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Traumatic brain injury (TBI) is a major contributor to morbidity and mortality in the United States as several million people visit the emergency department every year due to TBI exposures. Unfortunately, there is still no consensus on the pathology underlying mild TBI, the most common severity sub-type of TBI. Previous preclinical and post-mortem human studies have detailed the presence of diffuse axonal injury following TBI, suggesting that white matter pathology is the predominant pathology of diffuse brain injury. However, the inertial loading produced by TBI results in strain fields in both gray and white matter. In order to further characterize gray matter pathology in mild TBI, our lab used a pig model (n = 25) of closed-head rotational acceleration-induced TBI to evaluate blood-brain barrier disruptions, neurodegeneration, astrogliosis, and microglial reactivity in the cerebral cortex out to 1 year post-injury. Immunohistochemical staining revealed the presence of a hyper-ramified microglial phenotype-more branches, junctions, endpoints, and longer summed process length-at 30 days post injury (DPI) out to 1 year post injury in the cingulate gyrus ( p < 0.05), and at acute and subacute timepoints in the inferior temporal gyrus ( p < 0.05). Interestingly, we did not find neuronal loss or astroglial reactivity paired with these chronic microglia changes. However, we observed an increase in fibrinogen reactivity-a measure of blood-brain barrier disruption-predominately in the gray matter at 3 DPI ( p = 0.0003) which resolved to sham levels by 7 DPI out to chronic timepoints. Future studies should employ gene expression assays, neuroimaging, and behavioral assays to elucidate the effects of these hyper-ramified microglia, particularly related to neuroplasticity and responses to potential subsequent insults. Further understanding of the brain's inflammatory activity after mild TBI will hopefully provide understanding of pathophysiology that translates to clinical treatment for TBI.

Unbiased in vivo selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier (BBB), but little is known about the parameters of capsid-receptor interactions that govern their improved activity. This hampers broader efforts in precision capsid engineering and is a practical impediment to ensuring the translatability of capsid properties between preclinical animal models and human clinical trials. In this work, we utilize the adeno-associated virus (AAV)-PHP.B-Ly6a model system to better understand the targeted delivery and BBB penetration properties of AAV vectors. This model offers a defined capsid-receptor pair that can be used to systematically define relationships between target receptor affinity and in vivo activity of engineered AAV vectors. Here, we report a high-throughput method for quantifying capsid-receptor affinity and demonstrate that direct binding assays can be used to organize a vector library into families with varied affinity for their target receptor. Our data indicate that efficient central nervous system transduction requires high levels of target receptor expression at the BBB, but it is not a requirement for receptor expression to be limited to the target tissue. We observed that enhanced receptor affinity leads to reduced transduction of off-target tissues but can negatively impact on-target cellular transduction and penetration of endothelial barriers. Together, this work provides a set of tools for defining vector-receptor affinities and demonstrates how receptor expression and affinity interact to impact the performance of engineered AAV vectors in targeting the central nervous system. IMPORTANCE Novel methods for measuring adeno-associated virus (AAV)-receptor affinities, especially in relation to vector performance in vivo , would be useful to capsid engineers as they develop AAV vectors for gene therapy applications and characterize their interactions with native or engineered receptors. Here, we use the AAV-PHP.B-Ly6a model system to assess the impact of receptor affinity on the systemic delivery and endothelial penetration properties of AAV-PHP.B vectors. We discuss how receptor affinity analysis can be used to isolate vectors with optimized properties, improve the interpretation of library selections, and ultimately translate vector activities between preclinical animal models and humans., Competing Interests: The authors declare a conflict of interest. This research was supported by Amicus Therapeutics and Passage Bio. JMW is a paid advisor to and holds equity in iECURE, Scout Bio, Passage Bio, and the Center for Breakthrough Medicines (CBM). He also holds equity in the G2 Bio-associated asset companies. He has sponsored research agreements with Amicus Therapeutics, CBM, Elaaj Bio, FA212, G2 Bio, G2 Bio-associated asset companies, iECURE, Passage Bio, and Scout Bio, which are licensees of Penn technology. JMW and JJS are inventors on patents that have been licensed to various biopharmaceutical companies and for which they may receive payments.

