p75NGFR mRNA Research Articles

Suppressing Sema3A expression in muscle satellite cells affects terminal Schwann cells after muscle and nerve injury

To examine the role of Sema3A expression in satellite cells (SCs) and terminal Schwann cells (TSCs) in neuromuscular junction (NMJs) formation, expression was investigated during recovery from muscle- or nerve-crush injuries in muscle from mice with a SC-specific knockout of Sema3A. We tested the hypothesis that loss of SC-specific Sema3A expression would disrupt TSC gene and protein expression after both injuries. Gene expression in synaptic areas was studied using RNAscope multiplex fluorescence in situ hybridisation (ISH, Advanced Cell Diagnostics) to examine TSCs (Sema3A, S100B, P75NGFR), Pax7+ SCs, and Westerns to assay proteins (Sema3A and S100B, and γAchR, related to denervation). Muscle from transgenics with tamoxifen-induced conditional knockout, and two control groups (injured non-knockouts and no-surgery SC-specific knockouts) were examined 14 and 21days after nerve crush and 21 and 35days after muscle crush (ethics approval A30-142-0 (Kyushu U) and F14-15 UManitoba). Expression sites (number, area, and intensity) for Sema3A, S100B, P75NGFR and Pax7 mRNA were imaged, measured (Celleste software) and analyzed (ANOVA, linear regression, and Principal Component Analysis, PCA) as a function of regeneration time. After muscle crush, P75NGFR expression was higher in SC-specific Sema3A knockout mice (days 21 and 35) than in non-knockout controls (p<0.001 Tukey's, df=16) suggesting an increased number of TSCs. γAchR protein was highest at day 21 vs. controls (p<0.001 Tukey's df=32) and correlated to Pax7 intensity in SCs. S100B expression correlated with regeneration time (p<0.01, df=34). S100B protein decreased at day 21 (Tukey's, p<0.001), possibly due to loss of TSCs by out-migration or cell death, or lack of S100B promotor activity. After nerve crush, P75NGFR expression was higher at day 21 in knockouts than in controls and correlated to Sema3A expression intensity. γAchR protein correlated with P75NGFR and Pax7 expression, indicating SCs increase after denervation or that Pax7 expression/SC increases. S100B and Sema3A proteins in non-knockout controls were lower at day 14 after nerve injury (2-way Anova, p<0.001) and increased at day 21 (p<0.001), changes not seen in SC-specific Sema3A knockout mice. Combining all data for both injuries in PCAs, TSC sites (colocalized P75NGFR, Sema3A and S100B expression) increased with increased S100B and Sema3A proteins (Factors 1 and 2 accounted for 51.8% of variance). PCA of SC sites (overlap of Pax7, S100B, and Sema3A expression) showed Sema3A mRNA and protein and S100B accounted for 30.9% of variance in SCs. Loss of SC-specific Sema3A expression accelerated denervation after muscle injury. PCAs showed TSC function depends on Sema3A and S100B in reinnervation. Results advance our understanding of synaptic interactions of TSCs and SCs in vivo. This complex interaction between TSCs and SCs during reinnervation and regeneration opens an important new aspect of nerve-muscle interaction at the cellular level, with possible implications for diseases where those interactions decline, such as amyotrophic lateral sclerosis.

IFT80 Improves Invasion Ability in Gastric Cancer Cell Line via ift80/p75NGFR/MMP9 Signaling.

