Biomedical subjects
Publications and source records attributed to Louis F Reichardt.
NGF and NT-3 both signal through TrkA receptors on the axons of developing sympathetic neurons, but while NGF supports survival and differentiation, NT-3 does not. In this issue of Cell, the difference is explained as the ability of NGF, but not NT-3, to induce internalization and retrograde transport of activated TrkA.
Axons dictate whether or not they will become myelinated in both the central and peripheral nervous systems by providing signals that direct the development of myelinating glia. Here we identify the neurotrophin nerve growth factor (NGF) as a potent regulator of the axonal signals that control myelination of TrkA-expressing dorsal root ganglion neurons (DRGs). Unexpectedly, these NGF-regulated axonal signals have opposite effects on peripheral and central myelination, promoting myelination by Schwann cells but reducing myelination by oligodendrocytes. These findings indicate a novel role for growth factors in regulating the receptivity of axons to myelination and reveal that different axonal signals control central and peripheral myelination.
Tactile information is perceived by a heterogeneous population of specialized neurons. Neurotrophin receptors (the receptor tyrosine kinases, Trks) mark the major classes of these sensory neurons: TrkA is expressed in neurons that sense temperature and noxious stimuli, and TrkC is expressed in proprioceptive neurons that sense body position. Neurotrophin signaling through these receptors is required for cell survival. To test whether neurotrophins have an instructive role in sensory specification, we expressed rat TrkC from the TrkA (also known as Ntrk1) locus in mice. The surviving presumptive TrkA-expressing neurons adopted a proprioceptive phenotype, indicating that neurotrophin signaling can specify sensory neuron subtypes.
We have previously shown that mice with a CNS restricted knock-out of the integrin beta1 subunit gene (Itgb1-CNSko mice) have defects in the formation of lamina and folia in the cerebral and cerebellar cortices that are caused by disruption of the cortical marginal zones. Cortical structures in postnatal and adult Itgb1-CNSko animals are also reduced in size, but the mechanism that causes the size defect has remained unclear. We now demonstrate that proliferation of granule cell precursors (GCPs) is severely affected in the developing cerebellum of Itgb1-CNSko mice. In the absence of beta1 expression, GCPs lose contact with laminin in the meningeal basement membrane, cease proliferating, and differentiate prematurely. In vitro studies provide evidence that beta1 integrins act at least in part cell autonomously in GCPs to regulate their proliferation. Previous studies have shown that sonic hedgehog (Shh)-induced GCP proliferation is potentiated by the integrin ligand laminin. We show that Shh directly binds to laminin and that laminin-Shh induced cell proliferation is dependent on beta1 integrin expression in GCPs. Taken together, these data are consistent with a model in which beta1 integrin expression in GCPs is required to recruit a laminin-Shh complex to the surface of GCPs and to subsequently modulate the activity of signaling pathways that regulate proliferation.
A recent publication shows that two integrins, alpha6beta4 and alpha3beta1, interact with netrin-1, a protein best known for its role in axon guidance. Consistent with a role in pancreatic development in vivo, netrin is expressed together with these integrins in fetal pancreatic epithelium. The paper presents strong evidence that pancreatic epithelial cells adhere, spread and migrate on netrin-coated substrata through alpha6beta4. Data also indicate that alpha3beta1 mediates HGF-stimulated cell migration on netrin-coated substrata. These results indicate that netrins might have much broader functions than previously suspected.
We review the history of neurotrophins in the ear and the current understanding of the function of neurotrophins in ear innervation, development and maintenance. Only two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), and their receptors, tyrosine kinase B (TrkB) and TrkC, appear to provide trophic support for inner ear sensory neuron afferents. Mice lacking either both receptors or both ligands lose essentially all sensory innervation of targets in the vestibular and auditory systems of the ear. Analyzes of single mutants show less complete and differential effects on innervation of the different sensory organs within the ear. BDNF and TrkB are most important for survival of vestibular sensory neurons whereas NT-3 and TrkC are most important for survival of cochlear sensory neurons. The largely complementary roles of BDNF to TrkB and NT-3 to TrkC signaling do not reflect specific requirements for innervation of different classes of hair cells. Most neurons express both receptors. Instead, the losses observed in single mutants are related to the spatio-temporal expression pattern of the two neurotrophins. In an area where only one neurotrophin is expressed at a particular time in development, the other neurotrophin is not present to compensate for this absence, resulting in death of neurons innervating that region. Decisive evidence for this suggestion is provided by transgenic mice in which the BDNF coding region has been inserted into the NT-3 gene, resulting in expression of BDNF instead of NT-3. The expression of BDNF in the spatio-temporal pattern of NT-3 results in survival of almost all neurons that are normally lost in the NT-3 mutant. Thus, BDNF and NT-3 have a high level of functional equivalence for inner ear sensory neuron survival. Further analysis of the patterns of afferent fiber losses in mutations that do not develop differentiated hair cells shows that the expression of neurotrophins is remarkably strong and can support afferent innervation. Indeed, BDNF may be one of the earliest genes expressed selectively in hair cells and it appears to be regulated somewhat independently of the genes needed for hair cell differentiation.
