Glyphosate exposure induces synaptic impairment in hippocampal neurons and cognitive deficits in developing rats.

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.

Wnt7b signalling through Frizzled-7 receptor promotes dendrite development by coactivating CaMKII and JNK.

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.

Role of Wnt/ß-catenin pathway in the nucleus accumbens in long-term cocaine-induced neuroplasticity: a possible novel target for addiction treatment.

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/ß-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 ß-catenin (final effector of the pathway) in the nucleus accumbens (NAcc), together with a decrease in the activity of glycogen synthase kinase 3ß (GSK3ß). 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/ß-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.

Wnt/ß-catenin pathway in the prefrontal cortex is required for cocaine-induced neuroadaptations.

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 ß-catenin levels in the prefrontal cortex (PFC), amygdala (Amyg) and dorsal striatum (CPu) are decreased in animals that show sensitization. Accordingly, GSK3ß activity levels are increased in the same areas. Moreover, ß-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 ß-catenin levels with a systemic treatment of a GSK3ß inhibitor (Lithium Chloride) or inhibit Wnt/ß-catenin pathway with an intracerebral infusion of Sulindac before each cocaine injection. As expected, rescuing ß-catenin levels in the PFC as well as CPu and Amyg blocks cocaine-induced sensitization, while decreasing ß-catenin levels exclusively in the PFC exacerbates it. Therefore, our results demonstrate a new role for the Wnt/ß-catenin pathway as a required neuroadaptation in inducing behavioral sensitization.

Signaling across the synapse: a role for Wnt and Dishevelled in presynaptic assembly and neurotransmitter release.

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.

Exposure to glyphosate during pregnancy induces neurobehavioral alterations and downregulation of Wnt5a-CaMKII pathway.

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.

Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development.

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.

Neuronal development and axon growth are altered by glyphosate through a WNT non-canonical signaling pathway.

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.

Wnt signalling tunes neurotransmitter release by directly targeting Synaptotagmin-1.

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.

WNT signaling in neuronal maturation and synaptogenesis.

The Wnt signaling pathway plays a role in the development of the central nervous system and growing evidence indicates that Wnts also regulates the structure and function of the adult nervous system. Wnt components are key regulators of a variety of developmental processes, including embryonic patterning, cell specification, and cell polarity. In the nervous system, Wnt signaling also regulates the formation and function of neuronal circuits by controlling neuronal differentiation, axon outgrowth and guidance, dendrite development, synaptic function, and neuronal plasticity. Wnt factors can signal through three very well characterized cascades: canonical or ß-catenin pathway, planar cell polarity pathway and calcium pathway that control different processes. However, divergent downstream cascades have been identified to control neuronal morphogenesis. In the nervous system, the expression of Wnt proteins is a highly controlled process. In addition, deregulation of Wnt signaling has been associated with neurodegenerative diseases. Here, we will review different aspects of neuronal and dendrite maturation, including spinogenesis and synaptogenesis. Finally, the role of Wnt pathway components on Alzheimer's disease will be revised.

Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway.

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.