Activity-dependent extracellular potassium changes in unmyelinated versus myelinated areas in olfactory regions of the isolated female guinea-pig brain.

The potassium released in the extracellular space during neuronal activity is rapidly removed by glia and neurons to maintain tissue homeostasis. Oligodendrocyte-derived myelin axonal coating contributes to potassium buffering and is therefore crucial to control brain excitability. We studied activity-dependent extracellular potassium ([K+]o) changes in the piriform cortex (PC), a region that features highly segregated bundles of myelinated and unmyelinated fibers. Four-aminopyridine (4AP; 50 µM) treatment or patterned high-frequency stimulations (hfST) were utilized to generate [K+]o changes measured with potassium-sensitive electrodes in the myelinated lateral olfactory tract (LOT), in the unmyelinated PC layer I and in the myelinated deep PC layers in the ex vivo isolated guinea-pig brain. Seizure-like events induced by 4AP are initiated by the abrupt [K+]o rise in the layer I formed by unmyelinated fibers (Uva et al., 2017). Larger [K+]o shifts occurred in unmyelinated layers compared to the myelinated LOT. LOT hfST that mimicks pre-seizure discharges also generated higher [K+]o changes in unmyelinated PC layer I than in LOT and deep PC layers. The treatment with the Kir4.1 potassium channel blocker BaCl2 (100 µM) enhanced the [K+]o changes generated by hfST in myelinated structures. Our data show that activity-dependent [K+]o changes are intrinsically different in myelinated vs unmyelinated cortical regions. The larger [K+]o shifts generated in unmyelinated structures may represent a vehicle for seizure generation.

Heat shock protein amplification improves cerebellar myelination in the Npc1nih mouse model.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare prematurely fatal lysosomal lipid storage disease with limited therapeutic options. The prominent neuropathological hallmarks include hypomyelination and cerebellar atrophy. We previously demonstrated the efficacy of recombinant human heat shock protein 70 (rhHSP70) in preclinical models of the disease. It reduced glycosphingolipid levels in the central nervous system (CNS), improving cerebellar myelination and improved behavioural phenotypes in Npc1nih (Npc1-/-) mice. Furthermore, treatment with arimoclomol, a well-characterised HSP amplifier, attenuated lysosomal storage in NPC patient fibroblasts and improved neurological symptoms in Npc1-/- mice. Taken together, these findings prompted the investigation of the effects of HSP amplification on CNS myelination. METHODS: We administered bimoclomol daily or rhHSP70 6 times per week to Npc1-/- (BALB/cNctr-Npc1m1N/J, also named Npc1nih) mice by intraperitoneal injection from P7 through P34 to investigate the impact on CNS myelination. The Src-kinase inhibitor saracatinib was administered with/without bimoclomol twice daily to explore the contribution of Fyn kinase to bimoclomol's effects. FINDINGS: Treatment with either bimoclomol or rhHSP70 improved myelination and increased the numbers of mature oligodendrocytes (OLs) as well as the ratio of active-to-inactive forms of phosphorylated Fyn kinase in the cerebellum of Npc1-/- mice. Additionally, treatment with bimoclomol preserved cerebellar weight, an effect that was abrogated when co-administered with saracatinib, an inhibitor of Fyn kinase. Bimoclomol-treated mice also exhibited increased numbers of immature OLs within the cortex. INTERPRETATION: These data increase our understanding of the mechanisms by which HSP70 regulates myelination and provide further support for the clinical development of HSP-amplifying therapies in the treatment of NPC. FUNDING: Funding for this study was provided by Orphazyme A/S (Copenhagen, Denmark) and a Pathfinder Award from The Wellcome Trust.

APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes.

APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.

Myelination, axonal loss and Schwann cell characteristics in axonal polyneuropathy compared to controls.

