Physiology of PNS axons relies on glycolytic metabolism in myelinating Schwann cells.

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.

AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A.

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.

LKB1 specifies neural crest cell fates through pyruvate-alanine cycling.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

In vivo real-time dynamics of ATP and ROS production in axonal mitochondria show decoupling in mouse models of peripheral neuropathies.

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.

Assembly of juxtaparanodes in myelinating DRG culture: Differential clustering of the Kv1/Caspr2 complex and scaffolding protein 4.1B.

The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination.

Giant scaffolding protein AHNAK1 interacts with ß-dystroglycan and controls motility and mechanical properties of Schwann cells.

The profound morphofunctional changes that Schwann cells (SCs) undergo during their migration and elongation on axons, as well as during axon sorting, ensheathment, and myelination, require their close interaction with the surrounding laminin-rich basal lamina. In contrast to myelinating central nervous system glia, SCs strongly and constitutively express the giant scaffolding protein AHNAK1, localized essentially underneath the outer, abaxonal plasma membrane. Using electron microscopy, we show here that in the sciatic nerve of ahnak1(-) (/) (-) mice the ultrastructure of myelinated, and unmyelinated (Remak) fibers is affected. The major SC laminin receptor ß-dystroglycan co-immunoprecipitates with AHNAK1 shows reduced expression in ahnak1(-) (/) (-) SCs, and is no longer detectable in Cajal bands on myelinated fibers in ahnak1(-) (/) (-) sciatic nerve. Reduced migration velocity in a scratch wound assay of purified ahnak1(-) (/) (-) primary SCs cultured on a laminin substrate indicated a function of AHNAK1 in SC motility. This was corroborated by atomic force microscopy measurements, which revealed a greater mechanical rigidity of shaft and leading tip of ahnak1(-) (/) (-) SC processes. Internodal lengths of large fibers are decreased in ahnak1(-) (/) (-) sciatic nerve, and longitudinal extension of myelin segments is even more strongly reduced after acute knockdown of AHNAK1 in SCs of developing sciatic nerve. Together, our results suggest that by interfering in the cross-talk between the transmembrane form of the laminin receptor dystroglycan and F-actin, AHNAK1 influences the cytoskeleton organization of SCs, and thus plays a role in the regulation of their morphology and motility and lastly, the myelination process.

In vivo introduction of transgenes into mouse sciatic nerve cells in situ using viral vectors.

The myelin sheath is essential for the rapid and efficient propagation of action potentials. However, our understanding of the basic molecular mechanisms that regulate myelination, demyelination and remyelination is limited. Schwann cells produce myelin in the peripheral nervous system and remain associated with the axons of peripheral neurons throughout axonal migration to the target. Owing to the intimate relationship between these cell types it is difficult to fully reproduce their function in vitro. For this reason, we developed an approach based on the injection of an engineered virus into the sciatic nerve of mice to locally transduce peripheral nerve cells. This approach can be used as an alternative to germline transgenesis to facilitate the investigation of peripheral nerve biology in vivo. The detailed protocol, described here, requires 3 weeks to complete. In comparison with genetic modification strategies, this protocol is a fast, reproducible and straightforward method for introducing exogenous factors into myelinating Schwann cells and myelinated axons in vivo to investigate specific molecular mechanisms.

Dlg1-PTEN interaction regulates myelin thickness to prevent damaging peripheral nerve overmyelination.

The thickness of the myelin sheath that insulates axons is fitted for optimal nerve conduction velocity. Here, we show that, in Schwann cells, mammalian disks large homolog 1 (Dlg1) interacts with PTEN (phosphatase and tensin homolog deleted on chromosome 10) to inhibit axonal stimulation of myelination. This mechanism limits myelin sheath thickness and prevents overmyelination in mouse sciatic nerves. Removing this brake results also in myelin outfoldings and demyelination, characteristics of some peripheral neuropathies. Indeed, the Dlg1 brake is no longer functional in a mouse model of Charcot-Marie-Tooth disease. Therefore, negative regulation of myelination appears to be essential for optimization of nerve conduction velocity and myelin maintenance.

Pals1 is a major regulator of the epithelial-like polarization and the extension of the myelin sheath in peripheral nerves.

Diameter, organization, and length of the myelin sheath are important determinants of the nerve conduction velocity, but the basic molecular mechanisms that control these parameters are only partially understood. Cell polarization is an essential feature of differentiated cells, and relies on a set of evolutionarily conserved cell polarity proteins. We investigated the molecular nature of myelin sheath polarization in connection with the functional role of the cell polarity protein pals1 (Protein Associated with Lin Seven 1) during peripheral nerve myelin sheath extension. We found that, in regard to epithelial polarity, the Schwann cell outer abaxonal domain represents a basolateral-like domain, while the inner adaxonal domain and Schmidt-Lanterman incisures form an apical-like domain. Silencing of pals1 in myelinating Schwann cells in vivo resulted in a severe reduction of myelin sheath thickness and length. Except for some infoldings, the structure of compact myelin was not fundamentally affected, but cells produced less myelin turns. In addition, pals1 is required for the normal polarized localization of the vesicular markers sec8 and syntaxin4, and for the distribution of E-cadherin and myelin proteins PMP22 and MAG at the plasma membrane. Our data show that the polarity protein pals1 plays an essential role in the radial and longitudinal extension of the myelin sheath, likely involving a functional role in membrane protein trafficking. We conclude that regulation of epithelial-like polarization is a critical determinant of myelin sheath structure and function.

