Blocking Nerve Growth Factor Signaling Reduces the Neural Invasion Potential of Pancreatic Cancer Cells.

Perineural invasion (PNI) is thought to be one of the factors responsible for the high rate of tumor recurrence after surgery and the pain generation associated with pancreatic cancer. Signaling via the nerve growth factor (NGF) pathway between pancreatic cancer cells and the surrounding nerves has been implicated in PNI, and increased levels of these proteins have been correlated to poor prognosis. In this study, we examine the molecular mechanism of the NGF signaling pathway in PNI in pancreatic cancer. We show that knocking down NGF or its receptors, TRKA and p75NTR, or treatment with GW441756, a TRKA kinase inhibitor, reduces the proliferation and migration of pancreatic cancer cells in vitro. Furthermore, pancreatic cancer cells migrate towards dorsal root ganglia (DRG) in a co-culture assay, indicating a paracrine NGF signaling between the DRGs and pancreatic cancer cells. Knocking down the expression of NGF pathway proteins or inhibiting the activity of TRKA by GW441756 reduced the migratory ability of Mia PaCa2 towards the DRGs. Finally, blocking NGF signaling by NGF neutralizing antibodies or GW441756 inhibited the neurite formation in PC-12 cells in response to conditioned media from pancreatic cancer cells, indicating a reciprocal signaling pathway between the pancreatic cancer cells and nerves. Our results indicate that NGF signaling pathway provides a potential target for developing molecularly targeted therapies to decrease PNI and reduce pain generation. Since there are several TRKA antagonists currently in early clinical trials they could now be tested in the clinical situation of pancreatic cancer induced pain.

Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.

Retrograde communication from axonal targets to neuronal cell bodies is critical for both the development and function of the nervous system. Much progress has been made in recent years linking long-distance, retrograde signaling to a signaling endosome, yet the mechanisms governing the trafficking and signaling of these endosomes remain mostly uncharacterized. Here we report that in mouse sympathetic neurons, the target-derived nerve growth factor (NGF)-tropomyosin-related kinase type 1 (TrkA, also called Ntrk1) signaling endosome, on arrival at the cell body, induces the expression and recruitment of a new effector protein known as Coronin-1 (also called Coro1a). In the absence of Coronin-1, the NGF-TrkA signaling endosome fuses to lysosomes sixfold to tenfold faster than when Coronin-1 is intact. We also define a new Coronin-1-dependent trafficking event in which signaling endosomes recycle and re-internalize on arrival at the cell body. Beyond influencing endosomal trafficking, Coronin-1 is also required for several NGF-TrkA-dependent signaling events, including calcium release, calcineurin activation and phosphorylation of cAMP responsive element binding protein (CREB). These results establish Coronin-1 as an essential component of a feedback loop that mediates NGF-TrkA endosome stability, recycling and signaling as a critical mechanism governing developmental competition for survival.

Age-related changes of hippocampal synaptic plasticity in AßPP-null mice are restored by NGF through p75NTR.

Amyloid-ß protein precursor (AßPP) is a ubiquitous protein found in all cell types, suggesting basic and yet important roles, which still remain to be fully elucidated. Loss of function of AßPP has been linked to abnormal neuronal morphology and synaptic function within the hippocampus and alterations in spatial learning, suggesting a neurotrophic role for this protein. Besides AßPP, nerve growth factor (NGF) and other neurotrophins have also been shown to finely modulate neuronal excitability, synaptic plasticity, and cognitive functions. In addition, recent data support the hypothesis of a functional interconnection between AßPP and NGF pathway. Here, we demonstrated that loss of AßPP function, leading to progressive decrease of choline acetyltransferase expression in the septum, correlates with age-related impairment of long-term potentiation (LTP) in the dentate gyrus. We next addressed whether impaired hippocampal plasticity in AßPP-null mice can be restored upon NGF treatment. Notably, NGF, as well as Pro-NGF, can fully revert LTP deficits in AßPP-null mice through p75NTR and JNK pathway activation. Overall the present study may unveil a new mechanism by which, in the absence of AßPP, NGF treatment may preferentially direct p75-neurotrophin-dependent JNK activation toward regeneration and plasticity in functionally relevant brain circuits.