Cadherins and catenins are thought to promote adhesion between pre and postsynaptic elements in the brain. Here we show a role for beta-catenin in localizing the reserved pool of vesicles at presynaptic sites. Deletion of beta-catenin in hippocampal pyramidal neurons in vivo resulted in a reduction in the number of reserved pool vesicles per synapse and an impaired response to prolonged repetitive stimulation. This corresponded to a dispersion of vesicles along the axon in cultured neurons. Interestingly, these effects are not due to beta-catenin's involvement in cadherin-mediated adhesion or wnt signaling. Instead, beta-catenin modulates vesicle localization via its PDZ binding domain to recruit PDZ proteins such as Veli to cadherin at synapses. This study defines a specific role for cadherins and catenins in synapse organization beyond their roles in mediating cell adhesion.
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Targeted deletion of focal adhesion kinase (fak) in the developing dorsal forebrain resulted in local disruptions of the cortical basement membrane located between the neuroepithelium and pia-meninges. At disruption sites, clusters of neurons invaded the marginal zone. Retraction of radial glial endfeet, midline fusion of brain hemispheres, and gliosis also occurred, similar to type II cobblestone lissencephaly as seen in congenital muscular dystrophy. Interestingly, targeted deletion of fak in neurons alone did not result in cortical ectopias, indicating that fak deletion from glia is required for neuronal mislocalization. Unexpectedly, fak deletion specifically from meningeal fibroblasts elicited similar cortical ectopias in vivo and altered laminin organization in vitro. These observations provide compelling evidence that FAK plays a key signaling role in cortical basement membrane assembly and/or remodeling. In addition, FAK is required within neurons during development because neuron-specific fak deletion alters dendritic morphology in the absence of lamination defects.
We used deletion mutants to study beta-catenin function in axon arborization of retinal ganglion cells (RGCs) in live Xenopus laevis tadpoles. A deletion mutant betacatDeltaARM consists of the N- and C-terminal domains of wild-type beta-catenin that contain, respectively, alpha-catenin and postsynaptic density-95 (PSD-95)/discs large (Dlg)/zona occludens-1 (ZO-1) (PDZ) binding sites but lacks the central armadillo repeat region that binds cadherins and other proteins. Expression of DeltaARM in RGCs of live tadpoles perturbed axon arborization in two distinct ways: some RGC axons did not form arbors, whereas the remaining RGC axons formed arbors with abnormally long and tangled branches. Expression of the N- and C-terminal domains of beta-catenin separately in RGCs resulted in segregation of these two phenotypes. The axons of RGCs overexpressing the N-terminal domain of beta-catenin developed no or very few branches, whereas axons of RGCs overexpressing the C-terminal domain of beta-catenin formed arbors with long, tangled branches. Additional analysis revealed that the axons of RGCs that did not form arbors after overexpression of DeltaARM or the N-terminal domain of beta-catenin were frequently mistargeted within the tectum. These results suggest that interactions of the N-terminal domain of beta-catenin with alpha-catenin and of the C-terminal domain with PDZ domain-containing proteins are required, respectively, to initiate and shape axon arbors of RGCs in vivo.
