Differential activation of a novel non-canonical, ß-catenin-dependent WNT pathway controls axon guidance decisions at the midline

Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.También presentado al 3rd AXON-meeting, celebrado en Alicante (España) del 11 al 13 de septiembre de 2019.The Wnt pathway is an essential signaling cascade that regulates many developmental processes including cell proliferation, differentiation and migration as well as axon extension and pathfinding. However, it is still debated which of the two Wnt pathways ⎯ the canonical pathway that involves β-catenin-dependent transcriptional activity or the non-canonical that is independent of β-catenin activity during axon guidance decisions. Here, we show that Wnt5a expressed at the optic chiasm midline enhances the growth of contralaterally-projecting axons independently of β-catenin-mediated transcription and therefore of the canonical pathway. However, we also observe that Wnt5a signaling promotes the accumulation of β-catenin in the growth cone and that interfering with this accumulation stalls the axon of the contralateral neurons at the chiasm indicating that midline crossing requires β-catenin. Strikingly, this positive response of contralateral axons to Wnt5a is switched off upon ectopic expression of the transcription factor Zic2, the main determinant of axon midline avoidance. Transcriptome and chromatin occupancy screens verified that Zic2 controls this Wnt5a-response switch. Our analyses retrieve a set of genes, Fzd8, Apc2, Diversin and Lgr5, that convert the axonal response to Wnt5a from positive to negative. Overall we show that axon steering at the midline requires the inhibition of the Wnt5a-positive signaling to prevent crossing, a signal concomitant with the activation of the Eph/ephrin-repulsive signaling. Our findings clarify outstanding questions concerning the implication of Wnt signaling pathways in axon pathfinding and contribute to a better understanding of the participation of these pathways in other developmental and oncogenic scenarios.Peer reviewe

A Zic2-regulated switch in a non-canonical wnt/ß-catenin pathway is essential for the formation of bilateral circuits

Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celebrado de forma virtual del 18 al 20 de noviembre de 2020.Peer reviewe

A Zic2-regulated switch in a noncanonical Wnt/βcatenin pathway is essential for the formation of bilateral circuits

The Wnt pathway is involved in a wide array of biological processes during development and is deregulated in many pathological scenarios. In neurons, Wnt proteins promote both axon extension and repulsion, but the molecular mechanisms underlying these opposing axonal responses are unknown. Here, we show that Wnt5a is expressed at the optic chiasm midline and promotes the crossing of retinal axons by triggering an alternative Wnt pathway that depends on the accumulation of βcatenin but does not activate the canonical pathway. In ipsilateral neurons, the transcription factor Zic2 switches this alternative Wnt pathway by regulating the expression of a set of Wnt receptors and intracellular proteins. In combination with this alternative Wnt pathway, the asymmetric activation of EphB1 receptors at the midline phosphorylates βcatenin and elicits a repulsive response. This alternative Wnt pathway and its Zic2-triggered switch may operate in other contexts that require a two-way response to Wnt ligands.The laboratory of E.H. is funded with the following grants: PID2019-110535GB-I00 from the National Grant Research Program, PROMETEO Program (2020/007) from Generalitat Valenciana, RAF-20191956 from the Ramón Areces Foundation, and ERC-2011-20101109. M.T.L.-C. is the recipient of an FPI fellowship from the National Grant Research Program. J.P.L.-A. research is supported by grants RYC-2015-18056 and RTI2018-102260-B-100 from MICINN co-financed by ERDF. A.B. research is supported by grant SAF2017-87928-R from MICINN co-financed by ERDF. We also acknowledge the financial support received from the “Severo Ochoa” Program for Centers of Excellence in R&D (SEV-2013-0317).Peer reviewe

Recombinant Desmodus rotundus salivary plasminogen activator crosses the blood-brain barrier through a low-density lipoprotein receptor-related protein-dependent mechanism without exerting neurotoxic effects

