Animal Studies

Myalgic Encephalomyelitis, also known as Chronic Fatigue Syndrome (ME/CFS), is a multisystem illness characterized by extreme muscle fatigue associated with pain, neurocognitive impairment, and chronic inflammation. Despite intense investigation, the molecular mechanism of this disease is still unknown. Here we demonstrate that autophagy-related protein ATG13 is strongly upregulated in the serum of ME/CFS patients, indicative of impairment in the metabolic events of autophagy. A Thioflavin T-based protein aggregation assay, array screening for autophagy-related factors, densitometric analyses, and confirmation with ELISA revealed that the level of ATG13 was strongly elevated in serum samples of ME/CFS patients compared to age-matched controls. Moreover, our microglia-based oxidative stress response experiments indicated that serum samples of ME/CFS patients evoke the production of reactive oxygen species (ROS) and nitric oxide in human HMC3 microglial cells, whereas neutralization of ATG13 strongly diminishes the production of ROS and NO, suggesting that ATG13 plays a role in the observed stress response in microglial cells. Finally, an in vitro ligand binding assay provided evidence that ATG13 employs the Receptor for Advanced Glycation End-products (RAGE) to stimulate ROS in microglial cells. Collectively, our results suggest that an impairment of autophagy following the release of ATG13 into serum could be a pathological signal in ME/CFS.

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Deletion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-Cre: PTEN (PTEN CKO) mice with oral administration of tamoxifen, and CKO mice develop pathological itch and elevated itch responses on exposure to various pruritogens. PTEN deletion leads to ectopic expression of TRPV1 and MrgprA3 in IB4 non-peptidergic DRG neurons, and the TRPV1 is responsive to capsaicin. Importantly, the elevated itch responses are no longer present in Drg11-Cre: PTEN: TRPV1 (PTEN: TRPV1 dCKO) mice. In addition, thermal stimulation is enhanced in PTEN CKO mice but blunted in dCKO mice. PTEN-involved regulation of itch-related gene expression in DRG neurons provides insights for understanding molecular mechanism of itch and thermal sensation at the spinal level.

Metabolic adaptations can directly influence the scope and scale of macrophage activation and polarization. Here we explore the impact of type I interferon (IFNβ) on macrophage metabolism and its broader impact on cytokine signaling pathways. We find that IFNβ simultaneously increased the expression of immune-responsive gene 1 and itaconate production while inhibiting isocitrate dehydrogenase activity and restricting α-ketoglutarate accumulation. IFNβ also increased the flux of glutamine-derived carbon into the tricarboxylic acid cycle to boost succinate levels. Combined, we identify that IFNβ controls the cellular α-ketoglutarate/succinate ratio. We show that by lowering the α-ketoglutarate/succinate ratio, IFNβ potently blocks the JMJD3-IRF4-dependent pathway in GM-CSF and IL-4 activated macrophages. The suppressive effects of IFNβ on JMJD3-IRF4-dependent responses, including M2 polarization and GM-CSF-induced inflammatory pain, were reversed by supplementation with α-ketoglutarate. These results reveal that IFNβ modulates macrophage activation and polarization through control of the cellular α-ketoglutarate/succinate ratio.

Neuropathic pain is a debilitating chronic syndrome of the nervous system caused by nerve injury. In Drosophila, the Hedgehog (Hh) signaling pathway is related to increased pain sensitivity (hyperalgesia) but does not affect the baseline nociceptive threshold. In general, the contribution of the Hh signaling pathway to neuropathic pain in vertebrates is a highly debated issue. Alternatively, we investigated the potential role of Hh signaling in mechanical allodynia using a mouse model of neuropathic pain. Seven days after spinal nerve-transection (SNT) surgery, microglial activation increased in the ipsilateral spinal dorsal horn compared with that in the sham group; however, 21 days after surgery, microglial activation decreased. Contrastingly, astrocyte activation in the spinal cord did not differ between the groups. On day 21 of postsurgery, the SNT group showed marked upregulation of sonic hedgehog expression in peripheral glial cells but not in dorsal root ganglion (DRG) neurons. Intrathecal administration of the Hh signaling inhibitor vismodegib attenuated the mechanical allodynia observed on day 21 postsurgery. Conversely, intrathecal treatment with the Hh signaling activator smoothened agonist in naive mice induced mechanical allodynia, which was abolished by the ATP transporter inhibitor clodronate. Moreover, inhibition of Hh signaling by pretreatment with vismodegib significantly reduced ATP secretion and the frequency/number of spontaneous elevations of intracellular calcium ion levels in cultured DRG cells. Thus, the Hh signaling pathway appears to modulate the neural activity of DRG neurons via ATP release, and it plays an important role in sustaining mechanical allodynia and hypersensitivity in a mouse model of neuropathic pain.

Ample data support a prominent role of peripheral T-type calcium channels 3.2 (CaV3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of CaV3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from the natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. Here, we report the engineering of the potent and selective iPAs of CaV3.2 from the intrinsically disordered regions (IDR) of CaV3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in CaV3.2 protein and identified several CaV3.2iPA candidates that significantly reduced CaV3.2 current in HEK293 cells stably expressing human wide-type CaV3.2. Two prototype CaV3.2iPAs (iPA1 and iPA2) derived from the IDRs of CaV3.2 intracellular loop 2 and 3 respectively were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by CaV3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain following tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated CaV3.2iPA expression suppressed neuronal excitability, suggesting that CaV3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that CaV3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral CaV3.2-targeting strategy for clinical treatment of pain.