Relationship between tumor enhancement, edema, IDH1 mutational status, MGMT promoter methylation, and survival in glioblastoma.

BACKGROUND AND PURPOSE: Both IDH1 mutation and MGMT promoter methylation are associated with longer survival. We investigated the ability of imaging correlates to serve as noninvasive biomarkers for these molecularly defined GBM subtypes. MATERIALS AND METHODS: MR imaging from 202 patients with GBM was retrospectively assessed for nonenhancing tumor and edema among other imaging features. IDH1 mutational and MGMT promoter methylation status were determined by DNA sequencing and methylation-specific PCR, respectively. Overall survival was determined by using a multivariate Cox model and the Kaplan-Meier method with a log rank test. A logistic regression model followed by ROC analysis was used to classify the IDH1 mutation and methylation status by using imaging features. RESULTS: MGMT promoter methylation and IDH1 mutation were associated with longer median survival. Edema levels stratified survival for methylated but not unmethylated tumors. Median survival for methylated tumors with little/no edema was 2476 days (95% CI, 795), compared with 586 days (95% CI, 507-654) for unmethylated tumors or tumors with edema. All IDH1 mutant tumors were nCET positive, and most (11/14, 79%) were located in the frontal lobe. Imaging features including larger tumor size and nCET could be used to determine IDH1 mutational status with 97.5% accuracy, but poorly predicted MGMT promoter methylation. CONCLUSIONS: Imaging features are potentially predictive of IDH1 mutational status but were poorly correlated with MGMT promoter methylation. Edema stratifies survival in MGMT promoter methylated but not in unmethylated tumors; patients with methylated tumors with little or no edema have particularly long survival.

Differential gene expression in glioblastoma defined by ADC histogram analysis: relationship to extracellular matrix molecules and survival.

BACKGROUND AND PURPOSE: ADC histogram analysis can stratify outcomes in patients with GBM treated with bevacizumab. Therefore, we compared gene expression between high-versus-low ADC tumors to identify gene expression modules that could underlie this difference and impact patient prognosis. MATERIALS AND METHODS: Up-front bevacizumab-treated patients (N = 38) with newly diagnosed glioblastoma were analyzed by using an ADC histogram approach based on enhancing tumor. Using microarrays, we compared gene expression in high-versus-low ADC tumors in patients subsequently treated with bevacizumab. Tissue sections from a subset of tumors were stained for collagen and collagen-binding proteins. Progression-free and overall survival was determined by using Cox proportional hazard ratios and the Kaplan-Meier method with the log rank test. RESULTS: A total of 13 genes were expressed at 2-fold or greater levels in high- compared with low-ADC tumors at the P < .05 level. Of these, 6 encode for collagen or collagen-binding proteins. High gene expression for the collagen-binding protein decorin was associated with shorter survival (HR, 2.5; P = .03). The pattern and degree of collagen staining were highly variable in both high- and low-ADC tumors. CONCLUSIONS: High-ADC GBMs show greater levels of ECM protein gene expression compared with low-ADC GBMs. It is unclear whether this translates to the accumulation of higher levels of the encoded proteins. However, because ECM molecules could contribute to a proinvasive phenotype, this relationship merits further investigation.

Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria.

Tumor cells with stem cell-like properties can be cultured from human glioblastomas by using conditions that select for the expansion of neural stem cells. We generated cell lines from glioblastoma specimens with the goal to obtain model systems for glioma stem cell biology. Unsupervised analysis of the expression profiles of nine cell lines established under neural stem cell conditions yielded two distinct clusters. Four cell lines were characterized by the expression of neurodevelopmental genes. They showed a multipotent differentiation profile along neuronal, astroglial and oligodendroglial lineages, grew spherically in vitro, expressed CD133 and formed highly invasive tumors in vivo. The other five cell lines shared expression signatures enriched for extracellular matrix-related genes, had a more restricted differentiation capacity, contained no or fewer CD133+ cells, grew semiadherent or adherent in vitro and displayed reduced tumorigenicity and invasion in vivo. Our findings show that stable, multipotent glioblastoma cell lines with a full stem-like phenotype express neurodevelopmental genes as a distinctive feature, which may offer therapeutic targeting opportunities. The generation of another distinct cluster of cell lines showing similarly homogeneous profiling but restricted stem cell properties suggests that different phenotypes exist, each of which may lead to the typical appearance of glioblastoma.

Impaired water maze learning performance without altered dopaminergic function in mice heterozygous for the GDNF mutation.

