Neuronal expression of Fig4 is both necessary and sufficient to prevent spongiform neurodegeneration.

FIG4 is a ubiquitously expressed phosphatase that, in complex with FAB1/PIKFYVE and VAC14, regulates the biosynthesis of the signaling lipid PI(3,5)P(2). Null mutation of Fig4 in the mouse results in spongiform degeneration of brain and peripheral ganglia, defective myelination and juvenile lethality. Partial loss-of-function of human FIG4 results in a severe form of Charcot-Marie-Tooth neuropathy. Neurons from null mice contain enlarged vacuoles derived from the endosome/lysosome pathway, and astrocytes accumulate proteins involved in autophagy. Other cellular defects include astrogliosis and microgliosis. To distinguish the contributions of neurons and glia to spongiform degeneration in the Fig4 null mouse, we expressed Fig4 under the control of the neuron-specific enolase promoter and the astrocyte-specific glial fibrillary acidic protein promoter in transgenic mice. Neuronal expression of Fig4 was sufficient to rescue cellular and neurological phenotypes including spongiform degeneration, gliosis and juvenile lethality. In contrast, expression of Fig4 in astrocytes prevented accumulation of autophagy markers and microgliosis but did not prevent spongiform degeneration or lethality. To confirm the neuronal origin of spongiform degeneration, we generated a floxed allele of Fig4 and crossed it with mice expressing the Cre recombinase from the neuron-specific synapsin promoter. Mice with conditional inactivation of Fig4 in neurons developed spongiform degeneration and the full spectrum of neurological abnormalities. The data demonstrate that expression of Fig4 in neurons is necessary and sufficient to prevent spongiform degeneration. Therapy for patients with FIG4 deficiency will therefore require correction of the deficiency in neurons.

Taxane-containing induction chemotherapy followed by definitive chemoradiotherapy. Outcome in patients with locally advanced head and neck cancer.

BACKGROUND: Induction chemotherapy followed by definitive chemoradiotherapy is an intensified treatment approach for locally advanced squamous cell carcinoma of the head and neck (HNSCC) that might be associated with high rates of toxicity. MATERIALS AND METHODS: The data of 40 consecutive patients who underwent induction chemotherapy with docetaxel-containing regimens followed by intensity-modulated radiotherapy (IMRT) and concomitant systemic therapy for unresectable locally advanced HNSCC were retrospectively analyzed. Primary objectives were RT-related acute and late toxicity. Secondary objectives were response to induction chemotherapy, locoregional recurrence-free survival (LRRFS), overall survival (OS), and influencing factors for LRRFS and OS. RESULTS: The median follow-up for surviving patients was 21 months (range, 2-53 months). Patients received a median of three cycles of induction chemotherapy followed by IMRT to 72 Gy. Three patients died during induction chemotherapy and one during chemoradiotherapy. Acute RT-related toxicity was of grade 3 and 4 in 72 and 3 % of patients, respectively, mainly dysphagia and dermatitis. Late RT-related toxicity was mainly xerostomia and bone/cartilage necrosis and was of grade 3 and 4 in 15 % of patients. One- and 2-year LRRFS and OS were 72 and 49 % and 77 and 71 %, respectively. CONCLUSION: Induction chemotherapy followed by chemoradiotherapy using IMRT was associated with a high rate of severe acute and late RT-related toxicities in this selected patient cohort. Four patients were lost because of fatal complications. Induction chemotherapy did not compromise the delivery of full-dose RT; however, the use of three cycles of concomitant cisplatin was impaired.

Binding between the neural cell adhesion molecules axonin-1 and Nr-CAM/Bravo is involved in neuron-glia interaction.

Neural cell adhesion molecules of the immunoglobulin superfamily mediate cellular interactions via homophilic binding to identical molecules and heterophilic binding to other family members or structurally unrelated cell-surface glycoproteins. Here we report on an interaction between axonin-1 and Nr-CAM/Bravo. In search for novel ligands of axonin-1, fluorescent polystyrene microspheres conjugated with axonin-1 were found to bind to peripheral glial cells from dorsal root ganglia. By antibody blockage experiments an axonin-1 receptor on the glial cells was identified as Nr-CAM. The specificity of the interaction was confirmed with binding studies using purified axonin-1 and Nr-CAM. In cultures of dissociated dorsal root ganglia antibodies against axonin-1 and Nr-CAM perturbed the formation of contacts between neurites and peripheral glial cells. Together, these results implicate a binding between axonin-1 of the neuritic and Nr-CAM of the glial cell membrane in the early phase of axon ensheathment in the peripheral nervous system.

