsal root ganglia (DRG) neurons and the role of AT2R using EMA401. Primary sensory neurons were prepared from avulsed cervical human DRG and rat DRG; 24 h after plating, neurons were incubated for 24 to 96 h with synthetic mycolactone A/B, followed by immunostaining with antibodies to PGP9.5, Gap43, β tubulin, or Mitotracker dye staining. Acute functional effects were examined by measuring capsaicin responses with calcium imaging in DRG neuronal cultures treated with mycolactone.ResultsMorphological effects: Mycolactone-treated cultures showed dramatically reduced numbers of surviving neurons and non-neuronal cells, reduced Gap43 and β tubulin expression, degenerating neurites and reduced cell body diameter, compared with controls. Dose-related reduction of neurite length was observed in mycolactone-treated cultures. Mitochondria were distributed throughout the length of neurites and soma of control neurons, but clustered in the neurites and soma of mycolactone-treated neurons. Functional effects: Mycolactone-treated human and rat DRG neurons showed dose-related inhibition of capsaicin responses, which were reversed by calcineurin inhibitor cyclosporine and phosphodiesterase inhibitor 3-isobutyl-1-Methylxanthine, indicating involvement of cAMP/ATP reduction. The morphological and functional effects of mycolactone were not altered by Angiotensin II or AT2R antagonist EMA401.ConclusionMycolactone induces toxic effects in DRG neurons, leading to impaired nociceptor function, neurite degeneration, and cell death, resembling the cutaneous hypoalgesia and nerve damage in individuals with M. Ulcerans infection.

Sec61 blockade by mycolactone: A central mechanism in Buruli ulcer disease

“…In mice, neural pathology associated with hypoesthesia was induced by infection with M. ulcerans , or injection of purified mycolactone [En et al., ; Goto et al., ]. Short‐term exposure to mycolactone (24 h, 100 nM) induced significant neurite degeneration in rat and human primary dorsal root ganglion (DRG) sensory neurons [Anand et al., ]. Longer treatments (>48 h) induced massive mortality of primary DRGs in two studies [Anand et al., ; Isaac et al., ], although a third study reported a minimal loss of viability following exposure to mycolactone doses of up to 70 μM [Song et al., ].…”

Macrophage-to-sensory neuron crosstalk mediated by Angiotensin II type-2 receptor elicits neuropathic pain

“…In contrast, a myolactone toxin from Buruli ulcer-causing bacteria was shown to activate Gα i/o -coupled AT2R in sensory neurons, leading to analgesia in mice (11). Interestingly, a recent follow-up study demonstrated that the AT2R antagonist EMA401 was unable to prevent the myolactone toxin’s effect on sensory neurons in vitro (12). This raises the possibility that the analgesic actions of EMA401 could result from targeting non-neuronal AT2R, or could be entirely independent of AT2R antagonism.…”

Nociceptor‐like rat dorsal root ganglion neurons express the angiotensin‐II AT2 receptor throughout development

“…Recently, several synthetic drugs with antagonist effect on AT2R were reported to attenuate prostate cancer induced bone pain (Muralidharan et al, 2014), alleviate neuropathic pain (Anand et al, 2015; Rice et al, 2014; Smith et al, 2013) and reverse hypoesthesia induced by M. ulcerans in the Buruli ulcer (Anand et al, 2016; Marion et al, 2014). However, the molecular and cellular bases of these actions of AT2R remain poorly understood.…”

“…2015; Rice et al, 2014;Smith et al, 2013) and reverse hypoesthesia induced by M. ulcerans in the Buruli ulcer (Anand et al, 2016;Marion et al, 2014). However, the molecular and cellular bases of these actions of AT2R remain poorly understood.…”