Low-affinity Nerve Growth Factor Receptor Expression Research Articles

Efficient Oncoretroviral Gene Transfer in Primary, Secondary and Tertiary NOD/SCID Mice under Serum-Free Conditions.

Stable oncoretroviral gene transfer into hematopoietic stem cells (HSC) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSC to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells the role of ex-vivo expansion was investigated. For a possible clinical application all experiments were done according to good anufacturing practice (GMP) guidelines. The combination of Flt3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO), and Interleukin-6 (IL6) has been shown to stimulate proliferation and self-renewal of very primitive (SCID-repopulating cells, SRC) hematopoietic cells. We asked whether it is possible to efficiently transduce HSC with oncoretroviral vector, to expand them and whether transduced cells retain their self-renewal potential, as demonstrated by their capacity to efficiently and serially engraft NOD/SCID mice A Gibbon ape leukemia virus (GALV)-pseudotyped vectors already approved for clinical application has been used to efficiently and durably deliver a defective, non functional form of the cell surface marker truncated low affinity nerve growth factor receptor (LNGFR) into primitive cord blood (CB) HSC. The transduction was performed, following an up-to 24 hour exposure to FL, TPO, IL-6 and SCF, in the presence of the growth factors in serum-free (SF) medium on retronectin (RT) coated plates. At day 3 post-transduction, total cells and CD34+ cells were expanded 24-fold and 8.5-fold respectively. More than 40% of the cells were CD34+. Transduction efficiency was >55%. Serial transplantation is the most reliable method to assess the stable expression of a gene in cells with high proliferative potential. Mice transplanted with transduced or mock-transduced, expanded cells showed higher levels of human engraftment than those transplanted with unmanipulated cells (56.7%, 55% and 39.9% respectively). LNGFR expression of CD45+ cells was 14.35± 4.27%. All secondary mice transplanted with cells from primary recipient BM resulted engrafted (21.7%, 13.5 and 2.8% respectively). LNGFR expression was 47.54± 3.1% respectively of human CD45+ cells. BM cells from secondary recipients were used for tertiary transplants. Only mice transplanted with expanded cells were positively engrafted. Two mice out of five transplanted with secondary recipient BM cells derived from mice transplanted with transduced and expanded cells, showed good levels of human engraftment (6.15% CD45+). LNGFR expression was 49.1± 4.4% of human CD45+ cells. FACS analysis of the different subpopulations showed LNGFR expression within the progenitor (CD34+), B (CD19+), myeloid (CD14+), erythroid (GpA+) and megakaryocyte cells (CD41+) in equivalent proportion. BM of the engrafted mice was placed in a human colony assay. Human colonies also were generated from the murine BM. In conclusion, we have validated a SF-protocol for efficient gene transfer into human CB HSC using a retroviral vector. Under these conditions, transduced and expanded cells repopulated NOD/SCID mice for 3 generations with a human multilineage graft stably expressing the transgene. In a view of future clinical applications, this protocol represents a major step towards the achievement of this goal.

Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties

Myeloid lineage-derived dendritic cells (DCs) are considered the professional antigen-presenting cell type responsible for eliciting T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is a disease in which tumor antigens are expressed by the malignant clone that also has the potential to differentiate into DC-like cells (leukemic DCs) with antigen-presenting capacity. This study investigated whether the constitutive expression of the cytokine interleukin-7 (IL-7) in primary AML cells during their differentiation toward leukemic DCs results in superior antigen-presenting cells. A bicistronic retroviral vector encoding the IL-7 cytokine and the surface immunoselectable low-affinity nerve growth factor receptor (LNGFr) gene was constructed and used for transduction experiments. A serum-free system was used to transduce and differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the cytokine vector varied among patients, ranging from 5% to 30% as judged by LNGFr expression. The leukemic origin of the transduced cells was confirmed in a patient with a chromosomal translocation t(9:11) by fluorescence in situ hybridization analysis. Cytokine modified-cells consistently secreted IL-7 (mean, 415 pg +/- 190/10(6) cells/48 hours; n = 5). We demonstrate that IL-7-transduced cells are included in the differentiated leukemic DC subset, and, as shown in a particular case, that about half of the mature CD80(+) and CD83(+) populations coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and generate cytotoxic T lymphocytes against autologous leukemic blasts.

