Expression of Nerve Growth Factor in Human Pancreatic β Cells

Nerve growth factor (NGF) is an important modulator of rat pancreatic β-cell physiology in vitro. In this study, we analysed the expression of NGF, TrkA and insulin in human pancreatic islets from normal, ductal adenocarcinoma and insulinoma-afflicted samples, using double immunofluorescent labelling and confocal microscopy.

We found that in normal human pancreas, insulin and NGF are co-expressed in β cells. Moreover, similar to previous observations in rat, the high affinity NGF receptor TrkA is also expressed in β cells.

Pancreatic β cells in normal islets from adenocarcinoma and mucinous cystadenocarcinoma patients also expressed NGF. In 2 out of 15 exocrine tumour samples, NGF was detected also in the tissue surrounding the islets, while 2 out of 13 adenocarcinoma tumours expressed this growth factor.

In five insulinoma samples, we observed weaker immunofluorescent labelling of insulin and NGF in the neoplastic tissue, compared to the islets not afflicted by the tumour, which may be a consequence of increased hormone secretion rate.

We demonstrate that human β cells express TrkA and NGF. These findings are consistent with the hypothesis that NGF modulates insulin secretion through a paracrine/autocrine loop, similar to the one observed in cultured rat β cells.     

Age-dependent alterations in nerve growth factor (NGF)-related proteins, sortilin, and learning and memory in rats

The objective of this study was to evaluate the effects of aging on the performance of specific memory-related tasks in rats as well as to determine the levels of several nerve growth factor (NGF)-related proteins in relevant brain regions. The results indicated age-related impairments in spatial learning in a water maze task as well as deficits in recognition memory in a Spontaneous Novel Object Recognition task. In the prefrontal cortex and hippocampus, aged rats (compared to young controls) had elevated levels of the proneurotrophin, proNGF (+ 1.8-1.9 fold), p75^NTR receptors (+ 1.6-1.8 fold) and sortilin (+ 1.8-2.1 fold), and decreased levels of mature NGF (−36 to 44%), and phospho-TrkA receptors (−45 to 49%). The results of this study support the argument that NGF signaling is altered in the aging brain, and that such alterations may contribute to an age-related decline in cognitive function. These results may also help to identify specific components of the NGF-signaling pathway that could serve as targets for novel drug discovery and development for age-related disorders of cognition (e.g., Alzheimer's disease).     

夜香牛全草的生物活性成分研究

目的：研究广西产夜香牛生物活性化学成分。方法：用鼠性肾上腺髓质嗜铬细胞瘤PC-12细胞活性跟踪筛选，柱色谱方法分离化合物，谱学方法鉴定化合物结构。结果：分离得到4个化合物，根据谱学数据分析鉴定其结构分别为木脂素类化合物(+)-lirioresinol树脂醇B(1)，甾醇类化合物豆甾醇(2)和豆甾醇3-O-β-D-葡萄糖苷(3)，以及4-sulfo-benzocyclobutene(4)；用神经细胞体外模型试验，检测了分离得到化合物的NGF诱导活性。结论：化合物1，3,4为首次从该属植物中分离得到，化合物4为新的天然产物。     

Changes in nerve growth factor levels in dorsal root ganglia and spinal nerves in a rat neuropathic pain model

The purpose of this study was to measure the changes in levels of nerve growth factor (NGF) in dorsal root ganglia (DRG) and spinal nerves with the aim of investigating the role of NGF in a rat neuropathic pain model. Nerve injuries were made by tight ligation of the left L5 and L6 spinal nerves using 6-0 silk thread in male Sprague-Dawley rats. Before surgery and 1, 3, 5, 7, and 14 days after surgery, tissue samples collected included the L3-6 DRGs bilaterally, segments of the ipsilateral L5-6 spinal nerves proximal and distal to ligation sites, and corresponding sites of the contralateral L3-6 and the ipsilateral L3-4 spinal nerves. NGF levels in the DRGs of the injured spinal nerves (the left L5 and L6) did not change significantly from control values. The spinal nerve segments distal to ligation sites had higher levels of NGF than the control values. Unlesioned sites did not show any significant changes in NGF levels. The increase of NGF in distal segments of injured spinal nerves may be due to an accumulation of retrogradely transported NGF. The maintenance of NGF levels in the DRGs that had lost peripheral connections may reflect local synthesis after nerve injury.     