The assembly and maintenance of cilia depend on intraflagellar transport (IFT) proteins, which play an important role in development and homeostasis. IFT80 is a newly defined IFT protein and partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III, both characterized by abnormal skeletal development. However, the role and mechanism of IFT80 in the invasion of gastric cancer is unknown. We established SGC-7901 and MKN-45 gastric cancer cell lines that stably overexpressed IFT80, as verified by quantitative reverse transcription-PCR, Western blot, and immunofluorescence. Matrix metalloproteinase-9 (MMP9) plays an important role in tumor invasion, and its expression was assessed by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence. The invasion ability of IFT80 on SGC-7901 and MKN-45 cells was examined by the Matrigel invasion assay. The relationship between p75NGFR, and the p75NGFR antagonists, PD90780 and IFT80, were detected by quantitative reverse transcription-PCR and Western blotting. We first detected an IFT80 expression pattern, and found that IFT80 was highly expressed in gastric cancer clinical samples. Overexpression of IFT80 in the gastric cancer cell lines, SGC-7901 and MKN-45, led to lengthening cilia. Additionally, overexpression of IFT80 significantly improved proliferation and invasion, but inhibited apoptosis, in gastric cancer cells. We further found that overexpression of IFT80 increased p75NGFR and MMP9 mRNA and protein expression. Treatment with the p75NGFR antagonist PD90780 inhibited the increased invasion ability resulting from overexpression of IFT80 in SGC-7901 and MKN-45 gastric cancer cells. Thus, these results suggest that IFT80 plays an important role in invasion of gastric cancer through regulating the ift80/p75NGFR/MMP9 signal pathways.

Non-invasive neural stem cells become invasive in vitro by combinatorial FGF2 and BMP4 signaling

Neural stem cells (NSCs) typically show efficient self-renewal and selective differentiation. Their invasion potential, however, is not well studied. In this study, Sox2-positive NSCs from the E14.5 rat cortex were found to be non-invasive and showed only limited migration in vitro. By contrast, FGF2-expanded NSCs showed a strong migratory and invasive phenotype in response to the combination of FGF2 and BMP4. Invasive NSCs expressed Podoplanin (PDPN) and p75NGFR (Ngfr) at the plasma membrane after exposure to FGF2 and BMP4. FGF2 and BMP4 together upregulated the expression of Msx1, Snail1, Snail2, Ngfr, which are all found in neural crest (NC) cells during or after epithelial-mesenchymal transition (EMT), but not in forebrain stem cells. Invasive cells downregulated the expression of Olig2, Sox10, Egfr, Pdgfra, Gsh1/Gsx1 and Gsh2/Gsx2. Migrating and invasive NSCs had elevated expression of mRNA encoding Pax6, Tenascin C (TNC), PDPN, Hey1, SPARC, p75NGFR and Gli3. On the basis of the strongest upregulation in invasion-induced NSCs, we defined a group of five key invasion-related genes: Ngfr, Sparc, Snail1, Pdpn and Tnc. These genes were co-expressed and upregulated in seven samples of glioblastoma multiforme (GBM) compared with normal human brain controls. Induction of invasion and migration led to low expression of differentiation markers and repressed proliferation in NSCs. Our results indicate that normal forebrain stem cells have the inherent ability to adopt a glioma-like invasiveness. The results provide a novel in vitro system to study stem cell invasion and a novel glioma invasion model: tumoral abuse of the developmental dorsoventral identity regulation.

Dual response of BDNF to sublethal concentrations of β-amyloid peptides in cultured cortical neurons

Beta-amyloid (Aβ) deposition is one important pathological hallmark in Alzheimer's disease (AD). However, low levels of Aβ may modify critical endogenous protection systems before neurodegeneration occurs. We examined the time-course effect of sublethal concentrations of Aβ on total BDNF (panBDNF), BDNF transcripts (I, II, IV and VI), trkB.FL, trkB.T1 and p75NGFR mRNA expression in cultured cortical neurons. We have shown that Aβ exhibited a dual response on BDNF mRNA, i.e. an increase at short times (3–5 h) and a dramatic decrease at longer times (24 or 48 h). The early increase in BDNF expression seems to be driven by increased expression of transcripts I and IV. The BDNF drop was specific since did not occur for other mRNAs examined. The BDNF protein content showed a similar profile but did not follow the dramatic reduction as its encoding mRNA. These observations may help to explain cognitive deficits observed at initial stages of AD.

