Immunoablation of cells expressing theNG2 chondroitin sulphate proteoglycan (original) (raw)
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Immunoablation of cells expressing the NG2 chondroitin sulphate proteoglycan
Journal of Anatomy, 2013
Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron-glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 'knockout' mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.
NG2: a component of the glial scar that inhibits axon growth
Journal of anatomy, 2005
NG2 is a high-molecular-weight chondroitin sulphate proteoglycan found on the surfaces of oligodendrocyte precursor cells (OPCs). Here we review the history and biology of OPCs with an emphasis on their functions after experimentally induced CNS injury. Injury to brain or spinal cord results in the rapid accumulation of NG2-expressing OPCs in the glial scar that forms at the injury site. The glial scar is considered a biochemical and physical barrier to successful axon regeneration and the functional properties of NG2 suggest that it, along with other macromolecules, participates in the creation of this growth-inhibitory environment. NG2 is an important target for therapies designed to promote successful axon regrowth.
Journal of Neuroscience, 2010
The adult mammalian brain and spinal cord contain glial precursors that express platelet-derived growth factor receptor ␣ subunit (PDGFRA) and the NG2 proteoglycan. These "NG2 cells" descend from oligodendrocyte precursors in the perinatal CNS and continue to generate myelinating oligodendrocytes in the gray and white matter of the postnatal brain. It has been proposed that NG2 cells can also generate reactive astrocytes at sites of CNS injury or demyelination. To test this we examined the fates of PDGFRA/NG2 cells in the mouse spinal cord during experimental autoimmune encephalomyelitis (EAE)-a demyelinating condition that models some aspects of multiple sclerosis in humans. We administered tamoxifen to Pdgfra-CreER T2 :Rosa26R-YFP mice to induce yellow fluorescent protein (YFP) expression in PDGFRA/NG2 cells and their differentiated progeny. We subsequently induced EAE and observed a large (Ͼ4-fold) increase in the local density of YFP ϩ cells, Ͼ90% of which were oligodendrocyte lineage cells. Many of these became CC1-positive, NG2-negative differentiated oligodendrocytes that expressed myelin markers CNP and Tmem10/Opalin. PDGFRA/NG2 cells generated very few GFAP ϩ -reactive astrocytes (1-2% of all YFP ϩ cells) or NeuN ϩ neurons (Ͻ0.02%). Thus, PDGFRA/NG2 cells act predominantly as a reservoir of new oligodendrocytes in the demyelinated spinal cord.
2016
Classically, the central nervous system (CNS) was considered to contain neurons and three main types of glial cells - astrocytes, oligodendrocytes, and microglia. Now, it has been clearly established that NG2-glia are a fourth glial cell type that are defined by their expression of the NG2 chondroitin sulfate proteoglycan (Cspg4). NG2-glia are also known as oligodendrocyte precursor cells (OPCs) and express the alpha receptor for platelet-derived growth factor (Pdgfra) as well as other oligodendrocyte lineage markers. NG2-glia are most numerous during CNS development when they are responsible for massive generation of oligodendrocytes, the myelin-forming cells of the CNS. A significant population of NG2-glia persist in the adult CNS, where they generate oligodendrocytes throughout life. A unique feature of NG2-glia is that they receive synaptic inputs from neurons and are able to respond rapidly to neurotransmission via their specific ion channel and receptor profiles. Moreover, syn...
Astrocytes and NG2-glia: what's in a name?
Journal of Anatomy, 2005
Classic studies recognize two functionally segregated macroglial cell types in the central nervous system (CNS), namely astrocytes and oligodendrocytes. A third macroglial cell type has now been identified by its specific expression of the NG2 chondroitin sulphate proteoglycan (NG2-glia). These NG2-glia exist abundantly in both grey and white matter of the mature CNS and are almost as numerous as astrocytes. It is well established that NG2-glia give rise to oligodendrocytes. However, the majority of NG2-glia in the adult CNS proliferate very slowly and are nonmotile. Both astrocytes and NG2-glia display a stellate morphology and express ion channels and receptors to neurotransmitters used by neurons. Both types of glia make intimate contacts with neurons in grey and white matter, and their functional differences and similarities are only beginning to be unravelled. Recent observations emphasize the need to examine the relationship between astrocytes and NG2-glia, and address the question of whether they represent overlapping or two distinct glial cell populations. To be of any relevance, this classification must relate to specific functions in the neural network. At present, the balance of evidence is that NG2-glia and astrocytes are functionally segregated populations.
The roles of neuronal and glial precursors in overcoming chondroitin sulfate proteoglycan inhibition
Experimental Neurology, 2012
The extension of axons through the major inhibitory component of the glial scar, chondroitin sulfate proteoglycans (CSPG), remains a key obstacle for regeneration following spinal cord injury (SCI). We have previously shown that transplants composed of neuronal and glial restricted precursors (NRP and GRP respectively) promote regeneration and connectivity in the injured spinal cord (Bonner et al., 2010; 2011), however, little is known about the properties of these precursors at a cellular level. We now report that NRP-derived neurons, in contrast to dorsal root ganglion (DRG) neurons, have the ability to extend axons and cross over from a permissive substratum (laminin) onto inhibitory CSPG in vitro. Growth cones of neurons derived from NRP, compared to DRG, exhibit significantly lower levels of the CSPG receptors protein tyrosine phosphatase sigma (PTPσ) and leukocyte common antigen-related phosphatase (LAR). GRPconditioned medium prepared from the same cell densities did not affect the response of primary sensory neurons to CSPG confirming that the ability of NRP-derived neurons to cross onto CSPG is determined intrinsically. However, GRP-conditioned medium collected from high density cultures increased the probability of DRG axons to cross from LN onto CSPG and increased the length of DRG axons extending on CSPG. Collectively, these results suggest that (1) neurons derived from NRPs are intrinsically insensitive to CSPGs due to low levels of receptor expression, and (2) high levels of factors secreted by GRP can reduce the inhibitory effects of CSPG and promote axonal growth. These observations provide mechanistic insights into the specific roles of NRPs and GRPs in promoting regeneration and repair following SCI.
