Rosalind A. Segal

p75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp

In inhibiting neurite outgrowth, several myelin components, including the extracellular domain of Nogo-A (Nogo-66), oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG), exert their effects through the same Nogo receptor (NgR). The glycosyl phosphatidylinositol (GPI)-anchored nature of NgR indicates the requirement for additional transmembrane protein(s) to transduce the inhibitory signals into the interior of responding neurons. Here, we demonstrate that p75, a transmembrane protein known to be a receptor for the neurotrophin family of growth factors, specifically interacts with NgR. p75 is required for NgR-mediated signalling, as neurons from p75 knockout mice are no longer responsive to myelin and to each of the known NgR ligands. Blocking the p75–NgR interaction also reduces the activities of these inhibitors. Moreover, a truncated p75 protein lacking the intracellular domain, when overexpressed in primary neurons, attenuates the same set of inhibitory activities, suggesting that p75 is a signal transducer of the NgR–p75 receptor complex. Thus, interfering with p75 and its downstream signalling pathways may allow lesioned axons to overcome most of the inhibitory activities associated with central nervous system myelin.

A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors

The vast majority of brain tumors in adults exhibit glial characteristics. Brain tumors in children are diverse: Many have neuronal characteristics, whereas others have glial features. Here we show that activation of the Gi protein-coupled receptor CXCR4 is critical for the growth of both malignant neuronal and glial tumors. Systemic administration of CXCR4 antagonist AMD 3100 inhibits growth of intracranial glioblastoma and medulloblastoma xenografts by increasing apoptosis and decreasing the proliferation of tumor cells. This reflects the ability of AMD 3100 to reduce the activation of extracellular signal-regulated kinases 1 and 2 and Akt, all of which are pathways downstream of CXCR4 that promote survival, proliferation, and migration. These studies (i) demonstrate that CXCR4 is critical to the progression of diverse brain malignances and (ii) provide a scientific rationale for clinical evaluation of AMD 3100 in treating both adults and children with malignant brain tumors.

Neurotrophins use the Erk5 pathway to mediate a retrograde survival response

Growth factors synthesized and released by target tissues promote survival and differentiation of innervating neurons. This retrograde signal begins when growth factors bind receptors at nerve terminals. Activated receptors are then endocytosed and transported through the axon to the cell body. Here we show that the mitogen-activated protein kinase (MAPK) signaling pathways used by neurotrophins during retrograde signaling differ from those used following direct stimulation of the cell soma. During retrograde signaling, endocytosed neurotrophin receptors (Trks) activate the extracellular signal-related protein kinase 5 (Erk5) pathway, leading to nuclear translocation of Erk5, phosphorylation of CREB, and enhanced neuronal survival. In contrast, Erk1/2, which mediates nuclear responses following direct cell body stimulation, does not transmit a retrograde signal. Thus, the Erk5 pathway has a unique function in retrograde signaling. Differential activation of distinct MAPK pathways may enable an individual growth factor to relay information that specifies the location and the nature of stimulation.Growth factors synthesized and released by target tissues promote survival and differentiation of innervating neurons. This retrograde signal begins when growth factors bind receptors at nerve terminals. Activated receptors are then endocytosed and transported through the axon to the cell body. Here we show that the mitogen-activated protein kinase (MAPK) signaling pathways used by neurotrophins during retrograde signaling differ from those used following direct stimulation of the cell soma. During retrograde signaling, endocytosed neurotrophin receptors (Trks) activate the extracellular signal-related protein kinase 5 (Erk5) pathway, leading to nuclear translocation of Erk5, phosphorylation of CREB, and enhanced neuronal survival. In contrast, Erk1/2, which mediates nuclear responses following direct cell body stimulation, does not transmit a retrograde signal. Thus, the Erk5 pathway has a unique function in retrograde signaling. Differential activation of distinct MAPK pathways may enable an individual growth factor to relay information that specifies the location and the nature of stimulation.

Abnormal Cerebellar Development and Foliation in BDNF−/− Mice Reveals a Role for Neurotrophins in CNS Patterning

While target-derived neurotrophins are required for the survival of developing neurons in the PNS, the functions of neurotrophins in the CNS are unclear. Mice with a targeted gene deletion of brain-derived neurotrophic factor (BDNF) exhibit a wide-based gait. Consistent with this behavioral evidence of cerebellar dysfunction, there is increased death of granule cells, stunted growth of Purkinje cell dendrites, impaired formation of horizontal layers, and defects in the rostral-caudal foliation pattern. These abnormalities are accompanied by decreased Trk activation in granule and Purkinje cells of mutant animals, indicating that both cell types are direct targets for BDNF. These data suggest that BDNF acts as an anterograde or an autocrine-paracrine factor to regulate survival and morphologic differentiation of developing CNS neurons, and thereby affects neural patterning.

Blockade of Endogenous Ligands of TrkB Inhibits Formation of Ocular Dominance Columns

We have examined the hypothesis that the segregation of LGN axon terminals into ocular dominance (OD) patches in layer 4 of the visual cortex requires neurotrophins, acting as signals to modulate the pattern of synaptic connectivity. Neurotrophin receptor antagonists, composed of the extracellular domain of each member of the trk family of neurotrophin receptors fused to a human Fc domain, were infused directly into visual cortex during the peak phase of OD column formation. Infusion of trkB-IgG, which binds BDNF and NT-4/5, inhibited the formation of OD patches within layer 4, while trkA-IgG and trkC-IgG, which preferentially bind NGF and NT-3, respectively, had no effect. The autoradiographic labeling of LGN terminals in cortical layer 4 was reduced by trkB-IgG, in contrast with the increased labeling observed following NT-4/5 infusion. These data suggest that an endogenous ligand of trkB, normally present in limiting amounts within visual cortex, is necessary for the selective growth and remodeling of LGN axons into eye-specific patches.