Patterning of cortical efferent projections by semaphorin-neuropilin interactions.

Cortical neurons communicate with various cortical and subcortical targets by way of stereotyped axon projections through the white matter. Slice overlay experiments indicate that the initial growth of cortical axons toward the white matter is regulated by a diffusible chemorepulsive signal localized near the marginal zone. Semaphorin III is a major component of this diffusible signal, and cortical neurons transduce this signal by way of the neuropilin-1 receptor. These observations indicate that semaphorin-neuropilin interactions play a critical role in the initial patterning of projections in the developing cortex.

Engineering a platform for nerve regeneration with direct application to nerve repair technology.

The development of effective treatment options for repair of peripheral nerves is complicated by lack of knowledge concerning the interactions between cells and implants. A promising device, the multichannel scaffold, incorporates microporous channels, aligning glia and directing axonal growth across a nerve gap. To enhance clinical outcomes of nerve repair, a platform, representative of current implant technology, was engineered which 1) recapitulated key device features (porosity and linearity) and 2) demonstrated remyelination of adult neurons. The in vitro platform began with the study of Schwann cells on porous polycaprolactone (PCL) and poly(lactide co-glycolide) (PLGA) substrates. Surface roughness determined glial cell attachment, and an additional layer of topography, 40 µm linear features, aligned Schwann cells and axons. In addition, direct co-culture of sensory neurons with Schwann cells significantly increased neurite outgrowth, compared to neurons cultured alone (naive or pre-conditioned). In contrast to the control substrate (glass), on porous PCL substrates, Schwann cells differentiated into a mature myelinating phenotype, expressing Oct-6, MPZ and MBP. The direct applicability of this platform to nerve implants, including its response to physiological cues, allows for optimization of cell-material interactions, close observation of the regeneration process, and the study of therapeutics, necessary to advance peripheral nerve repair technology.

Neuropilin-2 is a receptor for semaphorin IV: insight into the structural basis of receptor function and specificity.

Neuropilins bind secreted members of the semaphorin family of proteins. Neuropilin-1 is a receptor for Sema III. Here, we show that neuropilin-2 is a receptor for the secreted semaphorin Sema IV and acts selectively to mediate repulsive guidance events in discrete populations of neurons. neuropilin-2 and semaIV are expressed in strikingly complementary patterns during neurodevelopment. The extracellular complement-binding (CUB) and coagulation factor domains of neuropilin-2 confer specificity to the Sema IV repulsive response, and these domains of neuropilin-1 are necessary and sufficient for binding of the Sema III semaphorin (sema) domain. The coagulation factor domains alone are necessary and sufficient for binding of the Sema III immunoglobulin- (Ig-) basic domain and the unrelated ligand, vascular endothelial growth factor (VEGF). Lastly, neuropilin-1 can homomultimerize and form heteromultimers with neuropilin-2. These results provide insight into how interactions between neuropilins and secreted semaphorins function to coordinate repulsive axon guidance during neurodevelopment.

Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.

Continuous renewal of the axonal pathway sensor apparatus by insertion of new sensor molecules into the growth cone membrane.

BACKGROUND: Growth cones at the tips of growing axons move along predetermined pathways to establish synaptic connections between neurons and their distant targets. To establish their orientation, growth cones continuously sample for, and respond to, guidance information provided by cell surfaces and the extracellular matrix. To identify specific guidance cues, growth cones have sensor molecules on their surface, which are expressed differentially during the temporospatial progress of axon outgrowth, at levels that depend on the pattern of neural activity. However, it has not been elucidated whether a change in gene expression can indeed change the molecular composition and, hence, the function of the sensor apparatus of growth cones. RESULTS: We have constructed adenoviral gene transfer vectors of the chicken growth cone sensor molecules axonin-1 and Ng-CAM. Using these vectors, we initiated the expression of axonin-1 and Ng-CAM in rat dorsal root ganglia explants during ongoing neurite outgrowth. Using specific surface immunodetection at varying time points after infection, we found that axonin-1 and Ng-CAM are transported directly to the growth cone and inserted exclusively in the growth cone membrane and not in the axolemma of the axon shaft. Furthermore, we found that axonin-1 and Ng-CAM do not diffuse retrogradely, suggesting that the sensor molecules are integrated into multimolecular complexes in the growth cone. CONCLUSIONS: During axon outgrowth, the pathway sensor apparatus of the growth cone is continuously updated by newly synthesized sensor molecules that originate directly from the transcription/translation machinery. Changes in the expression of sensor molecules may have a direct impact, therefore, on the exploratory function of the growth cone.

