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International audience; Charcot Marie Tooth disease is one of the most common inherited neurological disorders, affecting either axons from the motor and/or sensory neurons or Schwann cells of the peripheral nervous system, and caused by more than 100 genes. We previously identified mutations in FGD4, as responsible for CMT4H, an autosomal recessive demyelinating form of CMT. FGD4 encodes FRABIN, a GDP/GTP nucleotide exchange factor, particularly for the small GTPase cdc42. Remarkably, nerves from patients with CMT4H display excessive redundant myelin figures called outfoldings that arise from focal hypermyelination, suggesting that FRABIN could play a role in the control of PNS myelination. To gain insights into the role of FGD4/FRABIN in Schwann Cell myelination, we generated a knock-out mouse model (Fgd4SC-/-), with conditional ablation of Fgd4 in Schwann cells. We show that the specific deletion of FRABIN in Schwann cells leads to aberrant myelination in vitro, in dorsal root ganglia neurons/Schwann cells cocultures as well in vivo, in distal sciatic nerves from Fgd4SC-/- mice. We observed that those myelination defects are related to an upregulation of some interactors of the NRG1 type III/ERBB2/3 signaling pathway, which is known to ensure a proper level of myelination in the peripheral nervous system. Based on a yeast two- hybrid screen, we identified SNX3 as a new partner of FRABIN, which is involved in the regulation of endocytic trafficking. Interestingly, we showed that loss of FRABIN impairs endocytic trafficking which may contribute to the defective NRG1 type III/ERBB2/3 signaling and myelination. Using RNA-Seq in vitro, we have identified new potential effectors of the deregulated pathways, such as ERBIN, RAB11FIP2 and MAF, thereby providing cues to understand how FRABIN contributes to proper ERBB2 trafficking or even myelin membrane addition through cholesterol synthesis. Finally, we showed that the reestablishment of proper levels of the NRG1 type III/ERBB2/3 pathway using Niacin treatment reduces myelin outfoldings in nerves of CMT4H mice. Overall, our work reveals a new role of FRABIN in the regulation of NRG1 type III/ERBB2/3 NRG1signaling and myelination and opens future therapeutic strategies based on the modulation of the NRG1 type III/ERBB2/3 pathway to reduce CMT4H pathology and more generally others demyelinating Charcot Marie Tooth disease.

Charcot Marie Tooth disease (CMT) is one of the most common inherited neurological disorders, affecting either axons from the motor and/or sensory neurons or Schwann cells (SC) of the peripheral nervous system, and caused by more than 100 genes. We previously identified mutations in FGD4, as responsible for CMT4H, an autosomal recessive demyelinating form of CMT. FGD4 encodes FRABIN a GDP/GTP nucleotide exchange factor (GEF), particularly for the small GTPase cdc42. Remarkably, nerves from patients with CMT4H display excessive redundant myelin called outfoldings that arise from focal hypermyelination, suggesting that FRABIN could play a role in the control of PNS myelination. To gain insights into the role of FGD4/FRABIN in SC myelination, we generated a knock-out mouse model, with conditional ablation of fgd4 in SC. We showed that the specific deletion of FRABIN in SCs leads to aberrant myelination in vitro, in dorsal root ganglion (DRG)/SCs cocultures as well in vivo, in distal sciatic nerves. We observed that those myelination defects are related to an upregulation of some interactors of the NRG1 type III/ErbB2/3 signaling pathway, which is known to ensure a proper level of myelination in the PNS. Based on a yeast two-hybrid screen, we identified SNX3 as a new partner of FRABIN, which is involved in the regulation of endocytic trafficking. Interestingly, we showed that loss of FRABIN impairs endocytic trafficking which may contribute to the defective NRG1 type III/ErbB2/3 signaling and myelination. Finally, we showed that the reestablishment of proper levels of the NRG1 type III/ErbB2/3 pathway using Niacin treatment reduces myelin outfoldings in nerves of CMT4H mice. Overall, our work reveals a new role of FRABIN in the regulation of NRG1 type III/ErbB2/3 NRG1signaling and myelination and opens future therapeutic strategies based on the modulation of NRG1 type III/ErbB2/3 NRG1to reduce CMT4H pathology and more generally others demyelinating CMT.

SummaryDespite the lactate shuttle theory, 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, activatezd 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 mature myelinating Schwann cells (mSC) of mouse sciatic nerve. Deletion of this isoform in PLP-expressing cells in mice leads to a deficit of lactate in mSC and in peripheral nerves. This had no detectable consequences on the myelin sheath. However, 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 aerobic glycolysis is essential in mSC for the long-term maintenance of peripheral nerve axon physiology and function.