INTRODUCTION: Polyneuropathy is a debilitating condition characterized by distal sensory and motor deficits. Schwann cell dysfunction and axonal loss are integral factors in pathophysiology and disease progression of polyneuropathy. AIMS: The aim of this study was the assessment of Schwann cell characteristics, nerve fibers and myelination parameters in polyneuropathy patients compared to controls. METHODS: Nerve tissue was obtained from polyneuropathy patients (n = 10) undergoing diagnostic sural nerve biopsies. Biopsies of healthy peripheral nerves (n = 5) were harvested during elective sural nerve grafting for chronic peripheral nerve lesions. Exclusion criteria for the healthy control group were recent neurological trauma, diabetes, neurological and cardiovascular disease, as well as active malignancies and cytotoxic medication within the last 12 months. The over-all architecture of nerve sections and myelination parameters were histomorphometrically analyzed. Immunofluorescent imaging was used to evaluate Schwann cell phenotypes, senescence markers and myelination parameters. RESULTS: Histomorphometric analysis of nerve biopsies showed significant axonal loss in polyneuropathy patients compared to controls, which was in accordance with the neuropathological findings. Immunofluorescent staining of Schwann cells and myelin basic protein indicated a significant impairment of myelination and lower Schwann cell counts compared to controls. Phenotypic alterations and increased numbers of non-myelinating p75-positive Schwann cells were found in polyneuropathy patients. DISCUSSION: This study provided quantitative data of axonal loss, reduced myelination and Schwann cell dysfunction of polyneuropathy patients compared to neurologically healthy controls. Phenotypic alterations of Schwann cells were similar to those seen after peripheral nerve injury, highlighting the clinical relevance of Schwann cell dysfunction.

Human IPSC-Derived Model to Study Myelin Disruption.

Myelin is of vital importance to the central nervous system and its disruption is related to a large number of both neurodevelopmental and neurodegenerative diseases. The differences observed between human and rodent oligodendrocytes make animals inadequate for modeling these diseases. Although developing human in vitro models for oligodendrocytes and myelinated axons has been a great challenge, 3D cell cultures derived from iPSC are now available and able to partially reproduce the myelination process. We have previously developed a human iPSC-derived 3D brain organoid model (also called BrainSpheres) that contains a high percentage of myelinated axons and is highly reproducible. Here, we have further refined this technology by applying multiple readouts to study myelination disruption. Myelin was assessed by quantifying immunostaining/confocal microscopy of co-localized myelin basic protein (MBP) with neurofilament proteins as well as proteolipid protein 1 (PLP1). Levels of PLP1 were also assessed by Western blot. We identified compounds capable of inducing developmental neurotoxicity by disrupting myelin in a systematic review to evaluate the relevance of our BrainSphere model for the study of the myelination/demyelination processes. Results demonstrated that the positive reference compound (cuprizone) and two of the three potential myelin disruptors tested (Bisphenol A, Tris(1,3-dichloro-2-propyl) phosphate, but not methyl mercury) decreased myelination, while ibuprofen (negative control) had no effect. Here, we define a methodology that allows quantification of myelin disruption and provides reference compounds for chemical-induced myelin disruption.

Nociceptor subtypes are born continuously over DRG development.

Sensory neurogenesis in the dorsal root ganglion (DRG) occurs in two waves of differentiation with larger, myelinated proprioceptive and low-threshold mechanoreceptor (LTMR) neurons differentiating before smaller, unmyelinated (C) nociceptive neurons. This temporal difference was established from early birthdating studies based on DRG soma cell size. However, distinctions in birthdates between molecular subtypes of sensory neurons, particularly nociceptors, is unknown. Here, we assess the birthdate of lumbar DRG neurons in mice using a thymidine analog, EdU, to label developing neurons exiting mitosis combined with co-labeling of known sensory neuron markers. We find that different nociceptor subtypes are born on similar timescales, with continuous births between E9.5 to E13.5, and peak births from E10.5 to E11.5. Notably, we find that thinly myelinated Aδ-fiber nociceptors and peptidergic C-fibers are born more broadly between E10.5 and E11.5 than previously thought and that non-peptidergic C-fibers and C-LTMRs are born with a peak birth date of E11.5. Moreover, we find that the percentages of nociceptor subtypes born at a particular timepoint are the same for any given nociceptor cell type marker, indicating that intrinsic or extrinsic influences on cell type diversity are occurring similarly across developmental time. Overall, the patterns of birth still fit within the classical "two wave" description, as touch and proprioceptive fibers are born primarily at E10.5, but suggest that nociceptors have a slightly broader wave of birthdates with different nociceptor subtypes continually differentiating throughout sensory neurogenesis irrespective of myelination.