Establishment and characterization of rat dental epithelial derived ameloblast-lineage clones

Teeth are the hardest tissues covered with enamel produced by ameloblasts. The ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions during tooth morphogenesis. However, the molecular mechanism of ameloblast differentiation remains unclear. To address this question, we developed an in vitro assay system to evaluate the molecular mechanism of amelogenesis. First, we established dental epithelium-derived clones from 6-day-old rat incisors and established that cells of the clone SRE-G5 were the largest producers of amelogenin mRNA. Next, we analyzed the effects of several chemicals on the amelogenin expression in SRE-G5 cells. Only mitogen-activated protein kinase (MAPK) activators enhanced amelogenin mRNA expression. This finding corresponded to the immunohistochemical data showing the presence of phosphorylated forms of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) during ameloblast differentiation. To examine the roles of MAPK signals, we compared the effects of anisomycin and sodium salicylate on the expression of tooth-related differentiation markers. Both anisomycin and sodium salicylate induced amelogenin, Abcg2, and Bmp4 mRNA and down-regulated p75NGFR mRNA. On the other hand, ALP, ectodin, Bmp2 and Fgf8 mRNA were up-regulated only by anisomycin. These results indicate that MAPK signaling functions, at least in part, as the inducer of ameloblast differentiation.

Neurotrophin-4/5-depletion induces a delay in maturation of the periodontal Ruffini endings in mice

Neurotrophin-4/5 (NT-4/5) - a member of the neurotrophic factors - is a ligand for TrkB, which has been reported to be expressed in the mechanoreceptive Ruffini endings of the periodontal ligament. The present study examined developmental changes in the terminal morphology and neural density in homozygous mice with a targeted disruption of the nt-4/5 gene and wild-type mice by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by quantitative analysis using an image analyzer. Postnatal development of terminal Schwann cells was also investigated by enzymatic histochemistry for non-specific cholinesterase activity (ChE). Furthermore, the immuno-expression of TrkB and low affinity nerve growth factor receptor (p75-NGFR) was surveyed in the periodontal Ruffini endings as well as trigeminal ganglion. At postnatal 1 week, the lingual periodontal ligament of both types of mice contained PGP 9.5-positive nerve fibers showing a tree-like ramification with axonal swellings in their course. In both types of mice at 2 weeks of age, comparatively thick nerve fibers with a smooth outline increased in number, and frequently ramified to form nerve terminals with dendritic profiles. However, no typical Ruffini endings with irregular outlines observed in the adult wild-type mice were found in the periodontal ligament at this stage. At postnatal 3 weeks, typical Ruffini endings with irregular outlines were discernable in the periodontal ligament of the wild-type mice while the dendritic endings showing smooth outlines were restricted to the homozygous mice. After postnatal 8 weeks, both types of mice showed an increase in the number of Ruffini endings, but the morphology differed between the wild-type and NT-4/5 homozygous mice. In the wild-type mice, a major population of the Ruffini endings expanded their axonal branches and developed many microprojections, resulting in a reduction of endings with smooth outlines. In contrast, we failed to find such typical Ruffini endings in the periodontal ligament of the homozygous mice: A majority of the periodontal Ruffini endings continued to show smooth outlines at postnatal 12 weeks. Quantitative analysis on neural density demonstrated a reduction in the homozygous mice with a significant difference by postnatal 8 weeks. Enzymatic histochemistry for non-specific ChE did not exhibit a distinct difference in the distribution and density of terminal Schwann cells between wild-type and homozygous mice. Furthermore, TrkB and p75-NGFR mRNA and proteins did not differ in the trigeminal ganglion between the two types. The periodontal Ruffini endings also displayed immunoreactivities for TrkB and p75- NGFR in both phenotypes. These findings suggest that the nt-4/5 gene depletion caused a delay in the formation and maturation of the periodontal Ruffini endings in the mice by inhibiting the growth of the periodontal nerves at an early stage, and indicate that multiple neurotrophins such as NT- 4/5 and BDNF might play roles in the development and/or maturation of the periodontal Ruffini endings.

