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Retinal neurogenesis ceases by the early postnatal period, although retinal progenitor cells (RPCs) persist throughout life. In this study, we show that in the mammalian eye, the function of Toll-like receptor 4 (TLR4) extends beyond regulation of the innate immune response; it restricts RPC proliferation. In TLR4-deficient mice, enhanced proliferation of cells reminiscent of RPCs is evident during the early postnatal period. In vitro experiments demonstrate that TLR4 acts as an intrinsic regulator of RPC fate decision. Increased TLR4 expression in the eye correlates with the postnatal cessation of cell proliferation. However, deficient TLR4 expression is not sufficient to extend the proliferative period but rather contributes to resumption of proliferation in combination with growth factors. Proliferation in vivo is inhibited by both MyD88-dependent and -independent pathways, similar to the mechanisms activated by TLR4 in immune cells. Thus, our study attributes a novel role to TLR4 as a negative regulator of RPC proliferation.

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TENT [H.sub.c]). Morphological and kinetic analysis of TeNT [H.sub.c] retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT [H.sub.c] carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT [H.sub.c] and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor [p7.sup.5NTR]. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.

Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3 localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones. Key words: MRNA localization, beta-actin, neurotrophin, growth cone, neurite outgrowth

An amputated axon will regenerate if a growth cone forms at the tip of a transected axon, which transforms from a stable segment into a motile growth cone of irregular shape. However, the mechanisms involved in the regeneration process are little understood. A new study investigates the theory that the regeneration process may involve Ca2+ activated proteinases. It is suggested that localized proteolytic activity plays an important part in axotomy-induced growth cone formation.

A study on oligodendrocyte precursor cells was conducted to determine why precursor cells in the same age nerve have different proliferative properties. Results demonstrate that the differences in precursor cell maturation greatly affect the heterogeneous proliferative behavior of the cells. These suggest that maturation is an inherent property of the cells since these cells progressively mature as they proliferate. Findings also indicate that precursor cells have to reach threshold size before dividing. However, these cells sometimes divide unequally.

The role of sialic acid-binding epitope of myelin-associated glycoprotein (MAG) on neurite outgrowth was demonstrated through site-directed mutagenesis. Results show that the neurite inhibitory activity of MAG is distinct from its sialic acid site since by itself, sialic acid binding is not sufficient to effect inhibition of axonal growth. MAG's sialic acid-dependent binding is lost when it is mutated to either aspartic acid or alanine. Meanwhile MAG retains its ability to inhibit axonal regeneration when it is expressed at the surface of either Schwann or Chinese hamster ovary cells.

The motility and morphology of neuronal growth cones of gelsolin-null (GSn-) mice and wild-type mice were compared to determine the role of gelsolin in neurite elongation. Time-lapse video microscopy was used to record the behavior of the dynamic protrusions called filopodia in cultured hippocampal neurons. Results show that a greater number of filopodia is observed along neurites of GSn- mice. These support the view that gelsolin has a critical function in filopodial retraction.

In situ hybridization analysis of the mitotic motor protein CHO1/MKLP1 revealed that it is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Results suggest that the protein establishes a nonuniform microtubule polarity in the dendrite by transferring microtubules with leading minus ends. Data indicate that microtubule transport is common to both mitosis and neuronal process formation, with CHO1/MKLP1 used for both processes.

Compartmented cell cultures were utilized for the application of neurotrophic growth factor (NGF) to distal neurites and characterize NGF-induced tyrosine phosphorylation. The tyrosine phosphorylation of receptor tyrosine kinase (trk) and related proteins in the cell bodies or proximal neurites was induced by the binding of NGF to distal neurites. Furthermore, a propagation mechanism mediated the retrograde signals of NGF.

The effects of cytoskeleton-associated protein-23 (CAP-23) and growth-associated protein 43 (GAP-43) on neuronal growth and sprouting were analyzed in transgenic mice neurons expression CAP-23. The CAP-23-overexpressing mice exhibited spontaneous and greatly potentiated nerve sprouting at the neuromuscular junction. Furthermore, double-transgenic mice overexpressing CAP-23 and GAP-43 exhibited dramatic quantitative and qualitative potentiation of sprouting.

The role of axonin-1 and neuron-glia cell adhesion molecules (NgCAM) in mediating neural outgrowth was analyzed by the expression of NgCAM and axonin-1 in myeloma cells. NgCAM/axonin-1 heterodimers were detected by chemical cross-linking experiments with dorsal root ganglia neurons that were cultured on a laminin substratum. Furthermore, neurite outgrowth on NgCAM was inhibited by the suppression of axonin-1 translation indicating the role of growth cone membrane NgCAM/axonin-1 dimers in promoting neural outgrowth.