Retrograde neurotrophin signaling: Trk-ing along the axon

Target-derived neurotrophins are required for the growth and survival of innervating neurons. When released by postsynaptic targets, neurotrophins bind receptors (Trks) on nerve terminals. Activated Trks signal locally within distal axons and retrogradely through long axons to distant cell bodies in order to promote gene expression and survival. Although the mechanism of retrograde neurotrophin signaling is not fully elucidated, considerable evidence supports a model in which the vesicular transport of neurotrophin-Trk complexes transmits a survival signal that involves PI3K and Erk5. Other, non-vesicular modes of retrograde signaling are likely to function in parallel. Recent studies highlight the importance of the location of stimulation as a determinant of Trk signaling. Defects in signaling from distal axons to cell bodies may be causally related to neurodegenerative disorders.

Rapid Nuclear Responses to Target-Derived Neurotrophins Require Retrograde Transport of Ligand–Receptor Complex

Target-derived neurotrophins initiate signals that begin at nerve terminals and cross long distances to reach the cell bodies and regulate gene expression. Neurotrophin receptors, Trks, themselves serve as retrograde signal carriers. However, it is not yet known whether the retrograde propagation of Trk activation reflects movement of Trk receptors from neurites to cell bodies or reflects serial activation of stationary Trk molecules. Here, we show that neurotrophins selectively applied to distal neurites of sensory neurons rapidly induce phosphorylation of the transcription factor cAMP response element-binding protein (CREB) and also cause a slower increase in Fos protein expression. Both nuclear responses require activation of neurotrophin receptors (Trks) at distal nerve endings and retrograde propagation of Trk activation to the nerve cell bodies. Using photobleach and recovery techniques to follow biologically active, green fluorescent protein (GFP)-tagged BDNF receptors (TrkB-GFP) in live cells during retrograde signaling, we show that TrkB-GFP moves rapidly from neurites to the cell bodies. This rapid movement requires ligand binding, Trk kinase activity, and intact axonal microtubules. When they reach the cell bodies, the activated TrkB receptors are in a complex with ligand. Thus, the retrograde propagation of activated TrkB from neurites to cell bodies, although rapid, reflects microtubule-dependent transport of phosphorylated Trk–ligand complexes. Moreover, the relocation of activated Trk receptors from nerve endings to cell bodies is required for nuclear signaling responses. Together, these data support a model of retrograde signaling whereby rapid vesicular transport of ligand–receptor complex from the neurites to the cell bodies mediates the nuclear responses.

Studies on intercellular LETS glycoprotein matrices.

Intercellular matrices secreted by chick embryo fibroblasts in culture were studied by scanning electron microscopy. Cell-cell contact is a prerequisite for the expression of such matrices. The smallest fiber detected by transmission electron microscopy is 5--10 nm in diameter. These matrix fibers tend to cluster to form bundles. Immunofluorescence and immunoferritin procedures reveal that LETS protein is one of the components of the matrices. The matrices are isolated from other cellular organelles by detergent treatment. More than 90% of the proteins in cell-free matrices are LETS protein, suggesting that the matrices are probably made of only one component--LETS protein. Since the solubilization of matrices requires beta-mercaptoethanol, LETS protein matrices may be the first known polymer system in nature to use disulfide linkage as an intermolecular polymerization vehicle. Collagen does not appear to be involved in such matrices. The LETS protein matrix supports the morphological conversion of rounded cells into spindle-shaped, and also promotes myoblast fusion. It does not, however, exert an effect upon cell growth, the rate of glucose uptake or protease production.

Changes in neurotrophin responsiveness during the development of cerebellar granule neurons.

Neurotrophins and their receptors are widespread in the developing and mature CNS. Identifying the differentiation state of neurotrophin-responsive cells provides a basis for understanding the developmental functions of these factors. Studies using dissociated and organotypic cultures of rat cerebellum demonstrated that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) affect developing granule cells at distinct stages in differentiation. While early granule neurons in the external germinal layer responded to BDNF, more mature granule cells responded to NT-3. BDNF, but not NT-3, enhanced survival of granule cells in cultures of embryonic cerebella. Thus, BDNF and NT-3 have distinct sequential functions that are likely to be critical in the development of the cerebellum. BDNF may promote the initial commitment, while NT-3 may direct the subsequent maturation of granule cells.

Dynein motors transport activated Trks to promote survival of target-dependent neurons.

Mutations that alter dynein function are associated with neurodegenerative diseases, but it is not known why defects in dynein-dependent transport impair neuronal survival. Here we show that dynein function in axons is selectively required for the survival of neurons that depend on target-derived neurotrophins. Stimulation of axon terminals with neurotrophins causes internalization of neurotrophin receptors (Trks). Using real-time imaging of fluorescently tagged Trks, we show that dynein is required for rapid transport of internalized, activated receptors from axon terminals to remote cell bodies. When dynein-based transport is inhibited, neurotrophin stimulation of axon terminals does not support survival. These studies indicate that defects in dynein-based transport reduce trafficking of activated Trks and thereby obstruct the prosurvival effect of target-derived trophic factors, leading to degeneration of target-dependent neurons.