Exogenous glial cell line-derived neurotrophic factor (GDNF) exhibits potent survival-promoting effects on dopaminergic neurons of the nigrostriatal pathway that is implicated in Parkinson's disease and also protects neurons in forebrain ischemia of animal models. However, a role for endogenous GDNF in brain function has not been established. Although mice homozygous for a targeted deletion of the GDNF gene have been generated, these mice die within hours of birth because of deficits in kidney morphogenesis, and, thus, the effect of the absence of GDNF on brain function could not be studied. Herein, we sought to determine whether adult mice, heterozygous for a GDNF mutation on two different genetic backgrounds, demonstrate alterations in the nigrostriatal dopaminergic system or in cognitive function. While both neurochemical and behavioural measures suggested that reduction of GDNF gene expression in the mutant mice does not alter the nigrostriatal dopaminergic system, it led to a significant and selective impairment of performance in the spatial version of the Morris water maze. A standard panel of blood chemistry tests and basic pathological analyses did not reveal alterations in the mutants that could account for the observed performance deficit. These results suggest that endogenous GDNF may not be critical for the development and functioning of the nigrostriatal dopaminergic system but it plays an important role in cognitive abilities.

Mammalian-cell-produced neurturin (NTN) is more potent than purified Escherichia coli-produced NTN.

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor. Both factors promote the survival of dopaminergic (DA) neurons. We investigated the biological activity of mammalian-cell-produced NTN versus purified Escherichia coli-produced NTN. Baby hamster kidney cells were engineered to stably secrete mature human NTN. Mammalian-cell-derived NTN enhanced the activity of embryonic DA neurons in vitro, with greater potency (maximum effect achieved in the picogram range) than purified E. coli-produced NTN. Cell-based delivery of NTN (less than 10 ng/day) was also shown to be biologically active in vivo. These results suggest that mammalian-cell-derived NTN, synthesized de novo and delivered in small quantities to the parenchyma at the target site, may be as active as much larger quantities of purified, E. coli-produced NTN, delivered by other means.

The glial cell line-derived neurotrophic factor family receptor components are differentially regulated within sensory neurons after nerve injury.

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic effects on adult sensory neurons after nerve injury and is one of a family of proteins that includes neurturin, persephin, and artemin. Sensitivity to these factors is conferred by a receptor complex consisting of a ligand binding domain (GFRalpha1-GFRalpha4) and a signal transducing domain RET. We have investigated the normal expression of GDNF family receptor components within sensory neurons and the response to nerve injury. In normal rats, RET and GFRalpha1 were expressed in a subpopulation of both small- and large-diameter afferents projecting through the sciatic nerve [60 and 40% of FluoroGold (FG)-labeled cells, respectively]. GFRalpha2 and GFRalpha3 were both expressed principally within small-diameter DRG cells (30 and 40% of FG-labeled cells, respectively). Two weeks after sciatic axotomy, the expression of GFRalpha2 was markedly reduced (to 12% of sciatic afferents). In contrast, the proportion of sciatic afferents that expressed GFRalpha1 increased (to 66% of sciatic afferents) so that virtually all large-diameter afferents expressed this receptor component, and the expression of GFRalpha3 also increased (to 66% of sciatic afferents) so that almost all of the small-diameter afferents expressed this receptor component after axotomy. There was little change in RET expression. The changes in the proportions of DRG cells expressing different receptor components were mirrored by alterations in the total RNA levels within the DRG. The changes in GFRalpha1 and GFRalpha2 expression after axotomy could be largely reversed by treatment with GDNF.

Regulation of learning by EphA receptors: a protein targeting study.

EphA family receptor tyrosine kinases and their ephrin-A ligands are involved in patterning axonal connections during brain development, but until now a role for these molecules in the mature brain had not been elucidated. Here, we show that both the EphA5 receptor and its ephrin-A ligands (2 and 5) are expressed in the adult mouse hippocampus, and the EphA5 protein is present in a phosphorylated form. Because there are no pharmacological agents available for EphA receptors, we designed recombinant immunoadhesins that specifically bind to the receptor binding site of the ephrin-A ligand (antagonist) or the ligand binding site of the EphA receptor (agonist) and thus target EphA function. We demonstrate that intrahippocampal infusion of an EphA antagonist immunoadhesin leads to impaired performance in two behavioral paradigms, T-maze spontaneous alternation and context-dependent fear conditioning, sensitive to hippocampal function, whereas activation of EphA by infusion of an agonist immunoadhesin results in enhanced performance on these tasks. Because the two behavioral tasks have different motivational, perceptual, and motor requirements, we infer the changes were not caused by these performance factors but rather to cognitive alterations. We also find bidirectional changes in gene expression and in electrophysiological measures of synaptic efficacy that correlate with the behavioral results. Thus, EphA receptors and their ligands are implicated as mediators of plasticity in the adult mammalian brain.

Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson's disease after administration into the striatum or the lateral ventricle.

Both glial cell line-derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to exert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6-hydroxydopamine (6-OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6-OHDA injection. GDNF provided almost complete (90-92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion-induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6-OHDA-induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine-induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF-treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth-stimulating agent in the IS 6-OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.

Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.

Regulation of hippocampal synaptic plasticity by the tyrosine kinase receptor, REK7/EphA5, and its ligand, AL-1/Ephrin-A5.

The Eph-related tyrosine kinase receptor, REK7/EphA5, mediates the effects of AL-1/Ephrin-A5 and related ligands and is involved in the guidance of retinal, cortical, and hippocampal axons during development. The continued expression of REK7/EphA5 in the adult brain, in particular in areas associated with a high degree of synaptic plasticity such as the hippocampus, raises the question of its function in the mature nervous system. In this report we examined the role of REK7/EphA5 in synaptic remodeling by asking if agents that either block or activate REK7/EphA5 affect synaptic strength in hippocampal slices from adult mouse brain. We show that a REK7/EphA5 antagonist, soluble REK7/EphA5-IgG, impairs the induction of long-term potentiation (LTP) without affecting other synaptic parameters such as normal synaptic transmission or paired-pulse facilitation. In contrast, perfusion with AL-1/Ephrin-A5-IgG, an activator of REK7/EphA5, induces a sustained increase in normal synaptic transmission that partially mimics LTP. The sustained elevation of normal synaptic transmission could be attributable to a long-lasting binding of the AL-1/Ephrin-A5-IgG to the endogenous REK7/EphA5 receptor, as revealed by immunohistochemistry. Furthermore, maximal electrical induction of LTP occludes the potentiating effects of subsequent treatment with AL-1/Ephrin-A5-IgG. Taken together these results implicate REK7/EphA5 in the regulation of synaptic plasticity in the mature hippocampus and suggest that REK7/EphA5 activation is recruited in the LTP induced by tetanization.

Differential dependency of unmyelinated and A delta epidermal and upper dermal innervation on neurotrophins, trk receptors, and p75LNGFR.

The impact of the nerve growth factor (NGF) family of neurotrophins and their receptors was examined on the cutaneous innervation in the mystacial pads of mice. Ten sets of unmyelinated and thinly myelinated sensory and autonomic innervation were evaluated that terminated in the epidermis, upper dermis, and upper part of the intervibrissal hair follicles. Mystacial pads were analyzed from newborn to 4-week-old mice that had homozygous functional deletions of the genes for NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), tyrosine kinase (trk) A, trkB, trkC, or p75. Mystacial pads were also analyzed in adult transgenic mice that had overproduction of NGF, BDNF, or NT-3 driven by a keratin promoter gene. The innervation was revealed by using immunofluorescence and immunocytochemistry with antibodies for protein gene product (PGP) 9.5, calcitonin gene-related product (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), tyrosine hydroxylase (TH), and a neurofilament protein. The cumulative results indicated that NGF/trkA signaling plays a major role in the outgrowth and proliferation of sensory axons, whereas NT-3/ trkA signaling plays a major role in the formation of sensory endings. TrkC is also essential for the development of three sets of trkA-dependent sensory innervation that coexpress CGRP, SP, and GAL. Another set of sensory innervation that only coexpressed CGRP and SP was solely dependent upon NGF and trkA. Surprisingly, most sets of trkA-dependent sensory innervation are suppressed by trkB perhaps interacting with p75. BDNF and NT-4 appear to mediate this suppressing effect in the upper dermis and NT-4 in the epidermis. In contrast to sensory innervation, sympathetic innervation to the necks of intervibrissal hair follicles depends upon NGF/trkA signaling interacting with p75 for both the axon outgrowth and ending formation. Although NT-3/trkA signaling is essential for the full complement of sympathetic neurons, NT-3 is detrimental to the formation of sympathetic terminations to the necks of hair follicles. TrkB signaling mediated by BDNF but not NT-4 also suppresses these sympathetic terminations. One sparse set of innervation, perhaps parasympathetic, terminating at the necks of hair follicles is dependent solely upon NT-3 and trkC. Taken together, our results indicate that the innervation of the epidermis, upper dermis, and the upper portion of hair follicles is regulated by a competitive balance between promoting and suppressing effects of the various neurotrophins.