Loss of NGF-TrkA signaling from the CNS is not sufficient to induce cognitive impairments in young adult or intermediate-aged mice.

Many molecules expressed in the CNS contribute to cognitive functions either by modulating neuronal activity or by mediating neuronal trophic support and/or connectivity. An ongoing discussion is whether signaling of nerve growth factor (NGF) through its high-affinity receptor TrkA contributes to attention behavior and/or learning and memory, based on its expression in relevant regions of the CNS such as the hippocampus, cerebral cortex, amygdala and basal forebrain. Previous animal models carrying either a null allele or transgenic manipulation of Ngf or Trka have proved difficult in addressing this question. To overcome this problem, we conditionally deleted Ngf or Trka from the CNS. Our findings confirm that NGF-TrkA signaling supports survival of only a small proportion of cholinergic neurons during development; however, this signaling is not required for trophic support or connectivity of the remaining basal forebrain cholinergic neurons. Moreover, comprehensive behavioral analysis of young adult and intermediate-aged mice lacking NGF-TrkA signaling demonstrates that this signaling is dispensable for both attention behavior and various aspects of learning and memory.

TrkA gene ablation in basal forebrain results in dysfunction of the cholinergic circuitry.

Dysfunction of basal forebrain cholinergic neurons (BFCNs) is an early pathological hallmark of Alzheimer's disease (AD). Numerous studies have indicated that nerve growth factor (NGF) supports survival and phenotypic differentiation of BFCNs. Consistent with a potential link to AD pathogenesis, TrkA, a NGF receptor, is expressed in cholinergic forebrain neuronal populations including those in BF and striatum, and is markedly reduced in individuals with mild cognitive impairment (MCI) without dementia and early-stage AD. To investigate the role of TrkA in the development, connectivity, and function of the BF cholinergic system and its contribution to AD pathology, we have generated a forebrain-specific conditional TrkA knock-out mouse line. Our findings show a key role for TrkA signaling in establishing the BF cholinergic circuitry through the ERK pathway, and demonstrate that the normal developmental increase of choline acetyltransferase expression becomes critically dependent on TrkA signaling before neuronal connections are established. Moreover, the anatomical and physiological deficits caused by lack of TrkA signaling in BFCNs have selective impact on cognitive activity. These data demonstrate that TrkA loss results in cholinergic BF dysfunction and cognitive decline that is reminiscent of MCI and early AD.

Congenital insensitivity to pain with anhydrosis: report of a family case.

Congenital Insensitivity to pain with anhydrosis (CIPA) is a rare inherited disease. It is classified as hereditary sensory and autonomic neuropathy type IV. Pain insensitivity and autonomic deficits are present, but touch and pressure sensitivity are unimpaired. Mental retardation is usually present. We report a family case of a 5 years old girl and 2 years old boy with congenital insensitivity to pain, while discussing the clinical features and the anesthetic strategy of such patients. Patients with Congenital Insensitivity to Pain with anhydrosis may undergo surgery because of susceptibility to trauma due to absence of pain. The clinical features may intrinsically possess anesthetic challenges.

NTRK1 and NTRK2 receptors facilitate follicle assembly and early follicular development in the mouse ovary.

Recent studies have demonstrated that neurotrophins (NTs) and their NTRK tyrosine kinase receptors, thought to be exclusively required for the development of the nervous system, are also involved in controlling ovarian development. Here, we show that primordial follicle formation is decreased in the absence of nerve growth factor (NGF) or its receptor NTRK1, and in the absence of NTRK2, the receptor for neurotrophin-4 (NTF4) and brain-derived neurotrophic factor (BDNF). This deficiency is not due to premature oocyte loss, because the ovaries of Ntrk1(-/-) and Ntrk2(-/-) mice do not show an increased rate of oocyte death antedating the initiation of folliculogenesis. Moreover, exposure of NGF-deficient ovaries to NGF rescues the defect in follicular assembly, if NTRK1 receptors are present, suggesting that the absence of NTs causes a delay, and not an irretrievable loss, of follicle formation. Both the number of secondary follicles and FSH receptor (FSHR) expression are diminished in Ntrk1- and Ntrk2-null ovaries, but not in ovaries lacking the common NT receptor NGFR. Transient exposure of wild-type ovaries to NTF4 increases Fshr gene expression and enhances the ability of the ovary to respond to FSH with formation of cyclin D2, a cell cycle protein mediating the proliferative actions of FSH in the ovary. These results indicate that both NTRK1 and NTRK2 receptors are necessary for the timely assembly of primordial follicles and for sustaining early follicular development. They also suggest that a mechanism by which NTRK2 receptors facilitate subsequent follicle development is by inducing the formation of functional FSHR.

