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Neuronal cell fate decisions are directed in Drosophila by NUMB, a signaling adapter protein with two protein-protein interaction domains: a phosphotyrosinebinding domain and a proline-rich region (PRR) that functions as an SH3-binding domain. Here we show that there are at least four human NUMB isoforms and that these serve two distinct developmental functions in the neuronal lineage: differentiation (but not proliferation) is promoted by human NUMB protein isoforms with a type I (short) PRR. In contrast, proliferation (but not differentiation) is directed by isoforms that have a type II (long) PRR. The two types of PRR may promote distinct intracellular signaling pathways downstream of the NOTCH receptor during mammalian neurogenesis.During Drosophila peripheral nervous system development, a sensory organ precursor cell divides twice to produce the four cells that form a functional sensory organ (neuron, sheath, hair, and socket). NUMB functions in this lineage to direct specific binary cell fate choices: the IIb vs. IIa fate at the first division and at the second division, neuron vs. sheath as well as hair vs. socket (1). Absence of NUMB results in production of two IIa cells that then divide to give four sockets while ectopic expression of NUMB generates two IIb cells and, subsequently, four neurons and no hairs (2). The fate choices directed by Drosophila NUMB occur in a stereotypical lineage within which NUMB functions solely to control binary differentiation decisions and not the proliferation dynamics of the cells. NUMB has been hypothesized to function in directing cell fate choice by directly interacting with NOTCH and inhibiting NOTCH function (3).The recent cloning of a mammalian NUMB homologue suggested an evolutionarily conserved function for mammalian NUMB (4, 5). We showed that ectopic expression of this mammalian NUMB protein (mNUMB) promotes neuronal commitment in both Drosophila and cultured mammalian cells (4). Recently, it has been observed that a human NUMB homologue (hNUMB) is transported to the nucleus and associates with the modulator of mitogenesis, MDM2 (6, 7). Our previous studies showed no mitogenic component to murine NUMB function (4). Here we report the existence of four hNUMB isoforms. Based on the structure of the proline-rich region (PRR), the NUMB isoforms regulate either cell fate or cellular proliferation, but not both, during mammalian neurogenesis.

We have isolated a human cDNA for the signaling adapter molecule FRS-2/suc1-associated neurotrophic factor target and shown that it is tyrosine-phosphorylated in response to nerve growth factor (NGF) stimulation. Importantly, we demonstrate that the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the SH2 domain containing tyrosine phosphatase SH-PTP-2, the cyclindependent kinase substrate p13 suc1 , and the Src homology 3 (SH3) domain of Src, providing a functional link between TrkA, cell cycle, and multiple NGF signaling effectors. Importantly, overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA receptor mutant reconstitutes the ability of NGF to stop cell cycle progression and to stimulate neuronal differentiation.

Autonomic dysreflexia is a condition that develops after spinal cord injury in which potentially life-threatening episodic hypertension is triggered by stimulation of sensory nerves in the body below the site of injury. Central sprouting of small-diameter primary afferent fibers in the dorsal horn of the spinal cord occurs concurrently with the development of this condition. We propose a model for the development of autonomic dysreflexia in which increased nerve growth factor (NGF) in the injured cord stimulates small-diameter primary afferent fiber sprouting, thereby magnifying spinal sympathetic reflexes and promoting dysreflexia. We identified this population of afferent neurons using immunocytochemistry for calcitonin gene-related peptide. Blocking intraspinal NGF with an intrathecally-delivered neutralizing antibody to NGF prevented small-diameter afferent sprouting in rats 2 weeks after a high thoracic spinal cord transection. In the same rats, this anti-NGF antibody treatment significantly decreased (by 43%) the hypertension induced by colon stimulation. The extent of small-diameter afferent sprouting after cord transection correlated significantly with the magnitude of increases in arterial pressure during the autonomic dysreflexia. Neutralizing NGF in the spinal cord is a promising strategy to minimize the life-threatening autonomic dysreflexia that develops after spinal cord injury.

