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Myelin Basic Protein (MBP) is essential for both elaboration and maintenance of CNS myelin, and its reduced accumulation results in hypomyelination. How different Mbp mRNA levels affect myelin dimensions across the lifespan and how resident glial cells may respond to such changes are unknown. Here, to investigate these questions, we used enhancer-edited mouse lines that accumulate Mbp mRNA levels ranging from 8% to 160% of wild type. In young mice, reduced Mbp mRNA levels resulted in corresponding decreases in Mbp protein accumulation and myelin sheath thickness, confirming the previously demonstrated rate-limiting role of Mbp transcription in the control of initial myelin synthesis. However, despite maintaining lower line specific Mbp mRNA levels into old age, both MBP protein levels and myelin thickness improved or fully normalized at rates defined by the relative Mbp mRNA level. Sheath length, in contrast, was affected only when mRNA levels were very low, demonstrating that sheath thickness and length are not equally coupled to Mbp mRNA level. Striking abnormalities in sheath structure also emerged with reduced mRNA levels. Unexpectedly, an increase in the density of all glial cell types arose in response to reduced Mbp mRNA levels. This investigation extends understanding of the role MBP plays in myelin sheath elaboration, architecture, and plasticity across the mouse lifespan and illuminates a novel axis of glial cell crosstalk., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

Myelinating cells are sensitive to mechanical stimuli from their extracellular matrix. Ablation of YAP and TAZ mechanotransducers in Schwann cells abolishes the axon-Schwann cell recognition, myelination, and remyelination in the peripheral nervous system. It was unknown if YAP and TAZ are also required for myelination and remyelination in the central nervous system. Here we define the importance of oligodendrocyte (OL) YAP and TAZ in vivo, by specific deletion in oligodendroglial cells in adult OLs during myelin repair. Blocking YAP and TAZ expression in OL lineage cells did not affect animal viability or any major defects on OL maturation and myelination. However, using a mouse model of demyelination/remyelination, we demonstrate that YAP and TAZ modulate the capacity of OLs to remyelinate axons, particularly during the early stage of the repair process, when OL proliferation is most important. These results indicate that YAP and TAZ signaling is necessary for effective remyelination of the mouse brain., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Peripheral nerves and Schwann cells have to sustain constant mechanical constraints, caused by developmental growth as well as stretches associated with movements of the limbs and mechanical compressions from daily activities. In Schwann cells, signaling molecules sensitive to stiffness or stretch of the extracellular matrix, such as YAP/TAZ, have been shown to be critical for Schwann cell development and peripheral nerve regeneration. YAP/TAZ have also been suggested to contribute to tumorigenesis, neuropathic pain, and inherited disorders. Yet, the role of mechanosensitive ion channels in myelinating Schwann cells is vastly unexplored. Here we comprehensively assessed the expression of mechanosensitive ion channels in Schwann cells and identified that PIEZO1 and PIEZO2 are among the most abundant mechanosensitive ion channels expressed by Schwann cells. Using classic genetic ablation studies, we show that PIEZO1 is a transient inhibitor of radial and longitudinal myelination in Schwann cells. Contrastingly, we show that PIEZO2 may be required for myelin formation, as the absence of PIEZO2 in Schwann cells delays myelin formation. We found an epistatic relationship between PIEZO1 and PIEZO2, at both the morphological and molecular levels. Finally, we show that PIEZO1 channels affect the regulation of YAP/TAZ activation in Schwann cells. Overall, we present here the first demonstration that PIEZO1 and PIEZO2 contribute to mechanosensation in Schwann cells as well myelin development in the peripheral nervous system., (© 2022 Wiley Periodicals LLC.)

