Nerve-derived Factors Research Articles

Lingual Denervation Improves the Efficacy of Anti-PD-1 Immunotherapy in Oral Squamous Cell Carcinomas by Downregulating TGFβ Signaling.

Intratumoral nerve infiltration relates to tumor progression and poor survival in oral squamous cell carcinoma (OSCC). How neural involvement regulates antitumor immunity has not been well characterized. This study aims to investigate molecular mechanisms of regulating tumor aggressiveness and impairing antitumor immunity by nerve-derived factors. We performed the surgical lingual denervation in an immunocompetent mouse OSCC model to investigate its effect on tumor growth and the efficacy of anti-PD-1 immunotherapy. A trigeminal ganglion neuron and OSCC cell coculture system was established to investigate the proliferation, migration, and invasion of tumor cells and the PD-L1 expression. Both the neuron-tumor cell coculture in vitro model and the OSCC animal model were explored. Lingual denervation slowed down tumor growth and improved the efficacy of anti-PD-1 treatment in the OSCC model. Coculturing with neurons not only enhanced the proliferation, migration, and invasion but also upregulated TGFβ-SMAD2 signaling and PD-L1 expression of tumor cells. Treatment with the TGFβ signaling inhibitor galunisertib reversed nerve-derived tumor aggressiveness and downregulated PD-L1 on tumor cells. Similarly, lingual denervation in vivo decreased TGFβ and PD-L1 expression and increased CD8+ T-cell infiltration and the expression of IFNγ and TNFα within tumor. Neural involvement enhanced tumor aggressiveness through upregulating TGFβ signaling and PD-L1 expression in OSCC, while denervation of OSCC inhibited tumor growth, downregulated TGFβ signaling, enhanced activities of CD8+ T cells, and improved the efficacy of anti-PD-1 immunotherapy. This study will encourage further research focusing on denervation as a potential adjuvant therapeutic approach in OSCC. This study revealed the specific mechanisms for nerve-derived cancer progression and impaired antitumor immunity in OSCC, providing a novel insight into the cancer-neuron-immune network as well as pointing the way for new strategies targeting nerve-cancer cross-talk as a potential adjuvant therapeutic approach for OSCC.

Na,K-ATPase regulation in skeletal muscle.

Skeletal muscle contains one of the largest and the most dynamic pools of Na,K-ATPase (NKA) in the body. Under resting conditions, NKA in skeletal muscle operates at only a fraction of maximal pumping capacity, but it can be markedly activated when demands for ion transport increase, such as during exercise or following food intake. Given the size, capacity, and dynamic range of the NKA pool in skeletal muscle, its tight regulation is essential to maintain whole body homeostasis as well as muscle function. To reconcile functional needs of systemic homeostasis with those of skeletal muscle, NKA is regulated in a coordinated manner by extrinsic stimuli, such as hormones and nerve-derived factors, as well as by local stimuli arising in skeletal muscle fibers, such as contractions and muscle energy status. These stimuli regulate NKA acutely by controlling its enzymatic activity and/or its distribution between the plasma membrane and the intracellular storage compartment. They also regulate NKA chronically by controlling NKA gene expression, thus determining total NKA content in skeletal muscle and its maximal pumping capacity. This review focuses on molecular mechanisms that underlie regulation of NKA in skeletal muscle by major extrinsic and local stimuli. Special emphasis is given to stimuli and mechanisms linking regulation of NKA and energy metabolism in skeletal muscle, such as insulin and the energy-sensing AMP-activated protein kinase. Finally, the recently uncovered roles for glutathionylation, nitric oxide, and extracellular K(+) in the regulation of NKA in skeletal muscle are highlighted.

Salivary gland development and disease.

Mammalian salivary glands synthesize and secrete saliva via a vast interconnected network of epithelial tubes attached to secretory end units. The extensive morphogenesis required to establish this organ is dependent on interactions between multiple cell types (epithelial, mesenchymal, endothelial, and neuronal) and the engagement of a wide range of signaling pathways. Here we describe critical regulators of salivary gland development and discuss how mutations in these impact human organogenesis. In particular, we explore the genetic contribution of growth factor pathways, nerve-derived factors and extracellular matrix molecules to salivary gland formation in mice and humans.

