DRGE什么公司SMN deficiency causes pain hypersensitivity in a mild SMA mouse model through enhancing excitability of nociceptive dorsal root ganglion neurons

In light of a recent survey, chronic pain is a frequent issue in SMA children²⁵. The causes of pain are multifactorial and vary by medical conditions. To improve pain management in SMA patients, it is necessary to elucidate signaling pathways and molecules that induce chronic pain in the disease. In this study, we revealed that the mild Taiwanese SMA mouse model displays a pronounced increase in response to both noxious and innocuous stimuli including prominent sensitization to mechanical force. Using whole-cell patch clamp recording, we found that primary nociceptive neurons in L4–L6 DRGs are hyperexcitable, which involves enhanced Na⁺ currents due to increased expression of sodium channels, particularly of TTX-sensitive Na_v1.7 and TTX-resistant Na_v1.8. Furthermore, we uncovered that transcription factor NF-κB is both activated and upregulated in DRGs of SMA mice. Treatment with a selective NF-κB inhibitor PDTC in SMA mice confirmed the two VGSCs are downstream targets of NF-κB signaling. Our data is consistent with earlier studies that show NF-κB as a positive regulator of Na_v1.7 and Na_v1.8 in rats with diabetic neuropathy or motor fiber injury^41,42. In addition, screening with adrenergic blockers, we identified that enhanced adrenergic signaling, mediated by the β2 receptor, accounts for the increase in both phosphorylation and expression of p50 and p65 in primary nociceptive neurons of SMA mice. Finally, we unveiled that blood levels of NE, an important stress hormone and neurotransmitter as well as a pain inducer are significantly elevated in SMA mice. Therefore, we not only uncovered pain hypersensitivity as a pathological feature in a SMA mouse model, but more importantly, identified a detailed peripheral signaling cascade that induces pain hypersensitivity (Fig. 8).

We previously employed the same mild Taiwanese model in a preclinical study to first demonstrate the in vivo efficacy of nusinersen, the only approved drug to treat SMA, in which, an efficient correction of SMN2 splicing and rescue of tissue necrosis, a prominent phenotypic feature of this model, were achieved^27,52. However, to the best of our knowledge, no pathological changes in the nervous system and motor defects in the model have been previously documented; rotarod test revealed no differences in fall latency between SMA and heterozygous mice (Supplemental Fig. S3). Here we demonstrate that nociceptive DRG neurons in the model are hyperexcitable. Previously, hypoexcitability of cholinergic sensory neurons has been observed in Drosophila smn mutants^16,17. Though the underlying mechanisms are unknown, the discrepancy should be mainly due to differences in motor circuit features between the two species. For instance, sensory and motor neurons in Drosophila are cholinergic and glutamatergic, respectively, which are opposite as in humans and mice. Nonetheless, hyperexcitability of spinal cord motor neurons as well as activation of microglia and astrocytes in severe SMA mouse models has been well documented^21,23,53. A more recent study demonstrate that motor neurons derived from patients’ iPSCs are hyperexcitable⁵⁴. Interestingly, the profiles of motor neuron excitability in all the previous studies are similar to what we observed in nociceptive DRG neurons. Moreover, for both neuronal cell types, hyperexcitability involves robust increase in VGSC currents. These findings suggest that hyperexcitability may be a general feature in the mammalian nervous system when SMN is deficient.

NF-κB signaling is tightly coordinately with other signaling pathways and has been implicated as a cause in multiple pain models via modulating expression of multiple pain-related genes including some VGSCs. Our data show that activation of NF-κB signaling as well as upregulation of p50 and p65 also mediates the pain hypersensitivity in SMA mice. Interestingly, while our work was in progress, two studies described a reciprocal regulation between NF-κB signaling and SMN levels^19,55. Using mouse BV2 microglia cells, Kim and Choi reported that SMN inhibits NF-κB signaling through suppression of the E3 ubiquitin ligase activity of TRAF6 via physical interaction with the protein, a positive modulator in NF-κB signaling pathway⁵⁵. However, no activation of p65 was observed by them when SMN was depleted in the cultured cells without addition of IL-1 in the medium, suggesting that SMN has no effect on NF-κB activity under physiological conditions. On the other hand, Arumugam et al. reported that NF-κB signaling inhibits expression of SMN in cultured motor neurons isolated from mouse embryos⁵⁶. The authors also observed a reduction in phosphorylated RelA in cultured spinal cord motor neurons derived from a severe SMA mouse model, which contradicts with the result reported by Kim and Choi, which may reflect difference in cell types used in the two studies. We have recently shown that a severe mouse model develops systemic inflammation with enhanced NF-κB signaling in the liver, which is likely caused by both microbial infection and sterile stimuli⁴³. Currently, we do not know whether systemic inflammation also occurs in the mild SMA mouse model and whether enhanced NF-κB signaling involves cell-autonomous mechanisms. However, our data demonstrate a non-cell-autonomous mechanism for enhancing NF-κB signaling via the NE/ADRB2 pathway in SMA mice.