Regulation of sympathetic neuron differentiation by endogenous nerve growth factor and neurotrophin-3.

Nerve growth factor (NGF) and neurotrophin-3 (NT3) play distinctive roles in sympathetic axon growth and target field innervation and are required for sympathetic neuron survival in vivo. To ascertain if these neurotrophins selectively regulate the expression of genes that determine the functional characteristics of differentiated sympathetic neurons, we measured the mRNA levels for several such genes in the superior cervical ganglion of NGF(-/-), NT3(-/-) and wild type mouse embryos at a stage before excessive neuronal loss occurs in the absence of these neurotrophins. Despite the extensively documented ability of NGF to regulate the noradrenergic phenotype of sympathetic neurons, we found that tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) mRNA levels were normal in NGF(-/-) embryos, but significantly reduced in NT3(-/-) embryos. In contrast, the beta2 nicotinic acetylcholine receptor and PACAP receptor 1 mRNA levels were normal in NT3(-/-) embryos, but significantly reduced in NGF(-/-) embryos. Studies of mice lacking neurotrophin receptors suggested that the effects of NGF on gene expression require TrkA whereas those of NT3 require TrkA and p75(NTR). These findings demonstrate that endogenous NGF and NT3 have distinctive and separate effects on gene expression in early sympathetic neurons and that these selective effects on gene expression require a different combination of neurotrophin receptors.

Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.

Astrocytes promote the formation and function of excitatory synapses in the CNS. However, whether and how astrocytes modulate inhibitory synaptogenesis are essentially unknown. We asked whether astrocytes regulate the formation of inhibitory synapses between hippocampal neurons during maturation in vitro. Neuronal coculture with astrocytes or treatment with astrocyte-conditioned medium (ACM) increased the number of inhibitory presynaptic terminals, the frequency of miniature IPSCs, and the number and synaptic localization of GABA(A) receptor (GABA(A)R) clusters during the first 10 d in vitro. We asked whether neurotrophins, which are potent modulators of inhibitory synaptic structure and function, mediate the effects of astrocytes on inhibitory synapses. ACM from BDNF- or tyrosine receptor kinase B (TrkB)-deficient astrocytes increased inhibitory presynaptic terminals and postsynaptic GABA(A)R clusters in wild-type neurons, suggesting that BDNF and TrkB expression in astrocytes is not required for these effects. In contrast, although the increase in the number of inhibitory presynaptic terminals persisted, no increase was observed in postsynaptic GABA(A)R clusters after ACM treatment of hippocampal neurons lacking BDNF or TrkB. These results suggest that neurons, not astrocytes, are the relevant source of BDNF and are the site of TrkB activation required for postsynaptic GABA(A)R modulation. These data also suggest that astrocytes may modulate postsynaptic development indirectly by stimulating Trk signaling between neurons. Together, these data show that astrocytes modulate inhibitory synapse formation via distinct presynaptic and postsynaptic mechanisms.