SH3TC2/KIAA1985 protein is required for proper myelination and the integrity of the node of Ranvier in the peripheral nervous system.

Charcot-Marie-Tooth disease type 4C (CMT4C) is an early-onset, autosomal recessive form of demyelinating neuropathy. The clinical manifestations include progressive scoliosis, delayed age of walking, muscular atrophy, distal weakness, and reduced nerve conduction velocity. The gene mutated in CMT4C disease, SH3TC2/KIAA1985, was recently identified; however, the function of the protein it encodes remains unknown. We have generated knockout mice where the first exon of the Sh3tc2 gene is replaced with an enhanced GFP cassette. The Sh3tc2(DeltaEx1/DeltaEx1) knockout animals develop progressive peripheral neuropathy manifested by decreased motor and sensory nerve conduction velocity and hypomyelination. We show that Sh3tc2 is specifically expressed in Schwann cells and localizes to the plasma membrane and to the perinuclear endocytic recycling compartment, concordant with its possible function in myelination and/or in regions of axoglial interactions. Concomitantly, transcriptional profiling performed on the endoneurial compartment of peripheral nerves isolated from control and Sh3tc2(DeltaEx1/DeltaEx1) animals uncovered changes in transcripts encoding genes involved in myelination and cell adhesion. Finally, detailed analyses of the structures composed of compact and noncompact myelin in the peripheral nerve of Sh3tc2(DeltaEx1/DeltaEx1) animals revealed abnormal organization of the node of Ranvier, a phenotype that we confirmed in CMT4C patient nerve biopsies. The generated Sh3tc2 knockout mice thus present a reliable model of CMT4C neuropathy that was instrumental in establishing a role for Sh3tc2 in myelination and in the integrity of the node of Ranvier, a morphological phenotype that can be used as an additional CMT4C diagnostic marker.

Integrin-linked kinase is required for radial sorting of axons and Schwann cell remyelination in the peripheral nervous system.

During development, Schwann cells (SCs) interpret different extracellular cues to regulate their migration, proliferation, and the remarkable morphological changes associated with the sorting, ensheathment, and myelination of axons. Although interactions between extracellular matrix proteins and integrins are critical to some of these processes, the downstream signaling pathways they control are still poorly understood. Integrin-linked kinase (ILK) is a focal adhesion protein that associates with multiple binding partners to link integrins to the actin cytoskeleton and is thought to participate in integrin and growth factor-mediated signaling. Using SC-specific gene ablation, we report essential functions for ILK in radial sorting of axon bundles and in remyelination in the peripheral nervous system. Our in vivo and in vitro experiments show that ILK negatively regulates Rho/Rho kinase signaling to promote SC process extension and to initiate radial sorting. ILK also facilitates axon remyelination, likely by promoting the activation of downstream molecules such as AKT/protein kinase B.

P120 catenin is required for thickening of Schwann cell myelin.

Schwann cell myelin is comprised of compacted membrane regions and cytoplasmic regions with non-compacted membrane. While adherens junctions (AJ) are abundant in non-compact regions, their role in the myelination process is largely undefined. To explore this issue, a small inhibitory hairpin RNA directed against p120ctn has been delivered using adenovirus infection of Schwann cells at early stages of myelination in vivo. With strong and specific reduction in p120ctn levels for over 2 months, (a) the adherens junctions of the infected cells were reduced in size and immature with respect to recruitment of alpha-catenin; and (b) the formation of Schmidt-Lanterman incisures was prevented and there was a marked reduction in the thickness of the myelin sheath without a change in internodal length. These data show that p120ctn is necessary in the myelinating Schwann cell for the formation of mature adherens junctions and a normal myelin sheath.

Adherens junctions in myelinating Schwann cells stabilize Schmidt-Lanterman incisures via recruitment of p120 catenin to E-cadherin.

Schwann cell myelin contains highly compacted layers of membrane as well as noncompacted regions with a visible cytoplasm. One of these cytoplasmic compartments is the Schmidt-Lanterman incisure, which spirals through the compacted layers and is believed to help sustain the growth and function of compact myelin. Incisures contain adherens junctions (AJs), the key components of which are E-cadherin, its cytoplasmic partners called catenins, and F-actin. To explore in vivo the role of cadherin and catenins in incisures, E-cadherin mutant proteins that completely replace endogenous cadherin have been delivered to the cells using adenovirus. When the introduced cadherin lacked its extracellular domain, association of p120 catenin (p120ctn) with the cadherin did not occur, and incisures disappeared. Remarkably, the additional replacement of two phosphorylatable tyrosines by phenylalanine in the cytoplasmic tail of the mutant cadherin restored both p120ctn binding and incisure architecture, indicating that p120ctn recruitment is critical for incisures maintenance and might be regulated by phosphorylations. In addition, the ability of the p120ctn/cadherin complex to support incisures was blocked by mutation of the Rho GTPase regulatory region of the p120ctn, and downregulation of Rac1 activity at the junction reversed this inhibition. Because Rho GTPases regulate the state of the actin filaments, these findings suggest that one role of p120ctn in incisures is to organize the cytoskeleton at the AJ. Finally, developmental studies of Schwann cells demonstrated that p120ctn recruitment from the cytoplasm to the AJ occurs before the appearance of Rac1 GTPase and F-actin at the junction.