High Efficiency Gene Transfer to Human Hematopoietic SCID-Repopulating Cells Under Serum-Free Conditions

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34+ cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45+ cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions. © 1998 by The American Society of Hematology.

High Efficiency Gene Transfer to Human Hematopoietic SCID-Repopulating Cells Under Serum-Free Conditions

AbstractStable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34+ cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45+ cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.© 1998 by The American Society of Hematology.

Low affinity NGF receptor expression in the central nervous system during experimental allergic encephalomyelitis.

In the central nervous system (CNS), p75, or low-affinity nerve growth factor receptor (LNGFR), is assumed to play a critical role in mediating the effects of neurotrophins on neuronal survival. Recent studies have shown that nerve growth factor (NGF) can act also on immune cells through its binding to p75. Using immunohistochemistry, we have investigated the expression of the p75 receptor in the CNS during chronic relapsing experimental allergic encephalomyelitis (EAE) of the Lewis rat, an animal model of multiple sclerosis (MS). We report here a sequential expression of p75, first in Purkinje cells during the first attack, and secondly on both endothelial and perivascular cells in the latter stages of the disease. Moreover, starting from the second attack, p75 was also expressed on glial ensheathing cells, likely myelinating cells, located primarily in the dorsal roots. These data suggest that during EAE, LNGFR may play an important role in leukocyte-endothelial cell interactions and in the maintenance of Purkinje cells survival.

Low affinity NGF receptor expression in the central nervous system during experimental allergic encephalomyelitis

In the central nervous system (CNS), p75, or low-affinity nerve growth factor receptor (LNGFR), is assumed to play a critical role in mediating the effects of neurotrophins on neuronal survival. Recent studies have shown that nerve growth factor (NGF) can act also on immune cells through its binding to p75. Using immunohistochemistry, we have investigated the expression of the p75 receptor in the CNS during chronic relapsing experimental allergic encephalomyelitis (EAE) of the Lewis rat, an animal model of multiple sclerosis (MS). We report here a sequential expression of p75, first in Purkinje cells during the first attack, and secondly on both endothelial and perivascular cells in the latter stages of the disease. Moreover, starting from the second attack, p75 was also expressed on glial ensheathing cells, likely myelinating cells, located primarily in the dorsal roots. These data suggest that during EAE, LNGFR may play an important role in leukocyte-endothelial cell interactions and in the maintenance of Purkinje cells survival. J. Neurosci. Res. 52:83–92, 1998. © 1998 Wiley-Liss, Inc.

Loss of low-affinity nerve growth factor receptor during malignant transformation of the human prostate.

The low-affinity nerve growth factor receptor (LNGFR) exhibits an inverse association of epithelial expression with the degree of differentiation of prostate adenocarcinoma tissue. However, the stage at which loss of LNGFR expression is first manifested in the malignant prostate has not been determined. In order to characterize loss of LNGFR expression in the clinically localized malignant prostate of untreated patients, the pattern of expression of the LNGFR was examined in nonmalignant tissues, consisting of normal and prostatic intraepithelial neoplastic tissues, and in malignant tissues that had been graded by Gleason's scores and categorized into well, moderately, and poorly differentiated adenocarcinomas. In order to determine whether there was an inverse correlation between LNGFR expression and prostate-specific antigen (PSA) secretion, preoperative concentrations of PSA in the serum were also analyzed in relation to the differentiative state of the adenocarcinomas. Premalignant prostate tissues exhibited expression of the LNGFR on all epithelia, whereas in malignant prostate tissues a proportion of epithelia exhibited loss of expression of the LNGFR. An increase in Gleason score of the adenocarcinoma tissue was associated with an increase in the proportion of epithelia that exhibited loss of expression of the LNGFR. Moreover, the proportion of epithelia expressing the LNGFR was inversely correlated with an increase in the concentration of serum PSA. The loss of LNGFR expression was first manifested in epithelia that occurred toward the center of adenocarcinoma tissue. Furthermore, extensive loss of expression of the LNGFR of approximately 63% occurred in well-differentiated adenocarcinomas. These observations demonstrate that loss of LNGFR expression is first observed in well-differentiated malignant epithelia. Hence, loss of the LNGFR may be indicative of the initial stages of malignant transformation of prostate epithelia, as well as all subsequent stages of prostate adenocarcinomas.