Neurturin exerts potent actions on survival and function of midbrain dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent effects on survival and function of midbrain dopaminergic (DA) neurons in a variety of models. Although other growth factors expressed in the vicinity of developing DA neurons have been reported to support survival of DA neurons in vitro, to date none of these factors duplicate the potent and selective actions of GDNF in vivo. We report here that neurturin (NTN), a homolog of GDNF, is expressed in the nigrostriatal system, and that NTN exerts potent effects on survival and function of midbrain DA neurons. Our findings indicate that NTN mRNA is sequentially expressed in the ventral midbrain and striatum during development and that NTN exhibits survival-promoting actions on both developing and mature DA neurons. In vitro, NTN supports survival of embryonic DA neurons, and in vivo, direct injection of NTN into the substantia nigra protects mature DA neurons from cell death induced by 6-OHDA. Furthermore, administration of NTN into the striatum of intact adult animals induces behavioral and biochemical changes associated with functional upregulation of nigral DA neurons. The similarity in potency and efficacy of NTN and GDNF on DA neurons in several paradigms stands in contrast to the differential distribution of the receptor components GDNF Family Receptor alpha1 (GFRalpha1) and GFRalpha2 within the ventral mesencephalon. These results suggest that NTN is an endogenous trophic factor for midbrain DA neurons and point to the possibility that GDNF and NTN may exert redundant trophic influences on nigral DA neurons acting via a receptor complex that includes GFRalpha1.

Differential dependency of cutaneous mechanoreceptors on neurotrophins, trk receptors, and P75 LNGFR.

The impact of null mutations of the genes for the NGF family of neurotrophins and their receptors was examined among the wide variety of medium to large caliber myelinated mechanoreceptors which have a highly specific predictable organization in the mystacial pad of mice. Immunofluorescence with anti-protein gene product 9.5, anti-200-kDa neurofilament protein (RT97), and anti-calcitonin gene-related product was used to label innervation in mystacial pads from mice with homozygous null mutations for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), the three tyrosine kinase receptors (trkA, trkB, trkC), and the low-affinity nerve growth factor receptor p75. Specimens were sacrificed at birth and at 1, 2, and 4 weeks for each type of mutation as well as at 11 weeks and 1 year for p75 and trkC mutations, respectively. Our results demonstrate several major concepts about the role of neurotrophins in the development of cutaneous mechanoreceptors that are supplied by medium to large caliber myelinated afferents. First, each of the high-affinity tyrosine kinase receptors, trkA, trkB, and trkC, as well as the low-affinity p75 receptor has an impact on at least one type of mechanoreceptor. Second, consistent with the various affinities for particular trk receptors, the elimination of NGF, BDNF, and NT-3 has an impact comparable to or more complex than the absence of their most specific high-affinity receptors: trkA, trkB, and trkC, respectively. These complexities include potential NT-3 signaling through trkA and trkB to support some neuronal survival. Third, most types of afferents are dependent on a different combination of neurotrophins and receptors for their survival: reticular and transverse lanceolate afferents are dependent upon NT-3, NGF, and trkA; Ruffini afferents upon BDNF and trkB; longitudinal lanceolate afferents upon NGF, trkA, BDNF, and trkB; and Merkel afferents on NGF, trkA, NT-3, trkC, and p75. NT-4 has no obvious detrimental impact on the mechanoreceptor development in the presence of BDNF. Fourth, NT-4 and BDNF signaling through trkB may suppress Merkel innervation and NT-3 signaling through trkC may suppress Ruffini innervation. Finally, regardless of the neurotrophin/receptor dependency for afferent survival and neurite outgrowth, NT-3 has an impact on the formation of all the sensory endings. In the context of these findings, indications of competitive and suppressive interactions that appear to regulate the balance of innervation density among the various sets of innervation were evident.

Disruption of a single allele of the nerve growth factor gene results in atrophy of basal forebrain cholinergic neurons and memory deficits.