Increased extracellular fluid is associated with white matter fiber degeneration in CADASIL: in vivo evidence from diffusion magnetic resonance imaging.

BACKGROUND: White matter hyperintensities (WMHs) are one of the hallmarks of cerebral small vessel disease (CSVD), but the pathological mechanisms underlying WMHs remain unclear. Recent studies suggest that extracellular fluid (ECF) is increased in brain regions with WMHs. It has been hypothesized that ECF accumulation may have detrimental effects on white matter microstructure. To test this hypothesis, we used cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) as a unique CSVD model to investigate the relationships between ECF and fiber microstructural changes in WMHs. METHODS: Thirty-eight CADASIL patients underwent 3.0 T MRI with multi-model sequences. Parameters of free water (FW) and apparent fiber density (AFD) obtained from diffusion-weighted imaging (b = 0 and 1000 s/mm2) were respectively used to quantify the ECF and fiber density. WMHs were split into four subregions with four levels of FW using quartiles (FWq1 to FWq4) for each participant. We analyzed the relationships between FW and AFD in each subregion of WMHs. Additionally, we tested whether FW of WMHs were associated with other accompanied CSVD imaging markers including lacunes and microbleeds. RESULTS: We found an inverse correlation between FW and AFD in WMHs. Subregions of WMHs with high-level of FW (FWq3 and FWq4) were accompanied with decreased AFD and with changes in FW-corrected diffusion tensor imaging parameters. Furthermore, FW was also independently associated with lacunes and microbleeds. CONCLUSIONS: Our study demonstrated that increased ECF was associated with WM degeneration and the occurrence of lacunes and microbleeds, providing important new insights into the role of ECF in CADASIL pathology. Improving ECF drainage might become a therapeutic strategy in future.

The role of hyaluronan in myelination and remyelination after white matter injury.

Hyaluronan is one of the major components of the neural extracellular matrix (ECM) and provides structural support in physiological conditions. Altered hyaluronan regulation is implicated in the pathogenesis of white matter injury (WMI), such as perinatal WMI, multiple sclerosis (MS), traumatic brain injury (TBI). Early research reported diverse central nervous system (CNS) insults led to accumulated high-molecular-weight (HMW) hyaluronan in hypomyelinating/demyelinating lesions. Furthermore, recent findings have shown an elevated production of hyaluronan fragments in WMI, possibly resulting from HMW hyaluronan degradation. Subsequent in vitro studies identified bioactive hyaluronan fragments with a specific molecular weight (around 2x105 Da) regulating oligodendrocyte precursor cells (OPCs) maturation and myelination/remyelination in WMI. However, it is unclear about the effective hyaluronidases in generating bioactive hyaluronan fragments. Several hyaluronidases are proposed recently. Although PH20 is shown to block OPCs maturation by generating bioactive hyaluronan fragments in vitro, it seems unlikely to play a primary role in WMI with negligible expression levels in vivo. The role of other hyaluronidases on OPCs maturation and myelination/remyelination is still unknown. Other than hyaluronidases, CD44 and Toll-like receptors 2 (TLR2) are also implicated in HMW hyaluronan degradation in WMI. Moreover, recent studies elucidated bioactive hyaluronan fragments interact with TLR4, initiating signaling cascades to mediate myelin basic protein (MBP) transcription. Identifying key factors in hyaluronan actions may provide novel therapeutic targets to promote OPCs maturation and myelination/remyelination in WMI.

Interference of commissural connections through the genu of the corpus callosum specifically impairs sensorimotor gating.