Induction of interleukin 8 release from the HMC-1 mast cell line: synergy between stem cell factor and activators of the adenosine A(2b) receptor

The HMC-1 mast cell line has both adenosine A(3) and A(2b) receptors on its surface, but only agonists of the A(2b) receptor are effective at releasing interleukin 8. Object of this study was to look for co-factors for adenosine A(2b) receptor activation. There was a powerful and statistically significant synergy for release of IL-8, both at the mRNA level (measured after 4 hr) and protein level (measured after 24 hr), between adenosine A(2b) receptor agonists and stem cell factor (SCF). Suitable concentrations for showing synergy were 100 ng/mL SCF and 3 microM 5'-N-ethylcarboxamidoadenosine (NECA). At these concentrations, the IL-8 released into the culture medium after SCF and NECA together was typically 3-5-fold greater in amount than the sum of the amounts of IL-8 released after exposure to the same concentrations of NECA and SCF separately. Since mast cells may be exposed to both adenosine and stem cell factor in the diseased lung, the synergy observed in this model system may have implications for asthma.     

Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro

Microglia are thought to play a key role in the development and regeneration of the central nervous system although the mechanisms regulating their presence and activity are not fully understood. Substantial evidence suggests that members of the neurotrophin family such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4 (NT-3/4) have a dramatic effect on both neurons and perineuronal cells. This study employed two murine microglial lines, BV-2 and N9, to examine the action of these neurotrophins on the mitotic activity and survival of microglia in vitro. Neurotrophins were incorporated into the media at the time of plating and cell number and levels of mitochondrial dehydrogenase activity (MTT) were determined at various time points in vitro. NGF increased cell number and MTT levels of both cell lines in a dose-dependent manner. BV-2 was more sensitive to NGF than N9. Similar responses were elicited by BDNF, although the sensitivity of each cell line was different than that found for NGF. NT-3 and NT-4 had no effect on cell proliferation. However, NT-4 had an effect on the survival of BV-2 and N9 cells. The response of these cells to neurotrophins was blocked by K252a, a tyrosine kinase inhibitor, suggesting that actions of neurotrophins were mediated by high-affinity tyrosine kinase receptors (Trk). Immunolocalization studies revealed positive Trk (pan) reactivity in the above cell lines and in primary microglia, but an absence of the low-affinity p75 neurotrophin receptor. Western blot analysis supported the above observations. These studies suggest that in addition to their neurotrophic actions, NGF and BDNF may also regulate microglial dynamics, thereby influencing the surrounding milieu during neuronal regeneration.     

S-adenosyl-methionine-induced apoptosis in PC12 cells

Our previous studies showed that S-adenosyl-methionine (SAM) induced Parkinson's disease-like changes in rat. It caused death to dopamine neurons in the substantia nigra, which appeared shrunken and fragmented, indicative of apoptosis-like changes (Charlton and Crowell [1995] Mol. Chem. Neuropathol. 26:269-284; Charlton [1997] Life Sci. 61:495-502). In this study, we investigated whether SAM causes apoptosis in both undifferentiated PC12 (PC12) cells and nerve growth factor (NGF)-differentiated PC12 (D-PC12) cells. S-adenosyl-homocysteine (SAH), the nonmethyl analog of SAM, was also tested. SAM and SAH (1.0 nM to 10.0 microM) caused lactate dehydrogenase (LDH) release from the PC12 cells and D-PC12 cells; cells with morphological changes and fluorescent DNA fragmentation staining were detected among both PC12 cell and D-PC12 cell. Compared with the PC12 cell, the D-PC12 cell, a postmitotic cell, was more sensitive to the toxic effects of SAM or SAH and presented much greater LDH release, suggesting a lethal effect; surprisingly, the amounts of apoptotic cells did not differ significantly between the two kinds of cells. In medium deprived of exogenous methionine, a decline in LDH release was observed in PC12 and D-PC12 cells. Also, lower levels of intracellular SAM and SAH were observed in the methionine-deleted media, which were reversed by the addition of either SAM or SAH. An antivitamin B(12) monoclonal antibody was added to methionine-depleted medium, resulting in deficiency of both endogenous and exogenous methionine, which caused further decreases in LDH release and reduction in the levels of intracellular SAM and SAH. The preliminary data showed different sensitivities to SAM or SAH between PC12 cell and D-PC12 cells, which suggests that PC12 cell may be more stable as a metabolic model. Apoptosis of PC12 cells was also assessed by PARP cleavage detection, Western blot analysis of Bax and Bcl-2 proteins, and DNA laddering on agarose gel electrophoresis. The proapoptoic protein Bax was dominantly expressed, whereas Bcl-2 was slightly down-regulated by SAM. SAH weakly induced the expression of Bax and slightly decreased Bcl-2 levels. The effects of SAM and its analog, SAH, were demonstrated conclusively to induce apoptosis in PC12 cells.     