Administration of nerve growth factor (NGF) to aged or lesioned animals has been shown to reverse the atrophy of basal forebrain cholinergic neurons and ameliorate behavioral deficits. To examine the importance of endogenous NGF in the survival of basal forebrain cholinergic cells and in spatial memory, mice bearing a disruption mutation in one allele of the NGF gene were studied. Heterozygous mutant mice (ngf+/-) have reduced levels of NGF mRNA and protein within the hippocampus and were found to display significant deficits in memory acquisition and retention in the Morris water maze. The behavioral deficits observed in NGF-deficient mice were accompanied by both shrinkage and loss of septal cells expressing cholinergic markers and by a decrease in cholinergic innervation of the hippocampus. Infusions of NGF into the lateral ventricle of adult ngf+/- mice abolished the deficits on the water maze task. Prolonged exposure to NGF may be required to induce cognitive effects, because reversal of the acquisition deficit was seen after long (5 weeks) but not short (3 d) infusion. Although NGF administration did not result in any improvement in the number of septal cells labeled for choline acetyltransferase, this treatment did effectively correct the deficits in both size of cholinergic neurons and density of cholinergic innervation of the hippocampus. These findings demonstrate the importance of endogenous NGF for survival and function of basal forebrain cholinergic neurons and reveal that partial depletion of this trophic factor is associated with measurable deficits in learning and memory.

Csk and BatK show opposite temporal expression in the rat CNS: consistent with its late expression in development, BatK induces differentiation of PC12 cells.

BatK is a second member of the Csk family of regulatory kinases that phosphorylate a key inhibitory tyrosine on Src family kinases, leading to down-regulation. To investigate the roles of BatK and Csk, both of which are expressed in the brain, we compared their temporal expression patterns during development of the central nervous system (CNS) in rats. BatK mRNA is undetectable at embryonic day 12 (E12), appears in the developing nervous system at approximately E15, and its expression progressively increases up to the time of birth, thereafter remaining high throughout the adult brain. In striking contrast, Csk is highly expressed throughout embryonic development and remains high in the CNS until birth. It is then dramatically down-regulated in the adult brain except in the olfactory bulb. BatK and Csk thus exhibit complementary temporal expression patterns. Since BatK expression correlates with late-stage development and terminal differentiation, we speculated that it might be involved in regulating neuronal differentiation. Using PC12 cells as a model system, we show that overexpression of BatK is sufficient to induce neurite outgrowth in the absence of nerve growth factor. Further, overexpression of BatK activates the mitogen-activated protein kinase cascade. We propose a model suggesting that, despite overlapping in vitro activities, BatK and Csk regulate different targets in vivo and have different functions during and after neuronal development, BatK being the dominant regulator of Src kinases in the fully differentiated adult brain.

Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons.

The muscle-derived factors required for survival of embryonic motoneurons are not clearly identified. Cardiotrophin-1 (CT-1), a cytokine related to ciliary neurotrophic factor (CNTF), is expressed at high levels in embryonic limb bud and is secreted by differentiated myotubes. In vitro, CT-1 kept 43% of purified E14 rat motoneurons alive for 2 weeks (EC50 = 20 pM). In vivo, CT-1 protected neonatal sciatic motoneurons against the effects of axotomy. CT-1 action on motoneurons was inhibited by phosphatidylinositol-specific phospholipase C (PIPLC), suggesting that CT-1 may act through a GPI-linked component. Since no binding of CT-1 to CNTFR alpha was detected, CT-1 may use a novel cytokine receptor alpha subunit. CT-1 may be important in normal motoneuron development and as a potential tool for slowing motoneuron degeneration in human diseases.

Characterization of a multicomponent receptor for GDNF.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for central and peripheral neurons, and is essential for the development of kidneys and the enteric nervous system. Despite the potential clinical and physiological importance of GDNF, its mechanism of action is unknown. Here we show that physiological responses to GDNF require the presence of a novel glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrate that GDNF promotes the formation of a physical complex between GDNFR-alpha and the orphan tyrosin kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF uses a multi-subunit receptor system in which GDNFR-alpha and Ret function as the ligand-binding and signalling components, respectively.

Expression of the trk family of neurotrophin receptors in developing and adult dorsal root ganglion neurons.

Expression of trk receptors is a major determinant of neurotrophin responsiveness of sensory neurons. Although it has been apparent for some time that subpopulations of dorsal root and trigeminal ganglion neurons respond in vitro to each of the members of the neurotrophin family, the extent to which functionally distinct subclasses of sensory neurons are dependent on the actions of different neurotrophins for their development and function remains an active area of investigation. One step towards elucidating the role of various neurotrophins in development and function of sensory neurons has been to examine the distribution of trk receptors on sensory neurons. These studies have clearly revealed that members of the trk family are differentially expressed in functionally distinct populations of both developing and mature sensory neurons and, further, have provided evidence consistent with a shift in neurotrophin responsiveness during the development of sensory neurons.