Neurotrophins in cerebellar granule cell development and medulloblastoma

Medulloblastomas may be derived from granule cells of the developing cerebellum. Children with tumors expressing high levels of the neurotrophin-3 receptor, TrkC, have a more favorable outcome. During development, TrkC is expressed in the most mature granule cells. Favorable medulloblastomas may be derived from more highly differentiated granule cells.     

Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors

Neurotrophin-3 (NT-3) is a cystine knot growth factor that promotes the survival, proliferation, and differentiation of developing neurons and is a potential therapeutic for neurodegenerative diseases. To clarify the structural basis of receptor specificity and the role of neurotrophin dimerization in receptor activation, the structure of the NT-3 homodimer was determined using X-ray crystallography. The orthorhombic crystals diffract to 2.4 A, with dimer symmetry occurring about a crystallographic 2-fold axis. The overall structure of NT-3 resembles that of the other neurotrophins, NGF and BDNF; each protomer forms a twisted four-stranded beta sheet, with three intertwined disulfide bonds. There are notable differences, however, between NT-3 and NGF in the surface loops and in three functionally important regions, shown in previous mutagenesis studies to be critical for binding. One such difference implies that NT-3's binding affinity and specificity depend on a novel hydrogen bond between Gln 83, a residue important for binding specificity with TrkC, and Arg 103, a residue crucial for binding affinity with TrkC. NT-3's extensive dimer interface buries much of the otherwise solvent-accessible hydrophobic surface area and suggests that the dimeric state is stabilized through the formation of this hydrophobic core. A comparison of the dimer interface between the NT-3 homodimer and the BDNF/NT-3 heterodimer reveals similar patterns of hydrogen bonds and nonpolar contacts, which reinforces the notion that the evolutionarily conserved neurotrophin interface resulted from the need for receptor dimerization in signal initiation.     

Neurotrophins prevent death and differentially affect tyrosine hydroxylase of adult rat nigrostriatal neurons in vivo

Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) promote survival of mesencephalic dopaminergic neurons in vitro and affect normal and damaged ones in vivo. Here, these neurotrophins had markedly different potencies to prevent the death of axotomized nigrostriatal dopaminergic neurons when infused close to the rostral end of the nigral nucleus of adult rats (NT-4 > BDNF > NT-3; nerve growth factor or NGF without effect). With a high dose of BDNF (30 micrograms/day) complete protection was achieved in the rostral but not caudal nigral regions, consistent with its poor diffusion characteristics in brain tissue. Measurements of tyrosine hydroxylase immunoreactivity suggest that BDNF and NT-4 (presumably through their TrkB receptor) reduce the synthesis of this rate-limiting enzyme for dopamine synthesis in rescued as well as in normal neurons. In sharp contrast, survival-promoting doses of NT-3 (presumably through its TrkC receptor) maintained normal levels of tyrosine hydroxylase immunoreactivity in the rescued nigrostriatal neurons. These results suggest that for these adult central nervous system neurons, some neurotrophic factors are predominantly involved in facilitating cell survival, whereas others are more involved in regulating neurotransmitter function.     

Brain-derived neurotrophic factor and neurotrophin-3 enhance somatostatin gene expression through a likely direct effect on hypothalamic somatostatin neurons

