Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1-G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

There are no appropriate mouse models to study the pathophysiology of spontaneous disc herniations and associated pain pathology. We demonstrate that SM/J mice show a high incidence of age-associated lumbar disc herniations with neurovascular innervations. Transcriptomic comparisons of the SM/J annulus fibrosus with human tissues showed shared pathways related to immune cell activation and inflammation. Notably, aged SM/J mice showed increased pain sensitization and neuroinflammatory signatures associated with altered extracellular matrix regulation in the DRGs and spinal cord. There were increased T cells in the vertebral marrow, and CyTOF analysis showed increased splenic CD8+ T cells, nonspecific activation of CD8+ memory T cells, and enhanced IFN-γ production in the myeloid compartment. ScRNA-seq of PBMCs in SM/J showed more B cells, with lower proportions of T cells, monocytes, and granulocytes. This study identifies SM/J mice as a clinically-relevant model to study the pathophysiology of spontaneous disc herniations and highlights a causative axis for chronic discogenic pain with novel contributors from the primary lymphoid organs (spleen and vertebral marrow), circulation, and the nervous system.One-Sentence Summary The novel SM/J mouse model shows a neuroimmune axis drives chronic back pain, a leading cause of years lived with disability.### Competing Interest StatementThe authors have declared no competing interest.

Neuropathic pain (NP) is one of the most common and debilitating comorbidities of spinal cord injury (SCI). Current therapies are often ineffective due in part to an incomplete understanding of underlying pathogenic mechanisms. In particular, it remains unclear how SCI leads to dysfunction in the excitability of nociceptive circuitry. The immediate early gene c-Fos has long been used in pain processing locations as a marker of neuronal activation. We employed a mouse reporter line with fos-promoter driven Cre-recombinase to define neuronal activity changes in relevant pain circuitry locations following cervical spinal cord level (C)5/6 contusion (using both females and males), a SCI model that results in multiple forms of persistent NP-related behavior. SCI significantly increased activation of cervical dorsal horn (DH) projection neurons, as well as induced a selective reduction in the activation of a specific DH projection neuron subpopulation that innervates the periaqueductal gray (PAG), an important brain region involved in descending inhibitory modulation of DH pain transmission. SCI also increased the activation of both protein kinase C (PKC)γ and calretinin excitatory DH interneuron populations. Interestingly, SCI promoted a significant decrease in the activation selectively of neuronal nitric oxide synthase (nNOS)-expressing inhibitory interneurons of cervical DH. In addition, SCI altered activation of various supraspinal neuron populations associated with pain processing, including a large increase in thalamus and a significant decrease in PAG. These findings reveal a complex and diverse set of SCI-induced neuron activity changes across the pain circuitry neuraxis. Moving forward, these results can be used to inform therapeutic targeting of defined neuronal populations in NP.SIGNIFICANCE STATEMENTNeuropathic pain (NP) is one of the most common and highly debilitating comorbidities of spinal cord injury (SCI). Unfortunately, current therapies are often ineffective due in part to an incomplete understanding of underlying pathogenic mechanisms. In particular, it remains unclear how SCI leads to dysfunction in excitability of nociceptive circuitry. Using a FosTRAP2 reporter mouse line in a model of SCI-induced NP, we show SCI alters activation of a number of important interneuron and projection neuron populations across relevant spinal cord and brain locations of the pain circuitry neuraxis. These data suggest a role for maladaptive plasticity involving specific subpopulations of neurons and circuits in driving SCI-induced chronic pain. Moving forward, these results can be used to inform therapeutic targeting of defined neuronal populations in NP.

