Cytokeratin 18 as a Novel Biomarker in Patients with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a heart muscle disease associated with an increased risk for sudden cardiac death (SCD). Cytokeratin 18-based proteins, such as M30 and M65 antigens, are known cell-death biomarkers. M30 antigen is released from cells during apoptosis, and M65 antigen is released during cell death from any cause, such as apoptosis or necrosis. We aimed to study the expression of M30 and M65 antigens in peripheral blood obtained by 46 HCM patients and compare with 27 age- and sex-matched patients without HCM. We also investigated the CK18 expression in myocardium from postmortem HCM hearts. M30 and M65 antigens were significantly increased in the HCM vs. non-HCM group (Μ30: 338 ± 197 U/uL vs. 206 ± 166 U/uL, p = 0.003; M65: 428 ± 224 U/uL vs. 246 ± 214 U/uL, p = 0.001), and HCM patients with a higher expression of these markers (M30: 417 ± 208 vs. 271 ± 162 U/uL, p = 0.011; M65: 518 ± 242 vs. 351 ± 178 U/uL, p = 0.011) had a higher risk for SCD. In HCM, both apoptosis and necrosis are increased, but particularly necrosis (M30/M65 ratio: 0.75 ± 0.09 vs. 0.85 ± 0.02, p < 0.001). CK18 is expressed in the HCM myocardium (1.767 ± 0.412 vs. 0.537 ± 0.383, % of area, p = 0.0058). Therefore, M30 and M65 antigens may be novel biomarkers in HCM.

Microglia-derived extracellular vesicles trigger age-related neurodegeneration upon DNA damage.

DNA damage and neurodegenerative disorders are intimately linked but the underlying mechanism remains elusive. Here, we show that persistent DNA lesions in tissue-resident macrophages carrying an XPF-ERCC1 DNA repair defect trigger neuroinflammation and neuronal cell death in mice. We find that microglia accumulate dsDNAs and chromatin fragments in the cytosol, which are sensed thereby stimulating a viral-like immune response in Er1Cx/- and naturally aged murine brain. Cytosolic DNAs are packaged into extracellular vesicles (EVs) that are released from microglia and discharge their dsDNA cargo into IFN-responsive neurons triggering cell death. To remove cytosolic dsDNAs and prevent inflammation, we developed targeting EVs to deliver recombinant DNase I to Er1Cx/- brain microglia in vivo. We show that EV-mediated elimination of cytosolic dsDNAs is sufficient to prevent neuroinflammation, reduce neuronal apoptosis, and delay the onset of neurodegenerative symptoms in Er1Cx/- mice. Together, our findings unveil a causal mechanism leading to neuroinflammation and provide a rationalized therapeutic strategy against age-related neurodegeneration.

The Roles of Caloric Restriction Mimetics in Central Nervous System Demyelination and Remyelination.

The dysfunction of myelinating glial cells, the oligodendrocytes, within the central nervous system (CNS) can result in the disruption of myelin, the lipid-rich multi-layered membrane structure that surrounds most vertebrate axons. This leads to axonal degeneration and motor/cognitive impairments. In response to demyelination in the CNS, the formation of new myelin sheaths occurs through the homeostatic process of remyelination, facilitated by the differentiation of newly formed oligodendrocytes. Apart from oligodendrocytes, the two other main glial cell types of the CNS, microglia and astrocytes, play a pivotal role in remyelination. Following a demyelination insult, microglia can phagocytose myelin debris, thus permitting remyelination, while the developing neuroinflammation in the demyelinated region triggers the activation of astrocytes. Modulating the profile of glial cells can enhance the likelihood of successful remyelination. In this context, recent studies have implicated autophagy as a pivotal pathway in glial cells, playing a significant role in both their maturation and the maintenance of myelin. In this Review, we examine the role of substances capable of modulating the autophagic machinery within the myelinating glial cells of the CNS. Such substances, called caloric restriction mimetics, have been shown to decelerate the aging process by mitigating age-related ailments, with their mechanisms of action intricately linked to the induction of autophagic processes.

A class-specific effect of dysmyelination on the excitability of hippocampal interneurons.

