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Abstract Haplo-insufficiency of the gene encoding the myelin protein PMP22 leads to focal myelin overgrowth in the peripheral nervous system and hereditary neuropathy with liability to pressure palsies (HNPP). Conversely, duplication of PMP22 causes Charcot-Marie-Tooth disease type 1A (CMT1A), characterized by hypomyelination of medium to large caliber axons. The molecular mechanisms of abnormal myelin growth regulation by PMP22 have remained obscure. Here, we show in rodent models of HNPP and CMT1A that the PI3K/Akt/mTOR-pathway inhibiting phosphatase PTEN is correlated in abundance with PMP22 in peripheral nerves, without evidence for direct protein interactions. Indeed, treating DRG neuron/Schwann cell co-cultures from HNPP mice with PI3K/Akt/mTOR pathway inhibitors reduced focal hypermyelination. When we treated HNPP mice in vivo with the mTOR inhibitor Rapamycin, motor functions were improved, compound muscle amplitudes were increased and pathological tomacula in sciatic nerves were reduced. In contrast, we found Schwann cell dedifferentiation in CMT1A uncoupled from PI3K/Akt/mTOR, leaving partial PTEN ablation insufficient for disease amelioration. For HNPP, the development of PI3K/Akt/mTOR pathway inhibitors may be considered as the first treatment option for pressure palsies.

Abstract Recombinant human erythropoietin (rhEPO) has potent procognitive effects, likely hematopoiesis-independent, but underlying mechanisms and physiological role of brain-expressed EPO remained obscure. Here, we provide transcriptional hippocampal profiling of male mice treated with rhEPO. Based on ~108,000 single nuclei, we unmask multiple pyramidal lineages with their comprehensive molecular signatures. By temporal profiling and gene regulatory analysis, we build developmental trajectory of CA1 pyramidal neurons derived from multiple predecessor lineages and elucidate gene regulatory networks underlying their fate determination. With EPO as ‘tool’, we discover populations of newly differentiating pyramidal neurons, overpopulating to ~200% upon rhEPO with upregulation of genes crucial for neurodifferentiation, dendrite growth, synaptogenesis, memory formation, and cognition. Using a Cre-based approach to visually distinguish pre-existing from newly formed pyramidal neurons for patch-clamp recordings, we learn that rhEPO treatment differentially affects excitatory and inhibitory inputs. Our findings provide mechanistic insight into how EPO modulates neuronal functions and networks.

Neonatal hypoxia leads to white matter hypomyelination due to delayed oligodendrocyte maturation. The authors identify Sirt2 as a crucial regulator of oligodendrocyte differentiation during normal white matter development and in response to hypoxia.

Myelin is formed of proteins of long half-lives. The mechanisms of renewal of such a stable structure are unclear. Here, the authors show that myelin integrity requires continuous myelin synthesis at the inner tongue, contributing to the maintenance of a functional axon-myelin unit.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward-rectifying) and oligodendroglial Kir4.1 (inward-rectifying) potassium channels have important roles in regulating neuronal excitability at and around the nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE), with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs, and rescued neuronal pathology in oligodendrocyte–Kir4.1–deficient (OL-Kir4.1–deficient) mice. In summary, our findings indicate that neuron-OL compensatory interactions promoted resilience through Kv7 and Kir4.1 channels and identify pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination.

A key feature of advanced brain aging includes structural defects of intracortical myelin that are associated with secondary neuroinflammation. A similar pathology is seen in specific myelin mutant mice that model ‘advanced brain aging’ and exhibit a range of behavioral abnormalities. However, the cognitive assessment of these mutants is problematic because myelin-dependent motor-sensory functions are required for quantitative behavioral readouts. To better understand the role of cortical myelin integrity for higher brain functions, we generated mice lacking Plp1, encoding the major integral myelin membrane protein, selectively in ventricular zone stem cells of the mouse forebrain. In contrast to conventional Plp1 null mutants, subtle myelin defects were restricted to the cortex, hippocampus, and underlying callosal tracts. Moreover, forebrain-specific Plp1 mutants exhibited no defects of basic motor-sensory performance at any age tested. Surprisingly, several behavioral alterations reported for conventional Plp1 null mice (Gould et al., 2018) were absent and even social interactions appeared normal. However, with novel behavioral paradigms, we determined catatonia-like symptoms and isolated executive dysfunction in both genders. This suggests that loss of myelin integrity has an impact on cortical connectivity and underlies specific defects of executive function. These observations are likewise relevant for human neuropsychiatric conditions and other myelin-related diseases.

