Targeting neurotrophin receptors in the central nervous system.

Neurotrophic factors, and in particular the neurotrophins, restore the function of damaged neurons and prevent apoptosis in adults. The potential therapeutic property of the neurotrophins is however, complicated by the peptidergic structure of these trophic factors, which impairs their penetration into the brain parenchyma, and therefore makes their pharmaco-therapeutic properties difficult to evaluate. In this article we will focus on the neurotrophin Brain-derived neurotrophic factor (BDNF) and its receptors to address various therapeutic strategies that may overcome this problem. We will call this strategy "small molecule approach" because it relies on increasing the function of endogenous neurotrophins by pharmacological compounds that induce synthesis and release of neurotrophins in relevant brain areas or by small synthetic molecules that bind and activate specific neurotrophin receptors. The ability of small molecules to mimic BDNF has a potential therapeutic importance in preventing neuronal damage in several chronic neurodegenerative diseases including Parkinson's Disease, Alzheimer's Disease, and AIDS dementia.

Evolution of the neurotrophin signaling system in invertebrates.

Nucleotide sequences encoding orthologs of neurotrophins and their receptors, p75(NTR) and Trk receptors, have been identified in the sea urchin Strongylocentrotus purpuratus, and the acorn worm, Saccoglossus kowalevskii, whereas the ascidian (sea squirt) species Ciona intestinalis and Ciona savignii appear to lack such orthologs. These results suggest that a functional neurotrophin system was already present at the beginning of deuterostome evolution, but was lost in ascidians. Remarkably, it appears that evolution of a p75(NTR) ortholog represented one of the earliest events in the expansion of the tumor necrosis factor receptor superfamily.

Tracing the evolution and function of the Trk superfamily of receptor tyrosine kinases.

Most growth factors and their receptors have been strongly conserved during evolution. In contrast, Trks (Tropomyosin-related kinases) and related receptors in the Trk superfamily, Rors (receptor tyrosine kinase-like orphan receptors), Musks (muscle specific kinases) and Ddrs (discoidin domain receptor family), appear to be ancient, but their function has been lost in multiple lineages and the roles for the receptors have been modified over time. We will trace the evolution of the Trk superfamily and discuss possible conserved functional roles, including a unifying theme of target recognition by growing axons. We present an analogy between the evolution of G-protein-coupled receptors and receptor tyrosine kinases (RTKs), proposing that an important driving force for the divergence of receptors is the ease of divergence of their ligands.

Comparative analysis of neurotrophin receptors and ligands in vertebrate neurons: tools for evolutionary stability or changes in neural circuits?

To better understand the role of multiple neurotrophin ligands and their receptors in vertebrate brain evolution, we examined the distribution of trk neurotrophin receptors in representatives of several vertebrate classes. Trk receptors are largely expressed in homologous neuronal populations among different species/classes of vertebrates. In many neurons, trkB and trkC receptors are co-expressed. TrkB and trkC receptors are primarily found in neurons with more restricted, specialized dendritic and axonal fields that are thought to be involved in discriminative or 'analytical' functions. The neurotrophin receptor trkA is expressed predominantly in neurons with larger, overlapping dendritic fields with more heterogeneous connections ('integrative' or 'modulatory' systems) such as nociceptive and sympathetic autonomic nervous system, locus coeruleus and cholinergic basal forebrain. Surveys of trk receptor expression and function in the peripheral nervous system of different vertebrate classes reveal trends ranging from dependency on a single neurotrophin to a more complex dependency on increasing numbers of neurotrophins and their receptors, for example, in taste and inner ear innervation. Gene deletion studies in mice provide evidence for a complex regulation of neuronal survival of sensory ganglion cells by different neurotrophins. Although expression of neurotrophins and their receptors is predominantly conserved in most circuits, increasing diversity of neurotrophin ligands and their receptors and a more complex dependency of neurons on neurotrophins might have facilitated the formation of at least some new neuronal entities.

Each sensory nerve arising from the geniculate ganglion expresses a unique fingerprint of neurotrophin and neurotrophin receptor genes.

