Associate Professor Phil Jobling

Nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA) and the common neurotrophin receptor (NGFR/p75NTR), are increasingly implicated in cancer progression, but their clinicopathological significance in oesophageal cancer is unclear. In this study, the expression of NGF, NTRK1 and NGFR were analysed by immunohistochemistry in a cohort of 303 oesophageal cancers versus 137 normal adjacent oesophageal tissues. Immunostaining was digitally quantified and compared to clinicopathological parameters. NGF and NGFR staining were found in epithelial cells and at similar levels between oesophageal cancers and normal oesophageal tissue. NGFR staining was slightly increased with grade (p=0.0389). Interestingly, NTRK1 staining was markedly higher in oesophageal squamous cell carcinoma (OR 2.31, 95%CI 1.13¿4.38, p<0.0001) and significantly lower in adenocarcinoma (OR 0.50, 95%CI 0.44¿0.63, p<0.0001) compared to normal oesophageal tissue. In addition, NTRK1 staining was decreased in grade 2 and grade 3 (OR 0.51, 95%CI 0.21¿1.40, p<0.0001) compared to grade 1, suggesting a preferential involvement of this receptor in the more differentiated forms of oesophageal carcinomas. Together, these data point to NTRK1 as a biomarker and a candidate therapeutic target in oesophageal squamous cell carcinoma.

Nerves are emerging promoters of cancer progression, but the innervation of esophageal cancer and its clinicopathologic significance remain unclear. In this study, nerves were analyzed by immunohistochemistry in a cohort of 260 esophageal cancers, including 40 matched lymph node metastases and 137 normal adjacent esophageal tissues. Nerves were detected in 38% of esophageal cancers and were more associated with squamous cell carcinomas (P = 0.04). The surrounding or invasion of nerves by cancer cells (perineural invasion) was detected in 12% of esophageal cancers and was associated with reduced survival (P = 0.04). Nerves were found to express the following receptors for nerve growth factor (NGF): neurotrophic receptor tyrosine kinase 1 and nerve growth factor receptor. An association was suggested between high production of NGF by cancer cells and the presence of nerves (P = 0.02). In vitro, NGF production in esophageal cancer cells was shown by Western blot, and esophageal cancer cells were able to induce neurite outgrowth in the PC12 neuronal cells. The neurotrophic activity of esophageal cancer cells was inhibited by anti-NGF blocking antibodies. Together, these data suggest that innervation is a feature in esophageal cancers that may be driven by cancer cell¿released NGF.

Conspicuous coloured displays from ultraviolet to bright red have been documented in many species throughout the animal kingdom. These colours often occur as sexual signals and can be incorporated into different types of integuments (e.g. scales, feathers, skin). Two main mechanisms are known to produce coloured integuments: pigmentation and tissue structure. Although pigmental and structural coloration are separate mechanisms and can occur independently, some coloured displays might emerge from a combination of both. Here, we demonstrate, using biochemical, optical and morphological methodologies, that the yellow coloration of the skin located around the eye of Common (Indian) Mynas (Acridotheres tristis) is produced by both light-reflecting nanostructures and light-absorbing carotenoid pigments. Our analysis confirms that nanostructured collagen in the avian dermis work in combination with carotenoid pigments to produce vivid integumentary colours. Identifying the mechanisms behind the production of a coloured signal provides a basis for predicting how a signal¿s function might be influenced by environmental factors such as fledgling nutrition.

