Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside

Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12--14 days in vitro. Addition of 50 μg/ml exogenous Neu-NAcα2-3Galβ1-1′ ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [¹⁴C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [¹⁴C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [¹⁴C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O-2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Maturation-dependent apoptotic cell death of oligodendrocytes in myelin-deficient rats

Mutations in the proteolipid protein gene (PLP/plp), which encodes the major intrinsic membrane protein in central nervous system (CNS) myelin, cause inherited dysmyelination in mammals. One of these mutants, the myelin-deficient (md) rat, has severe dysmyelination that is associated with oligodendrocyte cell death. Using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay, which labels apoptotic cells, we find that cell death is increased in multiple white matter tracts of md rats. The tracts that myelinate the earliest show the earliest increase in cell death, and cell death persists for at least 22 days, the lifespan of these mutant animals. In all tracts, and at all developmental ages examined, apoptotic cells expressed the markers of mature oligodendrocytes, such as myelin basic protein, myelin-associated glycoprotein, and the Rip antigen, but not chondroitin sulfate proteoglycan, a marker of oligodendrocyte precursors. Mature oligodendrocytes fail to accumulate in md brain because they die before they fully mature. J. Neurosci. Res. 54:623–634, 1998. © 1998 Wiley-Liss, Inc.     

Axo‐glial communication through neurexin‐neuroligin signaling regulates myelination and oligodendrocyte differentiation

Axonal transsynaptic signaling between presynaptic neurexin (NX) and postsynaptic neuroligin (NL) is essential for many properties of synaptic connectivity. Here, we demonstrate the existence of a parallel axo‐glial signaling pathway between axonal NX and oligodendritic (OL) NL3. We show that this pathway contributes to the regulation of myelinogenesis, the maintenance of established myelination, and the differentiation state of the OL using in vitro models. We first confirm that NL3 mRNA and protein are expressed in OLs and in OL precursors. We then show that OLs in culture form contacts with non‐neuronal cells exogenously expressing NL3's binding partners NX1α or NX1β. Conversely, blocking axo‐glial NX‐NL3 signaling by saturating NX with exogenous soluble NL protein (NL‐ECD) disrupts interactions between OLs and axons in both in vitro and ex vivo assays. Myelination by OLs is tied to their differentiation state, and we find that blocking NX‐NL signaling with soluble NL protein also caused OL differentiation to stall at an immature stage. Moreover, in vitro knockdown of NL3 in OLs with siRNAs stalls their development and reduces branching complexity. Interestingly, inclusion of an autism related mutation in the NL blocking protein attenuates these effects; OLs differentiate and the dynamics of OL‐axon signaling occur normally as this peptide does not disrupt NX‐NL3 axo‐glial interactions. Our findings provide evidence not only for a new pathway in axo‐glial communication, they also potentially explain the correlation between altered white matter and autism. GLIA 2015;63:2023–2039     

The use of networks of dissociated rat dorsal root ganglion neurons to induce myelination by oligodendrocytes in culture

A neuronal culture system has been developed that has demonstrated to induce myelin formation by added oligodendrocytes. Networks of dissociated dorsal root ganglion neurons were prepared by suppressing non-neuronal cells (i.e. fibroblasts and Schwann cells) with a continuous 2 week exposure to 10⁻⁵ M fluorodeoxyuridine in the culture medium. After drug withdrawal, neuroglial cells were introduced in optic nerve implants from 1–2 week-old rats. These added glial cells migrated extensively over the unensheathed neurities and central myelin was formed by 2 weeks after the implant addition.     

Regulation and differential expression of tau mRNA isoforms as oligodendrocytes mature in vivo: implications for myelination

Oligodendrocytes and neurons derive from the same cell type but develop distinct morphologic and functional properties as they mature in vivo. Both cells express tau protein, a developmentally regulated protein in the central nervous system. The regulation of tau has been investigated extensively in neurons but not in oligodendrocytes, so we studied regulation of tau in oligodendrocytes in vivo. The amino-derived tau isoforms consist of isoforms with zero (A0), one (A1), or two (A2) inserts. We examined the developmental regulation of tau mRNA isoforms at the amino domain by comparing tau expression in oligodendrocytes (OLGs) isolated from 1- and 20-day-old rat brain and in age-matched cortex, which abounds in neurons. In the rat brain, myelination peaks at 20 days. By using semiquantitative RT-PCR, we found that OLGs and cortex from 1-day-old rat brain largely had amino-derived tau isoforms with no insert, whereas OLGs from 20-day-old rat brain had similar levels of amino-derived tau isoforms with no insert or with one insert. We also found that 20-day-old OLGs had twofold more tau mRNA levels than younger OLGs. In contrast to OLGs from 20-day-old rat brain, age-matched cortex had comparable levels of A0, A1, and A2 tau amino-derived isoforms. Further, younger and older OLGs had a reciprocal pattern of expression of both carboxy-derived tau mRNA isoforms with either three (3R) or four (4R) repeats. In contrast, younger and older cortex expressed either 3R or 4R tau. This study showed an upregulation of tau mRNA and cell-specific tau mRNA isoform expression in OLGs forming myelin.     