The adhesivity and inhibitory action of laminin beta2 chain during nuerite growth by motoneurons was analyzed in two native chick laminin isoforms such as laminin-2 (alpha2beta1gamma1) and laminin-4 (alpha2beta2gamma1). The native chick laminins promote the neural outgrowth of motor neurons during neural development. The activities of alpha2beta1gamma1 and alpha2beta2gamma1 were also mediated by neuronal integrins of the beta1 family. Furthermore, antibodies against the alpha subunits of laminin-1 receptors did not alter neurite outgrowth on chick laminin-2 and -4.

The signaling mechanisms that affect cortical ventricular zone neurogenesis were analyzed in cloned G-protein-coupled receptor genes that were derived from neocortical neuroblast cell lines. Cortical ventricular zone neurogenesis is mediated by the expression of the ventricular zone gene-1 (vzg-1) G-protein-coupled receptor gene. The restricted expression of vzg-1 in the cerebral cortical ventricular zone increased the levels of lysophosphatidic acid signaling molecules which promote high levels of non-specific binding.

Functional and molecular analysis of NrCAM neurofascin was conducted to determine its role in neurite development. Immobilized substrates of neurofascin act as promoting environments for the outgrowth of neurites. The NrCAM neuorfascin also mediates neurite outgrowth by acting as a cellular receptor for tectal neurons for the adhesion and elongation of neurites on the immobilized substrate. Furthermore, the F11 gene mediates neurite extension in the immobilized substrate.

A novel method was developed for the analysis of axonal tubulin transport during growth by utilizing compartmented cell cultures with newborn rat sympathetic neurons under silicone grease barriers. Immunoprecipitation with monoclonal antibodies was utilized to differentiate tubulin from other proteins. The quantitative analysis of the axoplasmic transport of labeled tubulin from the cell bodies to the proximal axons into the distal axons was conducted by utilizing the novel approach.

The heterophilic interactions between DM-GRASP and NgCAM are involved in the extension of neurites in the developing immune and nervous system. GRASP is a transmembrane protein belonging to the immunoglobulin super family of cell adhesion molecules. Two models have been proposed for the heterophilic interactions between GRASP and NgCAM. According to the trans-binding model, GRASP and NgCAM bind directly across cell membranes to other molecules. In the cis-binding model, the interactions occur in the plane of the membrane.

Neurotrophins regulate the survival and growth of neurons in the cerebellum of hypothyroid rats. Thyroid hormone deficiency increases the mRNA levels of nerve growth factor, neurotrophin (NT)-4 and NT-5, and decreases that of brain-derived neurotrophic factor (BDNF) and NT-3. The administration of NT-3 or BDNF prevents neuronal death and reverses the changes induced by hypothyroidism in the cerebellum. Granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo.

The protooncogene bcl-2 expresses a protein that prevents cell death induced by the presence of trans-retinoic acid in developing neuronal tissue. Retinoic acid induces apoptosis which is characterized by DNA cleavage, chromatin condensation, nuclear fragmentation and cytoplasmic vacuolation. However, retinoic acid and the signaling function of its receptors can also be inhibited by the addition of antisense oligonucleotides, cyclohexamide and actinomycin D.

The extracellular matrix protein thrombospondin-4 (TSP-4) is expressed by neurons and it stimulates the growth of neurites. The immunoreactivity of the TSP-4 protein in the nervous system indicates that this protein might participate in the development of the adult nervous system. This protein is also expressed in the skeletal muscle and in the heart. The TSP-4 protein binds to cell surfaces and promotes neurite activity towards sensory, motor, cerebellar, and retina neurons.

A study conducted on mouse N1E-115 neuroblastoma cells shows that the differentiation of neurons after mitosis is due to suppression of the activity of cyclin dependent kinase (cdk) and phosphorylation of the retinoblastoma gene product (pRb). The decrease in the activity of cdk2 is because of a reduction in its concentration while that of cdk4 is due to the activity of its inhibitor and a reduction in cdk4 phosphorylation. Overexpression of p27(super KIP1) or pRb restores the differentiation of neurons.

A study of the regulation of p75 mRNA and trkA mRNA expression in the growing sympathetic neurons of mouse embryo superior cervical sympathetic ganglion using quantitative reverse transcription shows that retinoic acid does not activate the expression of trkA mRNA or p75 mRNA. The expression of nerve growth factor (NGF) is independent of dipolarization, and sympathetic neurons become sensitive to up-regulate trk mRNA during the early developmental stages. With the increase in age, 90% of neurons start responding to NGF.

The immunoglobulin (Ig)-like protein F11 regulates axonal growth, in a complex interplay with tenascin-R (TN-R), NrCAM, NgCAM, and TN-C. The characterization of the binding regions of Ig and FNIII-like domains in these proteins could shed light on the process by which growth cones choose a specific pathway.

