Nerve growth factor regulates substance P in adult sensory neurons through both TrkA and p75 receptors

Expression of the nociceptive peptide, substance P (SP) is regulated by the neurotrophin, nerve growth factor (NGF), and exogenous exposure to high levels of NGF increases its cellular content and release. NGF utilizes two receptors, the NGF-specific tyrosine kinase receptor, TrkA, and also the non-specific neurotrophin receptor, p75(NTR) (p75). The purpose of this study is to determine the relative involvement of these receptors in nociception. To investigate the role of TrkA in SP signaling, sensory neurons from adult rats were grown in vitro and exposed to a TrkA-blocking antibody. Pretreatment with the antibody inhibited NGF-induced SP elevation. Furthermore, when neurons were exposed to K252a, a relatively specific TrkA kinase inhibitor, the NGF effect on SP was also inhibited. K252a did not prevent SP up-regulation in cells exposed to forskolin or glial cell line-derived neurotrophic factor (GDNF), two agents which increase SP expression independently of TrkA. When p75 was blocked by antiserum, SP up-regulation by NGF was also inhibited. The antiserum neither impacted neuronal survival or basal levels of SP expression, nor did it inhibit SP up-regulation induced by forskolin. Two other neurotrophins, which are also ligands for p75, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) did not block NGF-induced SP up-regulation, raising the possibility that activated p75 is able to cooperate in SP regulation regardless of which neurotrophin ligand occupies it. Our data suggest that NGF up-regulation of SP expression requires the involvement of both TrkA and p75, although the specific contribution of each receptor to SP signaling remains to be determined.     

Neurofibromin negatively regulates neurotrophin signaling through p21ras in embryonic sensory neurons

Embryonic sensory and sympathetic neurons that lack neurofibromin, the protein product of the neurofibromatosis type 1 (Nfl) gene, survive and extend neurites in the absence of neurotrophins. To determine whether neurofibromin negatively regulates neurotrophin signaling through its interaction with p21ras, we used Fab antibody fragments to block Ras function in DRG, trigeminal, nodose, and SCG neurons isolated from Nfl(-/-) and wild-type mouse embryos. We show that introduction of anti-Ras Fab fragments significantly reduces the ability of neurofibromin-deficient neurons to survive in the absence of neurotrophins. Moreover, addition of H-ras protein enhances the survival of Nfl(-/-), but not wild-type, DRG neurons. Our results are consistent with a major role for neurofibromin in modulating Trk signaling through p21ras during neuronal development.     

Noradrenergic facilitation of motor neurons: localization of adrenergic receptors in neurons and nonneuronal cells in the trigeminal motor nucleus.

Both alpha- and beta-adrenergic receptors (ARs) are involved in the facilitation of the monosynaptic jaw-closing reflex in the trigeminal motor nucleus (MoV) caused by norepinephrine (NE). The amplitude of muscle spindle afferent-evoked EPSPs in masseter motor neurons is 65% greater when noradrenergic axons to the motor nucleus are concomitantly activated and seems to be due to a presynaptic mechanism (Vornov, J. J., and J. Sutin. 1986. J. Neurosci. 6: 30-37). To determine the subtypes of ARs located on motor neurons and other cells, the cytotoxic lectin Ricin communis was injected into the masseter nerve of the trigeminal motor root to eliminate motor neurons in the masseter subnucleus of MoV. Autoradiography following incubation of tissue sections in the alpha 1 ligand 125IBE 2254 (125I-HEAT) or the nonselective beta ligand [125I]iodocyanopindolol (125ICYP) showed a decrease in alpha 1-AR binding related to the motor neuron degeneration and an increase in beta-AR binding associated with the glial reaction. To determine the extent to which glial proliferation was responsible for the increase in beta-ARs, cytosine arabinofuranoside (AraC) was administered to inhibit mitosis. Following AraC treatment, the total number of glial cells in the ricin-treated MoV was similar to that in normal MoV. Both beta-AR density and GFAP immunoreactivity remain increased, but to a lesser degree than following the ricin treatment alone. AraC also partially prevented the increase of immunolabeled or histochemically visualized microglia and capillary endothelial cells. The coincidence of the increases in beta-AR binding and GFAP in a region devoid of neurons argues that reactive astrocytes and other nonneuronal cells express beta-ARs in vivo. To determine whether the increase in astroglial beta-ARs was due to an up-regulation resulting from transynaptic degeneration of NE terminals, NE content was measured in MoV tissue punches, and NE terminals were visualized by immunocytochemical labeling of dopamine-beta-hydroxylase. NE content and NE terminal density remained unchanged following ricin-induced motor neuron degeneration.     