International audienceBackground and Purpose—Desmoteplase, a recombinant form of the plasminogen activator DSPAα1 from Desmodus rotundus, may offer improved clinical benefits for acute ischemic stroke treatment over the current therapy, recombinant tissue plasminogen activator (rtPA). Accumulating evidence suggests that clinical use of rtPA could be limited by unfavorable properties, including its ability to cross the blood-brain barrier (BBB), thus potentially adding to the pro-excitotoxic effect of endogenous tPA in cerebral parenchyma. Here, to investigate whether desmoteplase may display a safer profile than the structurally-related tPA, both agents were compared for their ability to cross the BBB and promote neurotoxicity. Methods—First, the passage of vascular DSPA and rtPA was investigated in vitro in a model of BBB, subjected or not to oxygen and glucose deprivation. Second, we studied DSPA- and rtPA-mediated effects in an in vivo paradigm of excitotoxic necrosis. Results—The rtPA and desmoteplase cross the intact BBB by LRP-mediated transcytosis. Under conditions of oxygen and glucose deprivation, translocation rates of both compounds increased; however, unlike rtPA, desmoteplase transport remained LRP-dependent. Additionally, neither intracerebral nor intravenous desmoteplase administration enhanced NMDA-induced excitotoxic striatal damage in vivo. Interestingly, intravenous but not intrastriatal coadministration of desmoteplase and rtPA reduced the pro-excitotoxic effect of rtPA. Conclusions—We show that desmoteplase crosses the BBB but does not promote neuronal death. Moreover, intravenous administration of desmoteplase antagonizes the neurotoxicity induced by vascular rtPA. This action may be caused by competition of desmoteplase with rtPA for LRP binding at the BBB, thus effectively blocking rtPA access to the brain parenchyma

Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor–related protein-mediated transcytosis

International audienceBackground—Accumulating evidence demonstrates a critical involvement of tissue-type plasminogen activator (tPA) in pathological and physiological brain conditions. Determining whether and how vascular tPA can cross the blood-brain barrier (BBB) to enter the brain is thus important, not only during stroke but also in physiological conditions. Methods and Results—In the present work, we provide evidence in vivo that intravenous injection of tPA increases NMDA-induced striatal lesion in the absence of BBB leakage. Accordingly, we show that tPA crosses the BBB both after excitotoxic lesion and in control conditions. Indeed, vascular injected tPA can be detected within the brain parenchyma and in the cerebrospinal fluid. By using an in vitro model of BBB, we have confirmed that tPA can cross the intact BBB. Its passage was blocked at 4°C, was saturable, and was independent of its proteolytic activity. We have shown that tPA crosses the BBB by transcytosis, mediated by a member of the LDL receptor–related protein family. Conclusions—We demonstrate that blood-derived tPA can reach the brain parenchyma without alteration of the BBB. The molecular mechanism of the passage of tPA from blood to brain described here could represent an interesting target to improve thrombolysis in stroke

Anti-NR1 N-terminal-domain vaccination unmasks the crucial action of tPA on NMDA-receptor-mediated toxicity and spatial memory.

International audienceFine-tuning of NMDA glutamatergic receptor signalling strategically controls crucial brain functions. This process depends on several ligands and modulators, one of which unexpectedly includes the serine protease tissue-type plasminogen activator (tPA). In vitro, tPA increases NMDA-receptor-mediated calcium influx by interacting with, and then cleaving, the NR1 subunit within its N-terminal domain. Owing to lack of in vivo evidence of the relevance and contribution of this mechanism in physiological and pathological brain processes, active immunisation was developed here in mice, to allow transient and specific prevention of the interaction of tPA with the NR1 subunit. Immunisation significantly reduced the severity of ischemic and excitotoxic insults in the mouse brain. Cognitive function was altered in some, but not all behavioural tasks affected in tPA-deficient mice. Our data demonstrate that in vivo, tPA controls neurotoxicity and the encoding of novel spatial experiences by binding to and cleaving the NMDA receptor NR1 subunit. Interesting therapeutic possibilities for several brain pathologies that involve excitotoxicity may now be envisaged