While lactate shuttle theory states that glial cells metabolize glucose into lactate to shuttle it to neurons, how glial cells support axonal metabolism and function remains unclear. Lactate production is a common occurrence following anaerobic glycolysis in muscles. However, several other cell types, including some stem cells, activated macrophages and tumor cells, can produce lactate in presence of oxygen and cellular respiration, using Pyruvate Kinase 2 (PKM2) to divert pyruvate to lactate dehydrogenase. We show here that PKM2 is also upregulated in myelinating Schwann cells (mSC) of mature mouse sciatic nerve versus postnatal immature nerve. Deletion of this isoform in PLP-expressing cells in mice leads to a deficit of lactate in mSC and in peripheral nerves. While the structure of myelin sheath was preserved, mutant mice developed a peripheral neuropathy. Peripheral nerve axons of mutant mice failed to maintain lactate homeostasis upon activity, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not altered but axonal mitochondria transport was slowed down, muscle axon terminals retracted and motor neurons displayed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment, which diverts pyruvate to mitochondrial oxidative phosphorylation, further aggravated motor dysfunction in mutant mice. Thus, lactate production through PKM2 enzyme and aerobic glycolysis is essential in mSC for the long-term maintenance of peripheral nerve axon physiology and function.

Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures.

Summary Whether glial cells use a particular metabolism to support axonal metabolism and function remains controversial. We show here that the deletion of PKM2, an enzyme essential for the Warburg effect, in mature myelinating Schwann cells (mSC) leads to a deficit of lactate in these cells and in peripheral nerves, and to motor defects despite no alteration of the myelin sheath. When electrically stimulated, peripheral nerve axons of mSC-PKM2 mutant mice failed to maintain lactate homeostasis, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not changed but axonal mitochondria transport was altered, muscle axon terminals retracted and motor neurons showed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment further aggravated axonal malfunction in mutant mice. Thus, cancer-like Warburg effect is essential in mSC for the long-term maintenance of peripheral nerve axons physiology and function. One Sentence Summary Lactate-dependent axons rely on Warburg effect in Schwann cells.

Charcot-Marie-Tooth disease 1A (CMT1A) results from a duplication of the gene leading to an excess of PMP22, a deficit of myelination and an instability of the myelin sheath in peripheral nerves. Patients present with reduced nerve conduction velocity, muscle waste, hand and foot deformations and foot drop walking problems. As gene silencing therapy has been shown to be effective in other monogenic neurological disorders, we evaluated the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9)-based gene therapy for CMT1A. AAV2/9-mediated delivery of eGFP and shRNAs targeting PMP22 mRNA in the sciatic nerve allowed widespread gene expression in myelinating Schwann cells in mouse, rat and nonhuman primate. The treatment restored wild-type PMP22 level, increased myelination and prevented motor and sensory impairment over 12 months in a rat model of CMT1A. Intra-nerve injection limited off-target transduction and immune response to barely detectable levels. A combination of previously characterized human skin biomarkers successfully discriminated treated animals from their untreated littermate controls indicating their potential use as part of outcome measures in future clinical trials. Our results support intra nerve injection of AAV2/9 as an effective strategy for the treatment of CMT1A as well as other demyelinating CMT diseases.

Affiliations: 1 INM, INSERM, Université de Montpellier, 80 Rue A. Fliche, 34090 Montpellier, France. 2 Aix-Marseille University, INSERM, MMG, 13385 Marseille, France 15 3 Departments of Clinical Neuroscience and Neuroscience, Karolinska Intitutet, Stockholm, Sweden. 4 Département de Biochimie et Génétique, Centre Hospitalier Universitaire, Angers, France. 5 Equipe Mitolab, MITOVASC, CNRS 6015, INSERM U1083, Université d'Angers, Angers, France. 20

Summary: Whether glial cells use a particular metabolism to support axonal metabolism and function remains controversial. We show here that the deletion of PKM2, an enzyme essential for the Warburg effect, in mature myelinating Schwann cells (mSC) leads to a deficit of lactate in these cells and in peripheral nerves, and to motor defects despite no alteration of the myelin 30 sheath. When electrically stimulated, peripheral nerve axons of mSC-PKM2 mutant mice failed to maintain lactate homeostasis, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not changed but axonal mitochondria transport was altered, muscle axon terminals retracted and motor neurons showed cellular stress. Further reducing lactate availability through dichloroacetate treatment definitely aggravated axonal malfunction in 35 mutant mice. Thus, cancer-like Warburg effect is essential in mSC for the long-term maintenance of peripheral nerve axons physiology and function.