PHENOTYPE OF PMP22 TRANSGENIC SCHWANN CELLS IN CULTURE

Charcot‐Marie‐Tooth type 1A (CMT1A) neuropathy is due to a duplication of the peripheral myelin protein 22 (PMP22) gene. In the sciatic nerves of PMP22 transgenic (PMP22tg) animals, Schwann cells (SC) switch on the myelinating program by producing detectable levels of myelin related proteins (PMP22, P0, MBP), but express also early maturational markers and antigens typical of non‐myelinating SC (p75NGFR).To further investigate PMP22tg SC behaviour, we established short‐term (five days) SC cultures from P30 normal and PMP22tg rats. Semiquantitative RT‐PCR was used to study PMP22, P0, MBP and p75NGFR mRNA expression, both in basal conditions and after forskolin (FSK) addition to mimic the axonal contact. SC proliferation rate was studied using a colorimetric assay (MTT based). Moreover, a gold particle motility assay was used to evaluate cell motility and shaping. Finally, preliminary experiments on SC migration in presence of IGFI were performed using a Boyden chamber.Homozygous and heterozygous PMP22tg SC, compared to normal ones, showed higher basal levels of PMP22 (0.82 vs 0.6 vs 0.09), P0 (2.24 vs 1.92 vs 0.53) and p75NGFR (1.33 vs 1.38 vs 0.34) mRNAs. FSK treatment (24 hrs) further increased PMP22 levels in homozygous (1.57 vs 0.82), heterozygous (0.93 vs 0.6) and normal (0.194 vs 0.09) cultures. PMP22tg SC showed a lower proliferation rate than normal ones. In basal conditions no differences were found between PMP22tg and normal SC in terms of cell area and motility, whereas FSK treatment determined, in PMP22tg SC, a significant decrease in cell area and motility. Finally, cell migration in the presence of a chemoattractant molecule did not differ between PMP22tg and normal SC.Cultured PMP22tg SC, besides maintaining the ability to overexpress PMP22, further increase its expression after FSK treatment. As PMP22tg SC in the presence of FSK also show a decreased motility and area, but migration seems to be identical between normal and transgenic cells, we conclude that axonal contact may increase PMP22 expression at a level that affects SC ability of forming myelin.

Real Time RT-PCR Shows Correlation between Retinoid-Induced Apoptosis and NGF-R mRNA Levels

Neurotrophins and retinoic acid have a critical role in the differentiation and the survival of neurons. All-trans-, 9-cis-retinoic acid (10−6 M) or NGF (50–100 ng/ml) induced morphologic differentiation and inhibited cell growth in SH-SY5Y neuroblastoma cells after 7 days of culture. Continuous treatment of undifferentiated cells with all-trans- or 9-cis-retinoic (10−6 M) did not induce apoptosis, whereas NGF-differentiated cells showed dramatic apoptosis after 2 to 4 days of retinoic acid treatment as evidenced by TUNEL reaction and flow cytometry analysis following propidium iodide staining. Addition of Ro41-5253 blocked all-trans-retinoic-induced apoptosis, suggesting that the apoptotic signaling pathway was mediated by RARs. The effects of all-trans- or 9-cis-retinoic acid on the expression of NGF receptors was evaluated using real-time fluorescence reverse transcription-PCR. A slight transient increase in the expression of p75NGFR mRNA was observed by 2 to 4 h after retinoid treatment of undifferentiated cells, whereas a larger increase in the expression of both TrkA and p75NGFR mRNA up to threefold the basal level, was observed by 2 to 6 h after retinoid treatment of NGF-differentiated cells. Our results suggest that NGF-differentiated cells may be more susceptible to retinoid-induced apoptosis that undifferentiated cells.

NGF prevents further atrophy of cholinergic cells of the nucleus basalis due to cortical infarction in adult post-hypothyroid rats but does not restore cell size compared to euthroid rats