Lee JM, Mayall JR, Chevalier A, McCarthy H, Van Helden D, Hansbro PM, Horvat JC, Jobling P. Chlamydia muridarum infection differentially alters smooth muscle function in mouse uterine horn and cervix. Am J Physiol Endocrinol Metab 318: E981 E994, 2020. First published April 21, 2020; doi:10.1152/ajpendo.00513. 2019. Chlamydia trachomatis infection is a primary cause of reproductive tract diseases including infertility. Previous studies showed that this infection alters physiological activities in mouse oviducts. Whether this occurs in the uterus and cervix has never been investigated. This study characterized the physiological activities of the uterine horn and the cervix in a Chlamydia muridarum (Cmu)-infected mouse model at three infection time points of 7, 14, and 21 days postinfection (dpi). Cmu infection significantly decreased contractile force of spontaneous contraction in the cervix (7 and 14 dpi; P < 0.001 and P < 0.05, respectively), but this effect was not observed in the uterine horn. The responses of the uterine horn and cervix to oxytocin were significantly altered by Cmu infection at 7 dpi (P < 0.0001), but such responses were attenuated at 14 and 21 dpi. Cmu infection increased contractile force to prostaglandin (PGF2_) by 53 83% in the uterine horn. This corresponded with the increased messenger ribonucleic acid (mRNA) expression of Ptgfr that encodes for its receptor. However, Cmu infection did not affect contractions of the uterine horn and cervix to PGE2 and histamine. The mRNA expression of Otr and Ptger4 was inversely correlated with the mRNA expression of Il1b, Il6 in the uterine horn of Cmu-inoculated mice (P < 0.01 to P < 0.001), suggesting that the changes in the Otr and Ptger4 mRNA expression might be linked to the changes in inflammatory cytokines. Lastly, this study also showed a novel physiological finding of the differential response to PGE2 in mouse uterine horn and cervix.

Prostate cancer progression has been shown to be dependent on the development of autonomic nerves into the tumour microenvironment. Sympathetic nerves activate adrenergic neurosignalling that is necessary in early stages of tumour progression and for initiating an angiogenic switch, whereas parasympathetic nerves activate cholinergic neurosignalling resulting in tumour dissemination and metastasis. The innervation of prostate cancer seems to be initiated by neurotrophic growth factors, such as the precursor to nerve growth factor secreted by tumour cells, and the contribution of brain-derived neural progenitor cells has also been reported. Current experimental, epidemiological and clinical evidence shows the stimulatory effect of tumour innervation and neurosignalling in prostate cancer. Using nerves and neurosignalling could have value in the management of prostate cancer by predicting aggressive disease, treating localized disease through denervation and relieving cancer-associated pain in bone metastases.

Nerves are emerging regulators of cancer progression. Cancer cells induce the outgrowth of nerves in the tumor microenvironment through the release of neu-rotrophic factors, and in return nerves liberate neurotransmitters that activate cancer growth and dissemination. Although sympathetic nerves drive tumor angiogenesis via the liberation of noradrena-line, sensory and parasympathetic nerves stimulate cancer stem cells. Interestingly, recent evidence indicates that parasympathetic nerves can eventually inhibit tumor progression, suggesting a yin¿yang type of regulation of cancer by nerves. From a broader perspective, the question of a higher level of control of cancer development by the central nervous system should be raised. Significance: Nerves are emerging regulators of cancer initiation, progression, and metastasis. Here, we review the evidence to date and explore the basic and clinical ramifications of these findings.

Nerve outgrowth in the tumor microenvironment (tumor neurogenesis) has recently been shown to be essential for cancer progression and the concept of nerve dependence is emerging in oncology. Neurotrophins such as nerve growth factor (NGF) have long been identified as drivers of neurogenesis during development and regeneration, but intriguingly they were also known to be expressed in human tumors where they can stimulate cancer cell growth. Recent findings have unraveled that NGF released by cancer cells is also a driver of tumor neurogenesis, via the stimulation of NGF receptors on nerve endings. In return, nerves infiltrated in the tumor microenvironment secrete neurotransmitters, which can stimulate both the growth of tumor cells and angiogenesis. This neurotrophic role of NGF in cancer is likely to be relevant to a large variety of human malignancies, as well as other neurotrophins, and may have ramifications in cancer pain. Therefore, pharmacological interventions against neurotrophin signaling have the potential not only to target cancer cells directly, but also to inhibit neurogenesis and its stimulatory impact on cancer progression and pain.