A proper protein orientation is often required in order to achieve specific protein-receptor interaction to elicit a desired biological response. Here, we present a Protein A-based biomimicking platform that is capable of efficiently orienting proteins for evaluating cellular behaviour. By absorbing Protein A onto aligned bio-mimicking polycaprolactone (PCL) fibers, we demonstrate that protein binding could be retained on these fibers for at least 7 days under physiologically relevant conditions. We further show that Protein A served as a molecular orientor to arrange the recombinant proteins in similar orientations. Such protein-orienting scaffolds were further verified to be biologically functional by using sensitive primary rat cortical neurons (CNs) and oligodendrocyte progenitor cells (OPCs), as model neural cells for a stringent proof of concept. Specifically, CNs that were seeded on fibers coated with Protein A and a known enhancer of neurite growth (L1 Cell Adhesion Molecular L1CAM) displayed the longest total neurite length (462.77 ± 100.79 μm, p < 0.001) as compared to the controls. Besides that, OPCs seeded on fibers coated with Protein A and Neuregulin-1 Type III (Nrg1 type III) (myelin enhancer) produced the longest myelin ensheathment length (19.8 ± 11.69 μm). These results demonstrate the efficacy of this platform for protein screening applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A key hallmark of many diseases, especially those in the central nervous system (CNS), is the change in tissue stiffness due to inflammation and scarring. However, how such changes in microenvironment affect the regenerative process remains poorly understood. Here, a biomimicking fiber platform that provides independent variation of fiber structural and intrinsic stiffness is reported. To demonstrate the functionality of these constructs as a mechanotransduction study platform, these substrates are utilized as artificial axons and the effects of axon structural versus intrinsic stiffness on CNS myelination are independently analyzed. While studies have shown that substrate stiffness affects oligodendrocyte differentiation, the effects of mechanical stiffness on the final functional state of oligodendrocyte (i.e., myelination) has not been shown prior to this. Here, it is demonstrated that a stiff mechanical microenvironment impedes oligodendrocyte myelination, independently and distinctively from oligodendrocyte differentiation. Yes-associated protein is identified to be involved in influencing oligodendrocyte myelination through mechanotransduction. The opposing effects on oligodendrocyte differentiation and myelination provide important implications for current work screening for promyelinating drugs, since these efforts have focused mainly on promoting oligodendrocyte differentiation. Thus, the platform may have considerable utility as part of a drug discovery program in identifying molecules that promote both differentiation and myelination., (© 2020 Wiley-VCH GmbH.)

The loss of oligodendrocytes (OLs) and subsequently myelin sheaths following injuries or pathologies in the CNS leads to debilitating functional deficits. Unfortunately, effective methods of remyelination remain limited. Here, we present a scaffolding system that enables sustained non-viral delivery of microRNAs (miRs) to direct OL differentiation, maturation, and myelination. We show that miR-219/miR-338 promoted primary rat OL differentiation and myelination in vitro. Using spinal cord injury as a proof-of-concept, we further demonstrate that miR-219/miR-338 could also be delivered non-virally in vivo using an aligned fiber-hydrogel scaffold to enhance remyelination after a hemi-incision injury at C5 level of Sprague-Dawley rats. Specifically, miR-219/miR-338 mimics were incorporated as complexes with the carrier, TransIT-TKO (TKO), together with neurotrophin-3 (NT-3) within hybrid scaffolds that comprised poly(caprolactone-co-ethyl ethylene phosphate) (PCLEEP)-aligned fibers and collagen hydrogel. After 1, 2, and 4 weeks post-treatment, animals that received NT-3 and miR-219/miR-338 treatment preserved a higher number of Olig2 + oligodendroglial lineage cells as compared with those treated with NT-3 and negative scrambled miRs (Neg miRs; p < 0.001). Additionally, miR-219/miR-338 increased the rate and extent of differentiation of OLs. At the host-implant interface, more compact myelin sheaths were observed when animals received miR-219/miR-338. Similarly within the scaffolds, miR-219/miR-338 samples contained significantly more myelin basic protein (MBP) signals (p < 0.01) and higher myelination index (p < 0.05) than Neg miR samples. These findings highlight the potential of this platform to promote remyelination within the CNS., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Important advances in our understanding of oligodendrocyte precursor cell biology and differentiation have stemmed from in vitro experiments using cultures of isolated primary oligodendrocyte precursor cells. To examine the process of myelination in the final stages of oligodendrocyte development, experimental systems have previously been limited to models utilizing neurons. Recent advances in three-dimensional culture systems, however, have opened the possibility to observe myelin sheath formation with only one cell type, the oligodendrocyte precursor cell. In this chapter, such a method is described for examining oligodendrocyte myelin sheath formation with isolated oligodendrocytes in the absence of neurons. This assay is ideal for gaining mechanistic insight into oligodendrocyte-specific regulation of myelin sheath formation. Oligodendrocyte heterogeneity can be readily assessed, determining whether different oligodendrocyte sources influence myelin sheath formation. As well, the direct impact of both physical and molecular cues on oligodendrocytes can be determined in this defined system. This assay extends the capability of two-dimensional oligodendrocyte cultures, permitting post-differentiation analysis of myelinating oligodendrocytes, the number of sheaths formed by individual oligodendrocytes, as well as the lengths of myelin sheaths formed.