White matter abnormalities in schizophrenic patients are characterized as regional tract-specific. Myelin loss at the genu of the corpus callosum (GCC) is one of the most consistent findings in schizophrenic patients across the different populations. We characterized the axons that pass through the GCC by stereotactically injecting an anterograde axonal tracing viral vector into the forceps minor of the corpus callosum in one hemisphere, and identified the homotopic brain structures that have commissural connections in the two hemispheres of the prefrontal cortex, including the anterior cingulate area, the prelimbic area, the secondary motor area, and the dorsal part of the agranular insular area, along with commissural connections with the primary motor area, caudoputamen, and claustrum. To investigate whether dysmyelination in these commissural connections is critical for the development of schizophrenia symptoms, we generated a mouse model with focal demyelination at the GCC by stereotactically injecting demyelinating agent lysolecithin into this site, and tested these mice in a battery of behavioral tasks that are used to model the schizophrenia-like symptom domains. We found that demyelination at the GCC influenced neither the social interest or mood state, nor the locomotive activity or motor coordination. Nevertheless, it specifically reduced the prepulse inhibition of acoustic startle that is a well-known measure of sensorimotor gating. This study advances our understanding of the pathophysiological contributions of the GCC-specific white matter lesion to the related disease, and demonstrates an indispensable role of interhemispheric communication between the frontal cortices for the top-down regulation of the sensorimotor gating.

Squalenoyl siRNA PMP22 nanoparticles are effective in treating mouse models of Charcot-Marie-Tooth disease type 1 A.

Charcot-Marie-Tooth disease type 1 A (CMT1A) lacks an effective treatment. We provide a therapy for CMT1A, based on siRNA conjugated to squalene nanoparticles (siRNA PMP22-SQ NPs). Their administration resulted in normalization of Pmp22 protein levels, restored locomotor activity and electrophysiological parameters in two transgenic CMT1A mouse models with different severity of the disease. Pathological studies demonstrated the regeneration of myelinated axons and myelin compaction, one major step in restoring function of myelin sheaths. The normalization of sciatic nerve Krox20, Sox10 and neurofilament levels reflected the regeneration of both myelin and axons. Importantly, the positive effects of siRNA PMP22-SQ NPs lasted for three weeks, and their renewed administration resulted in full functional recovery. Beyond CMT1A, our findings can be considered as a potent therapeutic strategy for inherited peripheral neuropathies. They provide the proof of concept for a new precision medicine based on the normalization of disease gene expression by siRNA.

A possible mechanism for neurofilament slowing down in myelinated axon: Phosphorylation-induced variation of NF kinetics.

Neurofilaments(NFs) are the most abundant intermediate filaments that make up the inner volume of axon, with possible phosphorylation on their side arms, and their slow axonal transport by molecular motors along microtubule tracks in a "stop-and-go" manner with rapid, intermittent and bidirectional motion. The kinetics of NFs and morphology of axon are dramatically different between myelinate internode and unmyelinated node of Ranvier. The NFs in the node transport as 7.6 times faster as in the internode, and the distribution of NFs population in the internode is 7.6 folds as much as in the node of Ranvier. We hypothesize that the phosphorylation of NFs could reduce the on-track rate and slow down their transport velocity in the internode. By modifying the '6-state' model with (a) an extra phosphorylation kinetics to each six state and (b) construction a new '8-state' model in which NFs at off-track can be phosphorylated and have smaller on-track rate, our model and simulation demonstrate that the phosphorylation-induced decrease of on-track rate could slow down the NFs average velocity and increase the axonal caliber. The degree of phosphorylation may indicate the extent of velocity reduction. The Continuity equation used in our paper predicts that the ratio of NFs population is inverse proportional to the ratios of average velocity of NFs between node of Ranvier and internode. We speculate that the myelination of axon could increase the level of phosphorylation of NF side arms, and decrease the possibility of NFs to get on-track of microtubules, therefore slow down their transport velocity. In summary, our work provides a potential mechanism for understanding the phosphorylation kinetics of NFs in regulating their transport and morphology of axon in myelinated axons, and the different kinetics of NFs between node and internode.

Small extracellular vesicles ameliorate peripheral neuropathy and enhance chemotherapy of oxaliplatin on ovarian cancer.