We have tested the hypotheses that nerve growth factor treatment in adult post-hypothyroid rats can: (1) restore cross-sectional area of cholinergic cells of the nucleus basalis and (2) prevent further atrophy of these neurons following cortical infaction. In addition, we assessed the expression of p75 NGFR and p140 trkA mRNAs in the nucleus basalis cells of post-hypothyroid rats. Rats were rendered hypothyroid by the addition of propylthiouracil to their diet beginning on embryonic day 19 until the age of 1 month. At this time both the pups and their dams continued to receive 0.05% propylthiouracil in their diet and the pups were thyroidectomized. At 60 days, propylthiouracil treatment was interrupted and thyroxine levels were restored to normal by daily subcutaneous administration of physiological levels of thyroxine. Morphometric analysis identified atrophied nucleus basalis magnocellularis cholinergic cells at two ages, days 75 and 105, identified by in situ hybridization for p75 NGFR and p140 trkA mRNAs in methylene blue stained cells (day 75) and choline acetyltransferase immunostaining (day 105). The mean number of silver grains (pixels) per μm 2 (mean±S.E.M.) of cell body cross-sectional area for p75 NGFR mRNA in the nucleus basalis magnocellularis of euthyroid rats was 3.43±0.89, which was not statistically different from post-hypothyroid animals (4.02±1.07). A similar finding was noted for p140 trkA mRNA: mean number of grains in the euthyroid group was 5.54±0.96 and was not statistically different from the post-hypothyroid group (6.32±1.45). Nerve growth factor treatment in adulthood (between days 75 and 82) did not restore cross-sectional area from early thyroid deprivation. However, it prevented further atrophy of nucleus basalis magnocellularis neurons following cortical devascularization inflicted in adulthood (day 75).

Expression of mRNAs for neurotrophic factors (NGF, BDNF, NT-3, and GDNF) and their receptors (p75NGFR, trkA, trkB, and trkC) in the adult human peripheral nervous system and nonneural tissues.

The steady-state mRNA levels of the four neurotrophic factors of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) and their receptors (p75NGFR, trkA, trkB and trkC) in the adult human peripheral nervous system (PNS) as well as nonneural tissues were examined using quantitative reverse transcription-polymerase chain reaction (RT-PCR). NGF and BDNF mRNA levels were high in the heart and spleen as well as in the dorsal root ganglia (DRG) and spinal cord, showing similar spatial expression patterns, while NT-3 mRNA levels were more pronounced in the liver and spleen. In contrast to these neurotrophins, GDNF mRNA expression occurred at the highest levels in the muscle, and it was also comparatively high in the spinal cord. p75NGFR mRNA was expressed extensively throughout the PNS tissues and in the spleen. The spatial expression patterns differed among trkA, and trkB and trkC mRNAs. trkA mRNA was greatly expressed in the DRG, sympathetic ganglia and spleen, while the trkB and trkC mRNA levels were high in the DRG, spinal cord and brain. The levels of trkB and trkC mRNAs with tyrosine kinase domain, compared to those with extracellular domain, were relatively high in the DRG, whereas they were low in the spinal cord and brain. The spatial patterns of the distributions of neurotrophic factors and their receptors mRNA levels in the adult human PNS and nonneural tissues are largely similar to those reported in other mammals, but these findings provide further, more specific, understanding relevant to the therapeutic approach to human diseases.

Expression of nerve growth factor and neurotrophin receptors in testicular cells suggest novel roles for neurotrophins outside the nervous system.

The present study was designed to clarify the non-neurotrophic role for neurotrophins in mouse testis. By means of SI nuclease protection assay we could demonstrate that the gene coding for the low-affinity nerve growth factor (NGF) receptor p75NGFR is transiently expressed during germ cell development. Gene expression for p75NGFR was detected in late-meiotic spermatocytes and early spermatids and was found to be co-expressed with trkB and trkC, two tyrosine kinase receptors, commonly regarded as the high-affinity receptors for brain-derived neurotrophic factor and neurotrophin-3. Gene transcripts for the high-affinity NGF receptor trkA were found exclusively in non-germ cells. Isolated Leydig cells, peritubular myoid cells and Sertoli cells, but not germ cells, could be identified as potential testicular NGF sources. Non-germ cells respond after incubation for several days with a sharp induction in NGF synthesis, which is accompanied by a loss of phenotypic expression patterns. The fact that p75NGFR mRNA expression was induced in cultured Sertoli cells and peritubular myoid cells suggests an autocrine mode of NGF action in these cells. Induction of NGF synthesis in cultured Leydig cells could be prevented by the glucocorticoid dexamethasone. Results indicate different roles for the individual neurotrophins in distinct testicular compartments and suggest that these neurotrophins might support testicular functions by signalling between individual cell types in an autocrine and paracrine manner.