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of a9- containing nicotinic acetylcholine (ACh) receptors (a9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and a9-subunit nAChR knockout (a9 -/- ) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of a9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the a9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from a9 -/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of a9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of a9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted efferent mechanism for altering hair cell membrane potential and decreasing membrane resistance that should reduce sensitivity to hair bundle displacements.

Animal models of maternal immune activation study the effects of infection, an environmental risk factor for schizophrenia, on brain development. Microglia activation and cytokine upregulation may have key roles in schizophrenia neuropathology. We hypothesised that maternal immune activation induces changes in microglia and cytokines in the brains of the adult offspring. Maternal immune activation was induced by injecting polyriboinosinic:polyribocytidylic acid into pregnant rats on gestational day (GD) 10 or GD19, with brain tissue collected from the offspring at adulthood. We observed no change in Iba1, Gfap, IL1-ß and TNF-a mRNA levels in the cingulate cortex (CC) in adult offspring exposed to maternal immune activation. Prenatal exposure to immune activation had a significant main effect on microglial IBA1-positive immunoreactive material (IBA1+IRM) in the corpus callosum; post-hoc analyses identified a significant increase in GD19 offspring, but not GD10. No change in was observed in the CC. In contrast, maternal immune activation had a significant main effect on GFAP+IRM in the CC at GD19 (not GD10); post-hoc analyses only identified a strong trend towards increased GFAP+IRM in the GD19 offspring, with no white matter changes. This suggests late gestation maternal immune activation causes subtle alterations to microglia and astrocytes in the adult offspring.

Neural and agonist-induced contractions of proximal (i.e. upper half adjacent to the cervix) and distal mouse vaginal smooth muscle strips were investigated. We hypothesised that nerve-mediated vaginal contractions arise through activity of cholinergic nerves. Nerve activation by bursts of electrical field stimulation (EFS) caused a primary transient contraction often accompanied by a secondary transient contraction, both larger in proximal than distal tissues (i.e. primary: 7-fold larger; secondary: 3-fold larger). Our hypothesis was supported as we found that cholinergic nerves mediated the primary transient contraction in both proximal and distal vaginal strips, as EFS responses were enhanced by neostigmine an anticholinesterase, massively inhibited by the competitive muscarinic receptor antagonist atropine and not affected by the non-selective a-adrenergic receptor antagonist phentolamine. Primary transient contractions were halved in amplitude by the L-type Ca2+ channel blocker nifedipine and markedly inhibited by the sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Resultant secondary transient contractions were abolished by nifedipine. Notably, the selective a1-adrenergic receptor agonist phenylephrine caused tonic contracture in distal but not proximal strips. Low-frequency EFS often initiated recurrent transient contractions similar to those elicited by CCh. Immunohistochemical studies demonstrated innervation of the smooth muscle. Findings of enhanced proximal cholinergic nerve-induced transient contractions, evidence that maintained nerve stimulation could cause recurrent contractions and the finding of distal phenylephrine-mediated tonic contraction have implications on insemination.

Nerve dependence has long been described in animal regeneration, where the outgrowth of axons is necessary to the reconstitution of lost body parts and tissue remodeling in various species. Recent discoveries have demonstrated that denervation can suppress tumor growth and metastasis, pointing to nerve dependence in cancer. Regeneration and cancer share similarities in regard to the stimulatory role of nerves, and there are indications that the stem cell compartment is a preferred target of innervation. Thus, the neurobiology of cancer is an emerging discipline that opens new perspectives in oncology.

Nerves are emerging as drivers of tumorigenesis, as demonstrated in the mouse where denervation suppresses prostate cancer; however, clinical evidence is needed. Patients with spinal cord injuries (SCIs) resulting in functional denervation of the prostate have a lower incidence of prostate cancer. This may constitute a clinical evidence for nerve dependence in human prostate tumorigenesis.