Mitochondria are critical for the function and maintenance of myelinated axons notably through Adenosine triphosphate (ATP) production. A direct by-product of this ATP production is reactive oxygen species (ROS), which are highly deleterious for neurons. While ATP shortage and ROS levels increase are involved in several neurodegenerative diseases, it is still unclear whether the real-time dynamics of both ATP and ROS production in axonal mitochondria are altered by axonal or demyelinating neuropathies. To answer this question, we imaged and quantified mitochondrial ATP and hydrogen peroxide (H2O2) in resting or stimulated peripheral nerve myelinated axons in vivo, using genetically-encoded fluorescent probes, two-photon time-lapse and CARS imaging. We found that ATP and H2O2 productions are intrinsically higher in nodes of Ranvier even in resting conditions. Axonal firing increased both ATP and H2O2 productions but with different dynamics: ROS production peaked shortly and transiently after the stimulation while ATP production increased gradually for a longer period of time. In neuropathic MFN2R94Q mice, mimicking Charcot-Marie-Tooth 2A disease, defective mitochondria failed to upregulate ATP production following axonal activity. However, elevated H2O2 production was largely sustained. Finally, inducing demyelination with lysophosphatidylcholine resulted in a reduced level of ATP while H2O2 level soared. Taken together, our results suggest that ATP and ROS productions are decoupled under neuropathic conditions, which may compromise axonal function and integrity.

Trio, a member of the Dbl family of guanine nucleotide exchange factors, activates Rac1 downstream of netrin 1/DCC signalling in axon outgrowth and guidance. Although it has been proposed that Trio also activates RhoA, the putative upstream factors remain unknown. Here, we show that Slit2 induces Trio-dependent RhoA activation, revealing a crosstalk between Slit and Trio/RhoA signalling. Consistently, we found that RhoA activity is hindered in vivo in Trio mutant mouse embryos. We next studied the development of the ventral telencephalon and thalamocortical axons, which have been previously shown to be controlled by Slit2. Remarkably, this analysis revealed that Trio knockout (KO) mice show phenotypes that bear strong similarities to the ones that have been reported in Slit2 KO mice in both guidepost corridor cells and thalamocortical axon pathfinding in the ventral telencephalon. Taken together, our results show that Trio induces RhoA activation downstream of Slit2, and support a functional role in ensuring the proper positioning of both guidepost cells and a major axonal tract. Our study indicates a novel role for Trio in Slit2 signalling and forebrain wiring, highlighting its role in multiple guidance pathways as well as in biological functions of importance for a factor involved in human brain disorders.

Mitochondria are critical for the function and maintenance of myelinated axons notably through ATP production. A by-product of this activity is reactive oxygen species (ROS), which are highly deleterious for neurons. While ROS and metabolism are involved in several neurodegenerative diseases, it is still unclear how axonal activity or myelin modulates ATP and ROS production in axonal mitochondria. We imaged and quantified mitochondrial ATP and hydrogen peroxide (H 2 O 2 ) in resting or stimulated peripheral nerve myelinated axons in vivo , using genetically-encoded fluorescent probes, two-photon time-lapse and CARS imaging. ATP and H 2 O 2 productions are intrinsically higher in nodes of Ranvier even in resting conditions. Axonal firing increased both ATP and H 2 O 2 productions but with different dynamics. In neuropathic MFN2 R94Q mice, mimicking Charcot-Marie-Tooth 2A disease, defective mitochondria failed to upregulate ATP production following axonal activity. However, H 2 O 2 production was dramatically sustained. Mimicking demyelinating peripheral neuropathy resulted in a reduced production of ATP while H 2 O 2 level soared. Taken together, our results suggest that ATP and ROS productions are decoupled under neuropathic conditions, which may compromise axonal function and integrity.