Our ability to rescue functional deficits after demyelinating diseases or spinal cord injuries is limited by our lack of understanding of the complex remyelination process, which is crucial to functional recovery. In this study, we developed an electrospun suspended poly(ε-caprolactone) microfiber platform to enable the screening of therapeutics for remyelination. As a proof of concept, this platform employed scaffold-mediated non-viral delivery of a microRNA (miR) cocktail to promote oligodendrocyte precursor cells (OPCs) differentiation and myelination. We observed enhanced OPCs differentiation when the cells were transfected with miR-219 and miR-338 on the microfiber substrates. Moreover, miRs promoted the formation of MBP + tubular extensions around the suspended fibers, which was indicative of myelination, instead of flat myelin membranes on 2D substrates. In addition, OPCs that were transfected with the cocktail of miRs formed significantly longer and larger amounts of MBP + extensions. Taken together, these results demonstrate the efficacy of this functional screening platform for understanding myelination., Statement of Significance: The lack of understanding of the complex myelination process has hindered the discovery of effective therapeutic treatments for demyelinating diseases. Hence, in vitro models that enable systematic understanding, visualization and quantification of myelination are valuable. Unfortunately, achieving reproducible in vitro myelination by oligodendrocytes (OLs) remains highly challenging. Here, we engineered a suspended microfiber platform that enables sustained non-viral drug/gene delivery to study OL differentiation and myelination. Sustained drug delivery permits the investigation of OL development, which spans several weeks. We show that promyelinogenic microRNAs promoted OL differentiation and myelination on this platform. Our engineered microfiber substrate could serve as a drug/gene screening platform and facilitate future translation into direct implantable devices for in vivo remyelination purposes., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The most prevalent neurological disorders of myelin include perinatal brain injury leading to cerebral palsy in infants and multiple sclerosis in adults. Although these disorders have distinct etiologies, they share a common neuropathological feature of failed progenitor differentiation into myelin-producing oligodendrocytes and lack of myelin, for which there is an unmet clinical need. Here, we reveal that a molecular pathology common to both disorders is dysregulation of activin receptors and that activin receptor signaling is required for the majority of myelin generation in development and following injury. Using a constitutive conditional knockout of all activin receptor signaling in oligodendrocyte lineage cells, we discovered this signaling to be required for myelination via regulation of oligodendrocyte differentiation and myelin compaction. These processes were found to be dependent on the activin receptor subtype Acvr2a, which is expressed during oligodendrocyte differentiation and axonal ensheathment in development and following myelin injury. During efficient myelin regeneration, Acvr2a upregulation was seen to coincide with downregulation of Acvr2b, a receptor subtype with relatively higher ligand affinity; Acvr2b was shown to be dispensable for activin receptor-driven oligodendrocyte differentiation and its overexpression was sufficient to impair the abovementioned ligand-driven responses. In actively myelinating or remyelinating areas of human perinatal brain injury and multiple sclerosis tissue, respectively, oligodendrocyte lineage cells expressing Acvr2a outnumbered those expressing Acvr2b, whereas in non-repairing lesions Acvr2b+ cells were increased. Thus, we propose that following human white matter injury, this increase in Acvr2b expression would sequester ligand and consequently impair Acvr2a-driven oligodendrocyte differentiation and myelin formation. Our results demonstrate dysregulated activin receptor signaling in common myelin disorders and reveal Acvr2a as a novel therapeutic target for myelin generation following injury across the lifespan.

