Reduction of APOE accounts for neurobehavioral deficits in fetal alcohol spectrum disorders.

A hallmark of fetal alcohol spectrum disorders (FASD) is neurobehavioral deficits that still do not have effective treatment. Here, we present that reduction of Apolipoprotein E (APOE) is critically involved in neurobehavioral deficits in FASD. We show that prenatal alcohol exposure (PAE) changes chromatin accessibility of Apoe locus, and causes reduction of APOE levels in both the brain and peripheral blood in postnatal mice. Of note, postnatal administration of an APOE receptor agonist (APOE-RA) mitigates motor learning deficits and anxiety in those mice. Several molecular and electrophysiological properties essential for learning, which are altered by PAE, are restored by APOE-RA. Our human genome-wide association study further reveals that the interaction of PAE and a single nucleotide polymorphism in the APOE enhancer which chromatin is closed by PAE in mice is associated with lower scores in the delayed matching-to-sample task in children. APOE in the plasma is also reduced in PAE children, and the reduced level is associated with their lower cognitive performance. These findings suggest that controlling the APOE level can serve as an effective treatment for neurobehavioral deficits in FASD.

Alternative splicing events as peripheral biomarkers for motor learning deficit caused by adverse prenatal environments.

Severity of neurobehavioral deficits in children born from adverse pregnancies, such as maternal alcohol consumption and diabetes, does not always correlate with the adversity's duration and intensity. Therefore, biological signatures for accurate prediction of the severity of neurobehavioral deficits, and robust tools for reliable identification of such biomarkers, have an urgent clinical need. Here, we demonstrate that significant changes in the alternative splicing (AS) pattern of offspring lymphocyte RNA can function as accurate peripheral biomarkers for motor learning deficits in mouse models of prenatal alcohol exposure (PAE) and offspring of mother with diabetes (OMD). An aptly trained deep-learning model identified 29 AS events common to PAE and OMD as superior predictors of motor learning deficits than AS events specific to PAE or OMD. Shapley-value analysis, a game-theory algorithm, deciphered the trained deep-learning model's learnt associations between its input, AS events, and output, motor learning performance. Shapley values of the deep-learning model's input identified the relative contribution of the 29 common AS events to the motor learning deficit. Gene ontology and predictive structure-function analyses, using Alphafold2 algorithm, supported existing evidence on the critical roles of these molecules in early brain development and function. The direction of most AS events was opposite in PAE and OMD, potentially from differential expression of RNA binding proteins in PAE and OMD. Altogether, this study posits that AS of lymphocyte RNA is a rich resource, and deep-learning is an effective tool, for discovery of peripheral biomarkers of neurobehavioral deficits in children of diverse adverse pregnancies.

Deletion of the Thrombin Proteolytic Site in Neurofascin 155 Causes Disruption of Nodal and Paranodal Organization.

In the central nervous system, myelin is attached to the axon in the paranodal region by a trimolecular complex of Neurofascin155 (NF155) in the myelin membrane, interacting with Caspr1 and Contactin1 on the axolemma. Alternative splicing of a single Neurofascin transcript generates several different Neurofascins expressed by several cell types, but NF155, which is expressed by oligodendrocytes, contains a domain in the third fibronectinIII-like region of the molecule that is unique. The immunoglobulin 5-6 domain of NF155 is essential for binding to Contactin1, but less is known about the functions of the NF155-unique third fibronectinIII-like domain. Mutations and autoantibodies to this region are associated with several neurodevelopmental and demyelinating nervous system disorders. Here we used Crispr-Cas9 gene editing to delete a 9 bp sequence of NF155 in this unique domain, which has recently been identified as a thrombin binding site and implicated in plasticity of the myelin sheath. This small deletion results in dysmyelination, eversion of paranodal loops of myelin, substantial enlargement of the nodal gap, a complete loss of paranodal septate junctions, and mislocalization of Caspr1 and nodal sodium channels. The animals exhibit tremor and ataxia, and biochemical and mass spectrometric analysis indicates that while NF155 is transcribed and spliced normally, the NF155 protein is subsequently degraded, resulting in loss of the full length 155 kDa native protein. These findings reveal that this 9 bp region of NF155 in its unique third fibronectinIII-like domain is essential for stability of the protein.