KEY POINTS: The hippocampal CA1 region is highly vulnerable to ischaemic stroke. Two forms of AMPA receptor (AMPAR) plasticity an anoxic form of long term potentiation and a delayed increase in Ca(2+) permeable (CP) AMPARs contribute to this susceptibility by increasing excitotoxicity. In CA1 the acid sensing ion channel 1a (ASIC1a) is known to facilitate LTP and contribute to ischaemic acidotoxicity. We have examined the role of ASIC1a in AMPAR ischaemic plasticity in organotypic hippocampal slice cultures exposed to oxygen glucose deprivation (a model of ischaemic stroke) and in hippocampal pyramidal neuron cultures exposed to acidosis. We find that ASIC1a activation promotes both forms of AMPAR plasticity and that neuroprotection by inhibiting ASIC1a circumvents any further benefit of blocking CP AMPARs. Our observations establish a new interaction between acidotoxicity and excitotoxicity and provide insight into the role of ASIC1a and CP AMPARs in neurodegeneration. Specifically we propose that ASIC1a activation drives certain post ischaemic forms of CP AMPAR plasticity. ABSTRACT: The CA1 region of the hippocampus is particularly vulnerable to ischaemic damage. While NMDA receptors play a major role in excitotoxicity it is thought to be exacerbated in this region by two forms of post ischaemic AMPA receptor (AMPAR) plasticity namely anoxic long term potentiation (a LTP) and a delayed increase in the prevalence of Ca(2+) permeable GluA2 lacking AMPARs (CP AMPARs). The acid sensing ion channel 1a (ASIC1a) which is expressed in CA1 pyramidal neurons is also known to contribute to post ischaemic neuronal death and to physiologically induced LTP. This raises the question does ASIC1a activation drive the post ischaemic forms of AMPAR plasticity in CA1 pyramidal neurons? We have tested this by examining organotypic hippocampal slice cultures (OHSCs) exposed to oxygen glucose deprivation (OGD) and dissociated cultures of hippocampal pyramidal neurons (HPNs) exposed to low pH (acidosis). We find that both a LTP and the delayed increase in the prevalence of CP AMPARs are dependent on ASIC1a activation during ischaemia. Indeed acidosis alone is sufficient to induce the increase in CP AMPARs. We also find that inhibition of ASIC1a channels circumvents any potential neuroprotective benefit arising from block of CP AMPARs. By demonstrating that ASIC1a activation contributes to post ischaemic AMPAR plasticity our results identify a functional interaction between acidotoxicity and excitotoxicity in hippocampal CA1 cells and provide insight into the role of ASIC1a and CP AMPARs as potential drug targets for neuroprotection. We thus propose that ASIC1a activation can drive certain forms of CP AMPAR plasticity and that inhibiting ASIC1a affords neuroprotection.

The effect of a 645 nm Light Emitting Diode (LED) light irradiation on the neurite growth velocity of adult Dorsal Root Ganglion (DRG) neurons with peripheral axon injury 4-10 days before plating and without previous injury was investigated. The real amount of light reaching the neurons was calculated by taking into account the optical characteristics of the light source and of media in the light path. The knowledge of these parameters is essential to be able to compare results of the literature and a way to reduce inconsistencies. We found that 4 min irradiation of a mean irradiance of 11.3 mW/cm(2) (corresponding to an actual irradiance reaching the neurons of 83 mW/cm(2)) induced a 1.6-fold neurite growth acceleration on non-injured neurons and on axotomized neurons. Although the axotomized neurons were naturally already in a rapid regeneration process, an enhancement was found to occur while irradiating with the LED light, which may be promising for therapy applications. Dorsal Root Ganglion neurons (A) without previous injury and (B) subjected to a conditioning injury.

Transient anoxia/hypoglycaemia in organotypic hippocampal slice cultures, a model of transient brain ischaemia, ultimately results in delayed cell death. Although the mechanisms underlying this delayed death remain unknown, an increase in excitatory drive has been postulated. We report here that transient anoxia/hypoglycaemia in rat hippocampal slice cultures resulted in a 70-80% enhancement of evoked, alpha-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) receptor-mediated, excitatory responses lasting over 60 min. This effect was prevented by blockade of N-methyl-d-aspartate (NMDA) receptors, did not involve changes of paired-pulse facilitation ratio, but was associated with a 50% increase in amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs). Consistent with this, paired recordings revealed the appearance of AMPA receptor-mediated EPSCs at previously silent synapses and occlusion by prior induction of long-term potentiation (LTP). Transient anoxia/hypoglycaemia further resulted in a 63% potentiation of evoked NMDA receptor-dependent synaptic responses, accounting for the 20% increase in ratio of AMPA to NMDA responses. No change in rectification properties of AMPA receptor-mediated currents could be detected within the first hour following anoxia/hypoglycaemia-induced potentiation. Western blot analyses of slice cultures exposed to either control conditions or a short anoxia/hypoglycaemia revealed a marked, 50-70% increase of GluR1, GluR2/3 and NR1 subunits 1 h, but not 15 min, after the anoxic/hypoglycaemic episode. This increase was blocked by an inhibitor of protein synthesis. Together these results indicate that a transient anoxia/hypoglycaemia is associated with a marked enhancement of excitatory transmission sharing similarities with the mechanisms underlying LTP, and is correlated with an increased synthesis of excitatory receptor subunits.