Basophil priming by neurotrophic factors. Activation through the trk receptor.

There is increasing evidence that nerve growth factor (NGF) acts on cells of the immune system, apart from its neurotrophic effects. In human basophils, NGF potentiates mediator release and primes the cells to produce leukotriene C4 in response to C5a. It is, however, unknown whether other homologous neurotrophins also act outside the nervous system, and whether activation of basophils by NGF requires interaction with trk tyrosine kinase receptors, the low affinity NGF receptor (LNGFR), or both. A triple mutant NGF designed to interrupt binding to the LNGFR was found to activate basophils with equal efficacy as wild-type NGF, demonstrating that the LNGFR is not necessary. Despite a 10 times lower potency of mutant NGF, no LNGFR expression was detected by FACS analysis. Brain-derived neurotrophic factor, which interacts with trkB, was inactive at concentrations up to 1000 ng/ml (> 30,000-fold lower potency than NGF), while neurotrophin-3, which is thought to interact with trkC, trkB, and more weakly with trk, induced a threshold effect at 300 ng/ml (approximately 10,000-fold lower potency), demonstrating that 1) the LNGFR cannot deliver a direct signal; and 2) basophils do not express functional trkB and trkC receptors. In agreement with the functional data, basophils (in contrast to other granulocyte types) expressed mRNA for trk, but not trkB or trkC, and no or minimal mRNA for LNGFR. These data demonstrate that human blood basophils express functional trk receptors that do not require the participation of LNGFR, and that, among the neurotrophin family, NGF is unique in priming basophils.

Differential effects of acth-related peptides and forskolin on rat schwann cell proliferation and low affinity NGF receptor expression

We investigated the effect of the neurotrophic peptides αMSH and Org 2766 on Schwann cell proliferation (BrdU incorporation) and low affinity nerve growth factor receptor (LNGFR) expression in vitro. Cells were cultured under four conditions: low or high density culture, with forskolin present or absent in the medium during one week before start of the experiment. Schwann cells were treated with αMSH, Org 2766 or forskolin. After 48h forskolin (10 μM) increased BrdU incorporation (32–151%) in all culture conditions and LNGFR production (25–194%) in 3 out of 4 conditions. αMSH and Org 2766 had no effect. This study shows that Schwann cell proliferation and LNGFR expression is under control of cAMP. Furthermore, the neurotrophic effect of αMSH and Org 2766 is probably not mediated through an increased cell division or LNGFR expression in Schwann cells.

Expression of Low Affinity NGF (p75) Receptors in Rat Superior Colliculus: Studies in Vivo, in Vitro, and in Fetal Tectal Grafts

The distribution and level of expression of the low affinity nerve growth factor receptor (LNGFR) in the rat visual system has been investigated under a number of experimental conditions. The aim was to determine the cellular location of the receptor and to study the factors which influence its expression. The monoclonal antibody 192-Ig and immunohistochemical techniques were used to examine LNGFR expression in (i) developing and adult rat superior colliculus (SC), (ii) fetal collicular tissue transplanted to the midbrain of newborn host rats, (iii) the SC of rats which had been unilaterally enucleated at birth, and (iv) mixed glial cell cultures from the neonatal SC. The effect of eye removal on LNGFR immunoreactivity in other normally retino-recipient areas was also assessed. Postnatal maturation of the rat SC was associated with an increase in LNGFR immunoreactivity. At birth, weak staining was seen ventral to the superficial gray layer. Staining gradually became located more dorsally until in the adult there was a dense band of immunoreactivity that extended 50-100 μm from the surface. Immunoreactive processes and cellular-like profiles were seen. Compared to adult host SC there was considerably more LNGFR immunoreactivity in transplanted tectal tissue, irrespective of whether the grafts were connected to the host. LNGFR expression in transplants was patchy and usually contiguous with the graft surface. Staining was not obviously related to the distribution of astrocytes or microglia and very few cells were LNGFR positive in tectal glial cell cultures. In SC and in tectal grafts it is probable that LNGFR immunoreactivity was primarily associated with intrinsic neurons. In support of this, LNGFR expression in the superficial SC was unaffected by neonatal eye removal; however, LNGFR staining in the pretectum and diencephalon was reduced or absent on the side contralateral to visual deafferentation. These conflicting sets of data suggest that (i) LNGFRs in central visual pathways are sometimes, but by no means always, associated with retinal innervation and (ii) LNGFR expression in visual target areas originates from diverse sources and is influenced and regulated by a variety of factors.