Abstract Up to one person in a population of 10,000 is diagnosed once in lifetime with an encephalitis, in 50–70% of unknown origin. Recognized causes amount to 20–50% viral infections. Approximately one third of affected subjects develops moderate and severe subsequent damage. Several neurotropic viruses can directly infect pyramidal neurons and induce neuronal death in cortex and hippocampus. The resulting encephalitic syndromes are frequently associated with cognitive deterioration and dementia, but involve numerous parallel and downstream cellular and molecular events that make the interpretation of direct consequences of sudden pyramidal neuronal loss difficult. This, however, would be pivotal for understanding how neuroinflammatory processes initiate the development of neurodegeneration, and thus for targeted prophylactic and therapeutic interventions. Here we utilized adult male NexCreERT2xRosa26-eGFP-DTA (= ‘DTA’) mice for the induction of a sterile encephalitis by diphtheria toxin-mediated ablation of cortical and hippocampal pyramidal neurons which also recruits immune cells into gray matter. We report multifaceted aftereffects of this defined process, including the expected pathology of classical hippocampal behaviors, evaluated in Morris water maze, but also of (pre)frontal circuit function, assessed by prepulse inhibition. Importantly, we modelled in encephalitis mice novel translationally relevant sequelae, namely altered social interaction/cognition, accompanied by compromised thermoreaction to social stimuli as convenient readout of parallel autonomic nervous system (dys)function. High resolution magnetic resonance imaging disclosed distinct abnormalities in brain dimensions, including cortical and hippocampal layering, as well as of cerebral blood flow and volume. Fluorescent tracer injection, immunohistochemistry and brain flow cytometry revealed persistent blood–brain-barrier perturbance and chronic brain inflammation. Surprisingly, blood flow cytometry showed no abnormalities in circulating major immune cell subsets and plasma high-mobility group box 1 (HMGB1) as proinflammatory marker remained unchanged. The present experimental work, analyzing multidimensional outcomes of direct pyramidal neuronal loss, will open new avenues for urgently needed encephalitis research.

Oligodendrocytes myelinate and metabolically support axons. The role of myelination in information processing beyond regulation of conduction velocity is unclear. Here, the authors show that myelination contributes to sustained stimulus perception in the auditory cortex, shaping neuronal responses.

Myelinating cells differentially myelinate axons of different diameters, however whether they can also restrict radial axonal growth remained unclear. Here, the authors show that CMTM6 in Schwann cells restricts axon diameters, affecting sensory nerve conduction and behavioral performance.

EPO treatment improves cognition, but underlying mechanisms were unknown. Here the authors describe a regulatory loop in which brain networks challenged by cognitive tasks drift into functional hypoxia that drives—via neuronal EPO synthesis—neurodifferentiation and dendritic spine formation.

Human myelin disorders are commonly studied in mouse models. Since both clades evolutionarily diverged approximately 85 million years ago, it is critical to know to what extent the myelin protein composition has remained similar. Here, we use quantitative proteomics to analyze myelin purified from human white matter and find that the relative abundance of the structural myelin proteins PLP, MBP, CNP, and SEPTIN8 correlates well with that in C57Bl/6N mice. Conversely, multiple other proteins were identified exclusively or predominantly in human or mouse myelin. This is exemplified by peripheral myelin protein 2 (PMP2), which was specific to human central nervous system myelin, while tetraspanin-2 (TSPAN2) and connexin-29 (CX29/GJC3) were confined to mouse myelin. Assessing published scRNA-seq-datasets, human and mouse oligodendrocytes display well-correlating transcriptome profiles but divergent expression of distinct genes, including Pmp2, Tspan2, and Gjc3. A searchable web interface is accessible via www.mpinat.mpg.de/myelin. Species-dependent diversity of oligodendroglial mRNA expression and myelin protein composition can be informative when translating from mouse models to humans.