Renal denervation prevents long-term sequelae of ischemic renal injury

Signals that drive interstitial fibrogenesis after renal ischemia reperfusion injury remain undefined. Sympathetic activation is manifest even in the early clinical stages of chronic kidney disease and is directly related to disease severity. A role for renal nerves in renal interstitial fibrogenesis in the setting of ischemia reperfusion injury has not been studied. In male 129S1/SvImJ mice, ischemia reperfusion injury induced tubulointerstitial fibrosis as indicated by collagen deposition and profibrotic protein expression 4 to 16 days after the injury.. Leukocyte influx, proinflammatory protein expression, oxidative stress, apoptosis, and cell cycle arrest at G2/M phase were enhanced after ischemia reperfusion injury. Renal denervation at the time of injury or up to 1 day post-injury improved histology, decreased proinflammatory/profibrotic responses and apoptosis, and prevented G2/M cell cycle arrest in the kidney. Treatment with afferent nerve-derived calcitonin gene-related peptide (CGRP) or efferent nerve-derived norepinephrine in denervated and ischemia reperfusion injury-induced kidneys mimicked innervation, restored inflammation and fibrosis, induced G2/M arrest, and enhanced TGF-β1 activation. Blocking norepinephrine or CGRP function using respective receptor blockers prevented these effects. Consistent with the in vivo study, treatment with either norepinephrine or CGRP induced G2/M cell cycle arrest in HK-2 proximal tubule cells, whereas antagonists against their respective receptors prevented G2/M arrest. Thus, renal nerve stimulation is a primary mechanism and renal nerve-derived factors drive epithelial cell cycle arrest and the inflammatory cascade causing interstitial fibrogenesis after ischemia reperfusion injury.

The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.

Perineural invasion is a form of cancer progression where cancer cells invade along nerves. This behavior is associated with poor clinical outcomes; therefore, it is critical to identify novel ligand-receptor interactions between nerves and cancer cells that support the process of perineural invasion. A proteomic profiler chemokine array was used to screen for nerve-derived factors secreted from tissue explants of dorsal root ganglion (DRG), and CCL2 was identified as a lead candidate. Prostate cancer cell line expression of CCR2, the receptor to CCL2, correlated closely with MAPK and Akt pathway activity and cell migration towards CCL2 and DRG. In vitro nerve and cancer coculture invasion assays of perineural invasion demonstrated that cancer cell CCR2 expression facilitates perineural invasion. Perineural invasion is significantly diminished in coculture assays when using DRG harvested from CCL2(-/-) knockout mice as compared with control CCL2(+/+) mice, indicating that CCR2 is required for perineural invasion in this murine model of perineural invasion. Furthermore, 20 of 21 (95%) patient specimens of prostate adenocarcinoma with perineural invasion exhibited CCR2 expression by immunohistochemistry, while just 3 of 13 (23%) lacking perineural invasion expressed CCR2. In summary, nerve-released CCL2 supports prostate cancer migration and perineural invasion though CCR2-mediated signaling. These results reveal CCL2-CCR2 signaling as a key ligand-receptor mechanism that mediates cancer cell communication with nerves during perineural invasion and highlight a potential future therapeutic target.

Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

BackgroundMicroarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration.ResultsConsiderable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences.ConclusionMany new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures

Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest “fractionation of movement” capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.

The promoter activity of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase in muscle: Suppressive roles of myogenesis and innervating nerve

The tetrameric globular form of acetylcholinesterase (G 4 AChE) is present and precisely controlled in muscles. The assembly and membrane targeting of G 4 AChE are directed by a proline-rich membrane anchor (PRiMA). It has been demonstrated that in muscle cells, the expression of PRiMA mRNA, as well as the level of G 4 AChE was suppressed by myogenesis and innervating nerve. A human PRiMA promoter-driven luciferase reporter was employed in this study to further reveal the activity of PRiMA transcription during myogenic differentiation and the influence of innervation. In parallel with PRiMA mRNA, the PRiMA promoter activity was suppressed by both myogenic regulatory factor(s) (MRFs) and nerve-derived factor(s). These results suggest that the regulation of PRiMA mRNA expression in muscle by MRFs and nerve-derived factors is due to a control system at the transcriptional level.

Transcriptional control of different subunits of AChE in muscles: Signals triggered by the motor nerve-derived factors

Acetylcholinesterase (AChE) is a highly polymorphic enzyme. Alternative splicing in the 3′ region of the primary transcript generates different subunits that contain the same catalytic domain but with distinct carboxyl termini. In mammals, the AChE R variant produces a soluble monomer that is up-regulated in the brain during stress. The AChE H variant produces a GPI-anchored dimer that is mainly expressed in blood cells, while AChE T variant is largely predominant in the brain and muscle. AChE T subunits associate with a collagen tail subunit (ColQ) forming asymmetric AChE species (A 4, A 8, and A 12 AChE) in muscle, and also form amphiphilic tetramers associated with a proline-rich membrane anchor (PRiMA) as globular AChE (G 4 AChE) in brain and muscle. The formation of these AChE forms depends on the physiological status of the muscles, and on the innervating nerves. The motor nerves achieve this regulation by two distinct mechanisms: release of the trophic factor calcitonin gene-related peptide (CGRP) and nerve-evoked electrical activity, which differentially regulate the expression levels of AChE T, PRiMA and ColQ via different downstream signaling cascades. The regulatory mechanisms provided by the nerve are important to account for the different expression patterns of AChE and associated proteins in fast- and slow-twitch muscles.