There are no effective treatments for chemotherapy induced peripheral neuropathy (CIPN). Small extracellular vesicles (sEVs) facilitate intercellular communication and mediate nerve function and tumour progression. We found that the treatment of mice bearing ovarian tumour with sEVs derived from cerebral endothelial cells (CEC-sEVs) in combination with a chemo-drug, oxaliplatin, robustly reduced oxaliplatin-induced CIPN by decreasing oxaliplatin-damaged myelination and nerve fibres of the sciatic nerve and significantly amplified chemotherapy of oxaliplatin by reducing tumour size. The combination therapy substantially increased a set of sEV cargo-enriched miRNAs, but significantly reduced oxaliplatin-increased proteins in the sciatic nerve and tumour tissues. Bioinformatics analysis revealed the altered miRNAs and proteins formed two distinct networks that regulate neuropathy and tumour growth, respectively. Intravenously administered CEC-sEVs were internalized by axons of the sciatic nerve and cancer cells. Reduction of CEC-sEV cargo miRNAs abolished the effects of CEC-sEVs on oxaliplatin-inhibited axonal growth and on amplification of the anti-cancer effect in ovarian cancer cells, suggesting that alterations in the networks of miRNAs and proteins in recipient cells contribute to the therapeutic effect of CEC-sEVs on CIPN. Together, the present study demonstrates that CEC-sEVs suppressed CIPN and enhanced chemotherapy of oxaliplatin in the mouse bearing ovarian tumour.

miR-142-3p regulates cortical oligodendrocyte gene co-expression networks associated with tauopathy.

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.

Rnd2 differentially regulates oligodendrocyte myelination at different developmental periods.

In the CNS, oligodendrocyte precursor cells differentiate into oligodendrocytes to wrap their plasma membranes around neuronal axons, generating mature neural networks with myelin sheaths according to spatial and temporal patterns. While myelination is known to be one of the most dynamic cell morphological changes, the overall intrinsic and extrinsic molecular cues controlling myelination remain to be fully clarified. Here, we describe the biphasic roles of Rnd2, an atypical branch of the Rho family GTPase, in oligodendrocyte myelination during development and after maturation in mice. Compared with littermate controls, oligodendrocyte-specific Rnd2 knockout mice exhibit decreased myelin thickness at the onset of myelination but increased myelin thickness in the later period. Larger proportions of Rho kinase and its substrate Mbs, the signaling unit that negatively regulates oligodendrocyte myelination, are phosphorylated at the onset of myelination, while their smaller proportions are phosphorylated in the later period. In addition, we confirm the biphasic role of Rnd2 through experiments with oligodendrocyte-specific Rnd2 transgenic mice. We conclude that Rnd2 positively regulates myelination in the early myelinating period and negatively regulates myelination in the later period. This unique modulator thus plays different roles depending on the myelination period.

The role of Sphingolipids in myelination and myelin stability and their involvement in childhood and adult demyelinating disorders.

Multiple sclerosis (MS) represents the most common demyelinating disease affecting the central nervous system (CNS) in adults as well as in children. Furthermore, in children, in addition to acquired diseases such as MS, genetically inherited diseases significantly contribute to the incidence of demyelinating disorders. Some genetic defects lead to sphingolipid alterations that are able to elicit neurological symptoms. Sphingolipids are essential for brain development, and their aberrant functionality may thus contribute to demyelinating diseases such as MS. In particular, sphingolipidoses caused by deficits of sphingolipid-metabolizing enzymes, are often associated with demyelination. Sphingolipids are not only structural molecules but also bioactive molecules involved in the regulation of cellular events such as development of the nervous system, myelination and maintenance of myelin stability. Changes in the sphingolipid metabolism deeply affect plasma membrane organization. Thus, changes in myelin sphingolipid composition might crucially contribute to the phenotype of diseases characterized by demyelinalization. Here, we review key features of several sphingolipids such as ceramide/dihydroceramide, sphingosine/dihydrosphingosine, glucosylceramide and, galactosylceramide which act in myelin formation during rat brain development and in human brain demyelination during the pathogenesis of MS, suggesting that this knowledge could be useful in identifying targets for possible therapies.

Protocol for pressure-clamped patch-clamp recording at the node of Ranvier of rat myelinated nerves.