Neurons in the geniculate ganglion, like those in other sensory ganglia, are dependent on neurotrophins for survival. Most geniculate ganglion neurons innervate taste buds in two regions of the tongue and two regions of the palate; the rest are cutaneous nerves to the skin of the ear. We investigated the expression of four neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and NT-4, and five neurotrophin receptors, trkA, trkB, trkC, p75, and truncated trkB (Trn-B) in single sensory neurons of the adult rat geniculate ganglion associated with the five innervation fields. For fungiform papillae, a glass pipette containing biotinylated dextran was placed over the target papilla and the tracer was iontophoresed into the target papilla. For the other target fields, Fluoro-Gold was microinjected. After 3 days, geniculate ganglia were harvested, sectioned, and treated histochemically (for biotinylated dextran) or immunohistochemically (for Fluoro-Gold) to reveal the neurons containing the tracer. Single labeled neurons were harvested from the slides and subjected to RNA amplification and RT-PCR to reveal the neurotrophin or neurotrophin receptor genes that were expressed. Neurons projecting from the geniculate ganglion to each of the five target fields had a unique expression profile of neurotrophin and neurotrophic receptor genes. Several individual neurons expressed more than one neurotrophin receptor or more than one neurotrophin gene. Although BDNF is significantly expressed in taste buds, its primary high affinity receptor, trkB, was not prominently expressed in the neurons. The results are consistent with the interpretation that at least some, perhaps most, of the trophic influence on the sensory neurons is derived from the neuronal somata, and the trophic effect is paracrine or autocrine, rather than target derived. The BDNF in the taste bud may also act in a paracrine or autocrine manner on the trkB expressed in taste buds, as shown by others.

Differing patterns of neurotrophin-receptor expressing neurons allow distinction of the transient Frorieps' ganglia from normal DRG before morphological differences appear.

The Frorieps' ganglia are dorsal root ganglia (DRG) that form and then degenerate during normal embryonic development of amniotes. Their degeneration or survival has been shown to be modulated by modifying expression of Hox-family and other genes involved in pattern formation, and by the mesodermal microenvironment of the cranial somites in which they develop. In ovo application of the neurotrophin NGF partially rescues DRG2 from degeneration. To further examine the potential role of neurotrophins in the life cycle of Frorieps' DRG we have now quantified the numbers of neurons expressing neurotrophin receptors trkA and trkC in avian Frorieps' ganglia (DRG2) and normal cervical DRG (DRG5). We have found that the Frorieps' DRG are different from normal DRG in terms of the numbers of neurons expressing these receptors. trkC-expressing neurons are generally lacking in DRG2, this is the earliest (St 18, E2.5) described difference between DRG2 and normal DRG, preceding morphological differences between these ganglia that appear at St 20. The difference between DRG2 and DRG5 in terms of numbers of trkA-expressing neurons is evident only at later embryonic stages, where DRG2 contains a higher proportion of trkA neurons than normal cervical DRG. The few trkC+ neurons present late in DRG2 development are not concentrated in the VL portion of the ganglion, the zone where trkC+ neurons are generally found in normal DRG. We also find that DRG2 neurons are smaller than those of normal DRG, this is true for both trkA+ and trkC+ populations. These data together therefore suggest that the neurons that survive in the Frorieps' ganglia at later stages belong almost exclusively to the trkA-expressing DM class DRG neurons. We further find that the differences in the populations of trkA/trkC between DRG2 and DRG5 result from signals from the mesodermal microenvironment, since DRG arising in cranial somites transplanted caudally contain few trkC+ neurons and a higher proportion of trkA+ cells than contralateral controls.

High affinity neurotrophin receptors in the human pre-term newborn, infant, and adult cerebellum.

