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Glyphosate is the active ingredient of several widely used herbicide formulations. Studies based on Glyphosate exposure in different experimental models have suggested that the nervous system represented a key target for its toxicity. Previously, we demonstrated that exposure to glyphosate during gestation induces deficits on behavioral and cognitive function in rats. The aim of the present work was to examine whether cognitive dysfunction induced by Glyphosate was connected to changes on synapse formation and maturation. To understand how glyphosate affects synaptic assembly, we performed in vitro assays on cultured hippocampal neurons that were exposed to the herbicide (0.5 or 1 mg/mL) for 5 or 10 days. Biochemical and immunocytochemical approaches revealed that Glyphosate treated neurons showed a decrease on dendritic complexity and synaptic spine formation and maturation. Moreover, results indicated that Glyphosate decreased synapse formation in hippocampal neurons. To evaluate these effects in vivo, pup rats were treated with 35 or 70 mg/kg of Glyphosate from PND 7 to PND 27, every 48 h. Results indicated that Glyphosate postnatal exposure induced cognitive impairments, since recognition and spatial memory were altered. To go further, we evaluated synaptic protein expression and synaptic organization in hippocampus. Images revealed that Glyphosate treatment downregulates synapsin-1, PSD-95, and CaMKII expression, and also decreased PSD-95 clustering in hippocampus. Taken together, these findings demonstrate for the first time that Glyphosate exposure affects synaptic assembly and reduced synaptic protein expression in hippocampus and that likely triggers the impairment of cognitive function and neuronal connectivity.

Glyphosate is the active ingredient of several widely used herbicide formulations. Studies based on Glyphosate exposure in different experimental models have suggested that the nervous system represented a key target for its toxicity. Previously, we demonstrated that exposure to glyphosate during gestation induces deficits on behavioral and cognitive function in rats. The aim of the present work was to examine whether cognitive dysfunction induced by Glyphosate was connected to changes on synapse formation and maturation. To understand how glyphosate affects synaptic assembly, we performed in vitro assays on cultured hippocampal neurons that were exposed to the herbicide (0.5 or 1 mg/mL) for 5 or 10 days. Biochemical and immunocytochemical approaches revealed that Glyphosate treated neurons showed a decrease on dendritic complexity and synaptic spine formation and maturation. Moreover, results indicated that Glyphosate decreased synapse formation in hippocampal neurons. To evaluate these effects in vivo, pup rats were treated with 35 or 70 mg/kg of Glyphosate from PND 7 to PND 27, every 48 h. Results indicated that Glyphosate postnatal exposure induced cognitive impairments, since recognition and spatial memory were altered. To go further, we evaluated synaptic protein expression and synaptic organization in hippocampus. Images revealed that Glyphosate treatment downregulates synapsin-1, PSD-95, and CaMKII expression, and also decreased PSD-95 clustering in hippocampus. Taken together, these findings demonstrate for the first time that Glyphosate exposure affects synaptic assembly and reduced synaptic protein expression in hippocampus and that likely triggers the impairment of cognitive function and neuronal connectivity.

Glyphosate-based formulations are the most popular herbicide used around the world. These herbicides are widely applied in agriculture to control weeds on genetically modified crops. Although there is much evidence showing that glyphosate-based herbicides induce toxic effect on reproductive and hepatic systems, and also cause oxidative damage on cells, studies from recent years revealed that the nervous system may represent a key target for their toxicity. In the present work, we evaluated the effect of glyphosate (without adjuvants) in neonate rats after gestational exposure. Particularly, we examined whether glyphosate during gestation affected the nervous system function at early development. Pregnant Wistar rats were treated with 24 or 35 mg/kg of pure glyphosate every 48 h and neurobehavioral studies were performed. Our results indicated that gestational exposure to glyphosate induced changes in reflexes development, motor activity and cognitive function, in a dose-dependent manner. To go further, we evaluated whether prenatal exposure to glyphosate affected the Ca+2-mediated Wnt non-canonical signaling pathway. Results indicated that embryos exposed to glyphosate showed an inhibition of Wnt5a-CaMKII signaling pathway, an essential cascade controlling the formation and integration of neural circuits. Taken together, these findings suggest that gestational exposure to glyphosate leads to a downregulation of Wnt/Ca+2 pathway that could induce a developmental neurotoxicity evidenced by deficits at behavioral and cognitive levels in rat pups.