This study assessed the¿effects of resveratrol, a polyphenol¿found in grapes and red wine¿on non-pregnant murine uteri under hypoxia. Resveratrol at 1, 3, 10, 30 and 100¿µM promoted uterine relaxation and decreased the amplitude and frequency of spontaneous uterine contractions. Assayed at 3, 10, 30¿µM, resveratrol inhibited the oxytocin-induced cumulative contractions reducing the maximum effect in a dose-dependent manner. In hypoxic uteri, resveratrol at 100¿µM restored the uterine contractions compromised by hypoxia. In addition, under hypoxia, resveratrol prevented the decrease in uterine contractions maintaining >75% of its contraction capability. The effects of resveratrol on uterine contractions under hypoxia were attenuated by tetraethylammonium (10¿mM) and almost abolished by glibenclamide (10¿µM). Our¿results show regenerative and protective effects of resveratrol in non-pregnant murine uteri under hypoxia and describes for the first time that these effects are mediated by blockade of ATP-sensitive potassium channels.

Interstitial neurons are located among white matter tracts of the human and rodent brain. Post-mortem studies have identified increased interstitial white matter neuron (IWMN) density in the fibre tracts below the cortex in people with schizophrenia. The current study assesses IWMN pathology in a model of maternal immune activation (MIA); a risk factor for schizophrenia. Experimental MIA was produced by an injection of polyinosinic:polycytidylic acid (PolyI:C) into pregnant rats on gestational day (GD) 10 or GD19. A separate control group received saline injections. The density of neuronal nuclear antigen (NeuN⁺) and somatostatin (SST⁺) IWMNs was determined in the white matter of the corpus callosum in two rostrocaudally adjacent areas in the 12week old offspring of GD10 (n=10) or GD19 polyI:C dams (n=18) compared to controls (n=20). NeuN⁺ IWMN density trended to be higher in offspring from dams exposed to polyI:C at GD19, but not GD10. A subpopulation of these NeuN⁺ IWMNs was shown to express SST. PolyI:C treatment of dams induced a significant increase in the density of SST⁺ IWMNs in the offspring when delivered at both gestational stages with more regionally widespread effects observed at GD19. A positive correlation was observed between NeuN⁺ and SST⁺ IWMN density in animals exposed to polyI:C at GD19, but not controls. This is the first study to show that MIA increases IWMN density in adult offspring in a similar manner to that seen in the brain in schizophrenia. This suggests the MIA model will be useful in future studies aimed at probing the relationship between IWMNs and schizophrenia.

The dorsal horn (DH) of the spinal cord contains a heterogenous population of neurons that process incoming sensory signals before information ascends to the brain. We have recently characterized calretinin-expressing (CR+) neurons in the DH and shown that they can be divided into excitatory and inhibitory subpopulations. The excitatory population receives high-frequency excitatory synaptic input and expresses delayed firing action potential discharge, whereas the inhibitory population receives weak excitatory drive and exhibits tonic or initial bursting discharge. Here, we characterize inhibitory synaptic input and neuromodulation in the two CR+ populations, in order to determine how each is regulated. We show that excitatory CR+ neurons receive mixed inhibition from GABAergic and glycinergic sources, whereas inhibitory CR+ neurons receive inhibition, which is dominated by glycine. Noradrenaline and serotonin produced robust outward currents in excitatory CR+ neurons, predicting an inhibitory action on these neurons, but neither neuromodulator produced a response in CR+ inhibitory neurons. In contrast, enkephalin (along with selective mu and delta opioid receptor agonists) produced outward currents in inhibitory CR+ neurons, consistent with an inhibitory action but did not affect the excitatory CR+ population. Our findings show that the pharmacology of inhibitory inputs and neuromodulator actions on CR+ cells, along with their excitatory inputs can define these two subpopulations further, and this could be exploited to modulate discrete aspects of sensory processing selectively in the DH.

