Impact of anti-CASPR2 autoantibodies from patients with autoimmune encephalitis on CASPR2/TAG-1 interaction and Kv1 expression.

Autoantibodies against CASPR2 (contactin-associated protein-like 2) have been linked to autoimmune limbic encephalitis that manifests with memory disorders and temporal lobe seizures. According to the growing number of data supporting a role for CASPR2 in neuronal excitability, CASPR2 forms a molecular complex with transient axonal glycoprotein-1 (TAG-1) and shaker-type voltage-gated potassium channels (Kv1.1 and Kv1.2) in compartments critical for neuronal activity and is required for Kv1 proper positioning. Whereas the perturbation of these functions could explain the symptoms observed in patients, the pathogenic role of anti-CASPR2 antibodies has been poorly studied. In the present study, we find that patient autoantibodies alter Caspr2 distribution at the cell membrane promoting cluster formation. We confirm in a HEK cellular model that the anti-CASPR2 antibodies impede CASPR2/TAG-1 interaction and we identify the domains of CASPR2 and TAG-1 taking part in this interaction. Moreover, introduction of CASPR2 into HEK cells induces a marked increase of the level of Kv1.2 surface expression and in cultures of hippocampal neurons Caspr2-positive inhibitory neurons appear to specifically express high levels of Kv1.2. Importantly, in both cellular models, anti-CASPR2 patient autoAb increase Kv1.2 expression. These results provide new insights into the pathogenic role of autoAb in the disease.

Contactin-associated protein-like 2, a protein of the neurexin family involved in several human diseases.

Contactin-associated protein-like 2 (CASPR2) is a cell adhesion protein of the neurexin family. Proteins of this family have been shown to play a role in the development of the nervous system, in synaptic functions, and in neurological diseases. Over recent years, CASPR2 function has gained an increasing interest as demonstrated by the growing number of publications. Here, we gather published data to comprehensively review CASPR2 functions within the nervous system in relation to CASPR2-related diseases in humans. On the one hand, studies on Cntnap2 (coding for CASPR2) knockout mice revealed its role during development, especially, in setting-up the inhibitory network. Consistent with this result, mutations in the CNTNAP2 gene coding for CASPR2 in human have been identified in neurodevelopmental disorders such as autism, intellectual disability, and epilepsy. On the other hand, CASPR2 was shown to play a role beyond development, in the localization of voltage-gated potassium channel (VGKC) complex that is composed of TAG-1, Kv1.1, and Kv1.2. This complex was found in several subcellular compartments essential for action potential propagation: the node of Ranvier, the axon initial segment, and the synapse. In line with a role of CASPR2 in the mature nervous system, neurological autoimmune diseases have been described in patients without neurodevelopmental disorders but with antibodies directed against CASPR2. These autoimmune diseases were of two types: central with memory disorders and temporal lobe seizures, or peripheral with muscular hyperactivity. Overall, we review the up-to-date knowledge on CASPR2 function and pinpoint confused or lacking information that will need further investigation.

The Syk kinases orchestrate cerebellar granule cell tangential migration.

The tyrosine kinases of the Syk family are essential components of the well-characterized immunoreceptor ITAM-based signaling pathway. However, ITAM-based signaling typically does not function in isolation. Instead, it is enmeshed in the molecular network controlling cellular adhesion and chemotaxis. Consistent with the increasing number of data involving ITAM-bearing molecules in neuronal functions, we previously depicted a role for Syk kinases in the establishment of neuronal connectivity. In the developing cerebellum, we found that Syk is essentially expressed in the granule cells (GC) and more importantly, phosphorylated on tyrosine residues representative of an active form of the kinase in tangentially migrating GC. In light of these findings, experiments were performed to establish the implication of Syk in this process. We showed that Syk state of phosphorylation is spatiotemporally regulated during GC ontogeny. Moreover, the analysis of external granular layer microexplants treated with a Syk pharmacological inhibitor together with the quantification of ectopic GC in Syk+/-; ZAP-70-/- mutant mice brought evidence of a requirement of Syk in GC tangential migration. Syk phosphorylation was induced by EphB2 engagement and locally turned down by a not yet identified factor that could in part explain the restricted pattern of Syk phosphorylation observed along GC migratory route. Whereas Syk kinase activity appeared not essential for ephrin/Eph-mediated axon extension, it might provide polarization signals required for proper nucleus translocation during GC migration. In conclusion, Syk kinase acts downstream of receptors controlling GC tangential migration.

Syk kinases are required for spinal commissural axon repulsion at the midline via the ephrin/Eph pathway.

