Glial lipid droplets and neurodegeneration in a Drosophila model of complex I deficiency.

Mitochondrial defects associated with respiratory chain complex I deficiency lead to heterogeneous fatal syndromes. While the role of NDUFS8, an essential subunit of the core assembly of the complex I, is established in mitochondrial diseases, the mechanisms underlying neuropathology are poorly understood. We developed a Drosophila model of NDUFS8 deficiency by knocking down the expression of its fly homologue in neurons or in glial cells. Downregulating ND23 in neurons resulted in shortened lifespan, and decreased locomotion. Although total brain ATP levels were decreased, histological analysis did not reveal any signs of neurodegeneration except for photoreceptors of the retina. Interestingly, ND23 deficiency-associated phenotypes were rescued by overexpressing the glucose transporter hGluT3 demonstrating that boosting glucose metabolism in neurons was sufficient to bypass altered mitochondrial functions and to confer neuroprotection. We then analyzed the consequences of ND23 knockdown in glial cells. In contrast to neuronal knockdown, loss of ND23 in glia did not lead to significant behavioral defects nor to reduced lifespan, but induced brain degeneration, as visualized by numerous vacuoles found all over the nervous tissue. This phenotype was accompanied by the massive accumulation of lipid droplets at the cortex-neuropile boundaries, suggesting an alteration of lipid metabolism in glia. These results demonstrate that complex I deficiency triggers metabolic alterations both in neurons and glial cells which may contribute to the neuropathology.

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.

Specific contactin N-glycans are implicated in neurofascin binding and autoimmune targeting in peripheral neuropathies.

Cell adhesion molecules (CAMs) play a crucial role in the formation of the nodes of Ranvier and in the rapid propagation of the nerve impulses along myelinated axons. These CAMs are the targets of autoimmunity in inflammatory neuropathies. We recently showed that a subgroup of patients with aggressive chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) shows autoantibodies to contactin (1). The complex of contactin·Caspr·neurofascin-155 (NF155) enables the formation of paranodal junctions, suggesting that antibody attack against paranodes may participate in the severity of CIDP. In the present study, we mapped the molecular determinants of contactin targeted by the autoantibodies. In three patients, immunoreactivity was directed against the Ig domains of contactin and was dependent on N-glycans. The serum of one patient was selectively directed against contactin bearing mannose-rich N-glycans. Strikingly, the oligomannose type sugars of contactin are required for association with its glial partner NF155 (2). To investigate precisely the role of contactin N-glycans, we have mutated each of the nine consensus N-glycosylation sites independently. We found that the mutation of three sites (N467Q/N473Q/N494Q) in Ig domain 5 of contactin prevented soluble NF155-Fc binding. In contrast, these mutations did not abolish cis-association with Caspr. Next, we showed that the cluster of N-glycosylation sites (Asn-467, Asn-473, and Asn-494) was required for immunoreactivity in one patient. Using cell aggregation assays, we showed that the IgGs from the four CIDP patients prevented adhesive interaction between contactin·Caspr and NF155. Importantly, we showed that the anti-contactin autoantibodies induced alteration of paranodal junctions in myelinated neuronal culture. These results strongly suggest that antibodies to CAMs may be pathogenic and induce demyelination via functional blocking activity.

Fibronectin type III-like domains of neurofascin-186 protein mediate gliomedin binding and its clustering at the developing nodes of Ranvier.

The cell adhesion molecules (CAMs) of the immunoglobulin superfamily (Ig-CAMs) play a crucial role in the organization of the node of Ranvier in myelinated axons. In the peripheral nervous system, Gliomedin (Gldn) secreted by Schwann cell microvilli binds NgCAM-related CAM (NrCAM) and Neurofascin-186 (NF186) and direct the nodal clustering of voltage-gated sodium channels (Nav). NF186 is the single axonal Gldn partner to ensure Nav clustering at nodes, whereas NrCAM is only required in glial cells (Feinberg, K., Eshed-Eisenbach, Y., Frechter, S., Amor, V., Salomon, D., Sabanay, H., Dupree, J. L., Grumet, M., Brophy, P. J., Shrager, P., and Peles, E. (2010) Neuron 65, 490-502). The olfactomedin domain of Gldn is implicated in the interaction with nodal Ig-CAMs. However, the interacting modules of NrCAM or NF186 involved in Gldn association are unknown. Here, we report that fibronectin type III-like (FnIII) domains of both Ig-CAMs mediate their interaction with Gldn in pulldown and cell binding assays. Using surface plasmon resonance assays, we determined that NrCAM and NF186 display similar affinity constant for their association with Gldn (K(D) of 0.9 and 5.7 nm, respectively). We characterized the FnIII domains 1 and 2 of NF186 as interacting modules that ensure association with Gldn. We found that the soluble FnIII domains of NF186 (FnIII-Fc) bind on Schwann cells and inhibit Gldn and Nav clustering at heminodes, the precursors of mature nodes in myelinating cultures. Our study reveals the unexpected importance of FnIII domains of Ig-CAMs in the organization of nodes of Ranvier in peripheral axons. Thus, NF186 utilizes distinct modules to organize the multimeric nodal complex.