2002
Several extracellular matrix (ECM) molecules have been identified as potent inhibitors of neurite outgrowth in vitro and are believed to limit axonal growth after CNS injury. Recent studies have shown that different members of the chondroitin sulfate proteoglycan (CSPG) class of putatively inhibitory ECM molecules are expressed after a number of CNS injuries. The purpose of this study was to evaluate the relative amounts of individual CSPGs expressed after spinal cord injury (SCI) and identify their cells of origin. Evaluation of total soluble CSPGs 2 weeks after dorsal column lesion in the rat demonstrated that NG2 is highly upregulated and is a major CSPG species. Immunocytochemical analysis further demonstrated that NG2 expression is upregulated within 24 hr of injury, peaks at 1 week, and remains elevated for at least an additional 7 weeks. NG2 expression results from a multicellular response to injury, in-cluding both reactive macrophages and oligodendrocyte progenitors; astrocytes were not identified as a major source of NG2. Immunocytochemical analysis of other CSPG family members 7 d after injury showed moderate upregulation of versican, brevican, and neurocan, and downregulation of phosphacan. Axonal tracing experiments demonstrated dense NG2 labeling adjacent to the forward processes of transected corticospinal tract axons in a spatial profile that could restrict axonal growth. Thus, NG2 is a major component of this putatively inhibitory class of ECM molecules expressed at sites of SCI and may restrict axonal regeneration.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999
Astrocytes, oligodendrocytes, and oligodendrocyte/type 2 astrocyte progenitors (O2A cells) can all produce molecules that inhibit axon regeneration. We have shown previously that inhibition of axon growth by astrocytes involves proteoglycans. To identify inhibitory mechanisms, we created astrocyte cell lines that are permissive or nonpermissive and showed that nonpermissive cells produce inhibitory chondroitin sulfate proteoglycans (CS-PGs). We have now tested these cell lines for the production and inhibitory function of known large CS-PGs. The most inhibitory line, Neu7, produces three CS-PGs in much greater amounts than the other cell lines: NG2, versican, and the CS-56 antigen. The contribution of NG2 to inhibition by the cells was tested using a function-blocking antibody. This allowed increased growth of dorsal root ganglion (DRG) axons over Neu7 cells and matrix and greatly increased the proportion of cortical axons able to cross from permissive A7 cells onto inhibitory Neu7 ...
Journal of Neuroscience, 2006
The NG2 chondroitin sulfate proteoglycan inhibits axon growth in vitro. Levels of NG2 increase rapidly in the glial scars that form at sites of CNS injury, suggesting that NG2 may inhibit axon regeneration. To determine the functions of NG2, we infused mixtures of neutralizing or non-neutralizing anti-NG2 monoclonal antibodies into the dorsally transected adult rat spinal cord and analyzed the regeneration of ascending mechanosensory axons anatomically. At 1 week after injury, ascending sensory axons in control animals terminated caudal to the lesion within an area containing dense deposits of NG2 immunoreactivity. In animals treated with the neutralizing anti-NG2 antibodies, labeled axons penetrated the caudal border of the lesion and grew into and beyond the lesion center. The low intrinsic growth capacity of adult neurons may also limit the ability of damaged axons to regenerate. To enhance growth, we combined antibody treatment with a peripheral nerve conditioning lesion. After a conditioning lesion and treatment with control, non-neutralizing antibodies, many sensory axons grew into the lesion core. These axons did not grow past the rostral border of the lesion; rather, they grew along the dorsal surface of the spinal cord and within any remaining pieces of the dorsal roots. In contrast, combining a peripheral nerve conditioning lesion with neutralizing anti-NG2 antibodies resulted in sensory axon regeneration past the glial scar and into the white matter rostral to the injury site. The combinatorial approach used here that neutralizes extrinsic inhibition and increases intrinsic growth results in anatomically correct axon regeneration, a prerequisite for functional recovery.
NG2 Glia: Novel Roles beyond Re-/Myelination
Neuroglia, 2018
Neuron-glia antigen 2-expressing glial cells (NG2 glia) serve as oligodendrocyte progenitors during development and adulthood. However, recent studies have shown that these cells represent not only a transitional stage along the oligodendroglial lineage, but also constitute a specific cell type endowed with typical properties and functions. Namely, NG2 glia (or subsets of NG2 glia) establish physical and functional interactions with neurons and other central nervous system (CNS) cell types, that allow them to constantly monitor the surrounding neuropil. In addition to operating as sensors, NG2 glia have features that are expected for active modulators of neuronal activity, including the expression and release of a battery of neuromodulatory and neuroprotective factors. Consistently, cell ablation strategies targeting NG2 glia demonstrate that, beyond their role in myelination, these cells contribute to CNS homeostasis and development. In this review, we summarize and discuss the adv...