In the hematopoietic system, Syk family tyrosine kinases are essential components of immunoreceptor ITAM-based signaling. While there is increasing data indicating the involvement of immunoreceptors in neural functions, the contribution of Syk kinases remains obscure. Previously, we identified phosphorylated forms of Syk kinases in specialized populations of migrating neurons or projecting axons. Moreover, we identified ephrin/Eph as guidance molecules utilizing the ITAM-bearing CD3zeta (Cd247) and associated Syk kinases for the growth cone collapse response induced in vitro Here, we show that in the developing spinal cord, Syk is phosphorylated in navigating commissural axons. By analyzing axon trajectories in open-book preparations of Syk(-/-); Zap70(-/-) mouse embryos, we show that Syk kinases are dispensable for attraction towards the midline but confer growth cone responsiveness to repulsive signals that expel commissural axons from the midline. Known to serve a repulsive function at the midline, ephrin B3/EphB2 are obvious candidates for driving the Syk-dependent repulsive response. Indeed, Syk kinases were found to be required for ephrin B3-induced growth cone collapse in cultured commissural neurons. In fragments of commissural neuron-enriched tissues, Syk is in a constitutively phosphorylated state and ephrin B3 decreased its level of phosphorylation. Direct pharmacological inhibition of Syk kinase activity was sufficient to induce growth cone collapse. In conclusion, Syk kinases act as a molecular switch of growth cone adhesive and repulsive responses.

Ectopic expression of the immune adaptor protein CD3zeta in neural stem/progenitor cells disrupts cell-fate specification.

Immune signaling and neuroinflammatory mediators have recently emerged as influential variables that regulate neural precursor/stem cell (NPC) behavior and function. In this study, we investigated whether the signaling adaptor protein CD3ζ, a transmembrane protein involved in T cell differentiation and function and recently shown to regulate neuronal development in the central nervous system (CNS), may have a role in NPC differentiation. We analyzed the expression profile of CD3ζ in embryonic rat brain during neurogenic periods and in neurosphere-derived neural cells, and we investigated the action of CD3ζ on cell differentiation. We found that CD3ζ expression coincided with neuronal commitment, but its forced expression in NPCs prevented the production of neurons and oligodendrocytes, but not astroglial cells. This blockade of neuronal differentiation was operated through an ITAM-independent mechanism, but required the Asp36 of the CD3ζ transmembrane domain involved in membrane receptor interaction. Together, our findings show that ectopic CD3ζ expression in NPCs impaired their normal cell-fate specification and suggest that variations of CD3ζ expression in the developing CNS might result in neurodevelopmental anomalies.

The immune molecule CD3zeta and its downstream effectors ZAP-70/Syk mediate ephrin signaling in neurons to regulate early neuritogenesis.

Recent studies have highlighted the key role of the immune protein CD3ζ in the maturation of neuronal circuits in the CNS. Yet, the upstream signals that might recruit and activate CD3ζ in neurons are still unknown. In this study, we show that CD3ζ functions early in neuronal development and we identify ephrinA1-dependent EphA4 receptor activation as an upstream regulator of CD3ζ. When newly born neurons are still spherical, before neurite extension, we found a transient CD3ζ aggregation at the cell periphery matching the initiation site of the future neurite. This accumulation of CD3ζ correlated with a stimulatory effect on filopodia extension via a Rho-GEF Vav2 pathway and a repression of neurite outgrowth. Conversely, cultured neurons lacking CD3ζ isolated from CD3ζ(-/-) mice showed a decreased number of filopodia and an enhanced neurite number. Stimulation with ephrinA1 induces the translocation of both CD3ζ and its activated effector molecules, ZAP-70/Syk tyrosine kinases, to EphA4 receptor clusters. EphrinA1-induced growth cone collapse was abrogated in CD3ζ(-/-) neurons and was markedly reduced by ZAP-70/Syk inhibition. Moreover, ephrinA1-induced ZAP-70/Syk activation was inhibited in CD3ζ(-/-) neurons. Altogether, our data suggest that CD3ζ mediates the ZAP-70/Syk kinase activation triggered by ephrinA-activated pathway to regulate early neuronal morphogenesis.

Syk kinase is phosphorylated in specific areas of the developing nervous system.

An increasing number of data involve immunoreceptors in brain development, synaptic plasticity and behavior. However it has yet to be determined whether these proteins in fact transmit an immunoreceptor-like signal in non-hematopoietic neuronal cells. The recruitment and activation of the Syk family tyrosine kinases, Syk and ZAP-70, being a critical step in this process, we conducted a thorough analysis of Syk/ZAP-70 expression pattern in nervous tissues. Syk/ZAP-70 is present in neurons of different structures including the cerebellum, the hippocampus, the visual system and the olfactory system. During the olfactory system ontogeny the protein is detected from the 16th embryonic day and persists in adulthood. Importantly, Syk was phosphorylated on tyrosine residues representative of an active form of the kinase in specialized neuronal subpopulations comprising rostral migratory stream neuronal progenitor cells, hippocampal pyramidal cells, retinal ganglion cells and cerebellar granular cells. Phospho-Syk staining was also observed in synapse-rich regions such as the olfactory bulb glomeruli and the retina inner plexiform layer. Furthermore, our work on cultured primary hippoccampal neurons indicates that as for hematopoietic cells, Syk phosphorylation is readily induced upon pervanadate treatment. Therefore, Syk appears to be a serious candidate in connecting immunoreceptors to downstream adaptor/effector molecules in neurons.