In mammals, thalamic axons are guided internally toward their neocortical target by corridor (Co) neurons that act as axonal guideposts. The existence of Co-like neurons in non-mammalian species, in which thalamic axons do not grow internally, raised the possibility that Co cells might have an ancestral role. Here, we investigated the contribution of corridor (Co) cells to mature brain circuits using a combination of genetic fate-mapping and assays in mice. We unexpectedly found that Co neurons contribute to striatal-like projection neurons in the central extended amygdala. In particular, Co-like neurons participate in specific nuclei of the bed nucleus of the stria terminalis, which plays essential roles in anxiety circuits. Our study shows that Co neurons possess an evolutionary conserved role in anxiety circuits independently from an acquired guidepost function. It furthermore highlights that neurons can have multiple sequential functions during brain wiring and supports a general role of tangential migration in the building of subpallial circuits.

Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.

Le fonctionnement du cortex cerebral repose sur la formation de circuits neuronaux mis en place au cours du developpement embryonnaire. Une unique connexion axonale relie le cortex cerebral au reste de l’organisme: la capsule interne. Ce faisceau axonal compact contient les Axones CorticoSubcerebraux (ACS), les Axones CorticoThalamiques (ACT) et les Axones reciproques ThalamoCorticaux (ATC) et traverse le Subpallium. Les ATC et ACS proviennent de neurones pyramidaux situes dans deux couches corticales distinctes, qui sont specifiees par l’expression de facteurs de transcription. Ces facteurs de transcription donnent une identite moleculaire aux neurones, notamment en determinant leur cible de projection. Cependant, les mecanismes cellulaires et moleculaires qui controlent la navigation des ACT versus ACS vers leur destination finale sont mal connus. Mes travaux montrent que le guidage des ACT pionniers a travers le subpallium necessite un controle a la fois temporel et spatial precis de leur navigation. J’etablis par une combinaison d’approches in vivo et in vitro, que les axones thalamocorticaux sont necessaires et suffisants pour guider les ACT pionniers, qui en leur absence adoptent par defaut la trajectoire des ACS. Ce processus repose sur la rencontre entre les ACT et ATC qui traversent le subpallium puis rejoignent les axones corticaux dans la partie dorsale, ou ceux-ci «pausent». De plus, je montre in vivo que la pause des axones corticaux est regulee par la voie de signalisation Sema3E/PlexinD1 et permettait d’orchestrer la rencontre entre les ATC et ACT. Ces travaux mettent en evidence que le controle temporel de la progression des axones determine egalement leur navigation spatiale

In mammals, the neocortex controls essential brain functions via a remarkable architecture of its internal microcircuits as well as via a specific wiring with the rest of the brain. In particular, reciprocal connections with the thalamus via the internal capsule are essential for the processing of sensory and motor information. During development, topographically organized thalamocortical (TCA) and corticothalamic (CTA) axons grow concomitantly through an intermediate target, the ventral telencephalon, where it has been proposed that they interact and guide each other (Handshake hypothesis, Molnar & Blakemore, Trends in Neurosci., 1995). However, the cascades of cellular and molecular events that ensure the wiring of the neocortex and thalamus remain to be fully deciphered. We have previously shown that the tangential migration of guidepost “corridor” neurons in the ventral telencephalon acts as a switch for the opening of a mammalian-specific internal route for TCA as well as provide positional information for their early topography (Lopez-Bendito, Cautinat et al., Cell, 2006; Bielle et al., Neuron, 2011; Bielle et al., unpublished). Here we show that in a second step, TCA regulate the pathfinding of reciprocal CTA within the ventral telencephalon. This process relies on a waiting period of CTA outgrowth, which is regulated by Sema3E/PlexinD1. Taken together, our results show that corridor neurons constitute a hub for TCA, which in turn regulate the navigation of reciprocal projections via a timely control of cortical axon outgrowth.

Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.