Inactivation and activation of Ras by the neurotrophin receptor p75

The neurotrophin receptor p75 induces neurotrophic and/or apoptotic signalling pathways and can also cooperate with the neurotrophic Trk receptor tyrosine kinases. Its intracellular part encloses a so-called 'death domain' with a segment similar to the wasp venom mastoparan which binds small GTPases such as Rho. To study possible interactions of p75 and Ras (and Rho) we used wild-type and mutant genes of p75 stably expressed by MDCK cells which normally have neither Trk nor p75. We found that p75 can directly bind the GTPases Ras and Rho and that the unstimulated p75 inactivates total cellular Ras through a differential influence on the dissociation of GDP and GTP from Ras and an exchange of bound Ras.GDP for free Ras.GTP. These properties of p75 could also be demonstrated in vitro and should therefore be cell type-independent. Stimulation of p75 with nerve growth factor causes Ras activation via adapter proteins known from Trk signalling and induces rapid outgrowth of cellular processes. Both inactivation and activation of Ras by p75 are controlled by the phosphorylation state of the receptor's two intracellular tyrosines. p75 also influences Rho activation and inactivation, and the combined interactions of the receptor with the two GTPases Ras and Rho can regulate neurite formation in an efficient, synergistic way.     

Developmental co-expression and functional redundancy of tyrosine phosphatases with neurotrophin receptors in developing sensory neurons

Receptor-type protein tyrosine phosphatases (RPTPs) have been implicated as direct or indirect regulators of neurotrophin receptors (TRKs). It remains less clear if and how such RPTPs might regulate TRK proteins in vivo during development. Here we present a comparative expression profile of RPTP genes and Trk genes during early stages of murine, dorsal root ganglion maturation. We find little if any specific, temporal mRNA co-regulation between individual RPTP and Ntrk genes between E12.5 and E14.5. Moreover, a double fluorescent in-situ hybridization and immunofluorescence study of seven Rptp genes with Ntrks revealed widespread co-expression of RPTPs in individual neurons, but no tight correlation with Trk expression profiles. No Rptp is expressed in 100% of Ntrk1-expressing neurons, whereas at least 6 RPTPs are expressed in 100% of Ntrk2- and Ntrk3-expressing neurons. An exception is Ptpro, which showed very selective expression. Short hairpin RNA suppression of Ptprf, Ptprs or Ptpro in primary, E13.5 DRG neurons did not alter TRK signalling. We therefore propose that TRK signalling may not be simply dependent on rate-limiting regulation by individual RPTP subtypes during sensory neuron development. Instead, TRK signalling has the potential to be buffered by concurrent inputs from several RPTPs in individual neurons.     

Effects of aging and axotomy on the expression of neurotrophin receptors in primary sensory neurons.