Vessels and Nerves: Marching to the Same Tune

The patterning of the nervous system is achieved through coordinated action of a variety of repulsive or attractive neuronal guidance factors that direct the growth of growing axons to specific pathways. Recent studies suggest that the same or similar sets of factors also guide migration of endothelial cells, helping to direct the stereotypical embryonic patterning of the developing vertebrate vascular system.

Neuroprotection as a treatment for glaucoma: pharmacological and immunological approaches.

Primary open-angle glaucoma is a chronic, progressive optic neuropathy associated with a gradual decline in visual functions, which may lead to blindness. In most cases, the optic neuropathy is associated with increased intraocular pressure. However, it is now generally accepted, that normalization of pressure, although necessary, is often not sufficient as a remedial measure. This is because of the existence of additional risk factors, some of which emerge as a consequence of the initial damage. This situation is reminiscent of the response to a traumatic axonal insult: some of the damage is immediate and is caused by the insult itself, while some is secondary and is caused by a deficiency of growth-supportive factors as well as by toxic factors derived from the damaged tissue. Accordingly, we have suggested that glaucoma may be viewed as a neurodegenerative disease and consequently is amenable to any therapeutic intervention applicable to these diseases. There is evidence that neuroprotection can be achieved both pharmacologically and immunologically. Pharmacological intervention (e.g. by using selective alpha-2 adrenergic receptor agonists) neutralizes some of the effects of the nerve-derived toxic factors and possibly increases the ability of the remaining healthy neurons, at any given time, to cope with the stressful conditions. Immunological intervention boosts the body's own repair mechanisms for counteracting the toxicity of physiological compounds acting as stress signals.

Guidance of regenerating motor axons in vivo by gradients of diffusible peripheral nerve-derived factors

During development of the central nervous system, neurons rely on target-derived factors to guide their outgrowing processes. Several CNS target-derived chemoattractive and repellant factors have been isolated and characterized, and their mechanism of action determined. For the peripheral nervous system, the results from numerous experiments suggest that during regeneration axons also respond to concentration gradients of target-derived factors leading to an oriented outgrowth up the gradient to the denervated target in vivo. The results from in vitro experiments have shown that diffusible concentration gradients of factors released from a length of denervated peripheral nerve, composed predominantly of Schwann cells, direct the outgrowth of sensory and motor neuron growth cones over distances of several hundred microns. However, a conclusive demonstration of a chemoattractive influence of diffusible concentration gradients on regenerating adult motor axons in vivo has remained elusive. The present experiments show that concentration gradients of denervated peripheral nerve-released factors direct the regeneration of adult motor axons in vivo, and that these gradients are effective over distances of more than 6.5 mm. Nonconditioned medium exerted no influence on the regenerating axons. Thus, results from in vivo experiments parallel those from in vitro experiments and indicate that isolated peripheral nerve-released factors that are effective in vitro will play a similar role on sensory and motor axons in vivo. Finally, the results show that diffusible concentration gradients of target-derived factors direct axon outgrowth both during both development and regeneration, as well as in vivo and in vitro. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 212–219, 2000

Guidance of regenerating motor axonsin vivo by gradients of diffusible peripheral nerve-derived factors

During development of the central nervous system, neurons rely on target-derived factors to guide their outgrowing processes. Several CNS target-derived chemoattractive and repellent factors have been isolated and characterized, and their mechanism of action determined. For the peripheral nervous system, the results from numerous experiments suggest that during regeneration axons also respond to concentration gradients of target-derived factors leading to an oriented outgrowth up the gradient to the denervated target in vivo. The results from in vitro experiments have shown that diffusible concentration gradients of factors released from a length of denervated peripheral nerve, composed predominantly of Schwann cells, direct the outgrowth of sensory and motor neuron growth cones over distances of several hundred microns. However, a conclusive demonstration of a chemoattractive influence of diffusible concentration gradients on regenerating adult motor axons in vivo has remained elusive. The present experiments show that concentration gradients of denervated peripheral nerve-released factors direct the regeneration of adult motor axons in vivo, and that these gradients are effective over distances of more than 6.5 mm. Nonconditioned medium exerted no influence on the regenerating axons. Thus, results from in vivo experiments parallel those from in vitro experiments and indicate that isolated peripheral nerve-released factors that are effective in vitro will play a similar role on sensory and motor axons in vivo. Finally, the results show that diffusible concentration gradients of target-derived factors direct axon outgrowth both during both development and regeneration, as well as in vivo and in vitro.