Localization and regulation of low affinity nerve growth factor receptor expression in the rat olfactory system during development and regeneration

Nerve growth factor (NGF), a classic neurotrophic factor, promotes neuronal survival, maintenance, regeneration and differentiation in the peripheral nervous system and parts of the central nervous system. NGF activity is mediated by cell surface bound receptors including the low affinity NGF receptor (LNGFr) which is expressed by some peripheral and central neurons and is present on peripheral nerve Schwann cells during development and regeneration. The olfactory system is a useful model for the study of the role of LNGFr in neuronal development and regeneration. The growth of olfactory axons into the brain begins in the embryo and continues through the first few postnatal weeks. In mature animals there is persistent turnover and generation of olfactory receptor neurons (ORNs) and continuous growth of new axons into the olfactory bulb. These new axons grow along the preexisting olfactory pathway. In the mature olfactory system, LNGFr has been observed in the glomerular layer of the olfactory bulb, the target of ORNs. However, neither the cellular localization nor the developmental expression of LNGFr has been characterized. Here, we tested the hypothesis that LNGFr expression is developmentally regulated in the olfactory nerve and is reinduced following injury to the mature olfactory nerve. LNGFr-immunoreactivity (IR) was first observed in the olfactory mucosa at embryonic day (E)13 and in the olfactory nerve at E14. LNGFr-IR increased in the nerve during embryonic development, began to decrease at around postnatal day (P)5 and was scarcely detectable in normal adults. The staining pattern suggests that LNGFr is located on the olfactory nerve Schwann cells. Streaks of LNGFr-IR were present in the adult olfactory nerve. We reasoned that these streaks might represent transient reexpression of LNGFr associated with normal olfactory neuron turnover and replacement. Consistent with this hypothesis, LNGFr was robustly reexpressed in the adult olfactory nerve following lesion of the olfactory epithelium. Starting late in development (E21) and in the adult, LNGFr-IR was also observed on fibers in deep layers of the olfactory bulb. LNGFr-IR was also observed in neurons of the nucleus of the diagonal band (NDB) in the basal forebrain. NDB is the sole source of cholinergic afferents of the olfactory bulb. Thus, we tested the hypothesis that LNGFr in the deep layers of the olfactory bulb is located on NDB axons by making lesions of NDB. Following the lesion, LNGFr-IR disappeared in the deep layers of the olfactory bulb but remained in the glomerular layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Peripheral Blood Lymphocytes as Target Cells of Retroviral Vector-Mediated Gene Transfer

AbstractPeripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T-cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector-mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

Peripheral blood lymphocytes as target cells of retroviral vector- mediated gene transfer

Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T- cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

Modulation of low‐affinity nerve growth factor receptor in injured adult rat spinal cord motoneurons

Spinal and brainstem motoneurons of the adult rat reexpress low-affinity nerve growth factor receptor (LNGFR) and its mRNA after axotomy. We have previously reported the time courses of this reexpression after cut (no regeneration) or crush (followed by regeneration) of the sciatic nerve. We have shown that the length of the different phases of this reexpression (appearance, maintenance and disappearance) can vary according to the type of axotomy. With the present study we expand our previous data and describe and analyze the modulation the LNGFR expression in adult spinal cord motoneurons following different lesion paradigms. In one approach we have imposed three traumatic injuries that still allow regeneration of the sciatic nerve but with a different time course with respect to the crush injury (application of a silicone regeneration chamber, multiple crushes and delayed repair of ligated nerves). In a second approach, we have determined the capability of three toxic or metabolic injuries to induce LNGFR expression without any direct trauma of the nerve (experimental diabetogenesis, botulinum and alpha-bungarotoxin intoxication and 2,5-hexanedione intoxication). In a third approach, we have investigated the effect of the block of the axoplasmic transport on the LNGFR expression following different topical applications of vincristine combined with a nerve crush. The results we present are consistent with the idea that: (1) LNGFR immunoreactivity in adult motoneurons is expressed by motoneurons that are attending to an axonal outgrowth and not a generic signal of cellular damage or impairment of the motor function; (2) LNGFR expression in these motoneurons is related to and parallels the outgrowth process time frame, and (3) the signal/s that trigger and sustain this reexpression may be retrogradely transported from the periphery.