Aging is accompanied by declined sensory perception, paralleled by widespread dystrophic and degenerative changes in both central and peripheral sensory pathways. Several lines of evidence indicate that neurotrophic interactions are of importance for a maintained plasticity in the adult and aging nervous system, and that changes in the expression of neurotrophins and/or their receptors may underpin senile neurodegeneration. We have here examined the expression of neurotrophin receptor (p75NTR, trkA, trkB, and trkC) mRNA and protein in intact and axotomized primary sensory neurons of young adult (3 months) and aged (30 months) rats. To examine possible differences among primary sensory neuron populations, we have studied trigeminal ganglia (TG) as well as cervical and lumbar dorsal root ganglia (DRG). In intact aged rats, a decrease in trk (A/B/C) mRNA labeling densities and protein-like immunoreactivities was observed. The decrease was most pronounced in lumbar DRG. In contrast, a small, not statistically significant, increase of p75NTR expression was observed in aged DRG neuron profiles. After axotomy, a down-regulation of mRNA and protein levels was observed for all neurotrophin receptors (p75NTR, trkA, trkB and trkC) in both young adult and aged rats. Consistent with the higher expression levels of neurotrophin receptors in unlesioned young adult primary sensory neurons, the relative effect of axotomy was more pronounced in the young adult than aged rats. Although a decrease in mean cell profile cross-sectional areas was found during aging and after axotomy, the characteristic distribution of neurotrophin receptor expression in different populations of NRG neurons was conserved. The present findings suggest an attenuation of neurotrophic signaling in primary sensory neurons with advancing age and that the expression of p75NTR and trks is regulated differently during aging. A similar dissociation of p75NTR and trk regulation has previously been reported in other neuronal systems during aging, suggesting that there may be a common underlying mechanism. Decreased access to ligands, disturbed axon function and systemic changes in androgen/estrogen levels are discussed as inducing and/or contributing factors.     

Small molecule modulation of p75 neurotrophin receptor functions

Ligand-independent and/or proNGF-induced p75(NTR) signaling has emerged as a potential major contributor to a number of pathological states, including axotomy-induced death, motor neuron degeneration, neuronal degeneration in Alzheimer's disease and oligodendrocyte death following spinal cord injury. A long standing goal in the neurotrophin field has been the development of non-peptide, small molecules capable of functioning as specific ligands at neurotrophin receptors such as p75(NTR) to promote desired biological outcomes. Synthetic peptides modeled on neurotrophin protein domains have been found to bind to and activate various neurotrophin receptors, raising the possibility that active, non-peptide, small molecule ligands might also be identified; however, traditional high-throughput screening approaches have been largely ineffective in identifying such compounds. Using pharmacophores derived from the structure of loop 1 of nerve growth factor, non-peptide, small molecules that function as p75(NTR) ligands to promote survival and block proNGF-induced death have recently been identified. Small molecule p75(NTR) ligands, with high potency and specificity, may provide novel therapeutic approaches for neurodegenerative diseases, neurotrauma and other pathologic states.     

The new wave of p75 neurotrophin receptor targeted therapies

Neurotrophins have been recognized for decades for their beneficial effects on growth,survival,and maintenance in the central nervous system,all of which suggest potential therapeutic utility.Although understanding and harnessing the activity of neurotrophins has proven difficult,the past several years have seen significant strides in the development of deliverable therapies that modulate neurotrophin activity(Shen et al.,2019;Yang et al.,2020;Xie et al.,2021).These recent studies have primarily focused on the multifunctional p75 neurotrophin receptor(p75NTR)which is upregulated in central nervous system disease and injury,thus offering a unique target for intervention.     

Experimental rabies virus infection of p75 neurotrophin receptor-deficient mice

The low-affinity neurotrophin (NT) receptor, p75^NTR, has complex biologic functions. A recent report provided evidence that the p75^NTR is a rabies virus receptor in cultured BSR cells. We studied the experimental infection of 6-day-old p75^NTR-deficient mice with the challenge virus standard strain of fixed rabies virus inoculated intracerebrally. The mice developed a fatal encephalitis. There were morphologic changes of apoptotic cell death involving neurons in widespread areas of the brain, which were associated with in situ evidence of oligonucleosomal DNA fragmentation. The findings were very similar to those that we previously reported in wild-type ICR mice of the same age. If the p75^NTR is an important receptor of rabies virus in animal hosts, then a greater effect on the clinical and pathologic features of rabies virus-infected p75^NTR-deficient mice would have been expected. Received: 9 March 1999 / Revised, accepted: 7 June 1999     

Brain-derived neurotrophic factor-immunoreactive primary sensory neurons in the rat trigeminal ganglion and trigeminal sensory nuclei