Summary: The mechanisms by which astrocytes modulate neural homeostasis, synaptic plasticity, and memory are still poorly explored. Astrocytes form large intercellular networks by gap junction coupling, mainly composed of two gap junction channel proteins, connexin 30 (Cx30) and connexin 43 (Cx43). To circumvent developmental perturbations and to test whether astrocytic gap junction coupling is required for hippocampal neural circuit function and behavior, we generate and study inducible, astrocyte-specific Cx30 and Cx43 double knockouts. Surprisingly, disrupting astrocytic coupling in adult mice results in broad activation of astrocytes and microglia, without obvious signs of pathology. We show that hippocampal CA1 neuron excitability, excitatory synaptic transmission, and long-term potentiation are significantly affected. Moreover, behavioral inspection reveals deficits in sensorimotor performance and a complete lack of spatial learning and memory. Together, our findings establish that astrocytic connexins and an intact astroglial network in the adult brain are vital for neural homeostasis, plasticity, and spatial cognition.

Generation of oligodendrocytes in the adult brain enables both adaptive changes in neural circuits and regeneration of myelin sheaths destroyed by injury, disease, and normal aging. This transformation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes requires processing of distinct mRNAs at different stages of cell maturation. Although mislocalization and aggregation of the RNA-binding protein, TDP-43, occur in both neurons and glia in neurodegenerative diseases, the consequences of TDP-43 loss within different stages of the oligodendrocyte lineage are not well understood. By performing stage-specific genetic inactivation of Tardbp in vivo, we show that oligodendrocyte lineage cells are differentially sensitive to loss of TDP-43. While OPCs depend on TDP-43 for survival, with conditional deletion resulting in cascading cell loss followed by rapid regeneration to restore their density, oligodendrocytes become less sensitive to TDP-43 depletion as they mature. Deletion of TDP-43 early in the maturation process led to eventual oligodendrocyte degeneration, seizures, and premature lethality, while oligodendrocytes that experienced late deletion survived and mice exhibited a normal lifespan. At both stages, TDP-43-deficient oligodendrocytes formed fewer and thinner myelin sheaths and extended new processes that inappropriately wrapped neuronal somata and blood vessels. Transcriptional analysis revealed that in the absence of TDP-43, key proteins involved in oligodendrocyte maturation and myelination were misspliced, leading to aberrant incorporation of cryptic exons. Inducible deletion of TDP-43 from oligodendrocytes in the adult central nervous system (CNS) induced the same progressive morphological changes and mice acquired profound hindlimb weakness, suggesting that loss of TDP-43 function in oligodendrocytes may contribute to neuronal dysfunction in neurodegenerative disease.

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here, we find that expression of the tetraspan-transmembrane protein CMTM5 (chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and central nervous system (CNS) myelin. Genetic disruption of the Cmtm5 gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5 deficiency causes an early-onset progressive axonopathy, which we also observe in global and tamoxifen-induced oligodendroglial Cmtm5 mutants. Presence of the WldS mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

Oligodendrocytes are the primary target of demyelinating disorders, and progressive neurodegenerative changes may evolve in the CNS. DNA damage and oxidative stress are considered key pathogenic events, but the underlying molecular mechanisms remain unclear. Moreover, animal models do not fully recapitulate human diseases, complicating the path to effective treatments. Here we report that mice with cell-autonomous deletion of the nuclear COP9 signalosome component CSN5 (JAB1) in oligodendrocytes develop DNA damage and defective DNA repair in myelinating glial cells. Interestingly, oligodendrocytes lacking JAB1 expression underwent a senescence-like phenotype that fostered chronic inflammation and oxidative stress. These mutants developed progressive CNS demyelination, microglia inflammation, and neurodegeneration, with severe motor deficits and premature death. Notably, blocking microglia inflammation did not prevent neurodegeneration, whereas the deletion of p21CIP1 but not p16INK4a pathway ameliorated the disease. We suggest that senescence is key to sustaining neurodegeneration in demyelinating disorders and may be considered a potential therapeutic target.