A selective thyroid hormone β receptor agonist enhances human and rodent oligodendrocyte differentiation

Nerve conduction within the mammalian central nervous system is made efficient by oligodendrocyte‐derived myelin. Historically, thyroid hormones have a well described role in regulating oligodendrocyte differentiation and myelination during development; however, it remains unclear which thyroid hormone receptors are required to drive these effects. This is a question with clinical relevance since nonspecific thyroid receptor stimulation can produce deleterious side‐effects. Here we report that GC‐1, a thyromimetic with selective thyroid receptor β action and a potentially limited side‐effect profile, promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. In addition, we used in vivo genetic fate tracing of oligodendrocyte progenitor cells via PDGFαR‐CreER;Rosa26‐eYFP double‐transgenic mice to examine the effect of GC‐1 on cellular fate and find that treatment with GC‐1 during developmental myelination promotes oligodendrogenesis within the corpus callosum, occipital cortex and optic nerve. GC‐1 was also observed to enhance the expression of the myelin proteins MBP, CNP and MAG within the same regions. These results indicate that a β receptor selective thyromimetic can enhance oligodendrocyte differentiation in vitro and during developmental myelination in vivo and warrants further study as a therapeutic agent for demyelinating models. GLIA 2014;62:1513–1529     

Investigation of neuromuscular abnormalities in neurotrophin‐3‐deficient mice

Neurotrophin‐3 (NT‐3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT‐3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency‐induced reduction in NT‐3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1‐43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve‐mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1‐43 were impaired in mature NT‐3‐deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype‐dependent variation in the number of synaptic vesicles near the active zone. NT‐3^+/? mice had normal soleus muscle fiber numbers but their fibers had smaller cross‐sectional areas and were more densely‐packed than wild‐type littermates. Moreover, the muscles of adult NT‐3‐deficient animals were weaker than those of wild‐type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT‐3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.     

GAT‐1 mediated GABA uptake in rat oligodendrocytes

Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT‐1, on oligodendrogenesis, we verified the possibility that GAT‐1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT‐1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT‐1 immunoreactivity was present in immature and mature oligodendrocytes. Co‐localization studies between GAT‐1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT‐1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine‐sensitive Na⁺‐dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT‐1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of “neuronal” and “glial” GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT‐1 have been implicated to date. GLIA 2017;65:514–522     

Caspase-3 activation in oligodendrocytes from the myelin-deficient rat

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.     

Monoclonal antibody Rip specifically recognizes 2',3'-cyclic nucleotide 3'-phosphodiesterase in oligodendrocytes

The antigen recognized with monoclonal antibody (mAb) Rip (Rip-antigen) has been long used as a marker of oligodendrocytes and myelin sheaths. However, the identity of Rip-antigen has yet to be elucidated. We herein identified the Rip-antigen. No signal recognized by mAb-Rip was detected by immunoblot analyses in the rat brain, cultured rat oligodendrocytes, or the oligodendrocyte cell line CG-4. As this antibody worked very well on immunocytochemistry and immunohistochemistry, Rip-antigen was immunopurified with mAb-Rip from the differentiated CG-4 cells. Eight strong-intensity bands thus appeared on 5-20% SDS-PAGE with SYPRO ruby fluorescence staining. To identify these molecules, each band extracted from the gel was analyzed by MALDI-QIT/TOF mass spectrometry. We found an interesting molecule in the oligodendrocytes from an approximately 44-kDa band as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). To test whether CNP was recognized by mAb-Rip, double-immunofluorescence staining was performed by using Alexa Fluor 488-conjugated mAb-Rip and Alexa Fluor 568-conjugated mAb-CNP in the rat cerebellum, mouse cerebellum, cultured rat oligodendrocytes, and CG-4 cells. The Rip-antigen was colocalized with CNP in these cells and tissues. To provide direct evidence that CNP was recognized by mAb-Rip, rat Cnp1-transfected HEK293T cells were used for double-immunofluorescence staining with mAb-Rip and mAb-CNP. The Rip-antigen was colocalized with CNP in rat Cnp1-transfected HEK293T cells, but the antigen was not detected by mAb-Rip and mAb-CNP in mock-transfected HEK293T cells. Overall, we have demonstrated that the antigen labeled with mAb-Rip is CNP in the oligodendrocytes.     