Immunohistochemistry for brain-derived neurotrophic factor (BDNF) was performed on the rat trigeminal ganglion (TG). The immunoreactivity (IR) was detected in 46% of TG neurons. These neurons were mostly small- or medium-sized (range, 149.7-1246.3 microm2; mean +/- SD = 373.4 +/- 151.6 microm2). A double immunofluorescence method also revealed that 54% of BDNF-immunoreactive (IR) neurons were immunoreactive for calcitonin-gene-related peptide. In addition, 93% of BDNF-IR TG neurons contained vanilloid receptor subtype 1. However, the co-expression of BDNF and vanilloid receptor 1-like receptor was very rare (less than 1%). In the trigeminal sensory nuclei, laminae II of the medullary dorsal horn was abundant in presumed BDNF-IR axon terminals. Such profiles were also detected in the dorsolateral part of the subnucleus oralis. The retrograde tracing and immunohistochemical methods demonstrated that BDNF-IR was common among cutaneous TG neurons (47%) but not tooth pulp TG neurons (13%). The present study indicates that BDNF-IR TG neurons have unmyelinated axons and project to the superficial medullary dorsal horn. It is likely that BDNF-containing neurons in both the trigeminal and spinal sensory systems have similarities in morphology and function. However, the content of BDNF in TG neurons probably depends on their peripheral targets. BDNF seems to convey nociceptive cutaneous input to the trigeminal sensory nuclei.     

Transganglionic degeneration in trigeminal primary sensory neurons.

In 16 kittens either the frontal or the inferior alveolar nerve was transected and in 17 adult rats either the supraorbital, the infraorbital or the mental nerve was divided. The postoperative survival periods were kept at 3-28 days for the kittens and 15-26 days for the rats. Sections from the caudal brain stem and the upper part of the cervical cord were impregnated according to the Fink-Heimer method, procedure II. In the kittens degeneration was found after the 8th postoperative day ipsilaterally in both the spinal and main sensory trigeminal nuclei and the spinal trigeminal tract. In the rats degeneration was found in all cases in the same ipsilateral structures as in the kittens. The amount of degeneration was relatively great in the rats, whereas it was very modest in the kittens. A somatotopical pattern was found for the degeneration both within the spinal and the main sensory nuclei. It was in agreement with what has been found in earlier studies, where other techniques have been used. By a comparison with the results of a previous study on the trigeminal nerve in the rat, where partial lesions of the ganglia had been made, it was found that the degeneration in the present study did not cover the whole area receiving primary trigeminal afferents. Possible explanations for this are discussed.     

Overexpression of nerve growth factor by murine smooth muscle cells: Role of the p75 neurotrophin receptor on sympathetic and sensory sprouting

Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle α‐actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, so these tissues display increased densities of NGF‐sensitive sympathetic efferents and sensory afferents. Here we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene‐related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexi around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF‐rich peripheral tissues. J. Comp. Neurol. 521:2621–2643, 2013. © 2013 Wiley Periodicals, Inc.     

Neuropeptides and neurotrophin receptor mRNAs primary sensory neurons of aged rats