Despite being a major health concern, little is known about the pathophysiological changes that underly concussion. Nonetheless, emerging evidence suggests that selective damage to white matter axons, or diffuse axonal injury (DAI), disrupts brain network connectivity and function. While voltage-gated sodium channels (NaChs) and their anchoring proteins at the nodes of Ranvier (NOR) on axons are key elements of the brain's network signaling machinery, changes in their integrity have not been studied in context with DAI. Here, we utilized a clinically relevant swine model of concussion that induces evolving axonal pathology, demonstrated by accumulation of amyloid precursor protein (APP) across the white matter. Over a two-week follow-up post-concussion with this model, we found widespread loss of NaCh isoform 1.6 (Nav1.6), progressive increases in NOR length, the appearance of void and heminodes and loss of βIV-spectrin, ankyrin G, and neurofascin 186 or their collective diffusion into the paranode. Notably, these changes were in close proximity, yet distinct from APP-immunoreactive swollen axonal profiles, potentially representing a unique, newfound phenotype of axonal pathology in DAI. Since concussion in humans is non-fatal, the clinical relevance of these findings was determined through examination of post-mortem brain tissue from humans with higher levels of acute traumatic brain injury. Here, a similar loss of Nav1.6 and changes in NOR structures in brain white matter were observed as found in the swine model of concussion. Collectively, this widespread and progressive disruption of NaChs and NOR appears to be a form of sodium channelopathy, which may represent an important substrate underlying brain network dysfunction after concussion., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Though hippocampal volume reduction is a pathological hallmark of schizophrenia, the molecular pathway(s) responsible for this degeneration remains unknown. Recent reports have suggested the potential role of impaired blood-brain barrier (BBB) function in schizophrenia pathogenesis. However, direct evidence demonstrating an impaired BBB function is missing. In this preliminary study, we used immunohistochemistry and serum immunoglobulin G (IgG) antibodies to investigate the state of BBB function in formalin-fixed postmortem samples from the hippocampus and surrounding temporal cortex of patients with schizophrenia ( n = 25) and controls without schizophrenia ( n = 27) matched for age, sex, and race. Since a functional BBB prevents the extravasation of IgGs, detection of IgGs in the parenchyma is used as direct evidence of BBB breakdown. We also developed a semi-quantitative approach to quantify the extent of IgG leak and therein BBB breach. Analysis of our immunohistochemistry data demonstrated a significantly higher incidence of IgG leak in patients with schizophrenia compared to controls. Further, BBB permeability was significantly higher in advanced-age patients with schizophrenia than both advanced-age controls and middle-aged patients with schizophrenia. Male patients with schizophrenia also demonstrated a significant increase in IgG permeability compared to control males. Interestingly, the extravasated IgGs also demonstrated selective immunoreactivity for neurons. Based on these observations, we suggest that BBB dysfunction and IgG autoantibodies could be two key missing pathoetiological links underwriting schizophrenia hippocampal damage., Competing Interests: LH has received or is planning to receive research funding or consulting fees from Mitsubishi, Your Energy Systems LLC, Neuralstem, Taisho, Heptares, Pfizer, Luye Pharma, Sound Pharma, Takeda, and Regeneron. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Goldwaser, Swanson, Arroyo, Venkataraman, Kosciuk, Nagele, Hong and Acharya.)