Astrocyte‐specific activation of TNFR2 promotes oligodendrocyte maturation by secretion of leukemia inhibitory factor

Tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2 have pleiotropic effects in neurodegenerative disorders. For example, while TNFR1 mediates neurodegenerative effects in multiple sclerosis, TNFR2 is protective and contributes to remyelination. The exact mode of TNFR2 action, however, is poorly understood. Here, we show that TNFR2‐mediated activation of the PI3K‐PKB/Akt pathway in primary astrocytes increased the expression of neuroprotective genes, including that encoding the neurotrophic cytokine leukemia inhibitory factor (LIF). To investigate whether intercellular signaling between TNFR2‐stimulated astrocytes and oligodendrocytes plays a role in oligodendrocyte maturation, we established an astrocyte–oligodendrocyte coculture model, composed of primary astrocytes from huTNFR2‐transgenic (tgE1335) mice and oligodendrocyte progenitor cells (OPCs) from wild‐type mice, capable of differentiating into mature myelinating oligodendrocytes. In this model, selective stimulation of human TNFR2 on astrocytes, promoted differentiation of cocultured OPCs to myelin basic protein‐positive mature oligodendrocytes. Addition of LIF neutralizing antibodies inhibited oligodendrocyte differentiation, indicating a crucial role of TNFR2‐induced astrocyte derived LIF for oligodendrocyte maturation. GLIA 2014;62:272–283     

Proline substitutions and threonine pseudophosphorylation of the SH3 ligand of 18.5-kDa myelin basic protein decrease its affinity for the Fyn-SH3 domain and alter process development and protein localization in oligodendrocytes

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92-99 (murine sequence -T(92)PRTPPPS(99)-) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP's SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca(2+) influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein-protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP's SH3 ligand domain. These results suggest that MBP's SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function.     

Sonic hedgehog promotes the generation of myelin proteins by transplanted oligosphere‐derived cells

The generation of large numbers of functionally relevant cells for transplantation remains central to the use of cell replacement as a therapeutic strategy for neurodegenerative diseases. In this study we have analyzed the effect of sonic hedgehog (Shh) pretreatment on the myelinating potential of transplanted oligosphere‐derived cells. The retina was chosen as a model for assessing this myelinating capability because 1) there is a lack of endogenous myelin in the normal rodent retina and 2) the retinal ganglion cell (RGC) axons are receptive to myelination, once myelinating cells have access to the retinal nerve fiber layer. Initially, oligospheres were generated in the presence of B104 CM but without the addition of Shh. After transplantation, 60% of the animals developed tumors in the eye that had received the transplant and were not analyzed for the presence of myelin. In the remaining retinas, the transplanted oligosphere‐derived cells were not myelin competent, as indicated by the absence of myelin proteins in the retinal nerve fiber layer. In contrast, when B104 CM oligospheres were generated in the presence of Shh, myelin proteins were found in the nerve fiber layer after transplantation. In addition, the amount of myelin proteins synthesized increased with time posttransplantation, with the majority of the nerve fiber layer immunoreactive for these proteins in some retinas after 2 months. This study has demonstrated that growth as oligospheres and endogenously derived growth/differentiation factors alone are not sufficient to induce the differentiation of B104‐treated oligosphere‐derived cells and that pretreating the oligospheres by growth in the presence of Shh before transplantation is essential to induce their myelinating competence. © 2009 Wiley‐Liss, Inc.     

Zn2+‐induced ERK activation mediates PARP‐1‐dependent ischemic‐reoxygenation damage to oligodendrocytes

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca²⁺]_i, loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia‐like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia‐induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O₂ (reperfusion‐like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high‐affinity Zn²⁺ chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high‐affinity Zn²⁺ probe, we showed that ischemia induced an intracellular Zn²⁺ rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia‐induced oligodendrocyte death. Moreover, UO126 blocked the ischemia‐induced increase in poly‐[ADP]‐ribosylation of proteins, and the poly[ADP]‐ribose polymerase 1 (PARP‐1) inhibitor DPQ significantly inhibited ischemia‐induced oligodendroglial cell death—demonstrating that PARP‐1 was required downstream in the Zn²⁺‐ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn²⁺, blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia‐reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult. © 2012 Wiley Periodicals, Inc.