Low-affinity nerve growth factor receptor expression in sciatic nerve during P 2-peptide induced experimental allergic neuritis

Northern blot, immunocytochemistry, and single nerve fiber immunostaining were used to determine the expression of low-affinity nerve growth factor (NGF) receptor (p75NGFR) in the peripheral nervous system of Lewis rats during the course of experimental allergic neuritis (EAN) induced with 100 μg of ‘SP26’ synthetic peptide, corresponding to residues 53–78 of bovine P 2 myelin protein. This severe reversible polyneuropathy, characterized by extensive multifocal demyelination in nerve roots, and axonal degeneration within sciatic nerve, represents a useful tool to investigate Schwann cell gene expression in these different pathological conditions. Our results showed that both p75NGFR mRNA and protein were induced within sciatic nerve by day 18 and 23 after ‘SP26’ immunization. By this time, however, p75NGFR was not detectable in roots of cauda equina. These data demonstrate that, during EAN induced by immunization with 100 μg of ‘SP26’, p75NGFR is up-regulated in Schwann cells and in nerve fibers distally along the nerve. This suggests that the induction of the receptor in sciatic nerve during P 2-peptide EAN is related to the loss of axonal contact by Schwann cells.

In vitro glial responses to nerve growth factor.

Nerve growth factor (NGF) stimulates expression of the low affinity neurotrophin receptor p75NGFR mRNA in primary cultures of neonatal rat cortical type I astrocytes. Nerve growth factor treatment altered glial morphology in glial fibrillary acidic protein positive (GFAP+) cell cultures derived from newborn (P0) and 3-day-old (P3) rat pups. When P0- or P3-derived primary glial cultures were serum-deprived, in the presence of 200 pM NGF for 5 days, the flat polygonal glia present in culture assumed a fibrous morphology, an effect not seen in the untreated serum-deprived controls. The NGF effect on astrocytic morphology was blocked by continuous serum treatment. Nerve growth factor did not stimulate astrocytic proliferation under these culture conditions, as assayed by cell cycle analysis using 3H thymidine autoradiography. P0-derived primary glial cultures expressed the signal transducing neurotrophin receptors p145trkB and p140trkA as determined by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were identified by sequencing or restriction enzyme analysis. Astrocytes internalized 125I-NGF at 37 degrees C but not at 4 degrees C, consistent with energy requirements for internalization. Also, internalization of 125I-NGF was abolished by the addition of a 300-1,000-fold excess of unlabeled NGF. Thus, astroglial cells in culture internalize NGF through a specific receptor-mediated process, express trkA and full-length trkB mRNAs at low levels, and respond to exogenous NGF by expressing a fibrous morphology under serum-free culture conditions.

Effects of acetyl- l-carnitine treatment and stress exposure on the nerve growth factor receptor (p75 NGFR) mRNA level in the central nervous system of aged rats

1. 1. There is growing evidence that the nerve growth factor protein (NGF), a neurotrophic factor for peripheral and central nervous system (CNS) neurons, may play a role in the modulation of the hypothalamo-pituitary-adrenocortical axis (HPAA). While NGF binding is decreased in rodent CNS after stress exposure, this reduction is prevented by treatment with Acetyl-L-Carnitine (ALCAR), a chemical substance able to prevent some degenerative events associated with aging. 2. 2. The authors studied the effect of cold stress on the low-affinity NGF receptor (p75 NGFR) mRNA levels in the basal forebrain and cerebellum of aged rats chronically treated with ALCAR. 3. 3. The present results show that ALCAR abolished the age-associated reduction of p75 NGFR mRNA levels in the basal forebrain of old animals, but did not affect the response to stress stimuli. 4. 4. Also, treatment with ALCAR mantained p75 NGFR mRNA levels in the cerebellum of old animals at levels almost identical to those observed in young control animals. 5. 5. These results suggest a neuroprotective effect for ALCAR on central cholinergic neurons exerted at the level of transcription of p75 NGFR. The restoration of p75 NGFR levels could increase trophic support by NGF of these CNS cholinergic neurons which are implicated in degenerative events associated with aging.