Behavioral sensitization is a progressive and enduring enhancement of the motor stimulant effects elicited by repeated administration of drugs of abuse. It can be divided into two distinct temporal and anatomical domains, termed initiation and expression, which are characterized by specific molecular and neurochemical changes. This study examines the role of the Wnt canonical pathway mediating the induction of cocaine sensitization. We found that β-catenin levels in the prefrontal cortex (PFC), amygdala (Amyg) and dorsal striatum (CPu) are decreased in animals that show sensitization. Accordingly, GSK3β activity levels are increased in the same areas. Moreover, β-catenin levels in nuclear fraction, mRNA expression of Axin2 and Wnt7b are decreased in the PFC of sensitized animals. Then, in order to demonstrate that changes in the PFC are crucial for initiation of sensitization, we either rescue β-catenin levels with a systemic treatment of a GSK3β inhibitor (Lithium Chloride) or inhibit Wnt/β-catenin pathway with an intracerebral infusion of Sulindac before each cocaine injection. As expected, rescuing β-catenin levels in the PFC as well as CPu and Amyg blocks cocaine-induced sensitization, while decreasing β-catenin levels exclusively in the PFC exacerbates it. Therefore, our results demonstrate a new role for the Wnt/β-catenin pathway as a required neuroadaptation in inducing behavioral sensitization.

The formation of complex dendritic arbors is crucial for the assembly of functional networks as abnormal dendrite formation underlies several neurodevelopmental and psychiatric disorders. Many extracellular factors have been postulated as regulators of dendritic growth. Wnt proteins play a critical role in neuronal development and circuit formation. We previously demonstrated that Wnt7b acts through the scaffold protein dishevelled 1 (Dvl1) to modulate dendrite arborisation by activating a non-canonical Wnt signalling pathway. Here, we identify the seven-transmembrane frizzled-7 (Fz7, also known as FZD7) as the receptor for Wnt7b-mediated dendrite growth and complexity. Importantly, Fz7 is developmentally regulated in the intact hippocampus, and is localised along neurites and at dendritic growth cones, suggesting a role in dendrite formation and maturation. Fz7 loss-of-function studies demonstrated that Wnt7b requires Fz7 to promote dendritic arborisation. Moreover, in vivo Fz7 loss of function results in dendritic defects in the intact mouse hippocampus. Furthermore, our findings reveal that Wnt7b and Fz7 induce the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and JNK proteins, which are required for dendritic development. Here, we demonstrate that Wnt7b-Fz7 signals through two non-canonical Wnt pathways to modulate dendritic growth and complexity.

Cocaine addiction is a chronic relapsing disorder characterized by the loss of control over drug-seeking and taking, and continued drug use regardless of adverse consequences. Despite years of research, effective treatments for psycho-stimulant addiction have not been identified. Persistent vulnerability to relapse arises from a number of long-lasting adaptations in the reward circuitry that mediate the enduring response to the drug. Recently, we reported that the activity of the canonical or Wnt/β-catenin pathway in the prefrontal cortex (PFC) is very important in the early stages of cocaine-induced neuroadaptations. In the present work, our main goal was to elucidate the relevance of this pathway in cocaine-induced long-term neuroadaptations that may underlie relapse. We found that a cocaine challenge, after a period of abstinence, induced an increase in the activity of the pathway which is revealed as an increase in the total and nuclear levels of β-catenin (final effector of the pathway) in the nucleus accumbens (NAcc), together with a decrease in the activity of glycogen synthase kinase 3β (GSK3β). Moreover, we found that the pharmacological modulation of the activity of the pathway has long-term effects on the cocaine-induced neuroplasticity at behavioral and molecular levels. All the results imply that changes in the Wnt/β-catenin pathway effectors are long-term neuroadaptations necessary for the behavioral response to cocaine. Even though more research is needed, the present results introduce the Wnt canonical pathway as a possible target to manage cocaine long-term neuroadaptations. Fil: Cuesta, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Batuecas, Jorgelina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Severin, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Funes, Alejandrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Rosso, Silvana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Pacchioni, Alejandra Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina

The growth and morphological differentiation of neurons are critical events in the establishment of proper neuronal connectivity and functioning. The developing nervous system is highly susceptible to damage caused by exposure to environmental contaminants. Glyphosate-containing herbicides are the most used agrochemicals in the world, particularly on genetically modified plants. Previous studies have demonstrated that glyphosate induces neurotoxicity in mammals. Therefore, its action mechanism on the nervous system needs to be determined. In this study, we report about impaired neuronal development caused by glyphosate exposure. Particularly, we observed that the initial axonal differentiation and growth of cultured neurons is affected by glyphosate since most treated cells remained undifferentiated after 1 day in culture. Although they polarized at 2 days in vitro, they elicited shorter and unbranched axons and they also developed less complex dendritic arbors compared to controls. To go further, we attempted to identify the cellular mechanism by which glyphosate affected neuronal morphology. Biochemical approaches revealed that glyphosate led to a decrease in Wnt5a level, a key factor for the initial neurite development and maturation, as well as inducing a down-regulation of CaMKII activity. This data suggests that the morphological defects would likely be a consequence of the decrease in both Wnt5a expression and CaMKII activity induced by glyphosate. Additionally, these changes might be reflected in a subsequent neuronal dysfunction. Therefore, our findings highlight the importance of establishing rigorous control on the use of glyphosate-based herbicides in order to protect mammals' health. Fil: Coullery, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Toxicología Experimental; Argentina Fil: Ferrari, María Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Toxicología Experimental; Argentina Fil: Rosso, Silvana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Toxicología Experimental; Argentina

Behavioral sensitization is a progressive and enduring enhancement of the motor stimulant effects elicited by repeated administration of drugs of abuse. It can be divided into two distinct temporal and anatomical domains, termed initiation and expression, which are characterized by specific molecular and neurochemical changes. This study examines the role of the Wnt canonical pathway mediating the induction of cocaine sensitization. We found that β-catenin levels in the prefrontal cortex (PFC), amygdala (Amyg) and dorsal striatum (CPu) are decreased in animals that show sensitization. Accordingly, GSK3β activity levels are increased in the same areas. Moreover, β-catenin levels in nuclear fraction, mRNA expression of Axin2 and Wnt7b are decreased in the PFC of sensitized animals. Then, in order to demonstrate that changes in the PFC are crucial for initiation of sensitization, we either rescue β-catenin levels with a systemic treatment of a GSK3β inhibitor (Lithium Chloride) or inhibit Wnt/β-catenin pathway with an intracerebral infusion of Sulindac before each cocaine injection. As expected, rescuing β-catenin levels in the PFC as well as CPu and Amyg blocks cocaine-induced sensitization, while decreasing β-catenin levels exclusively in the PFC exacerbates it. Therefore, our results demonstrate a new role for the Wnt/β-catenin pathway as a required neuroadaptation in inducing behavioral sensitization.

The functional assembly of the synaptic release machinery is well understood; however, how signalling factors modulate this process remains unknown. Recent studies suggest that Wnts play a role in presynaptic function. To examine the mechanisms involved, we investigated the interaction of release machinery proteins with Dishevelled-1 (Dvl1), a scaffold protein that determines the cellular locale of Wnt action. Here we show that Dvl1 directly interacts with Synaptotagmin-1 (Syt-1) and indirectly with the SNARE proteins SNAP25 and Syntaxin (Stx-1). Importantly, the interaction of Dvl1 with Syt-1, which is regulated by Wnts, modulates neurotransmitter release. Moreover, presynaptic terminals from Wnt signalling-deficient mice exhibit reduced release probability and are unable to sustain high-frequency release. Consistently, the readily releasable pool size and formation of SNARE complexes are reduced. Our studies demonstrate that Wnt signalling tunes neurotransmitter release and identify Syt-1 as a target for modulation by secreted signalling proteins., The mechanisms by which signalling proteins dynamically modulate neurotransmitter release remain poorly understood. Here, Ciani et al. show Wnt signalling influences vesicle pool availability in an activity-dependent manner via direct interactions with Dishevelled-1 and the synaptic vesicle calcium sensor Synaptotagmin-1.

Dendritic arborization is required for proper neuronal connectivity. Rho GTPases have been implicated in the regulation of dendrite development. However, the signaling pathways that impinge on these molecular switches remain poorly understood. Here we show that Wnt7b, which is expressed in the mouse hippocampus, increases dendritic branching in cultured hippocampal neurons. This effect is mimicked by the expression of Dishevelled (Dvl) and is blocked by Sfrp1, a secreted Wnt antagonist. Consistent with these findings, hippocampal neurons from mice lacking Dvl1 show reduced dendritic arborization. Activation of the canonical Wnt-Gsk3beta pathway does not affect dendritic development. In contrast, Wnt7b and Dvl activate Rac and JNK in hippocampal neurons. Dominant-negative Rac, dominant-negative JNK or inhibition of JNK blocks Dvl-mediated dendritic growth. These findings demonstrate a new function for the non-canonical Wnt pathway in dendrite development and identify Dvl as a regulator of Rho GTPases and JNK during dendritic morphogenesis.