The immunohistochemical occurrence of the high affinity neurotrophin (NT) receptors trkA, trkB, and trkC is shown in the pre-term newborn, infant, and adult human post-mortem cerebellum. Immunoreactive neuronal perikarya and processes were observed in all specimens examined, where they appeared unevenly distributed in the cerebellar cortical layers and deep nuclei, and showed regional differences among cerebellar lobules and folia. The trk receptor-antibodies, tested by Western blot on human cerebellum homogenates, revealed multiple immunoreactive bands for trkA and single bands for trkB and trkC. The results obtained show the tissue localization of the trk receptor-like immunoreactivity in the human cerebellum from prenatal to adult age. The analysis for codistribution of the receptors with the relevant ligand and among the receptors in discrete cortical and deep nuclei tissue fields shows a wide variety of conditions, from a good similarity in terms of type and density of labeled structures, to a lack of correspondence, and suggests the possibility of colocalization of trk receptors with the relevant neurotrophin and among them in the cerebellar cortex. These results sustain the concept that the neurotrophin trophic system participates in the development, differentiation, and maintenance of the human cerebellar connectivity and support the possibility of a multifactorial trophic support for the neurotrophins through target-derived and local mechanisms.

Selectivity in neurotrophin signaling: theme and variations.

Neurotrophins are a family of growth factors critical for the development and functioning of the nervous system. Although originally identified as neuronal survival factors, neurotrophins elicit many biological effects, ranging from proliferation to synaptic modulation to axonal pathfinding. Recent data indicate that the nature of the signaling cascades activated by neurotrophins, and the biological responses that ensue, are specified not only by the ligand itself but also by the temporal pattern and spatial location of stimulation. Studies on neurotrophin signaling have revealed variations in the Ras/MAP kinase, PI3 kinase, and phospholipase C pathways, which transmit spatial and temporal information. The anatomy of neurons makes them particularly appropriate for studying how the location and tempo of stimulation determine the signal cascades that are activated by receptor tyrosine kinases such as the Trk receptors. These signaling variations may represent a general mechanism eliciting specificity in growth factor responses.

The uniqueness of being a neurotrophin receptor.

Neurotrophins rely on Trk tyrosine kinase and p75 receptors for signal transduction. Recently, other roles for these receptors have been identified. Many questions have been raised about the mechanism by which these receptors mediate diverse cellular functions. Studies indicate a great deal of neurotrophin signaling specificity may stem from ligand-receptor selectivity and intracellular protein recruitment.

Neurotrophin receptors, tumor progression and tumor maturation.

The nerve growth factor receptor TrkA was initially isolated as a transforming oncogene, trk, in which most of the extracellular receptor part is replaced by the coding sequence for a tropomyosin-encoding gene. The impact that the identification of the first neurotrophin receptor has made on the entire field of developmental neurobiology cannot be overstated. Following a brief introduction to the biology of neurotrophins and their receptors, this review will focus on oncogenic Trk in human malignant disorders, discuss putative tumorigenic involvement of Trk family members in the childhood malignancy neuroblastoma, and point out potential neurotrophin-based treatment modalities for this and other neuroendocrine tumors.

Attenuation and recovery of nerve growth factor receptor mRNA in dorsal root ganglion neurons following axotomy.

The actions of nerve growth factor (NGF) are mediated by two receptor proteins, trk and p75. Recent evidence indicates that NGF upregulates the expression of both trk and p75 in responsive neurons including rat dorsal root ganglion (DRG) neurons. Axotomy by disconnecting the neuron from its source of target-derived NGF is predicted to lead to the downregulation of trk and p75 expression. However, previous studies of the effects of axotomy on trk and p75 mRNA expression in rat DRG have yielded discrepant results. We report that following sciatic nerve crush, trk and p75 mRNA levels in L4-L6 DRG decrease to approximately 50% of control levels at 4-14 days, return to control levels by 30 days, and are increased by approximately 30% at 60 days. Similar changes are observed following nerve transection although mRNA levels are slower in returning to normal and do not exceed control levels at later timepoints. Thus, trk and p75 expression decline early following target disconnection and later recover irrespective of target reinnervation. These observations indicate that target derived NGF is required for the maintenance of NGF receptor expression in adult rat DRG neurons and that non-target derived factors can appropriate this function following peripheral nerve injury.