[Radiation-induced xerostomia: prevention, treatment, perspectives]. / Xérostomie radio-induite: prevéntion, traitement, perspectives.

Most of head and neck cancer patients will undergo radiotherapy. Xerostomia is probably its most frequent side effect. Subjective and objective criteria allow evaluating and grading xerostomia. New radiotherapy techniques and use of cytoprotectants can help to preserve salivary gland function. Parasym-pathicomimetics and saliva substitutes reduce symptoms. Strict mouth cleaning and fluoride's use prevent teeth deterioration and infections. Important breakthroughs have been made in the pathophysiology of xerostomia and new treatments are developed.

Anatomy of rat semaphorin III/collapsin-1 mRNA expression and relationship to developing nerve tracts during neuroembryogenesis.

Semaphorin III/collapsin-1 (semaIII/coll-1) is a chemorepellent that exhibits a repulsive effect on growth cones of dorsal root ganglion neurons. To identify structures that express semaIII/coll-1 in developing mammals, we cloned the rat homologue and performed in situ hybridization on embryonic, neonatal, and adult rats. The relationship between semaIII/coll-1 mRNA distribution and developing nerve tracts was studied by combining in situ hybridization with immunohistochemistry for markers of growing nerve fibers. At embryonic day 11, semaIII/coll-1 expression was restricted to the olfactory pit, the basal and rostral surface of the telencephalic vesicle, the anlage of the eye, the epithelium of Rathke's pouch, and the somites. At later developmental stages, semaIII/coll-1 mRNA was found to be widely distributed in neuronal as well as in mesenchymal and epithelial structures outside the nervous system. Strong expression was found in the olfactory bulb, retina, lens, piriform cortex, amygdalostriatal area, pons, cerebellar anlage, motor nuclei of cranial nerves, and ventral spinal cord. After birth, mesenchymal staining decreased rapidly and expression became progressively restricted to specific sets of neurons in the central nervous system (CNS). In the mature CNS, semaIII/coll-1 mRNA remains detectable in mitral cells, neurons of the accessory bulb and cerebral cortex, cerebellar Purkinje cells, as well as a subset of cranial and spinal motoneurons. The temporal and spatial expression pattern of semaIII/coll-1 mRNA and its relationship to emerging nerve tracts suggests that semaIII/coll-1 is involved in guiding growing axons towards their targets by forming a molecular boundary that instructs axons to engage in the formation of specific nerve tracts.

Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and trigeminal axons.

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in vitro sensitivity of trigeminal axons to neurotrophins and target-derived diffusible factors and now report on these properties for geniculate axons. GDNF, BDNF, and NT-4, but not NT-3 or NGF, stimulate geniculate axon outgrowth during the ages investigated, embryonic days 12-14. Sensitivity to effective neurotrophins is developmentally regulated and different from that of the trigeminal ganglion. In vitro coculture studies revealed that geniculate axons were repelled by branchial arch explants that were previously shown to be repellent to trigeminal axons (Rochlin and Farbman [1998] J Neurosci 18:6840-6852). In addition, some branchial arch explants and untransfected COS7 cells repelled geniculate but not trigeminal axons. Sema3A, a ligand for neuropilin-1, is effective in repelling geniculate and trigeminal axons, and antineuropilin-1, but not antineuropilin-2, completely blocks the repulsion by arch explants that repel axon outgrowth from both ganglia. Sema3A mRNA is concentrated in branchial arch epithelium at the appropriate time to mediate the repulsion. In Sema3A knockout mice, geniculate and trigeminal afferents explore medial regions of the immature tongue and surrounding territories not explored in heterozygotes, supporting our previous hypothesis that Sema3A-based repulsion mediates the early restriction of sensory afferents away from midline structures.

The serotonin 5-HT4 receptor. 1. Design of a new class of agonists and receptor map of the agonist recognition site.