Although neurotrophins (NTs) have been extensively studied as neuronal survival factors in some areas of the central nervous system, little is known about their function or cellular targets in the hypothalamus. To understand their functional significance and sites of action on hypothalamic neurons, we examined the effects of their cognate ligands on neuropeptide content and messenger RNA (mRNA) expression in somatostatin neurons present in fetal rat hypothalamic cultures. Treatments were performed in defined insulin-free medium between days 6 and 8 of culture, since the maximal effects of NTs on somatostatin content and mRNA expression were observed after 48-h incubations. Brain-derived neurotrophic factor and NT-3, but not nerve growth factor, induced a dose-dependent increase in somatostatin content, which was influenced by plating density. The same treatment increased somatostatin mRNA and immunostaining intensity of somatostatin neurons, but had no effect on the number of these labeled neurons. The increased levels of somatostatin (peptide and mRNA) induced by NTs were not blocked by tetrodotoxin or by glutamate receptor antagonists, suggesting that endogenous neurotransmitters (e.g. glutamate) were not involved in these effects. In contrast, the stimulatory effects were completely blocked by K-252a, an inhibitor of tyrosine kinase (Trk) receptors, whereas the less active analog K-252b was ineffective. Double-labeling studies demonstrated that both TrkB or TrkC receptors were located on somatostatin neurons. Our results show that, in rat hypothalamic cultures, brain-derived neurotrophic factor, and NT-3 have a potent stimulatory effect on peptide synthesis in somatostatinergic neurons, likely through direct activation of TrkB and TrkC receptors.     

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms.     

A frequent activated smoothened mutation in sporadic basal cell carcinomas

Basal-cell carcinomas (BCCs) are the most common cancer in Caucasians. It has been reported that the patched gene is inactivated in 30-40% sporadic BCCs and 20% sporadic medulloblastomas via loss of heterozygosity and nonsense mutations. Recently, two activating smoothened mutations have been found in the sporadic basal cell carcinomas. One, at base pair 1604 (G-to-T transversion) of exon 9, changes codon 535 from tryptophan to leucine, and the other, at base pair 1685 (G-to-A transition) of exon 10, changes codon 562 from arginine to glutamine (Xie et al., 1998). In our study, 1604G-->T was found in 20 out of 97 (20.6%) sporadic BCCs. The high prevalence indicates that 1604G is the mutation hot spot in our tumor samples. This mutation was detected in all three histological subtypes of BCCs, suggesting that smoothened mutation is an early event during the development of the tumor. Our finding of a high smoothened mutation rate, together with high frequent patched gene mutations reported recently, indicates that activation of the hedgehog signal transduction pathway is the most common and early event in the development of sporadic BCCs. Additionally, to determine whether smoothened, like patched, is also involved in the carcinogenesis of medulloblastomas, we screened medulloblastoma samples for these two mutations by restriction analysis. We have found the 1604G-->T mutation in 1 out of 21 medulloblastomas. This result confirmed smoothened gene involvement in the carcinogenesis of medulloblastoma.     

A competitive enzyme-linked immunosorbent assay for measuring the levels of serum antibody to Haemophilus influenzae type b

1. The protein family of the neurotrophins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and Neurotrophin-3, -4/5, and -6 (NT-3; NT-4/5; NT-6) is well known to enhance the survival and to stabilize the phenotype of different populations of neurons in the central and the peripheral nervous system. These effects are mediated via binding to specific tyrosine kinase receptors (Trks) and to the low-affinity p75 neurotrophin receptor. 2. The neurotrophins NGF, BDNF, and NT-3 and the BDNF and NT-3 selective receptors (TrkB, TrkC) are expressed at high levels in neurons of the basal forebrain, the hippocampus, and the neocortex of the mammalian brain. The expression and secretion of NGF and BDNF in these brain areas is regulated by (physiological levels of) neuronal activity. 3. Exogenous application of the neurotrophins to hippocampal and neocortical neurons can enhance excitatory glutamatergic synaptic transmission via activation of Trk receptors. In addition, long-term potentiation (a potential cellular correlate for learning and memory formation in mammals) in the rodent hippocampus depends on endogenous supply of neurons with BDNF. 4. Judged by the analysis of electrophysiological data, the BDNF- and NT-3-induced enhancement of glutamatergic synapses is mediated by increasing the efficacy of glutamate release from the presynaptic neuron. However, neurotrophin-dependent postsynaptic enhancement of NMDA (but not AMPA) receptor responsiveness has also been shown. 5. On the molecular level, neither the pre- nor the postsynaptic modulation of glutamatergic synapses by neurotrophins is well understood. However, neurotrophins were shown to acutely affect intraneuronal Ca2+ levels and to influence molecular components of the transmitter release machinery, which could underly the presynaptic modifications, whereas BDNF-induced phosphorylation of NMDA-type glutamate receptors could account for the postsynaptic effects. 6. Taken together, these results suggest that the activity-dependent release of neurotrophins at frequently used synapses could modulate the synaptic efficacy at these junctions. Thus, neurotrophins might operate as locally released feedback modulators of synaptic transmission, and this could be a cellular correlate for certain aspects of information processing in the mammalian brain.     