The concept of adaptive myelination-myelin plasticity regulated by activity-is an important advance for the field. What signals set up the adaptable pattern in the first place? Here we review work that demonstrates an intrinsic pathway within oligodendrocytes requiring only an axon-shaped substrate to generate multilayered and compacted myelin sheaths of a physiological length. Based on this, we discuss a model we proposed in 2015 which argues that myelination has two phases-intrinsic and then adaptive-which together generate "smart wiring," in which active axons become more myelinated. This model explains why prior studies have failed to identify a signal necessary for central nervous system myelination and argues that myelination, like synapses, might contribute to learning by the activity-dependent modification of an initially hard-wired pattern. © 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 68-79, 2018., (© 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc.)

The Golgi complex is the Grand Central Station of intracellular membrane trafficking in the secretory and endocytic pathways. Anterograde and retrograde export of cargo from the Golgi complex involves a complex interplay between the formation of coated vesicles and membrane tubules, although much less is known about tubule-mediated trafficking. Recent advances using in vitro assays have identified several cytoplasmic phospholipase A2 (PLA2) enzymes that are required for the biogenesis of membrane tubules and their roles in the functional organization of the Golgi complex. In this chapter we describe methods for the cell-free reconstitution of PLA2-dependent Golgi membrane tubule formation. These methods should facilitate the identification of other proteins that regulate this process.

Since Río-Hortega's description of oligodendrocyte morphologies nearly a century ago, many studies have observed myelin sheath-length diversity between CNS regions. Myelin sheath length directly impacts axonal conduction velocity by influencing the spacing between nodes of Ranvier. Such differences likely affect neural signal coordination and synchronization. What accounts for regional differences in myelin sheath lengths is unknown; are myelin sheath lengths determined solely by axons or do intrinsic properties of different oligodendrocyte precursor cell populations affect length? The prevailing view is that axons provide molecular cues necessary for oligodendrocyte myelination and appropriate sheath lengths. This view is based upon the observation that axon diameters correlate with myelin sheath length, as well as reports that PNS axonal neuregulin-1 type III regulates the initiation and properties of Schwann cell myelin sheaths. However, in the CNS, no such instructive molecules have been shown to be required, and increasing in vitro evidence supports an oligodendrocyte-driven, neuron-independent ability to differentiate and form initial sheaths. We test this alternative signal-independent hypothesis--that variation in internode lengths reflects regional oligodendrocyte-intrinsic properties. Using microfibers, we find that oligodendrocytes have a remarkable ability to self-regulate the formation of compact, multilamellar myelin and generate sheaths of physiological length. Our results show that oligodendrocytes respond to fiber diameters and that spinal cord oligodendrocytes generate longer sheaths than cortical oligodendrocytes on fibers, co-cultures, and explants, revealing that oligodendrocytes have regional identity and generate different sheath lengths that mirror internodes in vivo., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Heterotrimeric G proteins transduce the ligand binding of transmembrane G protein coupled receptors into a variety of intracellular signaling pathways. Recently, heterotrimeric Gβγ subunit signaling at the Golgi complex has been shown to regulate the formation of vesicular transport carriers that deliver cargo from the Golgi to the plasma membrane. In addition to vesicles, membrane tubules have also been shown to mediate export from the Golgi complex, which requires the activity of cytoplasmic phospholipase A 2 (PLA 2 ) enzyme activity. Through the use of an in vitro reconstitution assay with isolated Golgi complexes, we provide evidence that Gβ1γ2 signaling also stimulates Golgi membrane tubule formation. In addition, we show that an inhibitor of Gβγ activation of PLA 2 enzymes inhibits in vitro Golgi membrane tubule formation. Additionally, purified Gβγ protein stimulates membrane tubules in the presence of low (sub-threshold) cytosol concentrations. Importantly, this Gβγ stimulation of Golgi membrane tubule formation was inhibited by treatment with the PLA 2 antagonist ONO-RS-082. These studies indicate that Gβ1γ2 signaling activates PLA 2 enzymes required for Golgi membrane tubule formation, thus establishing a new layer of regulation for this process.