The patch-clamp recording technique is indispensable for studying ion channel functions of cells but is challenging to apply to the node of Ranvier, a key site where action potentials are conducted along myelinated nerves. We have developed a pressure-clamped patch-clamp recording method applying to the node of Ranvier of rat myelinated nerves. The step-by-step protocol described here allows researchers to apply this approach to study mechanisms underlying saltatory conduction and information processing in myelinated nerves of mammals. For complete information on the generation and use of this protocol, please refer to Kanda et al. (2019).

Differential Contribution of Cadm1-Cadm3 Cell Adhesion Molecules to Peripheral Myelinated Axons.

Cell adhesion proteins of the Cadm (SynCAM/Necl) family regulate myelination and the organization of myelinated axons. In the peripheral nervous system (PNS), intercellular contact between Schwann cells and their underlying axons is believed to be mediated by binding of glial Cadm4 to axonal Cadm3 or Cadm2. Nevertheless, given that distinct neurons express different combinations of the Cadm proteins, the identity of the functional axonal ligand for Cadm4 remains to be determined. Here, we took a genetic approach to compare the phenotype of Cadm4 null mice, which exhibit abnormal distribution of Caspr and Kv1 potassium channels, with mice lacking different combinations of Cadm1-Cadm3 genes. We show that in contrast to mice lacking the single Cadm1, Cadm2, or Cadm3 genes, genetic ablation of all three phenocopies the abnormalities detected in the absence of Cadm4. Similar defects were observed in double mutant mice lacking Cadm3 and Cadm2 (i.e., Cadm3-/-/Cadm2-/-) or Cadm3 and Cadm1 (i.e., Cadm3-/-/Cadm1-/-), but not in mice lacking Cadm1 and Cadm2 (i.e., Cadm1-/-/Cadm2-/-). Furthermore, axonal organization abnormalities were also detected in Cadm3 null mice that were heterozygous for the two other axonal Cadms. Our results identify Cadm3 as the main axonal ligand for glial Cadm4, and reveal that its absence could be compensated by the combined action of Cadm2 and Cadm1.SIGNIFICANCE STATEMENT Myelination by Schwann cells enables fast conduction of action potentials along motor and sensory axons. In these nerves, Schwann cell-axon contact is mediated by cell adhesion molecules of the Cadm family. Cadm4 in Schwann cells regulates axonal ensheathment and myelin wrapping, as well as the organization of the axonal membrane, but the identity of its axonal ligands is not clear. Here, we reveal that Cadm mediated axon-glia interactions depend on a hierarchical adhesion code that involves multiple family members. Our results provide important insights into the molecular mechanisms of axon-glia communication, and the function of Cadm proteins in PNS myelin.

Atypical myelinogenesis and reduced axon caliber in the Scn1a variant model of Dravet syndrome: An electron microscopy pilot study of the developing and mature mouse corpus callosum.

Dravet Syndrome (DS) is a genetic neurodevelopmental disease. Recurrent severe seizures begin in infancy and co-morbidities follow, including developmental delay, cognitive and behavioral dysfunction. A majority of DS patients have an SCN1A heterozygous gene mutation. This mutation causes a loss-of-function in inhibitory neurons, initiating seizure onset. We have investigated whether the sodium channelopathy may result in structural changes in the DS model independent of seizures. Morphometric analyses of axons within the corpus callosum were completed at P16 and P50 in Scn1a heterozygote KO male mice and their age-matched wild-type littermates. Trainable machine learning algorithms were used to examine electron microscopy images of ~400 myelinated axons per animal, per genotype, including myelinated axon cross-section area, frequency distribution and g-ratios. Pilot data for Scn1a heterozygote KO mice demonstrate the average axon caliber was reduced in developing and adult mice. Qualitative analysis also shows micro-features marking altered myelination at P16 in the DS model, with myelin out-folding and myelin debris within phagocytic cells. The data has indicated, in the absence of behavioral seizures, factors that governed a shift toward small calibre axons at P16 have persisted in adult Scn1a heterozygote KO corpus callosum. The pilot study provides a basis for future meta-analysis that will enable robust estimates of the effects of the sodium channelopathy on axon architecture. We propose that early therapeutic strategies in DS could help minimize the effect of sodium channelopathies, beyond the impact of overt seizures, and therefore achieve better long-term treatment outcomes.