Oligodendrocyte involvement in Gulf War Illness.

Low level sarin nerve gas and other anti-cholinesterase agents have been implicated in Gulf War illness (GWI), a chronic multi-symptom disorder characterized by cognitive, pain and fatigue symptoms that continues to afflict roughly 32% of veterans from the 1990-1991 Gulf War. How disrupting cholinergic synaptic transmission could produce chronic illness is unclear, but recent research indicates that acetylcholine also mediates communication between axons and oligodendrocytes. Here we investigated the hypothesis that oligodendrocyte development is disrupted by Gulf War agents, by experiments using the sarin-surrogate acetylcholinesterase inhibitor, diisopropyl fluorophosphate (DFP). The effects of corticosterone, which is used in some GWI animal models, were also investigated. The data show that DFP decreased both the number of mature and dividing oligodendrocytes in the rat prefrontal cortex (PFC), but differences were found between PFC and corpus callosum. The differences seen between the PFC and corpus callosum likely reflect the higher percentage of proliferating oligodendroglia in the adult PFC. In cell culture, DFP also decreased oligodendrocyte survival through a non-cholinergic mechanism. Corticosterone promoted maturation of oligodendrocytes, and when used in combination with DFP it had protective effects by increasing the pool of mature oligodendrocytes and decreasing proliferation. Cell culture studies indicate direct effects of both DFP and corticosterone on OPCs, and by comparison with in vivo results, we conclude that in addition to direct effects, systemic effects and interruption of neuron-glia interactions contribute to the detrimental effects of GW agents on oligodendrocytes. Our results demonstrate that oligodendrocytes are an important component of the pathophysiology of GWI.

Treadmilling Model for Plasticity of the Myelin Sheath.

We propose a mechanism for myelin plasticity that would complement synaptic plasticity by adjusting conduction velocity for optimal spike-time arrival. In the proposed treadmilling model, myelin sheath thickness is a dynamic balance between the rates of new myelin deposited adjacent to the axon and removal of the outermost layer.

Regulation of myelin structure and conduction velocity by perinodal astrocytes.

The speed of impulse transmission is critical for optimal neural circuit function, but it is unclear how the appropriate conduction velocity is established in individual axons. The velocity of impulse transmission is influenced by the thickness of the myelin sheath and the morphology of electrogenic nodes of Ranvier along axons. Here we show that myelin thickness and nodal gap length are reversibly altered by astrocytes, glial cells that contact nodes of Ranvier. Thrombin-dependent proteolysis of a cell adhesion molecule that attaches myelin to the axon (neurofascin 155) is inhibited by vesicular release of thrombin protease inhibitors from perinodal astrocytes. Transgenic mice expressing a dominant-negative fragment of VAMP2 in astrocytes, to reduce exocytosis by 50%, exhibited detachment of adjacent paranodal loops of myelin from the axon, increased nodal gap length, and thinning of the myelin sheath in the optic nerve. These morphological changes alter the passive cable properties of axons to reduce conduction velocity and spike-time arrival in the CNS in parallel with a decrease in visual acuity. All effects were reversed by the thrombin inhibitor Fondaparinux. Similar results were obtained by viral transfection of tetanus toxin into astrocytes of rat corpus callosum. Previously, it was unknown how the myelin sheath could be thinned and the functions of perinodal astrocytes were not well understood. These findings describe a form of nervous system plasticity in which myelin structure and conduction velocity are adjusted by astrocytes. The thrombin-dependent cleavage of neurofascin 155 may also have relevance to myelin disruption and repair.

Cholinergic signaling in myelination.