Ferroptosis is a novel iron-dependent regulated cell death mechanism that affects cell metabolism; however, a detailed metabolomic analysis of ferroptotic cells is not yet available. Here, we elucidated the metabolome of H9c2 cardioblasts by gas chromatography-mass spectrometry during ferroptosis induced by RSL3, a GPX4 inhibitor, in the presence of ferrostatin-1 (a ferroptosis inhibitor), XJB-5-131 (a mitochondrial-targeted ROS scavenger), or TSM-1005-44 (a newly developed cellular ROS scavenger). Results demonstrated that RSL3 decreased the levels of amino acids involved in glutathione synthesis more than two-fold. In contrast, saturated fatty acids levels were markedly increased in RSL3-challenged cells, with no effects on unsaturated fatty acids. RSL3 significantly altered the levels of mitochondrial tricarboxylic acid cycle intermediates; isocitrate and 2-oxoglutarate were found to increase, whereas succinate was significantly decreased in RSL3-challenged cells. Ferrostatin-1, XJB-5-131, and TSM-1005-44 prevented RSL3-induced cell death and conserved the metabolomic profile of the cells. Since 2-oxoglutarate is involved in the regulation of ferroptosis, particularly through glutamine metabolism, we further assessed the role of glutaminolysis in ferroptosis in H9c2 cardioblasts. Genetic silencing of GLS1, which encodes the K-type mitochondrial glutaminase (glutaminase C), protected against ferroptosis in the early stage. In conclusion, our study demonstrates that RSL3-induced ferroptosis impairs the metabolome of H9c2 cardioblasts.

To better understand the spatiotemporal course of radiation-induced central nervous system (CNS) vascular necrosis and assess the therapeutic potential of approaches for protecting against radiation-induced necrosis, adult female Sprague Dawley rats received 40 Gy surface dose centered on the T9 thoracic spinal cord segment. Locomotor function, blood-spinal cord barrier (BSCB) integrity and histology were evaluated throughout the study. No functional symptoms were observed for several months postirradiation. However, a sudden onset of paralysis was observed at approximately 5.5 months postirradiation. The progression rapidly led to total paralysis and death within less than 48 h of symptom onset. Open-field locomotor scores and rotarod motor coordination testing showed no evidence of neurological impairment prior to the onset of overt paralysis. Histological examination revealed minimal changes to the vasculature prior to symptom onset. However, Evans blue dye (EvB) extravasation revealed a progressive deterioration of BSCB integrity, beginning at one week postirradiation, affecting regions well outside of the irradiated area. Minocycline treatment significantly delayed the onset of paralysis. The results of this study indicate that extensive asymptomatic disruption of the blood-CNS barrier may precede onset of vascular breakdown by several months and suggests that minocycline treatment has a therapeutic effect by delaying radiation-induced necrosis after CNS irradiation.

In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site [1]. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [2] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSCexos). In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR "hotspots" were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration.

TNFα is persistently elevated in many injury and disease conditions. Previous reports of cytotoxicity of TNFα for oligodendrocytes and their progenitors suggest that the poor endogenous remyelination in patients with traumatic injury or multiple sclerosis may be due in part to persistent inflammation. Understanding the effects of inflammatory cytokines on potential cell therapy candidates is therefore important for evaluating the feasibility of their use. In this study, we assessed the effects of long term exposure to TNFα on viability, proliferation, migration and TNFα receptor expression of cultured rat olfactory ensheathing cells (OECs) and Schwann cells (SCs). Although OECs and SCs transplanted into the CNS produce similar myelinating phenotypes, and might be expected to have similar therapeutic uses, we report that they have very different sensitivities to TNFα. OECs exhibited positive proliferative responses to TNFα over a much broader range of concentrations than SCs. Low TNFα concentrations increased proliferation and migration of both OECs and SCs, but SC number declined in the presence of 100 ng/ml or higher concentrations of TNFα. In contrast, OECs exhibited enhanced proliferation even at high TNFα concentrations (up to 1 µg/ml) and showed no evidence of TNF cytotoxicity even at 4 weeks post-treatment. Furthermore, while both OECs and SCs expressed TNFαR1 and TNFαR2, TNFα receptor levels were downregulated in OECs after exposure to100 ng/ml TNFα for 5-7 days, but were either elevated or unchanged in SCs. These results imply that OECs may be a more suitable cell therapy candidate if transplanted into areas with persistent inflammation.