STOP proteins contribute to the maturation of the olfactory system.

Regulation of microtubule dynamics is crucial for axon growth and guidance as well as for the establishment of synaptic connections. STOPs (Stable Tubule Only Polypeptides) are microtubule-associated proteins that regulate microtubule stabilization but are also able to interact with actin or Golgi membranes. Here, we have investigated the involvement of STOPs during the development of the olfactory system. We first describe the spatio-temporal expression patterns of N- and E-STOP, the two neuronal-specific isoforms of STOP. E- and N-STOP are expressed in the axonal compartment of olfactory sensory neurons, but are differentially regulated during development. Interestingly, each neuronal isoform displays a specific gradient distribution within the olfactory nerve layer. Then, we have examined the development of the olfactory system in the absence of STOPs. Olfactory axons display a normal outgrowth and targeting in STOP-null mice, but maturation of the synapses in the glomerular neuropil is altered.

Spatio-temporal expression pattern of receptors for myelin-associated inhibitors in the developing rat olfactory system.

The myelin-associated inhibitory proteins (Nogo-A, MAG and OMgp) that prevent axon regeneration in adult CNS, mediate their effects via a receptor referred as NgR1. Beside their inhibitory role in the adult CNS, Nogo-A and NgR1 might also be functionally involved in the developing nervous system. At the present time, no detailed study is available regarding either the onset of NgR1 expression during development or its spatio-temporal pattern of expression relative to the presence of Nogo-A. Two homologs of NgR1, NgR2 and NgR3, have been recently identified, but their function in the nervous system is still unknown in adult as well as during development. We have examined the spatio-temporal expression pattern of both NgR1, NgR2 and NgR3 mRNAs and corresponding proteins in the developing rat olfactory system using in situ hybridization and immunohistochemistry. From E15-E16 onwards, NgR1 mRNA was expressed by differentiating neurons in both the olfactory epithelium and the olfactory bulb. At all developmental stages, including adult animals, NgR1 protein was preferentially targeted to olfactory axons emerging from the olfactory epithelium. Using double-immunostainings in the postnatal olfactory mucosa, we confirm the neuronal localization of NgR1 and its preferential distribution along the olfactory axons. The NgR2 and NgR3 transcripts and their proteins display similar expression profiles in the olfactory system. Together, our data suggest that, in non-pathological conditions, NgR1 and its homologs may play a role in axon outgrowth in the rat olfactory system and may be relevant for the confinement of neural projections within the developing olfactory bulb.

Neuronal expression of Nogo-A mRNA and protein during neurite outgrowth in the developing rat olfactory system.

The major impediments to axonal regeneration in the central nervous system are growth-inhibitory proteins present in the myelin sheath, and Nogo-A is one of the most potent inhibitors synthesized by oligodendrocytes. However, neuronal expression of Nogo-A during development suggests that it may have an additional role. The spatio-temporal regulation of both Nogo-A mRNA and protein expression was examined by in situ hybridization and immunohistochemistry in the developing rat olfactory system. During embryonic and postnatal development (from E13 to P6), Nogo-A mRNA and protein were strongly expressed by differentiating neurons in the olfactory epithelium and in the olfactory bulb. From the second postnatal week, a progressive down-regulation of both Nogo-A mRNA and protein occurred, such that only a weak expression persisted in the adult olfactory system. Using double-immunostainings in the adult olfactory epithelium, we determined that Nogo-A was preferentially expressed by immature olfactory receptor neurons extending axonal processes toward the olfactory bulb. At all developmental stages, Nogo-A protein was preferentially targeted in olfactory axons emerging from the olfactory epithelium. Using an in vitro model of olfactory axon growth, we demonstrated that, in addition to its presence along the entire axon length, Nogo-A accumulated in axonal growth cone and at axonal branching points, with a distribution similar to that of microtubule-associated proteins. Moreover, Nogo-A was transiently expressed in dendritic processes in the postnatal olfactory bulb. Together, our data suggest that, in non-pathological conditions, Nogo-A may be involved in the processes of axonal growth and dendritic modeling through the regulation of microtubule dynamics.