Principles of hair cycle control.

The hair follicle (HF) undergoes life-long cyclic transformations between "resting" (telogen), growth (anagen), and apoptosis-driven regression (catagen). Contrary to conventional wisdom, cyclic remodelling affects even the distal HF epithelium; telogen is not a mere resting period, since it shows substantial metabolic and proliferative activity and may encompass a phase of controlled hair shaft-extrusion ("exogen"). Even under physiological circumstances, very few (malfunctioning?) HF may leave this cycle over time to be removed by inflammatory cells ("programmed organ deletion"). Although numerous systemic, metabolic, immunological, and nerve-derived factors (e.g. hormones, cytokines, neuropeptides, neurotransmitters, mast cells) can profoundly alter hair growth in vivo, neither vascular nor neural stimuli nor extrafollicular cells are essential for HF development or cycling. Rather, an intrafollicular "hair cycle clock" of as yet unknown nature drives the HF cycle. This elusive chronobiological timing device likely exploits secondary changes in the intra- and perifollicular signalling milieu for guiding the HF through its transformations. However, the supreme generator of cycling activity ("oscillator") that dictates any of these signalling switches is still as unknown as is its exact location. Since, clinically, the control of catagen is of paramount importance (too early anagen termination: alopecia, effluvium; catagen too late: hirsutism, hypertrichosis), the controls of catagen-associated keratinocyte apoptosis and of dermal papilla secretory activities are discussed as crucial targets for future therapeutic manipulations.

γ-AChR/ϵ-AChR Switch at Agrin-Induced Postsynaptic-like Apparatus in Skeletal Muscle

We transfected the extrajunctional region of denervated soleus muscles in adult rats with neural agrin cDNA to induce myofibers to form postsynaptic-like apparatus containing acetylcholine receptor (AChR) aggregates. By 1 week ≈30% of the AChR aggregates contained a mixture of ϵ-AChRs and γ-AChRs while ≈70% had only γ-AChRs. If the transfected muscles were reinnervated in the original junctional region, the postsynaptic-like apparatus, despite the absence of apposed axon terminals, gradually came to have only ϵ-AChRs. We conclude that at the postsynaptic apparatus of ectopic neuromuscular junctions formed by a foreign nerve implanted into the extrajunctional region of denervated muscles, agrin secreted by the axon terminal plays a direct role in the γ-AChR/ϵ-AChR switch that occurs as the apparatus reaches maturity. Our findings, together with results from other studies, indicate further that agrin and acetylcholine are the only nerve-derived factors required for this switch.

Regulation of muscle glucose transport and GLUT-4 by nerve-derived factors and activity-related processes.

Glucose transport and GLUT-4 were examined in muscles in which activity and nerve-derived factors were eliminated (denervation) and in muscles in which only muscle activity was eliminated but in which nerve-derived factors were maintained [tetrodotoxin (TTX) treatment]. After 3 days of denervation, insulin-stimulated 3-O-methylglucose transport was markedly lowered in perfused rat hindlimb muscles (soleus, plantaris, and red and white gastrocnemius; < or = 35%). GLUT-4 was also decreased by 11-65% in denervated muscles. Blocking muscle activity with TTX superfusion of the sciatic nerve for 3 days reduced the insulin-stimulated glucose transport to the same extent as in the denervated muscles (P > 0.05). However, in soleus, plantaris, and red gastrocnemius muscles, GLUT-4 expression was reduced much less by TTX treatment than by denervation (P < 0.05). GLUT-4 mRNA abundance was decreased in denervated muscles but not in TTX-treated muscles. These results suggest that muscle activity largely regulates the insulin-signaling mechanisms of glucose transport and that nerve-derived trophic factors affect pretranslational events to regulate GLUT-4 expression.

Specific stimulation of human T lymphocytes by substance P.

Substance P (SP) and SP(4-11) stimulated significantly the proliferation of human T lymphocytes in vitro at respective concentrations of 10(-11) M to 10(-7) M and 10(-9) M to 10(-7) M, as assessed by the uptake of [3H]thymidine and [3H]leucine. The proliferative response of human T lymphocytes to PHA was augmented by 10(-9) M to 10(-8) M SP and 10(-10) M to 10(-7) M SP(4-11). The inactive analogue [D-Pro2, D-Phe7, D-Trp9]-SP inhibited the effects of SP on T lymphocytes. The specific stimulation of T lymphocytes by SP suggests a unique mechanism for the regulation of immunologic responses by peripheral nerve-derived factors.