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for neurite extension and is known to occur mainly at the growth cone. This report examines whether this is a regulated event controlled by growth factors. The Golgi complex and nascent PPVs of hippocampal neurons in culture were pulse-labeled with fluorescent ceramide. We studied the dynamics of labeled PPVs upon arrival at the axonal growth cone. In controls and cultures stimulated with brain-derived neurotrophic factor (BDNF), PPV clusters persisted in growth cones with a half-life(t1/2) of >14 minutes. Upon challenge with IGF-1,however, fluorescent elements cleared from the growth cones with a t1/2 of only 6 minutes. Plasmalemmal expansion was measured directly as externalization of membrane glycoconjugates in resealed growth cone particles (GCPs) isolated from fetal forebrain. These assays demonstrated that membrane expansion could be stimulated by IGF-1 in a dose-dependent manner but not by BDNF, even though intact, functional BDNF receptor was present on GCPs. Because both BDNF and IGF-1 are known to enhance neurite growth, but BDNF did not stimulate membrane expansion at the growth cone, we studied the effect of BDNF on the IGF-1 receptor. BDNF was found to cause the translocation of the growth-cone-specific IGF-1 receptor subunitβ gc to the distal axon, in a KIF2-dependent manner. We conclude that IGF-1 stimulates axonal assembly at the growth cone, and that this occurs via regulated exocytosis of PPVs. This mechanism is affected by BDNF only indirectly, by regulation of the βgc level at the growth cone.

Initial axonal elongation is essential for neuronal polarization and requires polarized activation of IGF-1 receptors (IGF-1r) and the phosphatidylinositol 3 kinase (PI3k) pathway. Wingless-type family growth factors (Wnts) have also been implied in the regulation of axonal development. It is not known, however, if Wnts have any participation in the regulation of initial axonal outgrowth and the establishment of neuronal polarity. We used cultured hippocampal neurons and growth cone particles (GCPs) isolated from fetal rat brain to show that stimulation with the wingless family factor 3A (Wnt3a) was sufficient to promote neuronal polarization in the absence of IGF-1 or high insulin. We also show that Wnt3a triggered a strong activation of IGF-1r, PI3k, and Akt in developmental Stage 2 neurons and that the presence of activatable IGF-1r and PI3k activation were necessary for Wnt3a polarizing effects. Surface plasmon resonance (SPR) experiments show that Wnt3a did not bind specifically to the IGF-1r. Using crosslinking and immuno-precipitation experiments, we show that stimulation with Wnt3a triggered the formation of a complex including IGF-1r-Wnt3a-Frizzled-7. We conclude that Wnt3a triggers polarization of neurons via cross-activation of the IGF-1r/PI3k pathway upon binding to Fz7. Fil: Bernis, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Oksdath Mansilla, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Dupraz, Sebastian, Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Nieto Guil, Alvaro Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Fernández, Marisa Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Malchiodi, Emilio Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Rosso, Silvana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Quiroga, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Our results show that 2,4-dichlorophenoxyacetic acid (2,4-D) exposure through mother's milk during the period of rapid myelination (from the 15th to the 25th postnatal days) results in a myelin deficit in the pup's brain and demonstrates the vulnerability of the developing central nervous system (CNS) to 2,4-D. After 100 mg/kg 2,4-D administration to dams, brains of male and female rats show a significant diminution of myelin markers such as monohexosylceramide as well as phospholipids and free fatty acids (FFA) and an increase of cholesteryl esters. Histological studies revealed myelin deficit in some brain regions after 2,4-D treatment. These data indicate that 2,4-D, through the mother's milk, alters the myelination process during a specific postnatal period.

Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization., This work was supported by FONDECYT (PICT 05-01697) and grants from FONCyT (PICT 815), Agencia Cordoba Ciencia, and HHMI to A.C. J.A. was supported by a Plan National Grant (SAF 2006-02424), CIBERNED and Comunidad de Madrid (SAL 0202-2006). C.G.-B. was supported by Fondecyt 1095089 and ICM P05-001-F.