The role of myelination for axonal conduction is well-established in projection neurons but little is known about its significance in GABAergic interneurons. Myelination is discontinuous along interneuron axons and the mechanisms controlling myelin patterning and segregation of ion channels at the nodes of Ranvier have not been elucidated. Protein 4.1B is implicated in the organization of the nodes of Ranvier as a linker between paranodal and juxtaparanodal membrane proteins to the spectrin cytoskeleton. In the present study, 4.1B KO mice are used as a genetic model to analyze the functional role of myelin in Lhx6-positive parvalbumin (PV) and somatostatin (SST) neurons, two major classes of GABAergic neurons in the hippocampus. We show that 4.1B-deficiency induces disruption of juxtaparanodal K+ channel clustering and mislocalization of nodal or heminodal Na+ channels. Strikingly, 4.1B-deficiency causes loss of myelin in GABAergic axons in the hippocampus. In particular, stratum oriens SST cells display severe axonal dysmyelination and a reduced excitability. This reduced excitability is associated with a decrease in occurrence probability of small amplitude synaptic inhibitory events on pyramidal cells. In contrast, stratum pyramidale fast-spiking PV cells do not appear affected. In conclusion, our results indicate a class-specific effect of dysmyelination on the excitability of hippocampal interneurons associated with a functional alteration of inhibitory drive.

Nicotinamide enhances myelin production after demyelination through reduction of astrogliosis and microgliosis.

Caloric restriction is the chronic reduction of total caloric intake without malnutrition and has attracted a lot of attention as, among multiple other effects, it attenuates demyelination and stimulates remyelination. In this study we have evaluated the effect of nicotinamide (NAM), a well-known caloric restriction mimetic, on myelin production upon demyelinating conditions. NAM is the derivative of nicotinic acid (vitamin B3) and a precursor of nicotinamide adenine dinucleotide (NAD+), a ubiquitous metabolic cofactor. Here, we use cortical slices ex vivo subjected to demyelination or cultured upon normal conditions, a lysolecithin (LPC)-induced focal demyelination mouse model as well as primary glial cultures. Our data show that NAM enhances both myelination and remyelination ex vivo, while it also induces myelin production after LPC-induced focal demyelination ex vivo and in vivo. The increased myelin production is accompanied by reduction in both astrogliosis and microgliosis in vivo. There is no direct effect of NAM on the oligodendrocyte lineage, as no differences are observed in oligodendrocyte precursor cell proliferation or differentiation or in the number of mature oligodendrocytes. On the other hand, NAM affects both microglia and astrocytes as it decreases the population of M1-activated microglia, while reducing the pro-inflammatory phenotype of astrocytes as assayed by the reduction of TNF-α. Overall, we show that the increased myelin production that follows NAM treatment in vivo is accompanied by a decrease in both astrocyte and microglia accumulation at the lesion site. Our data indicate that NAM influences astrocytes and microglia directly, in favor of the remyelination process by promoting a less inflammatory environment.

Nogo-A and LINGO-1: Two Important Targets for Remyelination and Regeneration.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that causes progressive neurological disability in most patients due to neurodegeneration. Activated immune cells infiltrate the CNS, triggering an inflammatory cascade that leads to demyelination and axonal injury. Non-inflammatory mechanisms are also involved in axonal degeneration, although they are not fully elucidated yet. Current therapies focus on immunosuppression; however, no therapies to promote regeneration, myelin repair, or maintenance are currently available. Two different negative regulators of myelination have been proposed as promising targets to induce remyelination and regeneration, namely the Nogo-A and LINGO-1 proteins. Although Nogo-A was first discovered as a potent neurite outgrowth inhibitor in the CNS, it has emerged as a multifunctional protein. It is involved in numerous developmental processes and is necessary for shaping and later maintaining CNS structure and functionality. However, the growth-restricting properties of Nogo-A have negative effects on CNS injury or disease. LINGO-1 is also an inhibitor of neurite outgrowth, axonal regeneration, oligodendrocyte differentiation, and myelin production. Inhibiting the actions of Nogo-A or LINGO-1 promotes remyelination both in vitro and in vivo, while Nogo-A or LINGO-1 antagonists have been suggested as promising therapeutic approaches for demyelinating diseases. In this review, we focus on these two negative regulators of myelination while also providing an overview of the available data on the effects of Nogo-A and LINGO-1 inhibition on oligodendrocyte differentiation and remyelination.

Rac1 and Rac3 GTPases and TPC2 are required for axonal outgrowth and migration of cortical interneurons.

