Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin ...diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.
NG2 (nerve/glia antigen‐2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2‐expressing (NG2+) cells ...have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen‐inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2+ cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter+ astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen‐sensitive and NG2 gene locus‐dependent gene recombination could be detected in a small, but persistent population of cortical NeuN+ neurons starting from the second postnatal week. GLIA 2014;62:896–913
Main points
With the TgH(NG2‐CreERT2) mouse line, an improved genetic tool has been generated. Along lifespan NG2 glia mainly generate oligodendrocytes, while embryonic NG2 glia can also form astrocytes. No clear evidence for a neurogenic potential of NG2 glia could be established.
NG2 protein-expressing oligodendrocyte progenitor cells (OPC) are a persisting and major glial cell population in the adult mammalian brain. Direct synaptic innervation of OPC by neurons throughout ...the brain together with their ability to sense neuronal network activity raises the question of additional physiological roles of OPC, supplementary to generating myelinating oligodendrocytes. In this study we investigated whether OPC express neuromodulatory factors, typically synthesized by other CNS cell types. Our results show that OPC express two well-characterized neuromodulatory proteins: Prostaglandin D2 synthase (PTGDS) and neuronal Pentraxin 2 (Nptx2/Narp). Expression levels of the enzyme PTGDS are influenced in cultured OPC by the NG2 intracellular region which can be released by cleavage and localizes to glial nuclei upon transfection. Furthermore PTGDS mRNA levels are reduced in OPC from NG2-KO mouse brain compared to WT cells after isolation by cell sorting and direct analysis. These results show that OPC can contribute to the expression of these proteins within the CNS and suggest PTGDS expression as a downstream target of NG2 signaling.
The role of glia in modulating neuronal network activity is an important question. Oligodendrocyte precursor cells (OPC) characteristically express the transmembrane proteoglycan nerve-glia antigen 2 ...(NG2) and are unique glial cells receiving synaptic input from neurons. The development of NG2+ OPC into myelinating oligodendrocytes has been well studied, yet the retention of a large population of synapse-bearing OPC in the adult brain poses the question as to additional functional roles of OPC in the neuronal network. Here we report that activity-dependent processing of NG2 by OPC-expressed secretases functionally regulates the neuronal network. NG2 cleavage by the α-secretase ADAM10 yields an ectodomain present in the extracellular matrix and a C-terminal fragment that is subsequently further processed by the γ-secretase to release an intracellular domain. ADAM10-dependent NG2 ectodomain cleavage and release (shedding) in acute brain slices or isolated OPC is increased by distinct activity-increasing stimuli. Lack of NG2 expression in OPC (NG2-knockout mice), or pharmacological inhibition of NG2 ectodomain shedding in wild-type OPC, results in a striking reduction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) in pyramidal neurons of the somatosensory cortex and alterations in the subunit composition of their α-amino-3-hydroxy-5-methyl-4-isoxazolepr opionicacid (AMPA) receptors. In NG2-knockout mice these neurons exhibit diminished AMPA and NMDA receptor-dependent current amplitudes; strikingly AMPA receptor currents can be rescued by application of conserved LNS protein domains of the NG2 ectodomain. Furthermore, NG2-knockout mice exhibit altered behavior in tests measuring sensorimotor function. These results demonstrate for the first time a bidirectional cross-talk between OPC and the surrounding neuronal network and demonstrate a novel physiological role for OPC in regulating information processing at neuronal synapses.
The NG2 proteoglycan is expressed by oligodendrocyte precursor cells (OPCs) and is abundantly expressed by tumors such as melanoma and glioblastoma. Functions of NG2 include an influence on ...proliferation, migration and neuromodulation. Similar to other type-1 membrane proteins, NG2 undergoes proteolysis, generating a large ectodomain, a C-terminal fragment (CTF) and an intracellular domain (ICD) via sequential action of α- and γ-secretases which is enhanced by neuronal activity. Functional roles of NG2 have so far been shown for the full-length protein, the released ectodomain and CTF, but not for the ICD. In this study, we characterized the role of the NG2 ICD in OPC and Human Embryonic Kidney (HEK) cells. Overexpressed ICD is predominantly localized in the cell cytosol, including the distal processes of OPCs. Nuclear localisation of a fraction of the ICD is dependent on Nuclear Localisation Signals. Immunoprecipitation and Mass Spectrometry followed by functional analysis indicated that the NG2 ICD modulates mRNA translation and cell-cycle kinetics. In OPCs and HEK cells, ICD overexpression results in an mTORC1-dependent upregulation of translation, as well as a shift of the cell population toward S-phase. NG2 ICD increases the active (phosphorylated) form of mTOR and modulates downstream signaling cascades, including increased phosphorylation of p70S6K1 and increased expression of eEF2. Strikingly, levels of FMRP, an RNA-binding protein that is regulated by mTOR/p70S6K1/eEF2 were decreased. In neurons, FMRP acts as a translational repressor under activity-dependent control and is mutated in Fragile X Syndrome (FXS). Knock-down of endogenous NG2 in primary OPC reduced translation and mTOR/p70S6K1 phosphorylation in Oli-
. Here, we identify the NG2 ICD as a regulator of translation in OPCs via modulation of the well-established mTORC1 pathway. We show that FXS-related FMRP signaling is not exclusive to neurons but plays a role in OPCs. This provides a signal cascade in OPC which can be influenced by the neuronal network, since the NG2 ICD has been shown to be generated by constitutive as well as activity-dependent cleavage. Our results also elucidate a possible role of NG2 in tumors exhibiting enhanced rates of translation and rapid cell cycle kinetics.