Neurons in the superficial dorsal horn (SDH) of the spinal cord play an important role in nociceptive, thermal, itch and light touch sensations. Excitatory interneurons comprise ~65% of all SDH neurons but surprisingly few studies have investigated their role in spinal sensory processing. Here we use a transgenic mouse to study putative excitatory SDH neurons that express the calcium binding protein calretinin (CR). Our immunocytochemical, morphological and electrophysiological analysis identified two distinct populations of CR-expressing neurons, which we termed 'Typical' and 'Atypical'. Typical CR-expressing neurons comprised ~85% of the population and exhibited characteristic excitatory interneuron properties including delayed firing discharge, large rapid A-type potassium currents, and central, radial or vertical cell morphologies. Atypical neurons exhibited properties consistent with inhibitory interneurons, including tonic firing or initial bursting discharge, Ih currents, and islet cell morphology. Although both Typical and Atypical CR-expressing neurons responded to noxious peripheral stimulation, the excitatory drive onto Typical CR-expressing neurons was much stronger. Furthermore, Atypical CR-expressing cells comprise at least two functionally distinct subpopulations based on their responsiveness to noxious peripheral stimulation and neurochemical profile. Together our data suggest CR expression is not restricted to excitatory neurons in the SDH. Under normal conditions, the contribution of 'Typical' excitatory CR-expressing neurons to overall SDH excitability may be limited by the presence of A-type potassium currents, which limit the effectiveness of their strong excitatory input. Their contribution may, however, be increased in pathological situations where A-type potassium currents are decreased. By contrast, 'Atypical' inhibitory neurons with their excitable phenotype but weak excitatory input may be more easily recruited during increased peripheral stimulation.

Nerve infiltration is essential to prostate cancer progression, but the mechanism by which nerves are attracted to prostate tumors remains unknown. We report that the precursor of nerve growth factor (proNGF) is overexpressed in prostate cancer and involved in the ability of prostate cancer cells to induce axonogenesis. A series of 120 prostate cancer and benign prostate hyperplasia (BPH) samples were analyzed by IHC for proNGF. ProNGF was mainly localized in the cytoplasm of epithelial cells, with marked expression in cancer compared with BPH. Importantly, the proNGF level positively correlated with the Gleason score (n = 104, tB = 0.51). A higher level of proNGF was observed in tumors with a Gleason score of =8 compared with a Gleason score of 7 and 6 (P < 0.001). In vitro, proNGF was detected in LNCaP, DU145, and PC-3 prostate cancer cells and BPH-1 cells but not in RWPE-1 immortalized nontumorigenic prostate epithelial cells or primary normal prostate epithelial cells. Co-culture of PC12 neuronal-like cells or 50B11 neurons with PC-3 cells resulted in neurite outgrowth in neuronal cells that was inhibited by blocking antibodies against proNGF, indicating that prostate cancer cells can induce axonogenesis via secretion of proNGF. These data reveal that ProNGF is a biomarker associated with high-risk prostate cancers and a potential driver of infiltration by nerves.

Conclusions/Significance: Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca2+ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges.

Pain from the female reproductive tract (FRT) is a significant clinical problem for which there are few effective therapies. The complex neuroanatomy of pelvic organs not only makes diagnosis of pelvic pain disorders difficult but represents a challenge to development of targeted therapies. A number of potential therapeutic targets have been identified on sensory neurons supplying the FRT but our knowledge on the basic neurophysiology of these neurons is limited compared with other viscera. Until this is addressed we can only guess if the new experimental therapies proposed for somatic, gastrointestinal, or bladder pain will translate to the FRT. Once suitable therapeutic targets become clear, the next challenge is drug delivery. The FRT represents a promising system for topical drug delivery that could be tailored to act locally or systemically depending on formulation. Development of these therapies and their delivery systems will need to be done in concert with more robust in vivo and in vitro models of FRT pain. © 2014 Jobling, O'Hara and Hua.