Tissue pH is an indicator of altered cellular metabolism in diseases including stroke and cancer. Ischemic tissue often becomes acidic due to increased anaerobic respiration leading to irreversible cellular damage. Chemical exchange saturation transfer (CEST) effects can be used to generate pH-weighted magnetic resonance imaging (MRI) contrast, which has been used to delineate the ischemic penumbra after ischemic stroke. In the current study, a novel MRI ratiometric technique is presented to measure absolute pH using the ratio of CEST-mediated contrast from amine and amide protons: amine/amide concentration-independent detection (AACID). Effects of CEST were observed at 2.75 parts per million (p.p.m.) for amine protons and at 3.50 p.p.m. for amide protons downfield (i.e., higher frequency) from bulk water. Using numerical simulations and in vitro MRI experiments, we showed that pH measured using AACID was independent of tissue relaxation time constants, macromolecular magnetization transfer effects, protein concentration, and temperature within the physiologic range. After in vivo pH calibration using phosphorus ((31)P) magnetic resonance spectroscopy ((31)P-MRS), local acidosis is detected in mouse brain after focal permanent middle cerebral artery occlusion. In summary, our results suggest that AACID represents a noninvasive method to directly measure the spatial distribution of absolute pH in vivo using CEST MRI.

Neurotrophins signal via Trk tyrosine kinase receptors and a common receptor called p75. Nerve growth factor is the cognate ligand for TrkA, brain-derived neurotrophic factor for TrkB, and neurotrophin-3 (NT-3) for TrkC. NT-3 also binds TrkA and TrkB as a heterologous ligand. All neurotrophins bind p75, which regulates ligand affinity and Trk signals. Trk extracellular domain has five subdomains: a leucine-rich motif, two cysteinerich clusters, and immunoglobulin-like subdomains IgG-C1 and IgG-C2. The IgG-C1 subdomain is surface exposed in the tertiary structure and regulates ligandindependent activation. The IgG-C2 subdomain is less exposed but regulates cognate ligand binding and Trk activation. NT-3 as a heterologous ligand of TrkA and TrkB optimally requires the IgG-C2 but also binds other subdomains of these receptors. When p75 is co-expressed, major changes are observed; NGF-TrkA activation can occur also via the cysteine 1 subdomain, and brain-derived neurotrophic factor-TrkB activation requires the TrkB leucine-rich motif and cysteine 2 subdomains. We propose a two-site model of Trk binding and activation, regulated conformationally by the IgG-C1 subdomain. Moreover, p75 affects Trk subdomain utilization in ligand-dependent activation, possibly by conformational or allosteric control.

Two distinct nerve growth factor receptor (NGFR) complexes are present on NGF-responsive cell types; these correspond to 100 kDa and 158 kDa for the fast (fNGFR) and the slow (sNGFR) NGFRs, respectively. Previous studies indicate that each complex is derived from a separate gene product and that the sNGFR contains tyrosine kinase activity.The cDNA encoding the fNGFR has previously been cloned. In this report, a rat trk protooncogene cDNA has been isolated from PC12 cells and Trk has been shown to bind NGF, generating a complex of 158 kDa. Characterization of NGFTrk interactions indicates that Trk and NGF dissociate more slowly than do NGF and the fNGFR. Moreover, NGF-bound Trk is not destroyed by trypsin digestion whereas the NGFfNGFR complex is sensitive to trypsin digestion. These observations suggest that the trk protooncogene product, expressed in the absence of the fNGFR, binds NGF with properties characteristic of the sNGFR, which was identified as the high-affinity NGFR on primary neurons and PC12 cells.Previous work has established that two types of nerve growth factor receptors (NGFRs; type I and type II) can be distinguished on neurons of the peripheral nervous system (1, 2) and on NGF-responsive cell lines such as the rat pheochromocytoma cell line PC12 (3). More recent evidence indicates that both NGFR populations are also present in the central nervous system (4). Type I NGFRs are thought to mediate the biological responses of neuronal survival and neurite outgrowth (5, 6), whereas the role(s) of type II NGFRs is less clear.The most distinguishing feature of the NGFR subtypes is the difference in their rates of NGF dissociation. Kinetic measurements indicate that the rate of NGF dissociation from type I NGFRs is considerably slower than from type II NGFRs (1-3).

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