Onion bulbs are a hallmark of demyelinating peripheral neuropathies. Here the authors identify Neuregulin-1 type I expression in Schwann cells as an essential mechanism involved in the formation of these characteristic structures.

Summary: In adult cornu ammonis hippocampi, erythropoietin (EPO) expression drives the differentiation of new neurons, independent of DNA synthesis, and increases dendritic spine density. This substantial brain hardware upgrade is part of a regulatory circle: during motor-cognitive challenge, neurons experience “functional” hypoxia, triggering neuronal EPO production, which in turn promotes improved performance. Here, we show an unexpected involvement of resident microglia. During EPO upregulation and stimulated neurodifferentiation, either by functional or inspiratory hypoxia, microglia numbers decrease. Treating mice with recombinant human (rh)EPO or exposure to hypoxia recapitulates these changes and reveals the involvement of neuronally expressed IL-34 and microglial CSF1R. Surprisingly, EPO affects microglia in phases, initially by inducing apoptosis, later by reducing proliferation, and overall dampens microglia activity and metabolism, as verified by selective genetic targeting of either the microglial or pyramidal neuronal EPO receptor. We suggest that during accelerating neuronal differentiation, EPO acts as regulator of the CSF1R-dependent microglia.

Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity.

In several neurodegenerative disorders, axonal pathology may originate from impaired oligodendrocyte-to-axon support of energy substrates. We previously established transgenic mice that allow measuring axonal ATP levels in electrically active optic nerves. Here, we utilize this technique to explore axonal ATP dynamics in the Plpnull/y mouse model of spastic paraplegia. Optic nerves from Plpnull/y mice exhibited lower and more variable basal axonal ATP levels and reduced compound action potential (CAP) amplitudes, providing a missing link between axonal pathology and a role of oligodendrocytes in brain energy metabolism. Surprisingly, when Plpnull/y optic nerves are challenged with transient glucose deprivation, both ATP levels and CAP decline slower, but recover faster upon reperfusion of glucose. Structurally, myelin sheaths display an increased frequency of cytosolic channels comprising glucose and monocarboxylate transporters, possibly facilitating accessibility of energy substrates to the axon. These data imply that complex metabolic alterations of the axon-myelin unit contribute to the phenotype of Plpnull/y mice.

Abstract Enhanced NRG1‐ERBB4 signaling is a risk pathway in schizophrenia, and corresponding mouse models display several endophenotypes of the disease. Nonetheless, pathway‐directed treatment strategies with clinically applicable compounds have not been identified. Here, we applied a cell‐based assay using the split TEV technology to screen a library of clinically applicable compounds to identify modulators of NRG1‐ERBB4 signaling for repurposing. We recovered spironolactone, known as antagonist of corticosteroids, as an inhibitor of the ERBB4 receptor and tested it in pharmacological and biochemical assays to assess secondary compound actions. Transgenic mice overexpressing Nrg1 type III display cortical Erbb4 hyperphosphorylation, a condition observed in postmortem brains from schizophrenia patients. Spironolactone treatment reverted hyperphosphorylation of activated Erbb4 in these mice. In behavioral tests, spironolactone treatment of Nrg1 type III transgenic mice ameliorated schizophrenia‐relevant behavioral endophenotypes, such as reduced sensorimotor gating, hyperactivity, and impaired working memory. Moreover, spironolactone increases spontaneous inhibitory postsynaptic currents in cortical slices supporting an ERBB4‐mediated mode‐of‐action. Our findings suggest that spironolactone, a clinically safe drug, provides an opportunity for new treatment options for schizophrenia.