Rho GTPases, among them Rac1 and Rac3, are major transducers of extracellular signals and are involved in multiple cellular processes. In cortical interneurons, the neurons that control the balance between excitation and inhibition of cortical circuits, Rac1 and Rac3 are essential for their development. Ablation of both leads to a severe reduction in the numbers of mature interneurons found in the murine cortex, which is partially due to abnormal cell cycle progression of interneuron precursors and defective formation of growth cones in young neurons. Here, we present new evidence that upon Rac1 and Rac3 ablation, centrosome, Golgi complex and lysosome positioning is significantly perturbed, thus affecting both interneuron migration and axon growth. Moreover, for the first time, we provide evidence of altered expression and localization of the two-pore channel 2 (TPC2) voltage-gated ion channel that mediates Ca2+ release. Pharmacological inhibition of TPC2 negatively affected axonal growth and migration of interneurons. Our data, taken together, suggest that TPC2 contributes to the severe phenotype in axon growth initiation, extension and interneuron migration in the absence of Rac1 and Rac3.

Autophagic degradation of CNS myelin maintains axon integrity.

(Macro)autophagy is a major lysosome-dependent degradation mechanism which engulfs, removes and recycles unwanted cytoplasmic material, including damaged organelles and toxic protein aggregates. Although a few studies implicate autophagy in CNS demyelinating pathologies, its role, particularly in mature oligodendrocytes and CNS myelin, remains poorly studied. Here, using both pharmacological and genetic inhibition of the autophagic machinery, we provide evidence that autophagy is an essential mechanism for oligodendrocyte maturation in vitro. Our study reveals that two core myelin proteins, namely proteolipid protein (PLP) and myelin basic protein (MBP) are incorporated into autophagosomes in oligodendrocytes, resulting in their degradation. Furthermore, we ablated atg5, a core gene of the autophagic machinery, specifically in myelinating glial cells in vivo by tamoxifen administration (plp-Cre ERT2 ; atg5 f/f ) and showed that myelin maintenance is perturbed, leading to PLP accumulation. Significant morphological defects in myelin membrane such as decompaction accompanied with increased axonal degeneration are observed. As a result, the mice exhibit behavioral deficits. In summary, our data highlight that the maintenance of adult myelin homeostasis in the CNS requires the involvement of a fully functional autophagic machinery.

The developmental changes in intrinsic and synaptic properties of prefrontal neurons enhance local network activity from the second to the third postnatal weeks in mice.

The prefrontal cortex (PFC) is characterized by protracted maturation. The cellular mechanisms controlling the early development of prefrontal circuits are still largely unknown. Our study delineates the developmental cellular processes in the mouse medial PFC (mPFC) during the second and the third postnatal weeks and characterizes their contribution to the changes in network activity. We show that spontaneous inhibitory postsynaptic currents (sIPSC) are increased, whereas spontaneous excitatory postsynaptic currents (sEPSC) are reduced from the second to the third postnatal week. Drug application suggested that the increased sEPSC frequency in mPFC at postnatal day 10 (P10) is due to depolarizing γ-aminobutyric acid (GABA) type A receptor function. To further validate this, perforated patch-clamp recordings were obtained and the expression levels of K-Cl cotransporter 2 (KCC2) protein were examined. The reversal potential of IPSCs in response to current stimulation was significantly more depolarized at P10 than P20 while KCC2 expression is decreased. Moreover, the number of parvalbumin-expressing GABAergic interneurons increases and their intrinsic electrophysiological properties significantly mature in the mPFC from P10 to P20. Using computational modeling, we show that the developmental changes in synaptic and intrinsic properties of mPFC neurons contribute to the enhanced network activity in the juvenile compared with neonatal mPFC.

Cortical interneuron development: a role for small Rho GTPases.