Peroxisome proliferator-activated receptor gamma is a novel target of the nerve growth factor signaling pathway in PC12 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is subject to considerable interest because of its role in adipocyte differentiation, metabolic control, and anti-inflammatory action. PPARgamma research in brain cells is presently focused on glial PPARgamma because of its potential as a pharmacological target in the treatment of neurodegenerative diseases with an inflammatory component. In neurons PPARgamma function is far from clear, and PPARgamma agonist-dependent and -independent effects on cell survival or differentiation have been reported. We used PC12 cells, widely used to study neuronal signaling, such as nerve growth factor (NGF)-induced differentiation and survival or epidermal growth factor-dependent cell proliferation to dissect the possible involvement of PPARgamma in these pathways. We show that NGF but not epidermal growth factor increases the transcriptional activity of PPARgamma, and modulates the expression of this transcription factor. Because NGF signals through the tyrosine kinase (TrkA) NGF receptor and/or the p75NTR receptor, we used rescue experiments with a PC12 cell mutant lacking TrkA to show that NGF-induced PPARgamma activation is dependent on TrkA activation. Our results point out PPARgamma as a novel target of the TrkA-mediated neuronal cell survival and differentiating pathway and suggest a potential new inflammatory-independent therapeutic approach for pharmacological intervention in neurological disorders.

Ablation of TrkA function in the immune system causes B cell abnormalities.

The nerve growth factor (NGF) receptor TrkA is widely expressed in non-neural tissues suggesting pleiotropic functions outside the nervous system. Based on pharmacological and immuno-depletion experiments, it has been hypothesized that NGF plays an important role in the normal development and function of the immune system. However, attempts to unravel these functions by conventional gene targeting in mice have been hampered by the early postnatal lethality caused by null mutations. We have developed a novel 'reverse conditional' gene targeting strategy by which TrkA function is restored specifically in the nervous system. Mice lacking TrkA in non-neuronal tissues are viable and appear grossly normal. All major immune system cell populations are present in normal numbers and distributions. However, mutant mice have elevated serum levels of certain immunoglobulin classes and accumulate B1 cells with aging. These data, confirmed in a classical reconstitution model using embryonic fetal liver from TrkA-null mice, demonstrate that endogenous NGF modulates B cell development through TrkA in vivo. Furthermore, they demonstrate that many of the dramatic effects previously reported by pharmacological or immuno-depletion approaches do not reflect physiological developmental roles of TrkA in the immune system.

Ruffini endings are absent from the periodontal ligament of trkB knockout mice.

To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.

Testis developmental phenotypes in neurotropin receptor trkA and trkC null mutations: role in formation of seminiferous cords and germ cell survival.

The objective of the present study was to determine if the neurotropin receptors trkC and trkA are involved in embryonic testis development. These receptors bind neurotropin 3 and nerve growth factor, respectively. The hypothesis tested was that the absence of trkC or trkA receptors will have detrimental effects on testis development and morphology. The trkA and trkC homozygote knockout (KO) mice generally die either at or shortly after birth. Therefore, heterozygote mice were mated to obtain homozygote gene KO mice at Embryonic Day (E) 13, E14, E17, and E19 of gestation, with E0 being the plug date. Gonads from approximately 80 embryos were collected and fixed, and each embryo was genotyped. To determine gonadal characteristics for each genotype, the number of germ cells, number of seminiferous cords, seminiferous cord area, and interstitial area were calculated at each developmental age. Germ cell numbers varied in trkA gene KO mice from those of wild-type mice at each age evaluated. In trkC gene KO mice, differences were detected in germ cell numbers when compared to wild-type mice at E17 and E19. At E19, germ cell numbers were reduced in both trkA and trkC gene KO mice when compared to wild-type animals. Apoptosis was evaluated in testes of wild-type, trkC gene KO, and trkA gene KO mice to determine if the alteration in germ cell numbers at each developmental age was influenced by different patterns of germ cell survival or apoptosis. No differences were found in germ cell apoptosis during embryonic testis development. Interestingly, trkA gene KO mice that survived to Postnatal Day 19 had a 10-fold increase in germ cell apoptosis when compared to germ cells in wild-type mice. Evaluation of other morphological testis parameters demonstrated that trkC KO testes had reduced interstitial area at E13, reduced number of seminiferous cords at E14, and reduced seminiferous cord area at E19. The trkA gene KO testes had a reduction in the number of seminiferous cords at E14. Histology of both trkA and trkC gene KO testes demonstrated that these gonads appear to be developmentally delayed when compared to their wild-type testis counterparts at E13 during testis development. The current study demonstrates that both trkA and trkC neurotropin receptors influence germ cell numbers during testis development and events such as seminiferous cord formation.