Intravenous infusion of mesenchymal stem cells (MSCs) has been shown to reduce the severity of experimental spinal cord injury (SCI), but mechanisms are not fully understood. One important consequence of SCI is damage to the microvasculature and disruption of the blood spinal cord barrier (BSCB). In the present study we induced a contusive SCI at T9 in the rat and studied the effects of intravenous MSC infusion on BSCB permeability, microvascular architecture and locomotor recovery over a 10week period. Intravenously delivered MSCs could not be identified in the spinal cord, but distributed primarily to the lungs where they survived for a couple of days. Spatial and temporal changes in BSCB integrity were assessed by intravenous infusions of Evans blue (EvB) with in vivo and ex vivo optical imaging and spectrophotometric quantitation of EvB leakage into the parenchyma. SCI resulted in prolonged BSCB leakage that was most severe at the impact site but disseminated extensively rostral and caudal to the lesion over 6weeks. Contused spinal cords also showed an increase in vessel size, reduced vessel number, dissociation of pericytes from microvessels and decreases in von Willebrand factor (vWF) and endothelial barrier antigen (EBA) expression. In MSC-treated rats, BSCB leakage was reduced, vWF expression was increased and locomotor function improved beginning 1 week post-MSC infusion, i.e., 2weeks post-SCI. These results suggest that intravenously delivered MSCs have important effects on reducing BSCB leakage which could contribute to their therapeutic efficacy., (Copyright © 2015. Published by Elsevier Inc.)

Elevated nerve growth factor (NGF) is believed to play a role in many types of pain. An NGF-blocking antibody (muMab 911) has been shown to reduce pain and hyperalgesia in pain models, suggesting a novel therapeutic approach for pain management. Since NGF also plays important roles in peripheral nervous system development and sensory nerve outgrowth, we asked whether anti-NGF antibodies would adversely impact peripheral nerve regeneration. Adult rats underwent a unilateral sciatic nerve crush to transect axons and were subcutaneously dosed weekly for 8weeks with muMab 911 or vehicle beginning 1day prior to injury. Plasma levels of muMab 911 were assessed from blood samples and foot print analysis was used to assess functional recovery. At 8-weeks post-nerve injury, sciatic nerves were prepared for light and electron microscopy. In a separate group, Fluro-Gold was injected subcutaneously at the ankle prior to perfusion, and counts and sizes of retrogradely labeled and unlabeled dorsal root ganglion neurons were obtained. There was no difference in the time course of gait recovery in antibody-treated and vehicle-treated animals. The number of myelinated and nonmyelinated axons was the same in the muMab 911-treated crushed nerves and intact nerves, consistent with observed complete recovery. Treatment with muMab 911 did however result in a small decrease in average cell body size on both the intact and injured sides. These results indicate that muMab 911 did not impair functional recovery or nerve regeneration after nerve injury in adult rats., (Published by Elsevier Ltd.)

Intraneurally injected lysolecithin causes both segmental and paranodal demyelination. In demyelinated internodes, axonal components of nodes fragment and disappear, glial and axonal paranodal and juxtaparanodal proteins no longer cluster, and axonal Kv1.1/Kv1.2 K+ channels move from the juxtaparanodal region to appose the remaining heminodes. In paranodal demyelination, a gap separates two distinct heminodes, each of which contains the molecular components of normal nodes; paranodal and juxtaparanodal proteins are properly localized. As in normal nodes, widened nodal regions contain little or no band 4.1B. Lysolecithin also causes "unwinding" of paranodes: The spiral of Schwann cell membrane moves away from the paranodes, but the glial and axonal components of septate-like junctions remain colocalized. Thus, acute demyelination has distinct effects on the molecular organization of the nodal, paranodal, and juxtaparanodal region, reflecting altered axon-Schwann cell interactions.