Interactions of Estrogen with the Neurotrophins and Their Receptors during Neural Development

We are interested in examining mechanisms underlying estrogen actions during neuronal differentiation in the central nervous system (CNS). Our research has focused on one possible mechanism, the developmental interactions between estrogen and the neurotrophins (nerve growth factor [NGF], brain derived neurotrophic factor [BDNF] and neurotrophin-3 [NT-3]). Using combined isotopic and non-isotopic in situ hybridization, we found that neurons in developmental estrogen targets (e.g., the cerebral cortex), co-localized mRNAs for the neurotrophins (NGF or BDNF) with their cognate receptors (p75NGFR [the pan-neurotrophin receptor] and trkA or trkB [the tyrosine kinase receptors]), suggesting a localization of neurotrophin-autocrine loops to these estrogen-sensitive neurons. In contrast, the basal forebrain, which is estrogen-sensitive in the adult and during development, only expressed neurotrophin receptor mRNAs, suggesting that this region was not an autocrine neurotrophin target. We examined the potential for developmental estrogen-neurotrophin interactions, using a model neurotrophinsensitive system, i.e., differentiating PC12 cells. NGF significantly increased estrogen receptor density in PC12 cells. Reciprocally, estrogen up-regulated trkA mRNA and transiently down-regulated p75NGFR mRNA, suggesting that estrogen may increase the efficiency of NGF binding in PC12 cells. Similar estrogen-dependent regulation of NGF receptor mRNAs were also observed in the adult dorsal root ganglia, suggesting that estrogen may regulate NGF sensitivity in adult neurotrophin targets as well. Such estrogen-neurotrophin interactions may have an important role during differentiation and in the adult, following injury.

Effects of Estrogen Replacement on the Relative Levels of Choline Acetyltransferase, trkA, and Nerve Growth Factor Messenger RNAs in the Basal Forebrain and Hippocampal Formation of Adult Rats

Previous studies have shown that estrogen replacement can significantly affect the expression of choline acetyltransferase immunoreactivity (ChAT-IR) and low-affinity (p75 NGFR) nerve growth factor receptors within cholinergic neurons located in specific regions of the basal forebrain. To extend this work, we have examined the effects of estrogen replacement on relative levels of choline acetyltransferase (ChAT), trkA, and nerve growth factor (NGF) mRNAs within different regions of the basal forebrain and hippocampal formation using quantitative in situ hybridization techniques. Ovariectomized Sprague-Dawley rats received continuous estrogen replacement for 2 days, 1 week, or 2 weeks. The data show that estrogen replacement results in significant increases in relative cellular levels of ChAT mRNA in the medial septum (MS) and nucleus basalis magnocellularis (nBM), but not in the horizontal limb of the diagonal band of Broca (HDB) or the striatum. In contrast, estrogen replacement resulted in significant decreases in relative levels of NGF mRNA in the hippocampus and of trkA mRNA in the MS and nBM (but not in the HDB or the striatum). The time-course of these effects is consistent with estrogen having a direct effect on ChAT expression which is followed by indirect effects on trkA. The data are also consistent with previous findings in which estrogen replacement resulted in significant increases in ChAT-IR which were followed by significant decreases in p75 NGFR mRNA and protein and then a reduction in ChAT-IR back to control levels. Together, these data indicate that estrogen replacement can have significant effects on basal forebrain cholinergic function, and that some of these effects may be mediated by effects of estrogen replacement on the expression of NGF and NGF receptors.

Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells.

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. Using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF differentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75NGFR and trkA mRNA, may alter the ratio of the two NGF receptors, and, consequently, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintainance of neural function.