GABAergic interneurons control cortical excitation/inhibition balance and are implicated in severe neurodevelopmental disorders. In contrast to the multiplicity of signals underlying the generation and migration of cortical interneurons, the intracellular proteins mediating the response to these cues are largely unknown. We have demonstrated the unique and diverse roles of the Rho GTPases Rac1 and 3 in cell cycle and morphology in transgenic animals where Rac1 and Rac1/3 were ablated specifically in cortical interneurons. In the Rac1 mutant, progenitors delay their cell cycle exit, probably due to a prolonged G1 phase resulting in a cortex with 50% reductions in interneurons and an imbalance of excitation/inhibition in cortical circuits. This disruption in GABAergic inhibition alters glutamatergic function in the adult cortex, which could be reversed by enhancement of GABAergic functions during an early postnatal period. Furthermore, this disruption disturbs neuronal synchronization in the adult cortex. In the double mutant, additional severe cytoskeletal defects result in an 80% interneuron decrease. Both lines die postnatally from epileptic seizures. We have made progress towards characterizing the cell cycle defect in Rac1 mutant interneuron progenitors, determining the morphological and synaptic characteristics of single and double mutant interneurons and identifying some of the molecular players through which Racs exert their actions via proteomic analysis. In our present work, we review these studies and discuss open questions and future perspectives. We hope that our data will contribute to the understanding of the function of cortical interneurons, especially since preclinical models of interneuron-based cell therapies are being established.

Oligodendrocytes and Microglia: Key Players in Myelin Development, Damage and Repair.

Oligodendrocytes, the myelin-making cells of the CNS, regulate the complex process of myelination under physiological and pathological conditions, significantly aided by other glial cell types such as microglia, the brain-resident, macrophage-like innate immune cells. In this review, we summarize how oligodendrocytes orchestrate myelination, and especially myelin repair after damage, and present novel aspects of oligodendroglial functions. We emphasize the contribution of microglia in the generation and regeneration of myelin by discussing their beneficial and detrimental roles, especially in remyelination, underlining the cellular and molecular components involved. Finally, we present recent findings towards human stem cell-derived preclinical models for the study of microglia in human pathologies and on the role of microbiome on glial cell functions.

The beneficial role of the synthetic microneurotrophin BNN20 in a focal demyelination model.

BNN20, a C17-spiroepoxy derivative of the neurosteroid dehydroepiandrosterone, has been shown to exhibit strong neuroprotective properties but its role in glial populations has not been assessed. Our aim was to investigate the effect of BNN20 on glial populations by using in vitro and in vivo approaches, taking advantage of the well-established lysophosphatidylcholine (LPC)-induced focal demyelination mouse model. Our in vivo studies, performed in male mice, showed that BNN20 treatment leads to an increased number of mature oligodendrocytes (OLs) in this model. It diminishes astrocytic accumulation during the demyelination phase leading to a faster remyelination process, while it does not affect oligodendrocyte precursor cell recruitment or microglia/macrophage accumulation. Additionally, our in vitro studies showed that BNN20 acts directly to OLs and enhances their maturation even after they were treated with LPC. This beneficial effect of BNN20 is mediated, primarily, through the neurotrophin receptor TrkA. In addition, BNN20 reduces microglial activation and their transition to their pro-inflammatory state upon lipopolysaccharides stimulation in vitro. Taken together our results suggest that BNN20 could serve as an important molecule to develop blood-brain barrier-permeable synthetic agonists of neurotrophin receptors that could reduce inflammation, protect and increase the number of functional OLs by promoting their differentiation/maturation.

Neuronal, but not glial, Contactin 2 negatively regulates axon regeneration in the injured adult optic nerve.

Mammalian adult neurons of the central nervous system (CNS) display limited ability to regrow axons after trauma. The developmental decline in their regenerative ability has been attributed to both intrinsic and extrinsic factors, including postnatal suppression of transcription factors and non-neuronal inhibitory components, respectively. The cell adhesion molecule Contactin 2 (CNTN2) is expressed in neurons and oligodendrocytes in the CNS. Neuronal CNTN2 is highly regulated during development and plays critical roles in axon growth and guidance and neuronal migration. On the other hand, CNTN2 expressed by oligodendrocytes interferes with the myelination process, with its ablation resulting in hypomyelination. In the current study, we investigate the role of CNTN2 in neuronal survival and axon regeneration after trauma, in the murine optic nerve crush (ONC) model. We unveil distinct roles for neuronal and glial CNTN2 in regenerative responses. Surprisingly, our data show a conflicting role of neuronal and glial CNTN2 in axon regeneration. Although glial CNTN2 as well as hypomyelination are dispensable for both neuronal survival and axon regeneration following ONC, the neuronal counterpart comprises a negative regulator of regeneration. Specifically, we reveal a novel mechanism of action for neuronal CNTN2, implicating the inhibition of Akt signalling pathway. The in vitro analysis indicates a BDNF-independent mode of action and biochemical data suggest the implication of the truncated form of TrkB neurotrophin receptor. In conclusion, CNTN2 expressed in CNS neurons serves as an inhibitor of axon regeneration after trauma and its mechanism of action involves the neutralization of Akt-mediated neuroprotective effects.