Recent studies showed that the phospholipase subunits of Platelet Activating Factor Acetylhydrolase (PAFAH) Ib, α1 and α2 partially localize to the Golgi complex and regulate its structure and function. Using siRNA knockdown of individual subunits, we find that α1 and α2 perform overlapping and unique roles in regulating Golgi morphology, assembly, and secretory cargo trafficking. Knockdown of either α1 or α2 reduced secretion of soluble proteins, but neither single knockdown reduced secretion to the same degree as knockdown of both. Knockdown of α1 or α2 inhibited reassembly of an intact Golgi complex to the same extent as knockdown of both. Transport of VSV-G was slowed but at different steps in the secretory pathway: reduction of α1 slowed trans Golgi network to plasma membrane transport, whereas α2 loss reduced endoplasmic reticulum to Golgi trafficking. Similarly, knockdown of either subunit alone disrupted the Golgi complex but with markedly different morphologies. Finally, knockdown of α1, or double knockdown of α1 and α2, resulted in a significant redistribution of kinase dead protein kinase D from the Golgi to the plasma membrane, whereas loss of α2 alone had no such effect. These studies reveal an unexpected complexity in the regulation of Golgi structure and function by PAFAH Ib. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The mammalian Golgi complex, trans Golgi network (TGN) and ER-Golgi intermediate compartment (ERGIC) are comprised of membrane cisternae, coated vesicles and membrane tubules, all of which contribute to membrane trafficking and maintenance of their unique architectures. Recently, a new cast of players was discovered to regulate the Golgi and ERGIC: four unrelated cytoplasmic phospholipase A (PLA) enzymes, cPLA(2)α (GIVA cPLA(2)), PAFAH Ib (GVIII PLA(2)), iPLA(2)-β (GVIA-2 iPLA(2)) and iPLA(1)γ. These ubiquitously expressed enzymes regulate membrane trafficking from specific Golgi subcompartments, although there is evidence for some functional redundancy between PAFAH Ib and cPLA(2)α. Three of these enzymes, PAFAH Ib, cPLA(2)α and iPLA(2)-β, exert effects on Golgi structure and function by inducing the formation of membrane tubules. We review our current understanding of how PLA enzymes regulate Golgi and ERGIC morphology and function., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Previous studies have shown that membrane tubule-mediated export from endosomal compartments requires a cytoplasmic phospholipase A(2) (PLA(2)) activity. Here we report that the cytoplasmic PLA(2) enzyme complex platelet-activating factor acetylhydrolase (PAFAH) Ib, which consists of α1, α2, and LIS1 subunits, regulates the distribution and function of endosomes. The catalytic subunits α1 and α2 are located on early-sorting endosomes and the central endocytic recycling compartment (ERC) and their overexpression, but not overexpression of their catalytically inactive counterparts, induced endosome membrane tubules. In addition, overexpression α1 and α2 altered normal endocytic trafficking; transferrin was recycled back to the plasma membrane directly from peripheral early-sorting endosomes instead of making an intermediate stop in the ERC. Consistent with these results, small interfering RNA-mediated knockdown of α1 and α2 significantly inhibited the formation of endosome membrane tubules and delayed the recycling of transferrin. In addition, the results agree with previous reports that PAFAH Ib α1 and α2 expression levels affect the distribution of endosomes within the cell through interactions with the dynein regulator LIS1. These studies show that PAFAH Ib regulates endocytic membrane trafficking through novel mechanisms involving both PLA(2) activity and LIS1-dependent dynein function.

We report that platelet-activating factor acetylhydrolase (PAFAH) Ib, comprised of two phospholipase A(2) (PLA(2)) subunits, alpha1 and alpha2, and a third subunit, the dynein regulator lissencephaly 1 (LIS1), mediates the structure and function of the Golgi complex. Both alpha1 and alpha2 partially localize on Golgi membranes, and purified catalytically active, but not inactive alpha1 and alpha2 induce Golgi membrane tubule formation in a reconstitution system. Overexpression of wild-type or mutant alpha1 or alpha2 revealed that both PLA(2) activity and LIS1 are important for maintaining Golgi structure. Knockdown of PAFAH Ib subunits fragments the Golgi complex, inhibits tubule-mediated reassembly of intact Golgi ribbons, and slows secretion of cargo. Our results demonstrate a cooperative interplay between the PLA(2) activity of alpha1 and alpha2 with LIS1 to facilitate the functional organization of the Golgi complex, thereby suggesting a model that links phospholipid remodeling and membrane tubulation to dynein-dependent transport.