Involvement of the NGF receptors (Trka and p75lngfr) in the development and maintenance of the thymus.

Nerve growth factor (NGF) and its main low- (p75LNGFR) and high-affinity (TrkA) receptors have been found in the vertebrate thymus, thus suggesting they are involved in the control of thymic function. However, its role in this organ is poorly known. In the present study we used combined morphological and immunohistochemical techniques to analyze the distribution of TrkA and p75LNGFR in the rat thymus, as well as the structural changes in the thymus of p75 LNGFR or TrkA deficient mice. In adult rats both TrkA and p75LNGFR were localized in a subset of thymic epithelial cells found primarily in the subcapsular and medullary thymic regions, regarded to be endodermal-derived cells. Consistently, animals with a non-functional TrkA, but not those lacking p75LNGFR, showed structural changes consisting of a decrease in the density of thymocytes, absence of cortico-medullary border, and large cysts lined of endodermal epithelium. These results strongly suggest a function of the TrkA-NGF system in thymic functions mediated by epithelial cells, as well as a role of TrkA in the development of the murine thymus. The function of p75LNGFR remains to be established.

Embryonic neuronal death due to neurotrophin and neurotransmitter deprivation occurs independent of Apaf-1.

Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1. We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.

trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice.

To investigate the nerve growth factor requirement of developing oro-facial somatosensory afferents, we have studied the survival of sensory fibers subserving nociception, mechanoreception or proprioception in receptor tyrosine kinase (trkA) knockout mice using immunohistochemistry. trkA receptor null mutant mice lack nerve fibers in tooth pulp, including sympathetic fibers, and showed only sparse innervation of the periodontal ligament. Ruffini endings were formed definitively in the periodontal ligament of the trkA knockout mice, although calcitonin gene-related peptide- and substance P-immunoreactive fibers were reduced in number or had disappeared completely. trkA gene deletion had also no obvious effect on the formation of Meissner corpuscles in the palate. In the vibrissal follicle, however, some mechanoreceptive afferents were sensitive for trkA gene deletion, confirming a previous report [Fundin et al. (1997) Dev. Biol. 190, 94-116]. Moreover, calretinin-positive fibers innervating longitudinal lanceolate endings were completely lost in trkA knockout mice, as were the calretinin-containing parent cells in the trigeminal ganglion.These results indicate that trkA is indispensable for developing nociceptive neurons innervating oral tissues, but not for developing mechanoreceptive neurons innervating oral tissues (Ruffini endings and Meissner corpuscles), and that calretinin-containing, trkA dependent neurons in the trigeminal ganglion normally participate in mechanoreception through longitudinal lanceolate endings of the vibrissal follicle.

Non-TrkA-expressing small DRG neurons are lost in TrkA deficient mice.

Experiments over the past decade in which NGF/TrkA signaling has been abolished by antibodies or targeted gene mutations have shown that 70-85% of dorsal root ganglion (DRG) neurons require NGF for survival during development. There is consensus that many of the NGF-dependent neurons are small-diameter, peptidergic neurons subserving nociception. These neurons express the signaling receptor for NGF, TrkA. There is a major discrepancy, however, between the percentage of DRG neurons which require NGF for survival (70-85%) and percentage of DRG neurons expressing TrkA receptors (40-50%). The identity of these non-TrkA expressing, NGF-dependent neurons has not been established. A candidate group is a population of small DRG neurons with unmyelinated axons which bind BSI isolectins from the plant, Bandeiraea simplicifolia. We show here that most of these BSI-binding DRG neurons do not express TrkA in adult mice. However, in mutant mice in which NGF/TrkA signaling has been abolished by inactivation of the trkA gene, BSI-staining in the DRG and dorsal horn is completely eliminated. BSI-binding DRG cells are thus the first identified neuronal population in which cells do not express TrkA in maturity, but require NGF/TrkA signaling for survival during embryonic development. These neurons must either depend on NGF via a novel, indirect mechanism or alternatively, downregulate TrkA expression during development.