There is a long history of research on acetylcholine (ACh) function in myelinating glia, but a resurgence of interest recently as a result of the therapeutic potential of manipulating ACh signaling to promote remyelination, and the broader interest in neurotransmitter signaling in activity-dependent myelination. Myelinating glia express all the major types of muscarinic and nicotinic ACh receptors at different stages of development, and acetylcholinesterase and butyrylcholinesterase are highly expressed in white matter. This review traces the history of research on ACh signaling in Schwann cells, oligodendrocytes, and in the myelin sheath, and summarizes current knowledge on the intracellular signaling and functional consequences of ACh signaling in myelinating glia. Implications of ACh in diseases, such as Alzheimer's disease, multiple sclerosis, and white matter toxicity caused by pesticides are considered, together with an outline of major questions for future research. GLIA 2017;65:687-698.

Combinatorial actions of Tgfß and Activin ligands promote oligodendrocyte development and CNS myelination.

In the embryonic CNS, development of myelin-forming oligodendrocytes is limited by bone morphogenetic proteins, which constitute one arm of the transforming growth factor-ß (Tgfß) family and signal canonically via Smads 1/5/8. Tgfß ligands and Activins comprise the other arm and signal via Smads 2/3, but their roles in oligodendrocyte development are incompletely characterized. Here, we report that Tgfß ligands and activin B (ActB) act in concert in the mammalian spinal cord to promote oligodendrocyte generation and myelination. In mouse neural tube, newly specified oligodendrocyte progenitors (OLPs) are first exposed to Tgfß ligands in isolation, then later in combination with ActB during maturation. In primary OLP cultures, Tgfß1 and ActB differentially activate canonical Smad3 and non-canonical MAP kinase signaling. Both ligands enhance viability, and Tgfß1 promotes proliferation while ActB supports maturation. Importantly, co-treatment strongly activates both signaling pathways, producing an additive effect on viability and enhancing both proliferation and differentiation such that mature oligodendrocyte numbers are substantially increased. Co-treatment promotes myelination in OLP-neuron co-cultures, and maturing oligodendrocytes in spinal cord white matter display strong Smad3 and MAP kinase activation. In spinal cords of ActB-deficient Inhbb(-/-) embryos, apoptosis in the oligodendrocyte lineage is increased and OLP numbers transiently reduced, but numbers, maturation and myelination recover during the first postnatal week. Smad3(-/-) mice display a more severe phenotype, including diminished viability and proliferation, persistently reduced mature and immature cell numbers, and delayed myelination. Collectively, these findings suggest that, in mammalian spinal cord, Tgfß ligands and ActB together support oligodendrocyte development and myelin formation.

Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease.

In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.

On the occurrence of hypomyelination in a transgenic mouse model: a consequence of the myelin basic protein promoter?

Central nervous system hypomyelination is a feature common to a number of transgenic (Tg) mouse lines that express a variety of unrelated exogenous (i.e. non-central nervous system) transgenes. In this report, we document hypomyelination structurally by immunocytochemistry and functionally in the Tg line MBP-JE, which over expresses the chemokine CCL2 (MCP-1) within oligodendrocytes targeted by a myelin basic protein (MBP) promoter. Analysis of hypomyelinated optic nerves of Tg mice revealed progressive decrease in oligodendrocyte numbers with age (p < 0.01). Although molecular mechanisms underlying hypomyelination in this and other Tg models remain largely unknown, we present preliminary findings on oligodendrocyte progenitor cell (OPC) cultures in which, although OPC expressed CCR2, the receptor for CCL2, treatment with CCL2 had no significant effect on OPC proliferation, differentiation, or apoptosis. We suggest that hypomyelination in the MBP-JE model might not be due to CCL2 expression but rather the result of transcriptional dysfunction related to random insertion of the MBP promoter that disrupts myelinogenesis and leads to oligodendrocyte demise. Because an MBP promoter is a common denominator in most Tg lines displaying hypomyelination, we hypothesize that use of myelin gene sequences in the regulator region of Tg constructs might underlie this perturbation of myelination in such models.