Trk receptors are a family of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4 (NT3 and NT4). Neurotrophin signaling through these receptors regulates cell survival, proliferation, the fate of neural precursors, axon and dendrite growth and patterning, and the expression and activity of functionally important proteins, such as ion channels and neurotransmitter receptors. In the adult nervous system, the Trk receptors regulate synaptic strength and plasticity. The cytoplasmic domains of Trk receptors contain several sites of tyrosine phosphorylation that recruit intermediates in intracellular signaling cascades. As a result, Trk receptor signaling activates several small G proteins, including Ras, Rap-1, and the Cdc-42-Rac-Rho family, as well as pathways regulated by MAP kinase, PI 3-kinase and phospholipase-C-gamma (PLC-gamma). Trk receptor activation has different consequences in different cells, and the specificity of downstream Trk receptor-mediated signaling is controlled through expression of intermediates in these signaling pathways and membrane trafficking that regulates localization of different signaling constituents. Perhaps the most fascinating aspect of Trk receptor-mediated signaling is its interplay with signaling promoted by the pan-neurotrophin receptor p75NTR. p75NTR activates a distinct set of signaling pathways within cells that are in some instances synergistic and in other instances antagonistic to those activated by Trk receptors. Several of these are proapoptotic but are suppressed by Trk receptor-initiated signaling. p75NTR also influences the conformations of Trk receptors; this modifies ligand-binding specificity and affinity with important developmental consequences.
The ventromedial hypothalamic nucleus (VMN) is known to mediate autonomic responses in feeding and reproductive behaviors. To date, the most definitive molecular marker for the VMN is the orphan nuclear receptor steroidogenic factor-1 (SF-1). However, it is unclear whether SF-1 functions in the VMN as it does in peripheral endocrine organ development where loss of SF-1 results in organ agenesis due to apoptosis. Here, we provide evidence that SF-1 has a distinct role in later stages of VMN development by demonstrating the persistence of VMN precursors, the misexpression of an early marker (NKX2-1) concomitant with the absence of a late marker (BDNF neurotrophin), and the complete loss of projections to the bed nucleus of stria terminalis and the amygdala in sf-1 null mice. Our findings demonstrate that SF-1 is required for terminal differentiation of the VMN and suggest that transcriptional targets of SF-1 mediate normal circuitry between the hypothalamus and limbic structures in the telencephalon.
The melanocortin-4 receptor (MC4R) is critically involved in regulating energy balance, and obesity has been observed in mice with mutations in the gene for brain-derived neurotrophic factor (BDNF). Here we report that BDNF is expressed at high levels in the ventromedial hypothalamus (VMH) where its expression is regulated by nutritional state and by MC4R signaling. In addition, similar to MC4R mutants, mouse mutants that expresses the BDNF receptor TrkB at a quarter of the normal amount showed hyperphagia and excessive weight gain on higher-fat diets. Furthermore, BDNF infusion into the brain suppressed the hyperphagia and excessive weight gain observed on higher-fat diets in mice with deficient MC4R signaling. These results show that MC4R signaling controls BDNF expression in the VMH and support the hypothesis that BDNF is an important effector through which MC4R signaling controls energy balance.
A chimera of the nerve growth factor (NGF) receptor, TrkA, and green fluorescent protein (GFP) was engineered by expressing GFP in phase with the carboxyl terminus of TrkA. TrkA-GFP becomes phosphorylated on tyrosine residues in response to NGF and is capable of initiating signaling cascades leading to prolonged MAPK activation and differentiation in PC12 nnr5 cells. TrkA constructs, progressively truncated in the carboxyl-terminal domain, were prepared as GFP chimerae in order to identify which part of the receptor intracellular domain is involved in its trafficking. Immunofluorescence observations show that TrkA-GFP is found mainly in cell surface membrane ruffles and in endosomes. Biochemical analysis indicated that the cytoplasmic domain of TrkA is not necessary for correct maturation and cell surface translocation of the receptor. An antibody against the extracellular domain of TrkA (RTA) was used as ligand to stimulate internalization and phosphorylation of TrkA. Co-localization studies with anti-phosphorylated TrkA antibodies support a role for such complexes in the propagation of signaling from the cell surface, resulting in the activation of TrkA in areas of the endosome devoid of receptor-ligand complexes. Confocal time-lapse analysis reveals that the TrkA-GFP chimera shows highly dynamic trafficking between the cell surface and internal locations. TrkA-positive vesicles were estimated to move 0.46 +/- 0.09 microm/s anterograde and 0.48 +/- 0.07 microm/s retrograde. This approach and the fidelity of the biochemical properties of the TrkA-GFP demonstrate that real-time visualization of trafficking of tyrosine kinase receptors in the presence or absence of the ligand is feasible.