A significant portion of individuals living with traumatic spinal cord injury (SCI) experiences some degree of debilitating neuropathic pain (NP). This pain remains largely intractable in a majority of cases, due in part to an incomplete understanding of its underlying mechanisms. Central sensitization, an increase in excitability of pain transmission neurons located in superficial dorsal horn (sDH), plays a key role in development and maintenance of SCI-induced NP. Resident microglia and peripheral monocyte-derived macrophages (referred to collectively as MMΦ) are involved in promoting SCI-induced DH neuron hyperexcitability. Importantly, these MMΦ consist of populations of cells that can exert pro-inflammatory or anti-inflammatory signaling within injured spinal cord. It is critical to spatiotemporally characterize this heterogeneity to understand MMΦ contribution to NP after SCI. Given that a majority of SCI cases are cervical in nature, we used a model of unilateral C5/C6 contusion that results in persistent at-level thermal hyperalgesia and mechanical allodynia, two forms of NP-related behavior, in the forepaw. The aim of this study was to characterize the sDH MMΦ response within intact cervical spinal cord segments caudal to the lesion (i.e. the location of primary afferent nociceptive input from the forepaw plantar surface). Cervical SCI promoted a persistent MMΦ response in sDH that coincided with the chronic NP phenotype. Using markers of pro- and anti-inflammatory MMΦ, we found that the MMΦ population within sDH exhibited significant heterogeneity that evolved over time post-injury, including a robust and persistent increase in pro-inflammatory MMΦ that was especially pronounced at later times. C5/C6 contusion SCI also induced below-level thermal hyperalgesia and mechanical allodynia in the hindpaw; however, we did not observe a pronounced MMΦ response in sDH of L4/L5 spinal cord, suggesting that different inflammatory cell mechanisms occurring in sDH may be involved in at-level versus below-level NP following SCI. In conclusion, our findings reveal significant MMΦ heterogeneity both within and across pain transmission locations after SCI. These data also show a prominent and persistent pro-inflammatory MMΦ response, suggesting a possible role in DH neuron hyperexcitability and NP.

The voltage-gated potassium channel Kv3.4 is a crucial regulator of nociceptive signaling in the dorsal root ganglion (DRG) and the dorsal horn of the spinal cord. Moreover, Kv3.4 dysfunction has been linked to neuropathic pain. Although kinases and phosphatases can directly modulate Kv3.4 gating, the signaling mechanisms regulating the expression and stability of the Kv3.4 protein are generally unknown. We explored a potential role of PKCε and found an unexpected interaction that has a positive effect on Kv3.4 expression. Co-immunoprecipitation studies revealed a physical association between PKCε and Kv3.4 in both heterologous cells and rat DRG neurons. Furthermore, in contrast to the wild-type and constitutively active forms of PKCε, expression of a catalytically inactive form of the enzyme inhibits Kv3.4 expression and membrane localization through a dominant negative effect. Co-expression of Kv3.4 with the wild-type, constitutively active, or catalytically inactive forms of PKCε had no significant effects on Kv3.4 gating. These results suggest that a novel physical interaction of the Kv3.4 channel with functional PKCε primarily determines its stability and localization in DRG neurons. This interaction is akin to those of previously identified accessory ion channel proteins, which could be significant in neural tissues where Kv3.4 regulates electrical signaling.

Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar effects on the recombinant Kv3.4 channel heterologously expressed in Chinese hamster ovary cells, supporting our findings in DRG neurons. Elucidating the molecular basis of these effects, mutation of four previously characterized serines within the Kv3.4 N-terminal inactivation domain eliminated the effects of CaN inhibition on the Kv3.4 current. SCI similarly induced concurrent Kv3.4 current attenuation and slowing of inactivation. Although there was little change in CaN expression and localization after injury, SCI induced upregulation of the native regulator of CaN 1 (RCAN1) in the DRG at the transcript and protein levels. Consistent with CaN inhibition resulting from RCAN1 upregulation, overexpression of RCAN1 in naive DRG neurons recapitulated the effects of pharmacological CaN inhibition on the Kv3.4 current and the AP. Overall, these results demonstrate a novel regulatory pathway that links CaN, RCAN1, and Kv3.4 in DRG neurons. Dysregulation of this pathway might underlie a peripheral mechanism of pain sensitization induced by SCI. SIGNIFICANCE STATEMENT Pain sensitization associated with spinal cord injury (SCI) involves poorly understood maladaptive modulation of neuronal excitability. Although central mechanisms have received significant attention, recent studies have identified peripheral nerve hyperexcitability as a driver of persistent pain signaling after SCI. However, the ion channels and signaling molecules responsible for this change in primary sensory neuron excitability are still not well defined. To address this problem, this study used complementary electrophysiological and molecular methods to determine how Kv3.4, a voltage-gated K+ channel robustly expressed in dorsal root ganglion neurons, becomes dysfunctional upon calcineurin (CaN) inhibition. The results strongly suggest that CaN inhibition underlies SCI-induced dysfunction of Kv3.4 and the associated excitability changes through upregulation of the native regulator of CaN 1 (RCAN1).