We have examined the molecular organization of axons in the spinal cords of myelin-deficient (md) rats, which have profound CNS dysmyelination associated with oligodendrocyte cell death. Although myelin sheaths are rare, most large axons are at least partially surrounded by oligodendrocyte processes. At postnatal day 7 (P7), almost all node-like clusters of voltage-gated Na+ channels and ankyrinG are adjacent to axonal segments ensheathed by oligodendrocytes, but at P21, many node-like clusters are found in axonal segments that lack oligodendrocyte ensheathment. In P21 wild-type (WT) rats, the voltage-gated Na+ channels Na(v)1.2, Na(v)1.6, and Na(v)1.8, are found in different subpopulations of myelinated axons, and md rats have a similar distribution. The known molecular components of paranodes--contactin, Caspr, and neurofascin 155--are not clustered in md spinal cords, and no septate-like junctions between oligodendrocyte processes and axons are found by electron microscopy. Furthermore, Kv1.1 and Kv1.2 K+ channels are not spatially segregated from the node-like clusters of Na+ channels in md rats, in contrast to their WT littermates. These results suggest the following: node-like clusters of voltage-gated Na+ channels and ankyrinG form adjacent to ensheathed axonal segments even in the absence of a myelin sheath; these clusters persist after oligodendrocyte cell death; dysmyelination does not alter the expression of different nodal of voltage-gated Na+ channels; the absence of paranodes results in the mislocalization of neurofascin155, contactin, and Caspr, and the aberrant localization of Kv1.1 and Kv1.2.

Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells.

Ezrin, radixin, and moesin (ERM proteins), as well as the neurofibromatosis 2 (NF2) tumor suppressor merlin/schwannomin, all belong to the protein 4.1 family, yet only merlin is a tumor suppressor in Schwann cells. To gain insight into the possible functions of ERM proteins in Schwann cells, we examined their localization in peripheral nerve, because we have previously shown that merlin is found in paranodes and in Schmidt-Lanterman incisures. All three ERM proteins were highly expressed in the microvilli of myelinating Schwann cells that surround the nodal axolemma as well as in incisures and cytoplasmic puncta in the vicinity of the node. In all of these locations, ERM proteins were colocalized with actin filaments. In contrast, ERM proteins did not surround nodes in the CNS. The colocalization of ERM proteins with actin indicates that they have functions different from those of merlin in myelinating Schwann cells.

We examined the organization of the molecular components of the nodal region in spontaneously diabetic BB-Wistar rats. Frozen sections and teased fibers from the sciatic nerves were immunostained for nodal (voltage-gated Na(+) channels, ankyrin(G), and ezrin), paranodal (contactin, Caspr, and neurofascin 155 kDa), and juxtaparanodal (Caspr2, the Shaker-type K(+) channels Kv1.1 and Kv1.2, and their associated subunit Kvbeta2) proteins. All of these proteins were properly localized in myelinated fibers from rats that had been diabetic for 15-44 days, compared to age-matched, nondiabetic animals. These results demonstrate that the axonal membrane is not reorganized, so nodal reorganization is not likely to be the cause of nerve conduction slowing in this animal model of acute diabetes.

We have examined the localization of contactin-associated protein (Caspr), the Shaker-type potassium channels, Kv1.1 and Kv1.2, their associated beta subunit, Kvbeta2, and Caspr2 in the myelinated fibers of the CNS. Caspr is localized to the paranodal axonal membrane, and Kv1.1, Kv1.2, Kvbeta2 and Caspr2 to the juxtaparanodal membrane. In addition to the paranodal staining, an internodal strand of Caspr staining apposes the inner mesaxon of the myelin sheath. Unlike myelinated axons in the peripheral nervous system, there was no internodal strand of Kv1.1, Kv1.2, Kvbeta2, or Caspr2. Thus, the organization of the nodal, paranodal, and juxtaparanodal axonal membrane is similar in the central and peripheral nervous systems, but the lack of Kv1.1/Kv1.2/Kvbeta2/Caspr2 internodal strands indicates that the oligodendrocyte myelin sheaths lack a trans molecular interaction with axons, an interaction that is present in Schwann cell myelin sheaths.