The highly complex organization and computational capacity of the nervous system is essential for its function. This complexity is achieved during early development by the intrinsic property of each neuronal cell type and the influence of extracellular cues. The polarized morphology of neurons with elaborate dendritic arbors and long thin axons is critical for brain function. Neurite development is established after neurons extend multiple processes that soon become polarized when one or two processes differentiate into axons and the rest into dendrites. Both axons and dendrites extend and branch in search for their appropriate synaptic targets. Members of the Wnt family of proteins are well-known embryonic morphogens that regulate cell fate decisions during early embryonic patterning. However, new studies show that Wnts also regulate different stages of neurite development from axon outgrowth, pathfinding to axon branching and remodeling. Wnts also modulate the formation of complex dendritic arborization thus increasing the ability of neurons to receive multiple inputs. Finally, Wnts can function as target release molecules that modulate presynaptic axon remodeling and synaptic assembly. Here we discuss advances that shed new light into the function of Wnt signaling in neurite development with particular emphasis on the formation of the vertebrate nervous system.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a potent neurotoxic herbicide widely used in agriculture. The basic mechanisms by which 2,4-D produces cell damage have not yet been determined. In this study we have examined the effects of 2,4-D in primary cultures of cerebellar granule cells in order to obtain insights into the possible mechanisms underlying the toxic effects of this herbicide. The results obtained indicate that a 24-hour exposure to 2,4-D produces a striking and dose-dependent inhibition of neurite extension. This phenomenon is paralleled by a significant reduction in the cellular content of both dynamic and stable microtubules, a disorganization of the Golgi apparatus, and an inhibition in the synthesis of complex gangliosides. Interestingly, 2,4-D inhibits the in vitro polymerization of purified tubulin. Taken together, the present observations raise the possibility that at least one basic mechanism underlying 2,4-D neurotoxicity involves an inhibition of microtubule assembly. That event may cause a decreased neurite outgrowth response, and could also explain the observed differences in the pattern of ganglioside biosynthesis and/or the disorganization of the Golgi apparatus.

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 NH2-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 antisense 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.

The interaction of 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) with human serum albumin (HSA) was studied using fluorescence and differential scanning calorimetry (DSC). Fluorescence displacement of 1-anilino-8-naphtalenesulfonate (ANS) bound to HSA was used to evaluate the binding affinity of 2,4-D to HSA. The binding is associated to a high affinity site of HSA located in the IIIA subdomain. The association constant (Kass) of the herbicide was about 5 microM(-1), several times higher than the affinity found for pharmaceutical compounds. This relatively strong interaction with HSA was evidenced by the increase in HSA protein thermostability induced as consequence of herbicide interaction. 2,4-D induces an increase in the midpoint of thermal denaturation temperature from 60.1 degrees C in herbicide free solution to 75.6 degrees C in full ligand saturating condition. The calorimetric enthalpy and the excess heat capacity also increased upon 2,4-D binding. To investigate the possibility of other/s system/s of 2,4-D transport in blood, besides of HSA, the interaction of the herbicide with lipid monolayers was explored. No interaction was detected with any of the lipids tested. The overall results provided evidence that high affinity 2,4-D-HSA complex exhibits enhanced thermal stability and that HSA is the unique transport system for 2,4-D in blood.

Neonate rats were treated with 2,4-dichlorophenoxyacetic acid (2,4-D) from the 7th or 12th until the 17th or 25th postnatal day. Two drug dosages were used: 70 and 100 mg/kg body weight of 2,4-D. At the 17th day of age, no changes were observed in body weight, protein and DNA content. However, 25-day-old treated pups showed diminutions in body and brain weight, protein and DNA levels, depending on doses and period of treatment. With respect to ganglioside levels, few changes were observed in treated animals until the 17th day of age. However, at the 25th day, with higher dose and longer treatment a diminution in all parameters analyzed was observed. These results suggest a delay in CNS development when pups were exposed to a very severe chemical injury with 2,4-D. On the other hand, when the chemical injury was not too severe, the brain would be capable to trigger biochemical mechanisms producing a plasticity response which is expressed as changes in ganglioside content and composition.

Proper dialogue between presynaptic neurons and their targets is essential for correct synaptic assembly and function. At central synapses, Wnt proteins function as retrograde signals to regulate axon remodeling and the accumulation of presynaptic proteins. Loss of Wnt7a function leads to defects in the localization of presynaptic markers and in the morphology of the presynaptic axons. We show that loss of function of Dishevelled-1 (Dvl1) mimics and enhances the Wnt7a phenotype in the cerebellum. Although active zones appear normal, electrophysiological recordings in cerebellar slices from Wnt7a/Dvl1 double mutant mice reveal a defect in neurotransmitter release at mossy fiber–granule cell synapses. Deficiency in Dvl1 decreases, whereas exposure to Wnt increases, synaptic vesicle recycling in mossy fibers. Dvl increases the number of Bassoon clusters, and like other components of the Wnt pathway, it localizes to synaptic sites. These findings demonstrate that Wnts signal across the synapse on Dvl-expressing presynaptic terminals to regulate synaptic assembly and suggest a potential novel function for Wnts in neurotransmitter release.