Promoting myelin repair and return of function in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. Conduction block in demyelinated axons underlies early neurological symptoms, but axonal transection and neuronal loss are believed to be responsible for more permanent chronic deficits. Several therapies are approved for treatment of relapsing-remitting MS, all of which are immunoregulatory and clinically proven to reduce the rate of lesion formation and exacerbation. However, existing approaches are only partially effective in preventing the onset of disability in MS patients, and novel treatments to protect myelin-producing oligodendrocytes and enhance myelin repair may improve long-term outcomes. Studies in vivo in genetically modified mice have assisted in the characterization of mechanisms underlying the generation of neuropathology in MS patients, and have identified potential avenues for oligodendrocyte protection and myelin repair. However, no treatments are yet approved that target these areas directly, and in addition, the relationship between demyelination and axonal transection in the lesions of the disease remains unclear. Here, we review translational research targeting oligodendrocyte protection and myelin repair in models of autoimmune demyelination, and their potential relevance as therapies in MS.

Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory functions of IL-11.

Current therapies for multiple sclerosis target inflammation but do not directly address oligodendrocyte protection or myelin repair. The gp130 family cytokines ciliary neurotrophic factor, leukemia inhibitory factor, and IL-11 have been identified as oligodendrocyte growth factors, and IL-11 is also strongly immunoregulatory, but their underlying mechanisms of action are incompletely characterized. In this study, we demonstrate that these effects of IL-11 are mediated via differential regulation of apoptosis in oligodendrocytes versus Ag-presenting dendritic cells (DCs), and are dependent on lineage-specific activity of the transcription factors Stat1 versus Stat3. Focal demyelinating lesions induced in cerebral cortices of IL-11Rα(-/-) mice using stereotactic microinjection of lysolecithin were larger than in controls, and remyelination was delayed. In IL-11Rα(-/-) mice, lesions displayed extensive oligodendrocyte loss and axonal transection, and increased infiltration by inflammatory cells including CD11c(+) DCs, CD3(+) lymphocytes, and CD11b(+) phagocytes. In oligodendrocyte progenitor cell (OPC) cultures, IL-11 restricted caspase 9 activation and apoptosis, and it increased myelination in OPC-neuron cocultures. Importantly, siRNA inhibition of Stat1 enhanced the antiapoptotic effects of IL-11 on OPCs, but IL-11 induced apoptosis in the presence of Stat3 silencing. In contrast, IL-11 augmented caspase activation and apoptosis in cultures of CD11c(+) DCs, but not in CD11b(+) or CD3(+) cells. Inhibition of Stat3 exacerbated the proapoptotic effects of IL-11 on DCs, whereas they were ablated in Stat1(-/-) cultures. Collectively, these findings reveal novel mechanisms underlying the actions of a neuroprotective and immunoregulatory member of the gp130 cytokine family, suggesting avenues to enhance oligodendrocyte viability and restrict CNS inflammation in multiple sclerosis.

Targeting oligodendrocyte protection and remyelination in multiple sclerosis.

Multiple sclerosis is an inflammatory demyelinating disease of the brain and spinal cord with a presumed autoimmune etiology. Conduction block in demyelinated axons underlies early neurological symptoms, whereas axonal transection is believed responsible for more permanent later deficits. Approved treatments for the disease are immunoregulatory and reduce the rate of lesion formation and clinical exacerbation, but are only partially effective in preventing the onset of disability in multiple sclerosis patients. Approaches that directly protect myelin-producing oligodendrocytes and enhance remyelination may improve long-term outcomes and reduce the rate of axonal transection. Studies in genetically modified animals have improved our understanding of mechanisms underlying central nervous system pathology in multiple sclerosis models, and have identified pathways that regulate oligodendrocyte viability and myelin repair. However, although clinical trials are ongoing, many have been unsuccessful, and no treatments are yet approved that target these areas in multiple sclerosis. In this review, we examine avenues for oligodendrocyte protection and endogenous myelin repair in animal models of demyelination and remyelination, and their relevance as therapeutics in human patients.