Implication of Contactins in Demyelinating Pathologies.

Demyelinating pathologies comprise of a variety of conditions where either central or peripheral myelin is attacked, resulting in white matter lesions and neurodegeneration. Myelinated axons are organized into molecularly distinct domains, and this segregation is crucial for their proper function. These defined domains are differentially affected at the different stages of demyelination as well as at the lesion and perilesion sites. Among the main players in myelinated axon organization are proteins of the contactin (CNTN) group of the immunoglobulin superfamily (IgSF) of cell adhesion molecules, namely Contactin-1 and Contactin-2 (CNTN1, CNTN2). The two contactins perform their functions through intermolecular interactions, which are crucial for myelinated axon integrity and functionality. In this review, we focus on the implication of these two molecules as well as their interactors in demyelinating pathologies in humans. At first, we describe the organization and function of myelinated axons in the central (CNS) and the peripheral (PNS) nervous system, further analyzing the role of CNTN1 and CNTN2 as well as their interactors in myelination. In the last section, studies showing the correlation of the two contactins with demyelinating pathologies are reviewed, highlighting the importance of these recognition molecules in shaping the function of the nervous system in multiple ways.

Using the Allen gene expression atlas of the adult mouse brain to gain further insight into the physiological significance of TAG-1/Contactin-2.

The anatomic gene expression atlas (AGEA) of the adult mouse brain of the Allen Institute for Brain Science is a transcriptome-based atlas of the adult C57Bl/6 J mouse brain, based on the extensive in situ hybridization dataset of the Institute. This spatial mapping of the gene expression levels of mice under baseline conditions could assist in the formation of new, reasonable transcriptome-derived hypotheses on brain structure and underlying biochemistry, which could also have functional implications. The aim of this work is to use the data of the AGEA (in combination with Tabula Muris, a compendium of single cell transcriptome data collected from mice, enabling direct and controlled comparison of gene expression among cell types) to provide further insights into the physiology of TAG-1/Contactin-2 and its interactions, by presenting the expression of the corresponding gene across the adult mouse brain under baseline conditions and to investigate any spatial genomic correlations between TAG-1/Contactin-2 and its interacting proteins and markers of mature and immature oligodendrocytes, based on the pre-existing experimental or bibliographical evidence. The across-brain correlation analysis on the gene expression intensities showed a positive spatial correlation of TAG-1/Contactin-2 with the gene expression of Plp1, Myrf, Mbp, Mog, Cldn11, Bace1, Kcna1, Kcna2, App and Nfasc and a negative spatial correlation with the gene expression of Cspg4, Pdgfra, L1cam, Ncam1, Ncam2 and Ptprz1. Spatially correlated genes are mainly expressed by mature oligodendrocytes (like Cntn2), while spatially anticorrelated genes are mainly expressed by oligodendrocyte precursor cells. According to the data presented in this work, we propose that even though Contactin-2 expression during development correlates with high plasticity events, such as neuritogenesis, in adulthood it correlates with pathways characterized by low plasticity.

UniProt-Related Documents (UniReD): assisting wet lab biologists in their quest on finding novel counterparts in a protein network.

The in-depth study of protein-protein interactions (PPIs) is of key importance for understanding how cells operate. Therefore, in the past few years, many experimental as well as computational approaches have been developed for the identification and discovery of such interactions. Here, we present UniReD, a user-friendly, computational prediction tool which analyses biomedical literature in order to extract known protein associations and suggest undocumented ones. As a proof of concept, we demonstrate its usefulness by experimentally validating six predicted interactions and by benchmarking it against public databases of experimentally validated PPIs succeeding a high coverage. We believe that UniReD can become an important and intuitive resource for experimental biologists in their quest for finding novel associations within a protein network and a useful tool to complement experimental approaches (e.g. mass spectrometry) by producing sorted lists of candidate proteins for further experimental validation. UniReD is available at http://bioinformatics.med.uoc.gr/unired/.