Summary: The myelin sheath is a multilamellar plasma membrane extension of highly specialized glial cells laid down in regularly spaced segments along axons. Recent studies indicate that myelin is metabolically active and capable of communicating with the underlying axon. To be functionally connected to the neuron, oligodendrocytes maintain non-compacted myelin as cytoplasmic nanochannels. Here, we used high-pressure freezing for electron microscopy to study these cytoplasmic regions within myelin close to their native state. We identified 2,′3′-cyclic nucleotide 3′-phosphodiesterase (CNP), an oligodendrocyte-specific protein previously implicated in the maintenance of axonal integrity, as an essential factor in generating and maintaining cytoplasm within the myelin compartment. We provide evidence that CNP directly associates with and organizes the actin cytoskeleton, thereby providing an intracellular strut that counteracts membrane compaction by myelin basic protein (MBP). Our study provides a molecular and structural framework for understanding how myelin maintains its cytoplasm to function as an active axon-glial unit. : Snaidero et al. provide evidence that a system of cytoplasmic-rich channels is generated in myelin sheaths by the antagonist function of MBP and CNP. The authors suggest that these channels are required to provide trophic support to neurons and maintain functional axon-glial units over a long period of time. Keywords: myelin, oligodendrocytes, glia, axons, neurodegeneration, CNP, MBP, demyelinating diseases

Breakdown of myelin sheaths is a pathological hallmark of several autoimmune diseases of the nervous system. We employed autoantibody-mediated animal models of demyelinating diseases, including a rat model of neuromyelitis optica (NMO), to target myelin and found that myelin lamellae are broken down into vesicular structures at the innermost region of the myelin sheath. We demonstrated that myelin basic proteins (MBP), which form a polymer in between the myelin membrane layers, are targeted in these models. Elevation of intracellular Ca2+ levels resulted in MBP network disassembly and myelin vesiculation. We propose that the aberrant phase transition of MBP molecules from their cohesive to soluble and non-adhesive state is a mechanism triggering myelin breakdown in NMO and possibly in other demyelinating diseases.

The most common type of Charcot-Marie-Tooth disease is caused by a duplication of PMP22 leading to dysmyelination, axonal loss and progressive muscle weakness (CMT1A). Currently, no approved therapy is available for CMT1A patients. A novel polytherapeutic proof-of-principle approach using PXT3003, a low-dose combination of baclofen, naltrexone and sorbitol, slowed disease progression after long-term dosing in adult Pmp22 transgenic rats, a known animal model of CMT1A. Here, we report an early postnatal, short-term treatment with PXT3003 in CMT1A rats that delays disease onset into adulthood. CMT1A rats were treated from postnatal day 6 to 18 with PXT3003. Behavioural, electrophysiological, histological and molecular analyses were performed until 12 weeks of age. Daily oral treatment for approximately 2 weeks ameliorated motor deficits of CMT1A rats reaching wildtype levels. Histologically, PXT3003 corrected the disturbed axon calibre distribution with a shift towards large motor axons. Despite dramatic clinical amelioration, only distal motor latencies were improved and correlated with phenotype performance. On the molecular level, PXT3003 reduced Pmp22 mRNA overexpression and improved the misbalanced downstream PI3K-AKT / MEK-ERK signalling pathway. The improved differentiation status of Schwann cells may have enabled better long-term axonal support function. We conclude that short-term treatment with PXT3003 during early development may partially prevent the clinical and molecular manifestations of CMT1A. Since PXT3003 has a strong safety profile and is currently undergoing a phase III trial in CMT1A patients, our results suggest that PXT3003 therapy may be a bona fide translatable therapy option for children and young adolescent patients suffering from CMT1A.

Myelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS.

Glial support is critical for normal axon function and can become dysregulated in white matter (WM) disease. In humans, loss-of-function mutations of KCNJ10, which encodes the inward-rectifying potassium channel KIR4.1, causes seizures and progressive neurological decline. We investigated Kir4.1 functions in oligodendrocytes (OLs) during development, adulthood and after WM injury. We observed that Kir4.1 channels localized to perinodal areas and the inner myelin tongue, suggesting roles in juxta-axonal K+ removal. Conditional knockout (cKO) of OL-Kcnj10 resulted in late onset mitochondrial damage and axonal degeneration. This was accompanied by neuronal loss and neuro-axonal dysfunction in adult OL-Kcnj10 cKO mice as shown by delayed visual evoked potentials, inner retinal thinning and progressive motor deficits. Axon pathologies in OL-Kcnj10 cKO were exacerbated after WM injury in the spinal cord. Our findings point towards a critical role of OL-Kir4.1 for long-term maintenance of axonal function and integrity during adulthood and after WM injury.