Glial cell line-derived neurotrophic factor (GNDP) mRNA is a new neurotrophic factor pivotal in the development of peripheral tissues and the trophic activities of GNDP protein on populations of developing mammalian and avian peripheral neurons. The physiological trophic role of GNDP is revealed by the expression pattern of GNDP mRNA which correlates with sympathetic, sensory or motor innervation. Low level expressions of GNDP mRNA are also present in developing spinal cord, superior cervical ganglion, dorsal root ganglion and nodose ganglion of rat and embryonic chick.

A specific antisense oligonucleotide procedure helps delete the growth associated protein GAP-43 from primary sensory neurons, and helps define the function of GAP-43 in neurite outgrowth and growth cone activity. GAP-43 influences the induced growth cone spreading and branching. In the absence of GAP-43, the growth cones exhibit null concentration of local f-actin and poor adherence. GAP-43 is essential for stabilizing and promoting adhesion in growth cones.

Analysis of the role of microtubule dynamics in the growth of axons and motility of growth cones by injecting low doses of vinblastine to block the microtubule dynamics reveals that inhibition of microtubule dynamics prevents the forward and backward motility of the growth cones and affects microtubule bundling in the neurons. Inhibition of the microtubule dynamics also prevents entry of polymers into the peripheral lamella and the bundling of microtubules. These structural changes indicate that microtubule dynamics are essential for the axonal growth and growth cone mobility.

Experimental analysis of the function of microtubules during growth cone turning in neurons grown on a laminin-concentrated substrate reveals that the reorientation and bundling of microtubules is an essential step in turning when the growth cone attaches tightly to the substrate. The multi-step turning process requires an adhesion between the lamella and substrate to facilitate the protrusion of the microtubule bundle into the substrate. Recruitment, bundling and expulsion of the microtubule bundles occurs after the formation of stable lamella.

Analysis of the influence of local increases in the cyclic AMP-dependent activity on the neuron growth in cultured Xenopus reveals that local enhancement of cAMP-dependent activity induces a long-range suppressive effect on the distant neuron regions. Local perfusion of forskolin or 3',5'-cyclic monophosphate (8-br-cAMP) into the neuronal growth cones causes the increase in cAMP-dependent activity while soma-restricted local perfusion of 8-br-cAMP alone induces a reduced extension in the growth cones. Combined perfusion of a kinase inhibitor, H-8 and 8-br-cAMP into the growth cones eliminates the long-range inhibitory effect.

Experimental characterization of the signaling mechanisms that mediate integrins- and cadherins-induced neurite outgrowth shows that soluble N-cadherin and laminin (LN) enhance Ca2+ influx in growth cones and neuron cell bodies in a pertussis toxin-independent manner. Ni2+ and diltiazem cooperatively inhibit the voltage-dependent Ca2+ influx pathways in the ciliary ganglion neurons. Cadherin signaling thus includes cell type-specific Ca2+ alterations indicating the presence of different neuronal transduction pathways for the cell adhesion molecules.

Experimental analyses of neurons grown in a medium lacking microtubules due to colchicine or nocodazole drugs and in a medium with microtubule-stabilizing drug, taxol, reveal that neuron growth remains stable and that these neurons activate neurite outgrowth indicating that both processes are independent of microtubule assembly. Microtubules existing during growth of taxol-pretreated neurons comprise of acetylated alpha-tubulin. The microtubule assembly-independent neurite outgrowth indicates the possibility of microtubule assembly translocation to neurites.

Analysis of vinculin-deficient PC12 cell isolates to determine the contribution of the cytoskeletal protein vinculin to the modulation of integrin-dependent neurite outgrowth in the PC12 cells shows that vinculin deficiency reduces the neurite outgrowth, attenuates growth cone development and destabilizes filopodia and lamellipodia. The effects of vinculin deficiency prove that the protein is essential for the cellular processes. Interactions between actin-based cytoskeleton and the integrins are also essential for maintaining the lamellipodial and filopodiol stability.

Analysis of central nervous system in mouse brain extracts using monoclonal antibody 473HD reveals neural chondroitin sulfate proteoglycan DSD-1-PG on the nervous system. DSD-1-PG has a molecular mass ranging from 800 to 1,000 kD and a 350 to 400 kD core glycoprotein complex. DSD-1-PG enhances the neurite development and differentiation of neurons in the central nervous system.

A comparative study of different tyrosine kinase inhibitors' inhibition of neurite outgrowth over parental 3T3 monolayers indicates that the triggering of an erbstatin-sensitive tyrosine kinase is crucial for calcium channel activation, but not for integrin-dependent neurite outgrowth. Erbstatin analogue and tyrophostins 23 and 25 inhibit response triggered by three transfected cell adhesion molecules. Genistein, lavendustin and tyrphostins 34 and 47 do not affect neurite outgrowth.