The design and synthesis of a new class of potent and selective 5-HT4 receptor agonists containing an indole nucleus linked to a carbazimidamide are presented. A conformational study of the 5-HT4 receptor agonists serotonin and zacopride led to the identification of an initial pharmacophore and to the definition of a three-dimensional map of the 5-HT4 agonist recognition site. 1, a representative member of our new class of 5-HT4 receptor agonists, incorporates all reference structural features and matched perfectly with these models. 1 is a highly potent, full agonist at 5-HT4 receptors present in the isolated electrically stimulated guinea pig ileum preparation, with a pD2 value of 8.8, displaying selectivity (ranging from 40- to over 10,000-fold) versus other members of the serotonin receptor family.

The serotonin 5-HT4 receptor. 2. Structure-activity studies of the indole carbazimidamide class of agonists.

A number of substituted indole carbazimidamides were prepared and evaluated as 5-HT4 receptor agonists by using the isolated field-stimulated guinea pig ileum preparation. Their selectivity for the 5-HT4 receptor was established by examining their affinity for other 5-HT receptors using radioligand-binding techniques. Several selective and highly potent full as well as partial agonists emerged from this study. For example, 1b,d were found to be the most potent, full 5-HT4 receptor agonist described so far (EC50 = 0.5 and 0.8 nM, respectively), being 6 and 4 times more potent than serotonin itself. On the other hand, 5b and 1h appeared as partial 5-HT4 receptor agonists in the nonstimulated guinea pig ileum preparation with potencies, evaluated against serotonin action, respectively similar (5b, Ki = 12 nM) to and 300-fold higher (1h, Ki = 0.04 nM) than serotonin.

Adenoviral vector-mediated gene delivery to injured rat peripheral nerve.

Although much progress has been made, current treatments of peripheral nerve damage mostly result in only partial recovery. Local production of neurite outgrowth-promoting molecules, such as neurotrophins and/or cell adhesion molecules, at the site of damage may be used as a new means to promote the regeneration process. We have now explored the ability of an adenoviral vector encoding the reporter gene LacZ (Ad-LacZ) to direct the expression of a foreign gene to Schwann cells of intact and crushed rat sciatic nerves. Infusion of 8 x 10(7) PFU Ad-LacZ in the intact sciatic nerve resulted in the transduction of many Schwann cells with high levels of transgene expression lasting at least up to 12 days following viral vector administration. The efficacy of adenoviral vector delivery to a crushed nerve was investigated using three strategies. Injection of the adenoviral vector at the time of, or immediately after, a crush resulted in the transduction of only a few Schwann cells. Administration of the adenoviral vector the day after the crush resulted in the transduction of a similar number of Schwann cells 5 days after administration, as observed in uncrushed nerves. Regenerating nerve fibers were closely associated with beta-galactosidase-positive Schwann cells, indicating that the capacity of transduced Schwann cells to guide regenerating fibers was not altered. These results imply that the expression of growth-promoting proteins through adenoviral vector-mediated gene transfer may be a realistic option to promote peripheral nerve regeneration.

Adenovirus-mediated gene transfer in neurons: construction and characterization of a vector for heterologous expression of the axonal cell adhesion molecule axonin-1.

By homologous recombination, a first-generation adenovirus-based gene transfer vector, AdCMVax-1, was constructed as a means of manipulating the expression level of the axonal cell adhesion molecule axonin-1 in neurons and glial cells. AdCMVax-1 harbours the entire coding region of the chicken axonin-1 cDNA under the transcriptional control of the Cytomegalovirus enhancer/promoter in the early-region 1 of the viral genome. Characterization of AdCMVax-1 in vitro revealed highly efficient gene transfer and expression of recombinant axonin-1 in neurons and glial cells of dissociated rat dorsal root ganglia. Similar to its native counterpart, virus-derived axonin-1 was detected on the cell body, neurites, and growth cones of transduced neurons, occurred in a secreted and membrane-associated form, and could be cleaved from the membrane with phosphatidylinositol-specific phospholipase C. Functional characterization of recombinant axonin-1 revealed the same binding properties as previously reported for native axonin-1 isolated from the vitreous fluid of chicken embryos. In vivo gene transfer was studied by stereotactic injection of AdCMVax-1 in the dentate gyrus of the hippocampus and the facial nucleus in the brainstem of adult Wistar rats and revealed high level expression of recombinant axonin-1 in a subset of hippocampal neurons and motor neurons in the facial nucleus.

Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS.

In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied, following direct injection in several regions of the central and peripheral nervous system. The distribution of the cells expressing the transgene appears to be determined by natural anatomical boundaries and possibly by the degree of myelinization of a particular brain region. In highly myelinated areas with a compact cellular structure (e.g. the cortex and olfactory bulb) the spread of the viral vector is limited to the region close to the injection needle, while in areas with a laminar structure (e.g. the hippocampus and the eye) more widespread transgene expression is observed. Retrograde transport of the viral vector may serve as an attractive alternative route of transgene delivery. A time course of expression of beta-galactosidase in neural cells in the facial nucleus revealed high expression during the first week after AdLacZ injection. However, a significant decline in transgene expression during the second and third week was observed. This may be caused by an immune response against the transduced cells or by silencing of the cytomegalovirus promoter used to drive transgene expression. Taken together, the data underscore that for each application of adenoviral vectors as gene transfer agents in the nervous system it is important to examine vector spread in and infectability of the neural structure that is subject to genetic modification.

Differential expression of the mRNAs of the axonal glycoproteins axonin-1 and NgCAM in the developing chick retina.

Cell adhesion molecules expressed on the axonal membrane have been thought to be involved in the guidance of axons to their target area. In the chick, axonin-1 and NgCAM have been shown to promote, through reciprocal interactions, neurite outgrowth in vitro. We have recently shown that chick retinal ganglion cells (RGC) express both proteins as early as the axonal elongation begins. Their expression continues throughout the development of the retinotectal system synchronously with the chronotopic spread of axons. To further investigate the spatiotemporal distribution of axonin-1 and NgCAM in the retina, we have analysed the expression of their mRNAs in the present study. From stage 36 (E10) until hatching photoreceptors express axonin-1 but not NgCAM. In the inner nuclear layer groups of amacrine cells were strongly labelled with both probes but they seemed to belong to different subgroups. These patterns of expression might indicate a differential influence of the two proteins on the development of the local neural circuits of the retina.

Pharmacological profile of the abeorphine 201-678, a potent orally active and long lasting dopamine agonist.

The central dopaminergic effects of an abeorphine derivative 201-678 were compared to those of apomorphine and bromocriptine in different model systems. After oral administration, this compound induced contralateral turning in rats with 6-hydroxydopamine induced nigral lesions and exhibited strong anti-akinetic properties in rats with 6-hydroxydopamine induced hypothalamic lesions. It decreased dopamine metabolism in striatum and cortex, but did not modify noradrenaline and serotonin metabolism in the rat brain. 201-678 counteracted the in vivo increase of tyrosine hydroxylase activity induced by gamma-butyrolactone. In vitro it stimulated DA-sensitive adenylate cyclase and inhibited acetylcholine release from rat striatal slices. This compound had high affinity for 3H-dopamine and 3H-clonidine binding sites. These results indicate that 201-678 is a potent, orally active dopamine agonist with a long duration of action. Furthermore it appears more selective than other dopaminergic drugs.

Hepatic branch vagotomy enhances glucoprivic feeding in food-deprived old rats.

Glucoprivic feeding induced by intraperitoneal (IP) injection of 2-deoxy-D-glucose (2-DG, 250 mg/kg body weight) in the middle of the light phase was investigated in old (age: 15-16 months) and young (age; 2.5-3.5 months) hepatic branch-vagotomized (HBV) and sham-vagotomized (SV) rats. Rats were fed either a carbohydrate-rich diet or a fat-enriched diet with a moderate carbohydrate content. The glucoprivic feeding response was greater in 13-h food-deprived old HBV rats than in 13-h food-deprived old SV rats on both diets. 2-Deoxy-D-glucose produced a greater feeding response when rats were fed the fat-enriched diet. Independent of the diet, the transient hyperphagia induced by 2-DG was followed by a long-term hypophagia in old SV rats, but not in old HBV rats. In 13-h food-deprived young rats, hepatic branch vagotomy did not affect the changes in food intake induced by 2-DG. In undeprived old and young rats, the feeding response to 2-DG, exceeding that of deprived rats, was also not affected by HBV. It is concluded that under certain conditions hepatic branch vagotomy eliminates a 2-DG-induced signal inhibiting food intake and thus enhances glucoprivic feeding. The feeding response to 2-DG therefore seems to depend on stimuli affecting food intake in an antagonistic manner.