A new method of human cardiac troponin I and troponin T purification

Neurotrophins (NTFs) are a family of structurally related proteins with specific effects on the developing nervous system and a wide range of non-neuronal differentiating cells. To date, four NTFs have been characterized: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). To perform their biological effects, the NTFs must bind to appropriate receptors on the surface of responsive cells. High- and low-affinity receptors for NTFs have been identified. The high-affinity receptors are members of the trk protein tyrosine kinase receptor family. The low-affinity neurotrophin receptor gp75NTFR is a common receptor for all NTFs. Here we summarize some of our previous findings on the expression patterns of NGF, gp75NTFR, TrkB, and TrkC in the developing molar tooth of the rat. Both NGF and gp75NTFR are localized in dental epithelium and mesenchyme but often their expression patterns differ. Concomitant expression of NGF and gp75NTFR in mesenchyme is correlated with odontoblast differentiation. The trkB and trkC receptors show distinct cell-specific expression patterns in developing tooth, suggesting that other NTFs, apart from NGF, may be involved in odontogenesis. These data demonstrate that NTFs participate in the cascade of molecular events that direct tooth development, and support the notion that NTFs may have multiple and distinct roles in dental tissues.     

Age-dependent differences in the effects of GDNF and NT-3 on the development of neurons and glia from neural crest-derived precursors immunoselected from the fetal rat gut: expression of GFRalpha-1 in vitro and in vivo

No enteric neurons or glia develop in the gut below the rostral foregut in mice lacking glial cell line-derived neurotrophic factor (GDNF) or Ret. We analyzed the nature and age dependence of the effects of GDNF and, for comparison, those of NT-3, on the in vitro development of the precursors of enteric neurons and glia. Positive and negative immunoselection with antibodies to p75(NTR) were used to isolate crest-derived and crest-depleted populations of cells from the fetal rat bowel at E12, 14, and 16. Cells were typed immunocytochemically. GDNF stimulated the proliferation of nestin-expressing precursor cells isolated at E12, but not at E14-16. GDNF promoted the development of peripherin-expressing neurons (E12 > E14-16) and expression of TrkC. GDNF inhibited expression of S-100-expressing glia at E14-16. NT-3 did not affect cells isolated at E12, never stimulated precursors to proliferate, and promoted glial as well as neuronal development at E14-16. GFRalpha-1 was expressed both by crest- and non-crest-derived cells, although only crest-derived cells anchored GFRalpha-1 and GFRalpha-2 (GFRalpha-1 > GFRalpha-2). GDNF increased the number of neurons anchoring GFRalpha-1. GFRalpha-1 is immunocytochemically detectable in neurons of the E13 intestine and persists in adult neurons of both plexuses. We suggest that GDNF stimulates the proliferation of an early (E12) NT-3-insensitive precursor common to enteric neurons and glia; by E14, this common precursor is replaced by specified NT-3-responsive neuronal and glial progenitors. GDNF exerts a neurotrophic, but not a mitogenic, effect on the neuronal progenitor. The glial progenitor is not maintained by GDNF.     

