Serpinin in the Skin

The serpinins are relatively novel peptides generated by proteolytic processing of chromogranin A and they are comprised of free serpinin, serpinin-RRG and pGlu-serpinin. In this study, the presence and source of these peptides were studied in the skin. By Western blot analysis, a 40 kDa and a 50 kDa protein containing the sequence of serpinin were detected in the trigeminal ganglion and dorsal root ganglia in rats but none in the skin. RP-HPLC followed by EIA revealed that the three serpinins are present in similar, moderate amounts in rat dorsal root ganglia, whereas in the rat skin, free serpinin represents the predominant molecular form. There were abundant serpinin-positive cells in rat dorsal root ganglia and colocalization with substance P was evident. However, much more widespread distribution of the serpinins was found in dorsal root ganglia when compared with substance P. In the skin, serpinin immunoreactivity was found in sensory nerves and showed colocalization with substance P; as well, some was present in autonomic nerves. Thus, although not exclusively, there is evidence that serpinin is a constituent of the sensory innervation of the skin. The serpinins are biologically highly active and might therefore be of functional significance in the skin.

Cisplatin neurotoxicity targets specific subpopulations and K+ channels in tyrosine-hydroxylase positive dorsal root ganglia neurons

Among the features of cisplatin chemotherapy-induced peripheral neuropathy are chronic pain and innocuous mechanical hypersensitivity. The complete etiology of the latter remains unknown. Here, we show that cisplatin targets a heterogeneous population of tyrosine hydroxylase-positive (TH+) primary afferent dorsal root ganglion neurons (DRGNs) within the primary afferent dorsal root ganglia in mice, determined using single-cell transcriptome and electrophysiological analyses. TH+ DRGNs regulate innocuous mechanical sensation through C-low threshold mechanoreceptors. A differential assessment of wild-type and vitamin E deficient TH+ DRGNs revealed heterogeneity and specific functional phenotypes. The TH+ DRGNs comprise; fast-adapting eliciting one action potential (AP; 1-AP), moderately-adapting (>=2-APs), in responses to square-pulse current injection, and spontaneously firing (SF). Cisplatin increased the input resistance and AP frequency but reduced the temporal coding feature of 1-AP and >= 2-APs neurons. By contrast, cisplatin has no measurable effect on the SF neurons. Vitamin E reduced the cisplatin-mediated increased excitability, but did not improve the TH+ neuron temporal coding properties. Cisplatin mediates its effect by targeting outward K+ current, likely carried by through K2P18.1 (Kcnk18), discovered through the differential transcriptome studies and heterologous expression. Studies show a potential new cellular target for chemotherapy-induced peripheral neuropathy and implicate the possible neuroprotective effects of vitamin E in cisplatin chemotherapy.

Inhibition of phosphorylated calcium/calmodulin-dependent protein kinase IIα relieves streptozotocin-induced diabetic neuropathic pain through regulation of P2X3 receptor in dorsal root ganglia

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

Oncomodulin derived from regeneration-associated macrophages in dorsal root ganglia promotes axon regeneration in the spinal cord

Preconditioning peripheral nerve injury enhances axonal regeneration of dorsal root ganglia (DRG) neurons in part by driving pro-regenerative perineuronal macrophage activation. How these regeneration-associated macrophages influence the neuronal capacity of axon regeneration remains elusive. The present study reports that oncomodulin (ONCM) is an effector molecule derived from the regeneration-associated macrophages. ONCM was highly upregulated in DRG macrophages following preconditioning injury and necessary for the preconditioning-induced neurite outgrowth. ONCM-deficient macrophages failed to generate neurite outgrowth activity of the conditioned medium in the in vitro model of neuron-macrophage interaction. CCL2/CCR2 signaling is an upstream regulator of ONCM since the ONCM upregulation was dependent on CCR2 and CCL2 overexpression-mediated conditioning effects were attenuated in ONCM-deficient mice. Direct application of ONCM potently increased neurite outgrowth in cultured DRG neurons by activating a distinct gene set, particularly neuropeptide-related genes. AAV-mediated overexpression of ONCM construct with the signal sequence increased neuronal secretion of ONCM and enhanced neurite outgrowth in an autocrine manner. For a clinically relevant approach, we developed a nanogel-mediated system for localized delivery of recombinant ONCM to DRG tissue. Electrostatic encapsulation of ONCM by a reducible epsilon-poly(L-lysine)-nanogel (REPL-NG) resulted in a slow release of ONCM allowing sustained bioactivity. Intraganglionic injection of REPL-NG/ONCM complex achieved a remarkable long-range axonal regeneration beyond spinal cord lesion, surpassing the extent expected from the preconditioning effects. The NG-mediated ONCM delivery could be exploited as a therapeutic strategy for promoting sensory axon regeneration following spinal cord injury.

Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species

Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on Day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as Day 8 in rats (≥5x1013 vg/kg) and Day 14 in monkeys (≥3.3x1013 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species.

Sensitization of primary cultures from rat dorsal root ganglia with lipopolysaccharide (LPS) requires a robust inflammatory response

Objective We investigated whether it is possible to induce a state of “LPS-sensitization” in neurons of primary cultures from rat dorsal root ganglia by pre-treatment with ultra-low doses of LPS. Methods DRG primary cultures were pre-treated with low to ultra-low doses of LPS (0.001–0.1 µg/ml) for 18 h, followed by a short-term stimulation with a higher LPS-dose (10 µg/ml for 2 h). TNF-α in the supernatants was measured as a sensitive read out. Using the fura-2 340/380 nm ratio imaging technique, we further investigated the capsaicin-evoked Ca2+-signals in neurons from DRG, which were pre-treated with a wide range of LPS-doses. Results Release of TNF-α evoked by stimulation with 10 µg/ml LPS into the supernatant was not significantly modified by pre-exposure to low to ultra-low LPS-doses. Capsaicin-evoked Ca2+-signals were significantly enhanced by pre-treatment with LPS doses being above a certain threshold. Conclusion Ultra-low doses of LPS, which per se do not evoke a detectable inflammatory response, are not sufficient to sensitize neurons (Ca2+-responses) and glial elements (TNF-α-responses) of the primary afferent somatosensory system.

Sexual Dimorphism of Early Transcriptional Reprogramming in Dorsal Root Ganglia After Peripheral Nerve Injury

Peripheral nerve injury induces genome-wide transcriptional reprogramming of first-order neurons and auxiliary cells of dorsal root ganglia (DRG). Accumulating experimental evidence suggests that onset and mechanistic principles of post-nerve injury processes are sexually dimorphic. We examined largely understudied aspects of early transcriptional events in DRG within 24 h after sciatic nerve axotomy in mice of both sexes. Using high-depth RNA sequencing (>50 million reads/sample) to pinpoint sexually dimorphic changes related to regeneration, immune response, bioenergy, and sensory functions, we identified a higher number of transcriptional changes in male relative to female DRG. In males, the decline in ion channel transcripts was accompanied by the induction of innate immune cascades via TLR, chemokine, and Csf1-receptor axis and robust regenerative programs driven by Sox, Twist1/2, and Pax5/9 transcription factors. Females demonstrated nerve injury-specific transcriptional co-activation of the actinin 2 network. The predicted upstream regulators and interactive networks highlighted the role of novel epigenetic factors and genetic linkage to sex chromosomes as hallmarks of gene regulation post-axotomy. We implicated epigenetic X chromosome inactivation in the regulation of immune response activity uniquely in females. Sexually dimorphic regulation of MMP/ADAMTS metalloproteinases and their intrinsic X-linked regulator Timp1 contributes to extracellular matrix remodeling integrated with pro-regenerative and immune functions. Lexis1 non-coding RNA involved in LXR-mediated lipid metabolism was identified as a novel nerve injury marker. Together, our data identified unique early response triggers of sex-specific peripheral nerve injury regulation to gain mechanistic insights into the origin of female- and male-prevalent sensory neuropathies.

Sensory nerves enhance triple-negative breast cancer migration and metastasis via the axon guidance molecule PlexinB3

In breast cancer, nerve presence has been correlated with more invasive disease and worse prognosis, yet the mechanisms by which different types of peripheral nerves drive tumor progression remain poorly understood. In this study, we identified sensory nerves as more abundant in human triple-negative breast cancer (TNBC) tumors. Coinjection of sensory neurons isolated from the dorsal root ganglia (DRG) of adult female mice with human TNBC cells in immunocompromised mice increased the number of lung metastases. Direct in vitro co-culture of human TNBC cells with the dorsal root ganglia (DRG) of adult female mice revealed that TNBC cells adhere to sensory neuron fibers leading to an increase in migration speed. Species-specific RNA sequencing revealed that co-culture of TNBC cells with sensory nerves upregulates the expression of genes associated with cell migration and adhesion in cancer cells. We demonstrate that the axon guidance molecule Plexin B3 mediates cancer cell adhesion to and migration on sensory nerves. Together, our results identify a novel mechanism by which nerves contribute to breast cancer migration and metastasis by inducing a shift in TNBC cell gene expression and support the rationale for disrupting neuron-cancer cell interactions to target metastasis.SignificanceThe presence of nerves in breast tumors has been associated with poor outcome. Understanding the mechanisms by which nerves contribute to tumor progression could help identify novel strategies to target metastatic disease.