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila.

BACKGROUND: A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. RESULTS: We conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. CONCLUSION: The strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions.

AKT-sensitive or insensitive pathways of toxicity in glial cells and neurons in Drosophila models of Huntington's disease.

Huntington's disease (HD) is caused by an extended polyglutamine (polyQ) tract in the Huntingtin protein. Neuronal and glial dysfunction precedes the neurodegeneration and appears to be the primary cause for the early symptoms in HD. In recent years, development of Drosophila models of polyQ-related diseases facilitated research of candidate rescuer genes. In most cases, analysis in Drosophila was performed by assessing toxicity on retinal and/or brain neurons. However, none of the potential rescuers were evaluated on glial alterations. Here we used a genetic approach in Drosophila to characterize the phenotypic effects of mutant Huntingtin (mHtt) expressed in neurons or different glia subsets and we established a sensitive assay for evaluating modifiers of glial alterations. We determined the level of cell protection ensured by activation of the AKT and ERK anti-apoptotic kinases in the retina as well as in neurons and glia of the fly brain, compared with the rescuing effects of the HSP70 chaperone. We found that both AKT and HSP70 alleviated mHtt-induced toxicity in the retina. In contrast, their protective effects differed in the brain. HSP70 rescued neurodegeneration, locomotor defects and early lethality of flies expressing mHtt in neurons or glia. AKT failed to prevent brain neuronal death and lethality of flies, but significantly improved their locomotor performance when co-expressed with mHtt in glia. ERK had no beneficial effects in the retina or brain. These results indicate that mHtt activates distinct pathways of toxicity in Drosophila, either sensitive to AKT in retinal photoreceptors and glia, or independent in brain neurons.

NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts.

NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-beta-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.

The paranodal complex of F3/contactin and caspr/paranodin traffics to the cell surface via a non-conventional pathway.

During myelination, membrane-specialized domains are generated by complex interactions between axon and glial cells. The cell adhesion molecules caspr/paranodin and F3/contactin play a crucial role in the generation of functional septate-like junctions at paranodes. We have previously demonstrated that association with the glycosylphosphatidylinositol-linked F3/contactin is required for the recruitment of caspr/paranodin into the lipid rafts and its targeting to the cell surface. When transfected alone in neuroblastoma N2a cells, caspr/paranodin is retained in the endoplasmic reticulum (ER). Using chimerical constructs, we show that the cytoplasmic region does not contain any ER retention signal, whereas the ectodomain plays a crucial role in caspr/paranodin trafficking. A series of truncations encompassing the extracellular region of caspr/paranodin was unable to abolish ER retention. We show that N-glycosylation and quality control by the lectin-chaperone calnexin are required for the cell surface delivery of caspr/paranodin. Cell surface transport of F3/contactin and caspr/paranodin is insensitive to brefeldin A and the two glycoproteins are endoglycosidase H-sensitive when associated in complex, recruited into the lipid rafts, and expressed on the cell surface. Our results indicate a Golgi-independent pathway for the paranodal cell adhesion complex that may be implicated in the segregation of axonal subdomains.

Association of Caspr/paranodin with tumour suppressor schwannomin/merlin and beta1 integrin in the central nervous system.

Caspr/paranodin is an essential neuronal component of paranodal axoglial junctions, associated with contactin/F3. Its short intracellular domain contains a conserved motif (GNP motif) capable of binding protein 4.1 domains [FERM domains (four point one, ezrin, radixin, moesin)]. Schwannomin/merlin is a tumour suppressor expressed in many cell types, including in neurons, the function and partners of which are still poorly characterized. We show that the FERM domain of schwannomin binds to the paranodin GNP motif in glutathione S-transferase (GST)-pull down assays and in transfected COS-7 cells. The two proteins co-immunoprecipitated in brain extracts. In addition, paranodin and schwannomin were associated with integrin beta1 in transfected cells and in brain homogenates. The presence of paranodin increased the association between integrin beta1 and schwannomin or its N-terminal domain, suggesting that the interactions between these proteins are interdependent. In jimpy mutant mice, which display a severe dysmyelination with deficient paranodal junctions, the interactions between paranodin, schwannomin and integrin beta1 were profoundly altered. Our results show that schwannomin and integrin beta1 can be associated with paranodin in the central nervous system. Since integrin beta1 and schwannomin do not appear to be enriched in paranodes they may be quantitatively minor partners of paranodin in these regions and/or be associated with paranodin at other locations.