Axons are electrically excitable, cable-like neuronal processes that relay information between neurons within the nervous system and between neurons and peripheral target tissues. In the central and peripheral nervous systems, most axons over a critical diameter are enwrapped by myelin, which reduces internodal membrane capacitance and facilitates rapid conduction of electrical impulses. The spirally wrapped myelin sheath, which is an evolutionary specialisation of vertebrates, is produced by oligodendrocytes and Schwann cells; in most mammals myelination occurs during postnatal development and after axons have established connection with their targets. Myelin covers the vast majority of the axonal surface, influencing the axon's physical shape, the localisation of molecules on its membrane and the composition of the extracellular fluid (in the periaxonal space) that immerses it. Moreover, myelinating cells play a fundamental role in axonal support, at least in part by providing metabolic substrates to the underlying axon to fuel its energy requirements. The unique architecture of the myelinated axon, which is crucial to its function as a conduit over long distances, renders it particularly susceptible to injury and confers specific survival and maintenance requirements. In this review we will describe the normal morphology, ultrastructure and function of myelinated axons, and discuss how these change following disease, injury or experimental perturbation, with a particular focus on the role the myelinating cell plays in shaping and supporting the axon.

Michael W. Sereda was incorrectly associated with the Department of Cellular Neurophysiology, Hanover Medical School, Carl-Neuberg-Str. 1, 30625 Hanover, Germany. The correct affiliations for Michael W. Sereda are Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany and Department of Clinical Neurophysiology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.

Immunization with myelin components can elicit experimental autoimmune encephalomyelitis (EAE). EAE susceptibility varies between mouse strains, depending on the antigen employed. BL/6 mice are largely resistant to EAE induction with proteolipid protein (PLP), probably a reflection of antigen-specific tolerance. However, the extent and mechanism(s) of tolerance to PLP remain unclear. Here, we identified three PLP epitopes in PLP-deficient BL/6 mice. PLP-sufficient mice did not respond against two of these, whereas tolerance was “leaky” for an epitope with weak predicted MHCII binding, and only this epitope was encephalitogenic. In TCR transgenic mice, the “EAE-susceptibility-associated” epitope was “ignored” by specific CD4 T cells, whereas the “resistance-associated” epitope induced clonal deletion and Treg induction in the thymus. Central tolerance was autoimmune regulator dependent and required expression and presentation of PLP by thymic epithelial cells (TECs). TEC-specific ablation of PLP revealed that peripheral tolerance, mediated by dendritic cells through recessive tolerance mechanisms (deletion and anergy), could largely compensate for a lack of central tolerance. However, adoptive EAE was exacerbated in mice lacking PLP in TECs, pointing toward a non-redundant role of the thymus in dominant tolerance to PLP. Our findings reveal multiple layers of tolerance to a central nervous system autoantigen that vary among epitopes and thereby specify disease susceptibility. Understanding how different modalities of tolerance apply to distinct T cell epitopes of a target in autoimmunity has implications for antigen-specific strategies to therapeutically interfere with unwanted immune reactions against self.

Myelin is required for proper nervous system function. Schwann cells in developing nerves depend on extrinsic signals from the axon and from the extracellular matrix to first sort and ensheathe a single axon and then myelinate it. Neuregulin 1 type III (Nrg1III) and laminin α2β1γ1 (Lm211) are the key axonal and matrix signals, respectively, but how their signaling is integrated and if each molecule controls both axonal sorting and myelination is unclear. Here, we use a series of epistasis experiments to show that Lm211 modulates neuregulin signaling to ensure the correct timing and amount of myelination. Lm211 can inhibit Nrg1III by limiting protein kinase A (PKA) activation, which is required to initiate myelination. We provide evidence that excessive PKA activation amplifies promyelinating signals downstream of neuregulin, including direct activation of the neuregulin receptor ErbB2 and its effector Grb2-Associated Binder-1 (Gab1), thereby elevating the expression of the key transcription factors Oct6 and early growth response protein 2 (Egr2). The inhibitory effect of Lm211 is seen only in fibers of small caliber. These data may explain why hereditary neuropathies associated with decreased laminin function are characterized by focally thick and redundant myelin.