Analysis of neurite formation in disintegrated neurons of cultured chick dorsal root ganglion elucidates the role of growth-associated protein- (GAP) -43 in neurite outgrowth, development and spreading of growth cones. GAP-43 is a possible candidate for effecting nodulation of axonal cortex and growth cone activity. This nodulation renders transduction of external signal into growth cones, and elicits its response in initiating neurite formation and guidance.

Digitially improved video microtropy which concerns dynamic interactions between action filaments and microtubules is used to analyze the homophilic growth cone-target interactions between Aplysia bag cell neurons. Target contacts can stimulate focal F-action assembly and realignment, and subsequently controls target region-controlled microtubule redistribution.

The role of homeoproteins in the differentiation of chick motoneurons in culture was studied using a system that allowed perturbation of homeoprotein action within individual cells. Motoneurons incubated with the homeobox peptide of the Antennapedia gene (pAntp) took up the peptide and transported it into their nuclei, causing enhanced growth of the culture. A mutant of pAntp that could be internalized and transported into the nucleus but could not bind to homeoprotein cognate binding sites did not promote differentiation of the motoneuron culture.

Negative stain and freeze-etch electron microscopy were used to study the organization and polarity of actin filaments in the growth cones of neurons. The results showed that the three-dimensional structural network of expanding lamellipodia is composed of two subpopulations of actin filaments. One subpopulation is made up of 40-100 nanomete-wide filament bundles thar radiate from the lamellipodia leading edge. The other subpopulation is comprised by branching short filaments filling the space between the dorsal and ventralk membrane surfaces.

An experiment involving transfection of human L1 cells in mouse NIH-3T3 fibroblasts demonstrated that neural cell adhesion molecule and L1 induce neurite outgrowth by activation of a common cell adhesion molecule-specific second messenger pathway. Results alsoshowed that calcium influx directly activated the second messenger pathway thatmimics the neurite outgrowth induced by the cell adhesion molecule. This also indicated that cell adhesion was not an independent function in neurite outgrowth.

The expression of nerve growth factor (NGF) and of brain-derived neurotrophic factor (BDNF) in damaged peripheral nerves were compared. The results of transsection experiments on the rat sciatic nerve showed that BDNF mRNA levels in non-neuronal cells undergo a slow but continuous increase three days after nerve transsection which reaches a maximum about three to four weeks later. The time course and spatial pattern of BDNF mRNA increase differs from those of NGF, indicating that the synthesis of NGF and BDNF are controlled by different mechanisms.

In vitro studies were performed to determine whether a soluble form of the neuronal celladhesion molecule F3, which is normally anchored to membranes, can promote neurite extension in dorsal root ganglia neurons. The results showed that F3 promoted neurite initiation and extension in a dose-dependent manner. Addition of cerebrospinal fluid, a natural F3 source, promoted F3 effects, whereas addition of anti-F3 antibodies inhibited F3 effects. These findings suggest that the soluble form of F3 could act in a manner similar to growth and trophicfactors.

The behavior of axonal microtubules in vivo was studied by analyzing the movement of caged fluorescein-labeled tubulin in mouse sensory neurons and in nerve cells isolated from Xenopus embryos. It was observed that labeled tubulinwas stationary in mouse sensory neurons, but translocated en bloc in the case of Xenopus embryonal neurons. Further analysis of the position of polystyrene beads adhering to neurites indicate that the translocation mode of microtubulesand of some components of the axon surface are different in the two types of neurons, being stationary in the axons of mammalian neurons.

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class [I.sub.A] PI 3-kinase, overexpression of [Ruk.sub.I] (a natural inhibitor of Class [I.sub.A] PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-[x.sub.L] and anti-sense Bcl-2 or by overexpression of Bcl-[x.sub.S], Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an [NH.sub.2-]terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with anti sense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles. Key words: KIF4 * L1-glycoprotein * microtubule-based transport * axons * neuronal polarity

The promotion of neurite outgrowth of retinal ganglion cells is observed when protein tyrosine phosphatase mu (PTPmu) is used as a culture substrate. In addition, retinal cells yielded PTPmu in complex with N-cadherin. The enzymatic activity and the expression level of PTPmu were perturbed in retinal explant cultures to determine the physiological significance of the association between PTPmu and N-cadherin. The results suggested that PTPmu has a dual role in the regulation of the neurite outgrowth.

The mechanism of delivery and integration of new membrane to an axon is investigated using a method that visualizes the transport and exocytosis of vesicles derived from the cell body along the growing axon. Results reveal that local disassembly of microtubules leads to the addition of new membrane to the portion of the neurite where the tubules were disrupted. This indicates that the local destabilization of microtubules is responsible for the transport of membrane material to specific sites in the neuron.