Neuropeptides and neurotrophin receptors are regulated in primary sensory neurons in response to axonal injury, and axonal lesions are characteristic stigmata of aging primary sensory neurons. We have therefore examined the expression of neuropeptides and neurotrophin receptor mRNAs in 30-month-old (median survival age) Sprague-Dawley rats to see if similar adaptive mechanisms operate in senescence. The content of neuropeptides was examined with immunohistochemistry (IHC) and in situ hybridization (ISH), and the cellular mRNA expression of neurotrophin receptors was studied with ISH. All of the aged rats had symptoms of hind limb incapacity (posterior paralysis), but fore limbs did not seem affected. The size-distribution of neuronal profiles in cervical and lumbar dorsal root ganglia (DRGs) was similar in aged and young adult (2–3 months old) rats. In aged rats, the DRG neurons showed an increase in both immunolabelling and mRNA content of neuropeptide tyrosine (NPY), as well as an increased cellular expression of galanin mRNA. In the same animals, there were decreased cellular levels of calcitonin gene-related peptide (CGRP; IHC and ISH) and substance P (SP; IHC and ISH), while the difference in neuronal somatostatin (IHC and ISH) was small. The distribution of neuropeptide immunoreactivities in the dorsal horn of the corresponding spinal cord segments revealed a decreased labelling for CGRP-, SP-, and somatostatin-like immunoreactivities (LI) in the aged rats at both cervical and lumbar levels. NPY- and galanin-LI had a similar distribution in aged and young adult rats. NPY-immunoreactive fibers were also encountered in the dorsal column of aged but not young adult rats. ISH revealed that most of the primary sensory neurons express mRNA for the p75 low-affinity neurotrophin receptor (p75-LANR) and that there was no discernible difference between young adult and aged rats. The labelling intensity for mRNA encoding high-affinity tyrosine kinase receptors (TrkA, TrkB, and TrkC) was decreased in aged rat DRG neurons, while the percentage of neuronal profiles expressing mRNA for TrkA/B/C was similar in young adult and aged rats. The changed pattern of neuropeptide expression in primary sensory neurons of aged rats resembled that seen in young adult rats subjected to axonal injury of peripheral sensory nerves and may, thus, indicate aging-related lesions of sensory fibers. Since NPY is primarily present in large and galanin in small DRG neurons, the stronger effect on NPY as compared to galanin expression may indicate that aging preferentially affects neurons associated with mechanoreception (Aα and Aβ fibers) as compared to nociceptive units (A and C fibers). Furthermore, the observed changes in neuropeptide expression were most pronounced in lumbar DRGs, that harbors the sensory neurons supplying the affected hindlimbs of the rats. © 1996 Wiley-Liss, Inc.     

TrkA and mitogen-activated protein kinase phosphorylation are enhanced in sympathetic neurons lacking functional p75 neurotrophin receptor expression

This study examined the effects of hypomorphic p75 neurotrophin receptor (p75NTR) expression and high levels of nerve growth factor (NGF) on trkA phosphorylation and downstream activation of p44/42 mitogen-activated protein kinase (MAPK). Post-ganglionic sympathetic neurons from postnatal day 1 p75NTR exon III null mutant (p75(-/-)) and 129/SvJ mice were cultured in the presence of 50 ng/mL NGF and analysed by Western blotting. Levels of phosphorylated trkA are increased in p75(-/-) neurons compared with 129/SvJ neurons, and these higher levels are maintained with continuous exposure to NGF. MAPK is also phosphorylated to a greater extent in p75(-/-) neurons than in 129/SvJ neurons, both within 10 min of exposure to NGF, and with continuous NGF treatment for 5 days. These data provide new insight into the mechanism underlying enhanced neurite outgrowth in p75(-/-) neurons, demonstrating that trkA and MAPK signalling in sympathetic neurons are increased when p75NTR function is disrupted.     

Upregulation of p75 neurotrophin receptor after stroke in mice does not contribute to differential vulnerability of striatal neurons

The survival of different neuron types and the expression of the p75 neurotrophin receptor (p75(NTR)) after focal cerebral ischemia were studied in the mouse striatum using immunocytochemical and histochemical techniques and stereological procedures. As assessed at 1 week after 30 min of middle cerebral artery occlusion, the order of vulnerability was projection neurons > parvalbumin-expressing interneurons > nitric oxide synthase-containing interneurons > cholinergic interneurons. Within the ischemic lesion, projection neurons were almost completely lost whereas cholinergic interneurons were spared. Calretinin-immunoreactive interneurons also seemed resistant to the insult. Expression of p75(NTR) was induced in cholinergic interneurons within the lesioned area, raising the possibility of a protective action. However, the number of cholinergic interneurons was unaffected in p75(NTR) knockout mice subjected to the same ischemic insult. These quantitative data demonstrate that striatal neurons in the mouse are differentially susceptible to ischemic damage and argue against a significant role of p75(NTR) for the high resistance of cholinergic interneurons.