Pan-neurotrophin 1: a genetically engineered neurotrophic factor displaying multiple specificities in peripheral neurons in vitro and in vivo.

Pan-neurotrophin 1 (PNT-1) is a synthetic trophic factor engineered by combining active domains of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) into an NT-3 backbone. This molecule was produced in transiently transfected COS cells or in baculovirus-infected insect cells transfected COS cells or in baculovirus-infected insect cells and subsequently purified to homogeneity. Saturation binding in embryonic spinal sensory neurons demonstrated a greater number of high-affinity binding sites for PNT-1 than for its parental molecule NT-3. PNT-1 was shown to efficiently block the chemical crosslinking of NGF, BDNF, and NT-3 to their cognate Trk receptors and to the low-affintiy NGF receptor expressed on neuronal and nonneuronal cells. PNT-1 stimulated survival and proliferation of MG87 fibroblasts expressing either TrkA, TrkB, or TrkC. PNT-1 also promoted survival of a greater number of embryonic dorsal root ganglion neurons than any of the other neurotrophins alone, and its effects were equivalent to a combination of NGF, BDNF, and NT-3. Analysis of receptor-specific neurotrophic activities demonstrated that PNT-1 efficiently rescued TrkA mRNA-containing sympathetic neurons and TrkB and TrkC mRNA-containing sensory neurons from the dorsal root and nodose ganglia. Finally, PNT-1 showed robust retrograde transport to DRG neurons in vivo after injection into the sciatic nerve. Radiolabeled PNT-1 accumulated in small-, medium-, and large-sized neurons. Coinjection with different unlabeled neurotrophins inhibited PNT-1 transport in distinct subpopulations of neurons of different sizes, suggesting that this molecule affects sensory neurons of different modalities. These results indicate that PNT-1 is a potent and multispecific neurotrophic factor that may be useful in the treatment of peripheral neurophathies and nerve damage.

Expression of nuclear hormone receptors within the rat hippocampus: identification of novel orphan receptors.

Within the hippocampus, stimulus-transcriptional coupling plays an important role in post-seizure neuronal adaptation, post-ischemic cell death and the induction of long-term potentiation. To identify additional mediators of hippocampal transcriptional responses a targeted approach was developed and used to characterize the spectrum of nuclear hormone receptors expressed within this brain region. cDNAs encoding the DNA-binding domains of six different members of the nuclear hormone receptor superfamily were isolated. A majority were identical or closely related to receptors known to be expressed within the hippocampus. Two additional isolates, HZF-2 and HZF-3, encode the DNA-binding domain of novel members of the nuclear hormone receptor superfamily.

Colocalization of low- and high-affinity NGF receptors on PC12 cells, C6 glioma cells and dorsal root ganglion neurons.

The biological responsiveness of neural cells to nerve growth factor (NGF) appears to require expression and ligand binding to both the low-affinity NGF receptor (LNGFR) and the proto-oncogene product trk, the latter being a receptor tyrosine kinase. Immunolocalization of the LNGFR and the high-affinity component of the NGF receptor, trk (HNGFR) was studied by electron microscopic morphometric analysis on cultured PC12 pheochromocytoma cells, C6 glioma cells and neonatal rat dorsal root ganglia neurons using a double immunogold labeling technique. Two receptor-specific antibodies, anti-LNGFR monoclonal antibody 192-IgG and a polyclonal antibody against the 14 carboxy-terminal amino acids of the Trk protein, were utilized in conjunction with immunoglobulin conjugated to colloidal gold particles of different sizes. All cells treated with NGF (50 ng/ml) displayed significant colocalization of LNGFR/HNGFR-like immunoreactivity. Gold particles associated with LNGFR (LNGFR-like immunoreactivity) were frequently seen near 2 or 3 (or more) particles delineating the HNGFR on all cell surfaces. Positive Trk-like immunoreactivity (HNGFR) thus seems to localize in close proximity to LNGFRs in at least these cell types.