Growth hormone and prolactin regulate the expression of nerve growth factor receptors in INS-1 cells

We have previously demonstrated that beta-cells express both p75NGF-R and Trk-A, the low and high affinity nerve growth factor (NGF) receptors, respectively. In the current study, we provide evidences that in the beta-cell line INS-1, the expression of these receptors is tightly controlled by GH and PRL, two hormones implicated in beta-cell development and function. Within 24 h of treatment of INS-1 cells with human (h) GH, the numbers of low and high affinity NGF-binding sites, calculated after Scatchard analysis, increase 3- and 2.5-fold, respectively. The increase in the concentration of the high affinity NGF-binding sites is paralleled by an increase in Trk-A protein without any change at the mRNA steady state level, suggesting a translational/posttranslational effect. On the other hand, the increase in low affinity binding sites is paralleled by an increase in the p75NGF-R mRNA steady state level. The effect requires at least 8 h of treatment, and a dose of 50 ng/ml hGH is sufficient to induce an increase in the p75NGF-R mRNA steady state level. The effect of hGH can be mimicked in the same time- and dose-dependent manner by rat PRL and bovine GH, suggesting that the expression of NGF receptors can be transduced by both the somatogenic and lactogenic pathways. Finally, the increase in the p75NGF-R mRNA steady state level after PRL treatment is not due to mRNA stabilization, suggesting a transcriptional control, and requires concurrent protein synthesis. GH and PRL could thus be important regulators of the sensitivity of beta-cells to NGF.

Growth hormone and prolactin regulate the expression of nerve growth factor receptors in INS-1 cells.

We have previously demonstrated that beta-cells express both p75NGF-R and Trk-A, the low and high affinity nerve growth factor (NGF) receptors, respectively. In the current study, we provide evidences that in the beta-cell line INS-1, the expression of these receptors is tightly controlled by GH and PRL, two hormones implicated in beta-cell development and function. Within 24 h of treatment of INS-1 cells with human (h) GH, the numbers of low and high affinity NGF-binding sites, calculated after Scatchard analysis, increase 3- and 2.5-fold, respectively. The increase in the concentration of the high affinity NGF-binding sites is paralleled by an increase in Trk-A protein without any change at the mRNA steady state level, suggesting a translational/posttranslational effect. On the other hand, the increase in low affinity binding sites is paralleled by an increase in the p75NGF-R mRNA steady state level. The effect requires at least 8 h of treatment, and a dose of 50 ng/ml hGH is sufficient to induce an increase in the p75NGF-R mRNA steady state level. The effect of hGH can be mimicked in the same time- and dose-dependent manner by rat PRL and bovine GH, suggesting that the expression of NGF receptors can be transduced by both the somatogenic and lactogenic pathways. Finally, the increase in the p75NGF-R mRNA steady state level after PRL treatment is not due to mRNA stabilization, suggesting a transcriptional control, and requires concurrent protein synthesis. GH and PRL could thus be important regulators of the sensitivity of beta-cells to NGF.

A reverse transcription-polymerase chain reaction study of p75 nerve growth factor receptor gene expression in developing rat cerebellum

The actions of the neurotrophins are mediated through specific receptors. Nerve growth factor (NGF), the prototypic neurotrophin, binds to receptors of both high and low affinity. A protein 75 kDa in size (p75NGFR) binds NGF, as well as brain-derived neurotrophic factor and neurotrophin 3, with low affinity. Recent investigations suggest that this protein may also be a component of the high affinity NGF receptor complex. To study gene expression of the p75NGFR molecule, we used a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure levels of its messenger RNA (mRNA) in small samples of total RNA. The assay is based on using a shortened p75NGFR cRNA as an internal RNA standard to control for variability in reverse transcription and polymerase chain amplification. We measured p75NGFR mRNA levels in the rat cerebellum during ontogeny to further study the transient developmental increase in receptor gene expression known to occur in this brain region during the early postnatal period. We found that p75NGFRmRNA levels were most abundant at postnatal day 2, and then declined to lower levels throughout postnatal development and in the adult. Northern blot analysis of the same total RNA samples used in our RT-PCR assay verified that p75NGFR expression is highest in the early postnatal period. These results confirm those of previous studies accomplished with much larger amounts of RNA using ribonuclease protection or northern blot assays. The use of an RT-PCR assay that utilized an internal standard also controls against changes in RNA complexity which can affect the measurement of message abundance across developmental stages. These results again suggest that p75NGFR may play an important role in the postnatal development of cerebellum in rats