Effects of nerve growth factor and basic fibroblast growth factor on survival of cultured septal cholinergic neurons from adult rats

We have established a primary culture technique for neuronal cells from rat basal forebrain from postnatal day 58 (P58) to study the effects of neurotrophic factors on the neurons. The survival of acetylcholinesterase (AChE)-positive neurons of 2-week-old rat septum has already been reported to be strongly supported by nerve growth factor (NGF) in culture. In this culture study of neurons from adult rat brains, the survival of AChE-positive neurons from P58 rat septum was slightly improved by NGF, although low affinity NGF receptor expression was also observed on cultured P58 rat septum neurons as well as on those from 2-week-old rats. The addition of basic fibroblast growth factor (bFGF) improved the survival of AChE-positive neurons cultured from P58 rat septum, but did not promote the survival of neurons from P12 rat septum. These results suggest that NGF changes to a maintenance factor in adult rat brain from a survival factor in postnatal 2-week-old rats. The survival of cholinergic neurons in culture of adult rat septum might be supported by factor(s) other than NGF, such as bFGF.

Nerve growth factor (NGF)-mediated up-regulation of low-affinity NGF receptor gene expression in cultured basal forebrain cholinergic neurons from postnatal 3-day-old rats

We examined the effect of nerve growth factor (NGF) on the expression of low-affinity NGF receptor (LNGFR) in cultured P3 basal forebrain cholinergic neurons, on which NGF acts to promote differentiation. Based on the results of the RT-PCR (reverse transcriptase-polymerase chain reaction) method, over a 2-fold increase in the LNGFR mRNA level was found in NGF-treated cultures compared with control cultures at one day after the addition of 100 ng/ml NGF. This increase was maintained for up to 3 days after the addition of NGF. The increase in LNGFR mRNA was found even at 1 ng/ml NGF (= 40 pM), indicating that the up-regulation of the LNGFR mRNA level in cultured cholinergic neurons occurred at an NGF concentration near the K d value for the high-affinity NGF receptor. In addition, immunohistochemical staining showed stronger staining with a monoclonal anti-LNGFR antibody of the cholinergic neurons in NGF-treated cultures than those in control cultures. Our results suggest that NGF can up-regulate LNGFR expression at the mRNA and protein levels in cultured P3 basal forebrain cholinergic neurons and that this mechanism may play an important role in potentiating the effect of NGF on these neurons.

Expression of neurotrophins and the low-affinity NGF receptor in septal and hippocampal reaggregate cultures: local physiologic effects of NGF synthesized in the septal region

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of a family of trophic factors designated the neurotrophins, each of which can bind to the low-affinity NGF receptor (LNGFR). To investigate the mechanisms that regulate the expression of the neurotrophins and the LNGFR in the developing brain, we grew cells from the embryonic mouse septum and hippocampus in reaggregating cell culture and compared neurotrophin and LNGFR expression in developing reaggregates with that seen in the developing septum and hippocampus in situ. NGF, BDNF, NT-3 and LNGFR were each expressed in septal and hippocampal reaggregates as well as the native septum and hippocampus. Additionally, the temporal expression profiles observed in reaggregates were generally similar to those seen in the respective brain regions in situ. In order to determine whether NGF can modulate neurotrophin or LNGFR expression, reaggregates were cultured in the continual presence of either exogenous NGF or anti-NGF antibodies. NGF-treated septal cultures expressed twice the level of LNGFR mRNA as was seen in untreated septal cultures; on the other hand, septal cultures grown in the presence of anti-NGF antibodies, to neutralized endogenously synthesized NGF, displayed a 3-fold decrease in LNGFR mRNA expression compared to untreated cultures. No effects of NGF or anti-NGF were observed on LNGFR expression in hippocampal reaggregates, or on neurotrophin mRNA expression in either reaggregate type. These results suggest that regulatory mechanisms intrinsic to the septal and hippocampal regions control neurotrophin and LNGFR expression. NGF is likely to be one of these regulatory cues since it acts locally in septal reaggregates to control the developmental expression of LNGFR mRNA. The possible roles of locally synthesized NGF and other neurotrophins in the development of septal neurons are discussed.

Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS

Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septa) or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.