Receptors that mediates NE-induced pain could be different from case to case. Either α1 or β2 antagonists have been frequently shown to attenuate pain hypersensitivity in different conditions^46,57,58. Here we show that enhanced adrenergic signaling mediated by the β2 receptor is involved in pain hypersensitivity in SMA mice; and this is attributed in part to increase in the blood NE level rather than upregulation or phosphorylation of ADRB2. Interestingly, a recent study reported that agonists of β2 adrenergic signaling alleviated pain when injected into CSF⁵⁹, suggesting the roles of β2 signaling are location-dependent.

The effects of NE/ADRB2 on NE-κB activity are complex, and could be inhibitory or stimulatory depending on cell types⁵⁰. For example, NE inhibits NF-κB signaling through activating IkBα in rat primary cortical neurons and astrocytes^51,60. On the contrary, NE activates NF-κB homodimer p50/p50 through promoting its nuclear translocation in rat cultured pinealocytes⁶¹. In the present study, we show that NE/ADRB2 signaling promotes expression as well as phosphorylation of p65 and p50 in SMA mouse DRG neurons. It has been well established that NE is a critical factor in causing allodynia and hyperalgesia. However, peripheral NE has no effect on pain in healthy subjects but aggravates pain in abnormal conditions. Indeed, intraperitoneal administration of NE has no pain-inducing effect on heterozygous SMA mice (Fig. 7f). This suggests that NE acts as an accomplice in pain hypersensitivity. In the case of SMA, lack of SMN presumably creates a permissive state that allows NE and potentially other agents to exert their pro-nociceptive roles. Our observation that administration of propranolol and butoxamine relieved allodynia and NE exacerbated allodynia in SMA mice in 30 min is in agreement with a previous report that epinephrine-induced hyperalgesia develops rapidly⁴⁶. Take together with our finding that β2-adrenergic signaling promotes expression of NF-κB subunits, these data suggest two peripheral mechanisms present in the NE/ADRB2-induced pain hypersensitivity in SMA mice: a fast path and a slow path. The former mechanism should involve post-translational modifications of critical proteins including NF-κB subunits as well as PKA or PKC⁴⁶, leading to a quick conformational alteration of VGSCs. The latter mechanism should involve gene expression alterations in the signaling nodes including upregulation of NF-κB, Na_v1.7 and Na_v1.8, and might play a more important role in persistent pain hypersensitivity in SMA mice.

Increase of plasma levels of NE, a major stress hormone, has profound effects on various tissues. For example, in the pancreas, it promotes release of glucagon, leading to glucose rise. In line with this, Bowenman et al. observed that blood glucagon levels are elevated in SMA mice⁶². Though the authors attribute the observation to an unbalance in populations of α- and β-cells in the islet, a persistent high level of blood NE is probably a cause as well⁶². The sympathetic nervous system and the hypothalamic-pituitary-adrenal axis are two sources of plasma NE. However, the fact that plasma E levels are not altered suggests activation of sympathetic nerves in SMA mice as the main cause. Although disturbance in the autonomic nervous system is a long-recognized feature in both SMA patients and mouse models, its role in SMA pathogenesis has been underappreciated¹¹. Taking into account the model used in our study being a very mild one, autonomic dysfunction may be a serious issue in the context of SMA.

Although we revealed a cell-non-autonomous pathway that mediates pain hypersensitivity in mild SMA mice through activating primary nociceptive neurons (Fig. 8), the mechanisms driving SMA-associated pain hypersensitivity are undoubtedly complex. For example, we found that expression of several pain-associated potassium channel genes is downregulated in the DRGs of the SMA mouse model (Supplemental Fig. S4), which may also contribute to increased excitability of the DRG neurons.

In light of our data, several key questions remain to be addressed. First, the source of increased plasma NE levels needs to clarified. If indeed an increase in release from autonomic nerves occurs, then what is the trigger? Neuropathic or inflammatory? It is also intriguing to find out whether psychological factors such as anxiety play a role. In addition, it is important to understand to what extent the observations made in the mild mouse model are directly relevant to SMA patients. Nonetheless, considering that SMA mouse models have been widely used for drug development, leading to identification of nusinersen and many others that are in clinical trials, key genes in our study may provide potential drug targets for pain relief in patients with SMA. For example, high-throughput screens or targeted approaches can be designed to identify molecules that suppress NF-κB activities and/or excitability of primary nociceptive neurons in DRGs. Particularly, the SCN9A gene represents an ideal target for treating chronic pain in the context of SMA as well other diseases as disruption of this gene alone leads to lack of pain but appears not to cause other health issues except anosmia^63,64.