A growing body of evidence indicates a close relationship between tyrosine kinase receptor trafficking and signaling. Biochemical and molecular analyses of the expression, fate, and kinetics of membrane trafficking of the nerve growth factor (NGF) receptor TrkA were performed in PC12 cells. Pulse-chase experiments indicate that TrkA is synthesized as a 110-kDa N-glycosylated precursor that leads to the mature 140-kDa form of the receptor with a half-life of conversion of approximately 24 +/- 0.5 min. Neuraminidase digestion shows that modification of the carbohydrate moiety of the receptor by sialylation occurs during maturation. The 140-kDa form is rapidly translocated to the cell surface as assessed by cell surface biotinylation performed on intact PC12 cells. Mature receptor half-life is approximately 138 +/- 4 min and is shortened to 86 +/- 8 min by NGF treatment. Flow cytometric analysis indicates that NGF induces clearing of this receptor from the cell surface within minutes of treatment. The addition of NGF decreases the half-life of cell surface gp140(TrkA) from 100 to 35 min and leads to enhanced lysosomal degradation of the receptor. The process of NGF-induced TrkA internalization is clearly affected by interfering with ligand binding to p75(NTR). An analysis of receptor activation kinetics also shows that receptor signaling primarily takes place from an intracellular location. Together, these data show that the primary effect of NGF treatment is a p75(NTR)-modulated decrease in TrkA transit time at the cell surface.
In dystrophic mice, a model of merosin-deficient congenital muscular dystrophy, laminin-2 mutations produce peripheral nerve dysmyelination and render Schwann cells unable to sort bundles of axons. The laminin receptor and the mechanism through which dysmyelination and impaired sorting occur are unknown. We describe mice in which Schwann cell-specific disruption of beta1 integrin, a component of laminin receptors, causes a severe neuropathy with impaired radial sorting of axons. beta 1-null Schwann cells populate nerves, proliferate, and survive normally, but do not extend or maintain normal processes around axons. Interestingly, some Schwann cells surpass this problem to form normal myelin, possibly due to the presence of other laminin receptors such as dystroglycan and alpha 6 beta 4 integrin. These data suggest that beta 1 integrin links laminin in the basal lamina to the cytoskeleton in order for Schwann cells to ensheath axons, and alteration of this linkage contributes to the peripheral neuropathy of congenital muscular dystrophy.
To study the development of the cerebellum, we generated a transgenic mouse line Tg(malpha6-cre)B1LFR that expresses CRE recombinase under the GABA(A) receptor alpha6 subunit promoter. In this line, recombination of an R26R reporter allele occurred postnatally in granule cells of the cerebellum and dorsal cochlear nucleus, as well as in a subset of precerebellar nuclei in the brainstem. All neurons in which recombination occurred originated during embryogenesis from the rhombic lip. This might be explained by a very early specification event at the rhombic lip that primes cells derived from this structure to express the transgene during neuronal maturation. As no recombination occurred in the inferior olive, it may be derived from a distinct subset of precursors at the rhombic lip. No recombination occurred in any of the interneurons in the cerebellum (stellate cells, basket cells, and Golgi cells), consistent with the hypothesis that they are not derived from the same embryonic tissue as the granule cells.
Neurotrophins are essential to the normal development and maintenance of the nervous system. Neurotrophin signaling is mediated by Trk family tyrosine kinases such as TrkA, TrkB and TrkC, as well as by the pan-neurotrophin receptor p75NTR. Here we have deleted the trkB gene in cerebellar precursors by Wnt1-driven Cre--mediated recombination to study the function of the TrkB in the cerebellum. Despite the absence of TrkB, the mature cerebellum of mutant mice appears similar to that of wild type, with all types of cell present in normal numbers and positions. Granule and Purkinje cell dendrites appear normal and the former have typical numbers of excitatory synapses. By contrast, inhibitory interneurons are strongly affected: although present in normal numbers, they express reduced amounts of GABAergic markers and develop reduced numbers of GABAergic boutons and synaptic specializations. Thus, TrkB is essential to the development of GABAergic neurons and regulates synapse formation in addition to its role in the development of axon terminals.