In the vertebrate nervous system, myelination of axons for rapid impulse propagation requires the synthesis of large amounts of lipids and proteins by oligodendrocytes and Schwann cells. Myelin membranes are thought to be cell-autonomously assembled by these axon-associated glial cells. Here, we report the surprising finding that in normal brain development, a substantial fraction of the lipids incorporated into central nervous system (CNS) myelin are contributed by astrocytes. The oligodendrocyte-specific inactivation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), an essential coactivator of the transcription factor SREBP and thus of lipid biosynthesis, resulted in significantly retarded CNS myelination; however, myelin appeared normal at 3 months of age. Importantly, embryonic deletion of the same gene in astrocytes, or in astrocytes and oligodendrocytes, caused a persistent hypomyelination, as did deletion from astrocytes during postnatal development. Moreover, when astroglial lipid synthesis was inhibited, oligodendrocytes began incorporating circulating lipids into myelin membranes. Indeed, a lipid-enriched diet was sufficient to rescue hypomyelination in these conditional mouse mutants. We conclude that lipid synthesis by oligodendrocytes is heavily supplemented by astrocytes in vivo and that horizontal lipid flux is a major feature of normal brain development and myelination.

Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels.

In several neurodegenerative diseases and myelin disorders, the degeneration profiles of myelinated axons are compatible with underlying energy deficits. However, it is presently impossible to measure selectively axonal ATP levels in the electrically active nervous system. We combined transgenic expression of an ATP-sensor in neurons of mice with confocal FRET imaging and electrophysiological recordings of acutely isolated optic nerves. This allowed us to monitor dynamic changes and activity-dependent axonal ATP homeostasis at the cellular level and in real time. We find that changes in ATP levels correlate well with compound action potentials. However, this correlation is disrupted when metabolism of lactate is inhibited, suggesting that axonal glycolysis products are not sufficient to maintain mitochondrial energy metabolism of electrically active axons. The combined monitoring of cellular ATP and electrical activity is a novel tool to study neuronal and glial energy metabolism in normal physiology and in models of neurodegenerative disorders.

Neuregulin-1 (NRG1) gene variants are associated with increased genetic risk for schizophrenia. It is unclear whether risk haplotypes cause elevated or decreased expression of NRG1 in the brains of schizophrenia patients, given that both findings have been reported from autopsy studies. To study NRG1 functions in vivo, we generated mouse mutants with reduced and elevated NRG1 levels and analyzed the impact on cortical functions. Loss of NRG1 from cortical projection neurons resulted in increased inhibitory neurotransmission, reduced synaptic plasticity, and hypoactivity. Neuronal overexpression of cysteine-rich domain (CRD)-NRG1, the major brain isoform, caused unbalanced excitatory-inhibitory neurotransmission, reduced synaptic plasticity, abnormal spine growth, altered steady-state levels of synaptic plasticity-related proteins, and impaired sensorimotor gating. We conclude that an “optimal” level of NRG1 signaling balances excitatory and inhibitory neurotransmission in the cortex. Our data provide a potential pathomechanism for impaired synaptic plasticity and suggest that human NRG1 risk haplotypes exert a gain-of-function effect.

Background: Studying astrocytes in higher brain functions has been hampered by the lack of genetic tools for the efficient expression of inducible Cre recombinase throughout the CNS, including the neocortex. Methods: Therefore, we generated BAC transgenic mice, in which CreERT2 is expressed under control of the Aldh1l1 regulatory region. Results: When crossbred to Cre reporter mice, adult Aldh1l1-CreERT2 mice show efficient gene targeting in astrocytes. No such Cre-mediated recombination was detectable in CNS neurons, oligodendrocytes, and microglia. As expected, Aldh1l1-CreERT2 expression was evident in several peripheral organs, including liver and kidney. Conclusions: Taken together, Aldh1l1-CreERT2 mice are a useful tool for studying astrocytes in neurovascular coupling, brain metabolism, synaptic plasticity and other aspects of neuron-glia interactions.