In the central nervous system, oligodendroglial expression of myelin basic protein (MBP) is crucial for the assembly and structure of the myelin sheath. MBP synthesis is tightly regulated in space ...and time, particularly at the post-transcriptional level. We have identified the DEAD-box RNA helicase DDX5 (also known as p68) in a complex with
mRNA in oligodendroglial cells. Expression of DDX5 is highest in progenitor cells and immature oligodendrocytes, where it localizes to heterogeneous populations of cytoplasmic ribonucleoprotein (RNP) complexes associated with
mRNA in the cell body and processes. Manipulation of the amount of DDX5 protein inversely affects the level of MBP. We present evidence that DDX5 is involved in post-transcriptional regulation of MBP protein synthesis, with implications for oligodendroglial development. In addition, knockdown of DDX5 results in an increased abundance of MBP isoforms containing exon 2 in immature oligodendrocytes, most likely by regulating alternative splicing of
Our findings contribute to the understanding of the complex nature of MBP post-transcriptional control in immature oligodendrocytes where DDX5 appears to affect the abundance of MBP proteins via distinct but converging mechanisms.
The obtained single cell suspension was resuspended in panning buffer and transferred to a bacterial culture dish coated with Anti-BSL1 Griffonia simplicifolia lectin (Vector Labs, L-1100) for ...negative selection, followed by a positive selection step using rat anti-mouse CD140a (Biolegend, 135902) as primary antibody and AffiniPure goat anti-rat IgG (H + L) (Dianova, 112-005-003) as secondary antibody enabling a purity of 95% OPC. Since recent data demonstrated that loss of NG2 lead to downregulation of pro-inflammatory cytokines in a focal demyelinating model 33 we analyzed the expression levels of Il1β and Infg in the NG2-/- and NG2+/+ mice after de- and remyelination. In both these models where striking differences between NG2-/- and NG2+/+ mice were seen, there is an extensive inflammation with blood brain barrier damage and it is conceivable that the NG2 proteoglycan itself, possibly as a shed ectodomain, modulates the inflammatory response. Since it has been shown that cleavage of the extracellular NG2 domain regulates neuronal networks 24 it might be also possible that neuronal signaling in turn modulates oligodendroglial properties, such as proliferation or differentiation. Cuprizone is a copper chelator that induces oligodendroglial cell death followed by demyelination, astrogliosis and microglia activation. Since the blood brain barrier is not affected only few blood derived monocytes and T cells invade the CNS; furthermore axonal damage is less pronounced that in inflammatory demyelinating models such as EAE 59, 60.
Central nervous system myelination requires the synthesis of large amounts of myelin basic protein (MBP) at the axon-glia contact site. MBP messenger RNA (mRNA) is transported in RNA granules to ...oligodendroglial processes in a translationally silenced state. This process is regulated by the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binding to the cis-acting A2 response element (A2RE). Release of this repression of MBP mRNA translation is thus essential for myelination. Mice deficient in the Src family tyrosine kinase Fyn are hypomyelinated and contain reduced levels of MBP. Here, we identify hnRNP A2 as a target of activated Fyn in oligodendrocytes. We show that active Fyn phosphorylates hnRNP A2 and stimulates translation of an MBP A2RE-containing reporter construct. Neuronal adhesion molecule L1 binding to oligodendrocytes results in Fyn activation, which leads to an increase in hnRNP A2 phosphorylation. These results suggest that Fyn kinase activation results in the localized translation of MBP mRNA at sites of axon-glia contact and myelin deposition.
NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular ...Ca(2+)-concentration (Ca(2+)(i)) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca(2+)-signaling pathways we combined patch-clamp recordings, Ca(2+)-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca(2+)-channels, Ca(2+)-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke Ca(2+)(i)-elevations in NG2 cells. The Ca(2+)-influx is amplified by Ca(2+)-induced Ca(2+)-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic Ca(2+)(i) elevations, suggesting that Ca(2+)(i) elevations in NG2 cells might be restricted to their processes. Local Ca(2+)-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells.
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