Impairing committed cholesterol biosynthesis in white matter astrocytes, but not grey matter astrocytes, enhances in vitro myelination.

Remyelination is a regenerative process that is essential to recover saltatory conduction and to prevent neurodegeneration upon demyelination. The formation of new myelin involves the differentiation of oligodendrocyte progenitor cells (OPCs) toward oligodendrocytes and requires high amounts of cholesterol. Astrocytes (ASTRs) modulate remyelination by supplying lipids to oligodendrocytes. Remarkably, remyelination is more efficient in grey matter (GM) than in white matter (WM), which may relate to regional differences in ASTR subtype. Here, we show that a feeding layer of gmASTRs was more supportive to in vitro myelination than a feeding layer of wmASTRs. While conditioned medium from both gmASTRs and wmASTRs accelerated gmOPC differentiation, wmOPC differentiation is enhanced by secreted factors from gmASTRs, but not wmASTRs. In vitro analyses revealed that gmASTRs secreted more cholesterol than wmASTRs. Cholesterol efflux from both ASTR types was reduced upon exposure to pro-inflammatory cytokines, which was mediated via cholesterol transporter ABCA1, but not ABCG1, and correlated with a minor reduction of myelin membrane formation by oligodendrocytes. Surprisingly, a wmASTR knockdown of Fdft1 encoding for squalene synthase (SQS), an enzyme essential for the first committed step in cholesterol biosynthesis, enhanced in vitro myelination. Reduced secretion of interleukin-1ß likely by enhanced isoprenylation, and increased unsaturated fatty acid synthesis, both pathways upstream of SQS, likely masked the effect of reduced levels of ASTR-derived cholesterol. Hence, our findings indicate that gmASTRs export more cholesterol and are more supportive to myelination than wmASTRs, but specific inhibition of cholesterol biosynthesis in ASTRs is beneficial for wmASTR-mediated modulation of myelination.

Transcriptional heterogeneity between primary adult grey and white matter astrocytes underlie differences in modulation of in vitro myelination.

BACKGROUND: Multiple sclerosis (MS) is an inflammation-mediated demyelinating disease of the central nervous system that eventually results in secondary axonal degeneration due to remyelination failure. Successful remyelination is orchestrated by astrocytes (ASTRs) and requires sequential activation, recruitment, and maturation of oligodendrocyte progenitor cells (OPCs). In both MS and experimental models, remyelination is more robust in grey matter (GM) than white matter (WM), which is likely related to local differences between GM and WM lesions. Here, we investigated whether adult gmASTRs and wmASTRs per se and in response to MS relevant Toll-like receptor (TLR) activation differently modulate myelination. METHODS: Differences in modulation of myelination between adult gmASTRs and wmASTRs were examined using an in vitro myelinating system that relies on a feeding layer of ASTRs. Transcriptional profiling and weighted gene co-expression network analysis were used to analyze differentially expressed genes and gene networks. Potential differential modulation of OPC proliferation and maturation by untreated adult gmASTRs and wmASTRs and in response to TLR3 and TLR4 agonists were assessed. RESULTS: Our data reveal that adult wmASTRs are less supportive to in vitro myelination than gmASTRs. WmASTRs more abundantly express reactive ASTR genes and genes of a neurotoxic subtype of ASTRs, while gmASTRs have more neuro-reparative transcripts. We identified a gene network module containing cholesterol biosynthesis enzyme genes that positively correlated with gmASTRs, and a network module containing extracellular matrix-related genes that positively correlated with wmASTRs. Adult wmASTRs and gmASTRs responding to TLR3 agonist Poly(I:C) distinctly modulate OPC behavior, while exposure to TLR4 agonist LPS of both gmASTRs and wmASTRs results in a prominent decrease in myelin membrane formation. CONCLUSIONS: Primary adult gmASTRs and wmASTRs are heterogeneous at the transcriptional level, differed in their support of in vitro myelination, and their pre-existing phenotype determined TLR3 agonist responses. These findings point to a role of ASTR heterogeneity in regional differences in remyelination efficiency between GM and WM lesions.