Nerve growth factor inhibits sympathetic neurons’ response to an injury cytokine

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors

In postnatal tissues, angiogenesis occurs in nontumoral conditions on appropriate stimuli. In the nervous tissue, hypoxia, neural graft, increased neural function, and synaptic activity are associated with neoangiogenesis. We have investigated the occurrence of neoangiogenesis in the superior cervical ganglia (scg) of newborn rats treated for 8–21 days with 6-hydroxy-dopamine (6-OHDA), nerve growth factor (NGF), or 6-OHDA + NGF. The two latter treatments induced a significant increase in scg size. However, the increase after combined treatment far exceeded that of NGF alone. Similarly, histological and histochemical analysis revealed neuronal hypertrophy and endothelial cell hyperplasia associated with stromal hypertrophy (as described by laminin immunostaining) and increased vascular bed (as revealed by platelet/endothelial cell adhesion molecule-1 immunostaining) in 6-OHDA + NGF-treated pups. NGF, either alone or associated with 6-OHDA, also induced a significant up-regulation of NADPH diaphorase, neuronal nitric oxide synthase, and vascular endothelial growth factor expression in scg neurons. The present investigation suggests that the increase of scg size induced by NGF and 6-OHDA + NGF is associated with neoangiogenesis, and that the induction of vasoactive and angiogenic factors in neurons represents a further and previously undisclosed effect of NGF.

Particles move along actin filament bundles in nerve growth cones.

Organelle movement along actin filaments has been demonstrated in dissociated squid axoplasm [Kurznetsov, S. A., Langford, G.M. & Weiss, D. G. (1992) Nature (London) 356, 722-725 and Bearer, E.L., DeGiorgis, J.A., Bodner, R.A., Kao, A.W. & Reese, T.S. (1993) Proc. Natl. Acad. Sci. USA 90, 11252-11256] but has not been shown to occur in intact neurons. Here we demonstrate that intracellular transport occurs along actin filament bundles in intact neuronal growth cones. We used video-enhanced differential interference contrast microscopy to observe intracellular transport in superior cervical ganglion neurons cultured under conditions that enhance the visibility of actin bundles within growth cone lamellipodia. Intracellular particles, ranging in size from < 0.5-1.5 microns, moved along linear structures (termed transport bundles) at an average maximum rate of 0.48 micron/sec. After particle movement had been viewed, cultures were preserved by rapid perfusion with chemical fixative. To determine whether particle transport occurred along actin, we then used fluorescence microscopy to correlate this movement with actin and microtubule distributions in the same growth cones. The observed transport bundles colocalized with actin but not with microtubules. The rates of particle movement and the association of moving particles with actin filament bundles suggest that myosins may participate in the transport of organelles (or other materials) in intact neurons.

Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents

Experimental lemurs either were infected orally with the agent of bovine spongiform encephalopathy (BSE) or were maintained as uninfected control animals. Immunohistochemical examination for proteinase-resistant protein (prion protein or PrP) was performed on tissues from two infected but still asymptomatic lemurs, killed 5 months after infection, and from three uninfected control lemurs. Control tissues showed no staining, whereas PrP was detected in the infected animals in tonsil, gastrointestinal tract and associated lymphatic tissues, and spleen. In addition, PrP was detected in ventral and dorsal roots of the cervical spinal cord, and within the spinal cord PrP could be traced in nerve tracts as far as the cerebral cortex. Similar patterns of PrP immunoreactivity were seen in two symptomatic and 18 apparently healthy lemurs in three different French primate centers, all of which had been fed diets supplemented with a beef protein product manufactured by a British company that has since ceased to include beef in its veterinary nutritional products. This study of BSE-infected lemurs early in their incubation period extends previous pathogenesis studies of the distribution of infectivity and PrP in natural and experimental scrapie. The similarity of neuropathology and PrP immunostaining patterns in experimentally infected animals to those observed in both symptomatic and asymptomatic animals in primate centers suggests that BSE contamination of zoo animals may have been more widespread than is generally appreciated.

A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat

Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter–IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.

Retardation of skeletal development and cervical abnormalities in transgenic mice expressing a dominant-negative retinoic acid receptor in chondrogenic cells

Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen α1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

Pancreatic β cells synthesize and secrete nerve growth factor

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

On the actions that one nerve cell can have on another: Distinguishing “drivers” from “modulators”

When one nerve cell acts on another, its postsynaptic effect can vary greatly. In sensory systems, inputs from “drivers” can be differentiated from those of “modulators.” The driver can be identified as the transmitter of receptive field properties; the modulator can be identified as altering the probability of certain aspects of that transmission. Where receptive fields are not available, the distinction is more difficult and currently is undefined. We use the visual pathways, particularly the thalamic geniculate relay for which much relevant evidence is available, to explore ways in which drivers can be distinguished from modulators. The extent to which the distinction may apply first to other parts of the thalamus and then, possibly, to other parts of the brain is considered. We suggest the following distinctions: Cross-correlograms from driver inputs have sharper peaks than those from modulators; there are likely to be few drivers but many modulators for any one cell; and drivers are likely to act only through ionotropic receptors having a fast postsynaptic effect whereas modulators also are likely to activate metabotropic receptors having a slow and prolonged postsynaptic effect.

Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population.

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA. In vitro reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.

Cocaine- and amphetamine-regulated transcript in the rat vagus nerve: A putative mediator of cholecystokinin-induced satiety

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the central nervous system. Recent studies have pointed to a role for CART-derived peptides in inhibiting feeding behavior. Although these actions have generally been attributed to hypothalamic CART, it remains to be determined whether additional CART pathways exist that link signals from the gastrointestinal tract to the central control of food intake. In the present study, we have investigated the presence of CART in the rat vagus nerve and nodose ganglion. In the viscerosensory nodose ganglion, half of the neuron profiles expressed CART and its predicted peptide, as determined by in situ hybridization and immunohistochemistry. CART expression was markedly attenuated after vagotomy, but no modulation was observed after food restriction or high-fat regimes. A large proportion of CART-labeled neuron profiles also expressed cholecystokinin A receptor mRNA. CART-peptide-like immunoreactivity was transported in the vagus nerve and found in a dense fiber plexus in the nucleus tractus solitarii. Studies on CART in the spinal somatosensory system revealed strong immunostaining of the dorsal horn but only a small number of stained cell bodies in dorsal root ganglia. The present results suggest that CART-derived peptides are present in vagal afferent neurons sensitive to cholecystokinin, suggesting that the role of these peptides in feeding may be explained partly by mediating postprandial satiety effects of cholecystokinin.

Nerve growth factor is an autocrine factor essential for the survival of macrophages infected with HIV

Nerve growth factor (NGF) is a neurotrophin with the ability to exert specific effects on cells of the immune system. Human monocytes/macrophages (M/M) infected in vitro with HIV type 1 (HIV-1) are able to produce substantial levels of NGF that are associated with enhanced expression of the high-affinity NGF receptor (p140 trkA) on the M/M surface. Treatment of HIV-infected human M/M with anti-NGF Ab blocking the biological activity of NGF leads to a marked decrease of the expression of p140 trkA high-affinity receptor, a concomitant increased expression of p75NTR low-affinity receptor for NGF, and the occurrence of apoptotic death of M/M. Taken together, these findings suggest a role for NGF as an autocrine survival factor that rescues human M/M from the cytopathic effect caused by HIV infection.

Improved cervical smear assessment using antibodies against proteins that regulate DNA replication

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.