Acute cholinergic rescue of synaptic plasticity in the neurodegenerating cortex of anti-nerve-growth-factor mice

Deficits in cholinergic systems innervating cerebral cortex are associated with cognitive impairment during senescence and in age-related neurodegenerative pathologies. However, little is known about the role of cholinergic pathways in modulating cortical plasticity. Basal forebrain cholinergic neurons are a major target for nerve-growth factor (NGF). In order to investigate the relationship between cholinergic innervation and cortical synaptic plasticity, we exploited a transgenic mouse model in which the activity of NGF in the adult nervous system is neutralized by the expression of blocking antibodies to NGF itself (anti-NGF mice) [Ruberti, F. et al. (2000). J. Neurosci. 20, 2589-2601]. In 6-month-old anti-NGF mice, we show that the reduction in cholinergic innervation of the cortex is associated with different forms of synaptic plasticity impairment. A local, acute increase in the availability of acetylcholine rescues these synaptic plasticity deficits, thus indicating that a cholinergic system mediates the impairment of cortical plasticity at this early stage of the neurodegenerative process triggered by NGF neutralization. Our results represent an important step in unveiling the pivotal role of cholinergic transmission in modulating adult cortical plasticity.     

Growth Control of C6 glioma in vivo by Nerve Growth Factor

Treatment with nerve growth factor (NGF) causes differentiation of rat C6 glioma cells and strongly inhibits their proliferation in vitro. This suggests that induction of NGF-mediated differentiation may provide a novel therapeutic approach to growth control of glial tumors. We examined the effects of NGF treatment on the growth potential of C6 glioma, which expressed NGF receptor in vivo. C6 glioma cells (1 × 10⁶ cells/10 l) were transplanted into the rat striatum. After 4 days, the animals were given successive injections of 100 ng NGF into the growing tumor at every 4 days (n = 10 rats). Controls were subjected to identical procedures with vehicle which did not contain NGF (n = 10 rats). At 14 days after transplantation, we evaluated the tumor volume, proliferative cell index (PCI) based on the MIB-1 immunoreactivity and enzyme activities related to energy metabolism by enzyme histochemistry. We found that the NGF treatment markedly reduced the tumor volume of the C6 glioma (34.00 ± 8.47 mm³ to 7.22 ± 4.92 mm³, p < 0.01). NGF treatment also decreased the PCI (33.34 ± 9.57% to 3.85 ± 3.56%, p < 0.01) with a negative correlation with tumor volume (r = 0.972, p < 0.01), and the hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) activities (p < 0.01 and p < 0.01, respectively) which reflect the demand for nucleic acid synthesis for proliferation through the glycolytic and pentose phosphate pathways. The present results demonstrate for the first time that inhibition of tumor cell proliferation of C6 glioma by NGF occurs in vivo, probably through the NGF-mediated differentiation of C6 glioma cells which has been observed in in vitro studies.     

Distinct usages of phospholipase C gamma and Shc in intracellular signaling stimulated by neurotrophins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrophin family, bind to and activate TrkA, TrkB and TrkC, respectively, members of the Trk receptor tyrosine kinase family, to exert various effects including promotion of differentiation and survival, and regulation of synaptic plasticity in neuronal cells. Many reports have suggested that different neurotrophins show distinct biological functions, although molecular mechanisms by which neurotrophins exert their different functions remain unclear. In the present study, we found distinct usages of phospholipase Cgamma (PLCgamma) and Shc in intracellular signaling stimulated by neurotrophins. BDNF stimulated much stronger interactions of PLCgamma with Trk than NGF and NT-3 in PC12 cells stably expressing TrkB and cultured cerebral cortical neurons, respectively, although BDNF, NGF and NT-3 induced similar levels of tyrosine phosphorylation of Trk. Furthermore, the cultured cortical neurons showed large PLCgamma-dependent increases in intracellular Ca(2+) levels in response to BDNF compared with NT-3. In Shc signaling, NGF, but not BDNF, displayed interactions between Trk and Shc in a phenylarsine oxide (PAO; an inhibitor of tyrosine phosphatase)-dependent manner in TrkB-expressing PC12 cells. These results indicated that neurotrophins stimulate distinct kinds of interactions between Trk and PLCgamma and between Trk and Shc. These differences may lead to the distinct biological functions of neurotrophins.