Opposing roles for endogenous BDNF and NT-3 in regulating cortical dendritic growth

Neurons within each layer of cerebral cortex express multiple members of the neurotrophin family and their corresponding receptors. This multiplicity could provide functional redundancy; alternatively, different neurotrophins may direct distinct aspects of cortical neuronal growth and differentiation. By neutralizing endogenous neurotrophins in organotypic slices of developing cortex with Trk receptor bodies (Trk-IgGs), we found that BDNF and NT-3 oppose one another in regulating the dendritic growth of pyramidal neurons. In layer 4, both endogenous and exogenous NT-3 inhibited the dendritic growth stimulated by BDNF. In contrast, in layer 6 both endogenous and exogenous BDNF inhibited dendritic growth stimulated by NT-3. These antagonistic actions of endogenous BDNF and NT-3 provide a mechanism by which dendritic growth and retraction can be dynamically regulated during cortical development, and suggest that the multiple neurotrophins expressed in developing cortex represent distinct components of an extracellular signaling system for regulating dendritic growth.     

Neurotrophic factors and their receptor expressions in human neuropathies

Currently, many neurotrophic factors are known to promote peripheral nerve regeneration after nerve injury or nerve degeneration. Among such neurotrophic factors, neurotrophin family (NGF, BDNF, NT-3, NT-4) and GDNF are most potent ones, and they have been demonstrated to express in the distal stump of the experimental nerve transection. In this study, we assessed the expression levels of neurotrophin mRNAs and GDNF mRNAs and their receptor mRNAs for p75, TrkA, TrkB, TrkC, GDNFR alpha and Ret in the sural nerve biopsy specimens from various human neuropathies. All the neurotrophin mRNAs and GDNF mRNAs were significantly elevated in the diseased nerves and their expression levels well correlated to the extent of T-cell and macrophage invasion. p75 and GDNFR alpha mRNAs were upregulated in correlation to the extent of axonal degeneration. On the other hand, the TrkB and TrkC mRNAs were likely downregulated. These findings suggest that the regulatory mechanism for expression of neurotrophins and GDNF mRNAs and their receptor mRNAs are different. Significant upreguration of the neurotrophic factor mRNAs in the nerve lesion would be the compensation mechanism reacting for the lesion, even in the lesions which are progressing in severity. Extrinsic supplement of these neurotrophins would be beneficial for promotion of repair of the peripheral nerve lesion particularly when they are progressive lesions.     

Brain-derived neurotrophic factor and neurotrophin-4 increase neurotrophin-3 expression in the rat hippocampus

Hippocampal levels of mRNA encoding nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are rapidly induced by enhanced neuronal activity following seizures and glutamate or muscarinic receptor activation. However, the levels of neurotrophin-3 (NT-3) mRNA acutely decrease after limbic seizures suggesting that a different mode of regulation may exist for these neurotrophins. Here we show that BDNF and neutrotrophin-4 (NT-4), but not NT-3 itself, up-regulate NT-3 mRNA in cultured hippocampal neurons. In the rat hippocampus, the muscarinic receptor agonist, pilocarpine increased BDNF mRNA levels rapidly and those of NT-3 with a delay of several hours. Injection of BDNF into neonatal rats elevated NT-3 mRNA in the hippocampus which demonstrates that BDNF is able to enhance NT-3 expression in vivo. The regulation of NT-3 by BDNF and NT-4 enlargens the neurotrophic spectrum of these neurotrophins to include neuron populations responsive primarily to NT-3.     

Sporadic medulloblastomas contain PTCH mutations

Nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin's syndrome, is an autosomal dominant disorder that predisposes to developmental defects and various forms of cancer. PTCH was recently proposed as a candidate gene for NBCCS due to its frequent mutation in basal cell carcinomas, the cancer most often associated with this syndrome. Another NBCCS-associated cancer is medulloblastoma, a common central nervous system tumor in children. Most medulloblastomas, however, occur without indication of an inherited predisposition. We have examined 24 sporadic medulloblastomas for loss of heterozygosity (LOH) at loci flanking as well as within PTCH. In cases with LOH, single-strand conformational polymorphism and sequencing analysis were performed to determine the status of the remaining PTCH allele. Microsatellite analysis indicated LOH of PTCH in 5 of 24 tumors, and in three of these cases a mutation of the remaining allele was identified. Two of the mutations were duplication insertions, and the third consisted of a single base deletion. It is interesting that all three mutations occur in exon 17 of the PTCH gene. These data suggest that inactivation of PTCH function is involved in the development of at least a subset of sporadic medulloblastomas.     

Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus

The neurotrophins are signaling factors important for the differentiation and survival of distinct neuronal populations during development. To test whether the neurotrophins also function in the mature nervous system, the effects of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophic factor 3 (NT-3) on the strength of synaptic transmission in hippocampal slices were determined. Application of BDNF or NT-3 produced a dramatic and sustained (2 to 3 hours) enhancement of synaptic strength at the Schaffer collateral-CA1 synapses; NGF was without significant effect. The enhancement was blocked by K252a, an inhibitor of receptor tyrosine kinases. BDNF and NT-3 decreased paired-pulse facilitation, which is consistent with a possible presynaptic modification. Long-term potentiation could still be elicited in slices previously potentiated by exposure to the neurotrophic factors, which implies that these two forms of plasticity may use at least partially independent cellular mechanisms.     

A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control

Neurotrophins exert many biological effects not directly targeted at neurons, including modulation of keratinocyte proliferation and apoptosis in vitro. Here we exploit the cyclic growth and regression activity of the murine hair follicle to explore potential nonneuronal functions of neurotrophins in the skin, and analyze the follicular expression and hair growth-modulatory function of BDNF, NT-4, and their high-affinity receptor, TrkB. The cutaneous expression of BDNF and NT-4 mRNA was strikingly hair cycle dependent and peaked during the spontaneous, apoptosis-driven hair follicle regression (catagen). During catagen, BDNF mRNA and immunoreactivity, as well as NT-4-immunoreactivity, were expressed in the regressing hair follicle compartments, whereas TrkB mRNA and immunoreactivity were seen in dermal papilla fibroblasts, epithelial strand, and hair germ. BDNF or NT-4 knockout mice showed significant catagen retardation, whereas BDNF-overexpressing mice displayed acceleration of catagen and significant shortening of hair length. Finally, BDNF and NT-4 accelerated catagen development in murine skin organ culture. Together, our data suggest that BDNF and NT-4 play a previously unrecognized role in skin physiology as agents of hair growth control. Thus, TrkB agonists and antagonists deserve exploration as novel hair growth-modulatory drugs for the management of common hair growth disorders.     

The intrinsic radioresistance of glioblastoma-derived cell lines is associated with a failure of p53 to induce p21(BAX) expression

Radiation is the primary modality of therapy for all commonly occurring malignant brain tumors, including medulloblastoma and glioblastoma. These two brain tumors, however, have a distinctly different response to radiation therapy. Medulloblastoma is very sensitive to radiation therapy, whereas glioblastoma is highly resistant, and the long-term survival of medulloblastoma patients exceeds 50%, while there are few long-term survivors among glioblastoma patients. p53-mediated apoptosis is thought to be an important mechanism mediating the cytotoxic response of tumors to radiotherapy. In this study, we compared the response to radiation of five cell lines that have wild-type p53: three derived from glioblastoma and two derived from medulloblastoma. We found that the medulloblastoma-derived cell lines underwent extensive radiation-induced apoptotic cell death, while those from glioblastomas did not exhibit significant radiation-induced apoptosis. p53-mediated induction of p21(BAX) is thought to be a key component of the pathway mediating apoptosis after the exposure of cells to cytotoxins, and the expression of mRNA encoding p21(BAX) was correlated with these cell lines undergoing radiation-induced apoptosis. The failure of p53 to induce p21(BAX) expression in glioblastoma-derived cell lines is likely to be of biologic significance, since inhibition of p21(BAX) induction in medulloblastoma resulted in a loss of radiation-induced apoptosis, while forced expression of p21(BAX) in glioblastoma was sufficient to induce apoptosis. The failure of p53 to induce p21(BAX) in glioblastoma-derived cell lines suggests a distinct mechanism of radioresistance and may represent a critical factor in determining therapeutic responsiveness to radiation in glioblastomas.