Ablation of CNTN2+ Pyramidal Neurons During Development Results in Defects in Neocortical Size and Axonal Tract Formation.

Corticothalamic axons express Contactin-2 (CNTN2/TAG-1), a neuronal recognition molecule of the immunoglobulin superfamily involved in neurogenesis, neurite outgrowth, and fasciculation. TAG-1, which is expressed transiently by cortical pyramidal neurons during embryonic development, has been shown to be fundamental for axonal recognition, cellular migration, and neuronal proliferation in the developing cortex. Although Tag-1 -/- mice do not exhibit any obvious defects in the corticofugal system, the role of TAG-1+ neurons during the development of the cortex remains elusive. We have generated a mouse model expressing EGFP under the Tag-1 promoter and encompassing the coding sequence of Diptheria Toxin subunit A (DTA) under quiescence with no effect on the expression of endogenous Tag-1. We show that while the line recapitulates the expression pattern of the molecule, it highlights an extended expression in the forebrain, including multiple axonal tracts and neuronal populations, both spatially and temporally. Crossing these mice to the Emx1-Cre strain, we ablated the vast majority of TAG-1+ cortical neurons. Among the observed defects were a significantly smaller cortex, a reduction of corticothalamic axons as well as callosal and commissural defects. Such defects are common in neurodevelopmental disorders, thus this mouse could serve as a useful model to study physiological and pathophysiological cortical development.

Models and treatments for traumatic optic neuropathy and demyelinating optic neuritis.

Pathologies of the optic nerve could result as primary insults in the visual tract or as secondary deficits due to inflammation, demyelination, or compressing effects of the surrounding tissue. The extent of damage may vary from mild to severe, differently affecting patient vision, with the most severe forms leading to complete uni- or bilateral visual loss. The aim of researchers and clinicians in the field is to alleviate the symptoms of these, yet uncurable pathologies, taking advantage of known and novel potential therapeutic approaches, alone or in combinations, and applying them in a limited time window after the insult. In this review, we discuss the epidemiological and clinical profile as well as the pathophysiological mechanisms of two main categories of optic nerve pathologies, namely traumatic optic neuropathy and optic neuritis, focusing on the demyelinating form of the latter. Moreover, we report on the main rodent models mimicking these pathologies or some of their clinical aspects. The current treatment options will also be reviewed and novel approaches will be discussed.

Selective Axonal Expression of the Kv1 Channel Complex in Pre-myelinated GABAergic Hippocampal Neurons.

In myelinated fibers, the voltage-gated sodium channels Nav1 are concentrated at the nodal gap to ensure the saltatory propagation of action potentials. The voltage-gated potassium channels Kv1 are segregated at the juxtaparanodes under the compact myelin sheath and may stabilize axonal conduction. It has been recently reported that hippocampal GABAergic neurons display high density of Nav1 channels remarkably in clusters along the axon before myelination (Freeman et al., 2015). In inhibitory neurons, the Nav1 channels are trapped by the ankyrinG scaffold at the axon initial segment (AIS) as observed in pyramidal and granule neurons, but are also forming "pre-nodes," which may accelerate conduction velocity in pre-myelinated axons. However, the distribution of the Kv1 channels along the pre-myelinated inhibitory axons is still unknown. In the present study, we show that two subtypes of hippocampal GABAergic neurons, namely the somatostatin and parvalbumin positive cells, display a selective high expression of Kv1 channels at the AIS and all along the unmyelinated axons. These inhibitory axons are also highly enriched in molecules belonging to the juxtaparanodal Kv1 complex, including the cell adhesion molecules (CAMs) TAG-1, Caspr2, and ADAM22 and the scaffolding protein 4.1B. Here, taking advantage of hippocampal cultures from 4.1B and TAG-1 knock-out mice, we observed that 4.1B is required for the proper positioning of Caspr2 and TAG-1 along the distal axon, and that TAG-1 deficiency induces alterations in the axonal distribution of Caspr2. However, the axonal expression of Kv1 channels and clustering of ankyrinG were not modified. In conclusion, this study allowed the analysis of the hierarchy between channels, CAMs and scaffolding proteins for their expression along hippocampal inhibitory axons before myelination. The early steps of channel compartmentalization preceding myelination may be crucial for stabilizing nerve impulses switching from a continuous to saltatory conduction during network development.