Schwann cells and oligodendrocytes make the myelin sheaths of the PNS and CNS, respectively. Their myelin sheaths are structurally similar, consisting of multiple layers of specialized cell membrane that spiral around axons, but there are several differences. (1) CNS myelin has a "radial component" composed of a tight junction protein, claudin-11/oligodendrocyte-specific protein. (2) Schwann cells have a basal lamina and microvilli. (3) Although both CNS and PNS myelin sheaths have incisures, those in the CNS lack the structural as well as the molecular components of "reflexive" adherens junctions and gap junctions. In spite of their structural differences, the axonal membranes of the PNS and CNS are similarly organized. The nodal axolemma contains high concentrations of voltage-dependent sodium channels that are linked to the axonal cytoskeleton by ankyrin(G). The paranodal membrane contains Caspr/paranodin, which may participate in the formation of axoglial junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated beta2 subunit, as well as Caspr2, which is closely related to Caspr. The myelin sheath probably organizes these axonal membrane-related proteins via trans interactions.

Among higher metazoans, echinoderms exhibit the most impressive capacity for regeneration. Holothurians, or sea cucumbers, respond to adverse stimuli by autotomizing and ejecting their visceral organs, which are then regenerated. Neuronal fibers and cell bodies are present within the viscera, but previous regeneration studies have not accounted for the nervous component. We used light microscopic immunocytochemistry and ultrastructural studies to describe the regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima. This study provides evidence that the enteric nervous system of this echinoderm regenerates after evisceration and that in 3-5 weeks the regenerated system is virtually identical to that of noneviscerated animals. The regeneration of the enteric nervous system occurs parallel to the regeneration of other organ components. Nerve fibers and cells are observed within the mesenterial thickenings that give rise to the new intestine and within the internal connective tissue prior to lumen formation. We also used bromodeoxyuridine incorporation to show that proliferation of the neuronal population occurs in the regenerating intestine. The regeneration of the nervous system commands high interest because members of the closely related phylum Chordata either lack or have a very limited capacity to regenerate their nervous system. Thus, holothurians provide a model system to study enteric nervous system regeneration in deuterostomes.

We examined the localization of Caspr and the K(+) channels Kv1.1 and Kv1.2, all of which are intrinsic membrane proteins of myelinated axons in the PNS. Caspr is localized to the paranode; Kv1. 1, Kv1.2 and their beta2 subunit are localized to the juxtaparanode. Throughout the internodal region, a strand of Caspr staining is flanked by a double strand of Kv1.1/Kv1.2/Kvbeta2 staining. This tripartite strand apposes the inner mesaxon of the myelin sheath, and forms a circumferential ring that apposes the innermost aspect of Schmidt-Lanterman incisures. The localization of Caspr and Kv1.2 are not disrupted in mice with null mutations of the myelin associated glycoprotein, connexin32, or Kv1.1 genes. At all of these locations, Caspr and Kv1.1/Kv1.2/Kvbeta2 define distinct but interrelated domains of the axonal membrane that appear to be organized by the myelin sheath.

Tst-1/SCIP/Oct-6, a POU domain transcription factor, is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. In tst-1/scip/oct-6-null sciatic nerves, Schwann cells are transiently arrested at the "promyelinating" stage, when they have a one-to-one relationship with an axon but before they have elaborated a myelin sheath. To determine when Schwann cells express Tst-1/SCIP/Oct-6, we examined beta-galactosidase (beta-gal) expression in heterozygous tst-1/scip/oct-6 mice, in which one copy of the tst-1/scip/oct-6 gene has been replaced with the LacZ gene. beta-Gal expression from the LacZ gene seems to parallel Tst-1/SCIP/Oct-6 expression from the endogenous tst-1/scip/oct-6 gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of 5bromo-4-chloro-3-indolyl-beta-D-galactopyranoside- (X-gal) and halogenated indolyl-beta-D-galactoside- (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels of beta-gal, both in developing and in regenerating nerves. Thus, the expression of beta-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested in tst-1/scip/oct-6-null mice, indicating that Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann cells.