Glial Cells and Their Function in the Adult Brain: A Journey through the History of Their Ablation

Glial cells, consisting of microglia, astrocytes, and oligodendrocyte lineage cells as their major components, constitute a large fraction of the mammalian brain. Originally considered as purely non-functional glue for neurons, decades of research have highlighted the importance as well as further functions of glial cells. Although many aspects of these cells are well characterized nowadays, the functions of the different glial populations in the brain under both physiological and pathological conditions remain, at least to a certain extent, unresolved. To tackle these important questions, a broad range of depletion approaches have been developed in which microglia, astrocytes, or oligodendrocyte lineage cells (i.e., NG2-glia and oligodendrocytes) are specifically ablated from the adult brain network with a subsequent analysis of the consequences. As the different glial populations are very heterogeneous, it is imperative to specifically ablate single cell populations instead of inducing cell death in all glial cells in general. Thanks to modern genetic manipulation methods, the approaches can now directly be targeted to the cell type of interest making the ablation more specific compared to general cell ablation approaches that have been used earlier on. In this review, we will give a detailed summary on different glial ablation studies, focusing on the adult mouse central nervous system and the functional readouts. We will also provide an outlook on how these approaches could be further exploited in the future

Oligodendrocyte Precursor Cells Synthesize Neuromodulatory Factors

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

Daily Eastern News: March 03, 2017

https://thekeep.eiu.edu/den_2017_mar/1002/thumbnail.jp

Reactive Glia in the Injured Brain Acquire Stem Cell Properties in Response to Sonic Hedgehog

SummaryAs a result of brain injury, astrocytes become activated and start to proliferate in the vicinity of the injury site. Recently, we had demonstrated that these reactive astrocytes, or glia, can form self-renewing and multipotent neurospheres in vitro. In the present study, we demonstrate that it is only invasive injury, such as stab wounding or cerebral ischemia, and not noninvasive injury conditions, such as chronic amyloidosis or induced neuronal death, that can elicit this increase in plasticity. Furthermore, we find that Sonic hedgehog (SHH) is the signal that acts directly on the astrocytes and is necessary and sufficient to elicit the stem cell response both in vitro and in vivo. These findings provide a molecular basis for how cells with neural stem cell lineage emerge at sites of brain injury and imply that the high levels of SHH known to enter the brain from extraneural sources after invasive injury can trigger this response

Conditional deletion of β1‐integrin in astroglia causes partial reactive gliosis

Astrocytes play many pivotal roles in the adult brain, including their reaction to injury. A hallmark of astrocytes is the contact of their endfeet with the basement membrane surrounding blood vessels, but still relatively little is known about the signaling mediated at the contact site. Here, we examine the role of β1‐integrin at this interface by its conditional deletion using different Cre lines. Thereby, the protein was reduced only at postnatal stages either in both glia and neurons or specifically only in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and up‐regulation of the intermediate filaments GFAP and vimentin as well as pericellular components, such as Tenascin‐C and the DSD‐1 proteoglycan. In addition, we also observed to a certain degree a non‐cell autonomous activation of microglial cells after conditional β1‐integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of β1‐integrin‐mediated signaling is not sufficient to elicit proliferation of these cells as observed after brain injury. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood brain barrier disturbances. As these effects did not appear after neuron‐specific deletion of β1‐integrin, we conclude that β1‐integrin‐mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in β1‐integrin‐mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury. © 2009 Wiley‐Liss, Inc

The NG2 ICD is located in the nucleus.

<p><b>A</b> Cartoon of the NG2 full length protein and the major cleavage fragments (ectodomain, CTF, ICD). Ectodomain cleavage (indicated by the α) has been reported by others [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.ref028" target="_blank">28</a>], while intracellular cleavage (indicated by the γ) has been found by our group [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.ref021" target="_blank">21</a>]. Expression constructs NG2_del leading to high NG2 CTF and lower ICD levels by proteolytic processing and the NG2_ICD construct leading to high levels of NG2 ICD are both shown in red. <b>B</b> Cytoplasmic (cyto), crude membrane (CM) and nuclear fractions of HEK cells transfected with empty plasmid (control), NG2_ICD, or NG2_del are shown. NG2_del full-length (FL) protein, the membrane bound NG2 CTF and the NG2 ICD are shown in WB (schematically shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.g002" target="_blank">Fig 2D</a>). The NG2 ICD shows the highest levels when expressed as a recombinant protein (NG2_ICD). This is present in all fractions but highest in the nuclear fraction. NG2_del-derived NG2 ICD (generated by proteolysis) is only detectable in the nuclear fraction and runs at the same size as the recombinant NG2 ICD.</p

NG2 dependent regulation of PTGDS and Nptx2 <i>in vivo</i>.

<p><b>A&B</b> mRNA levels of target-genes were directly analyzed by qRT-PCR from FACS isolated OPC and other cells (negative (neg.) sort). Single cell suspensions from total brains of postnatal day 9 (P9) NG2-KO and WT mice were used for FACS. <b>A</b> Enrichment of target mRNA in OPC in comparison to other cell-types is shown for WT (black bar) and NG2-KO (grey bar), (ΔΔCT = [ΔCT OPC]–[ΔCT other cells]). Enrichment of PDGFRα mRNA validates OPC enrichment. Nptx2 mRNA was enriched within OPC of both genotypes, while PTGDS mRNA was only enriched in WT OPC. <b>B</b> Genotype-specific mRNA enrichment in OPC (black bar) and other cells (grey bar) is plotted (ΔΔCT = [ΔCT WT cells]–[ΔCT KO cells]). PTGDS was the sole target gene analyzed exhibiting differential expression between WT and KO genotypes. Expression was highly increased in WT-derived OPC and down-regulated in the other cells (neg. sort) from these mice. <b>C</b> Western Blot of soluble protein fractions. Soluble fractions were extracted from P9 mouse brain of WT and NG2-KO mice. Total PTGDS and Nptx2 protein levels show no difference between genotypes. <b>D</b> Protein levels of post nuclear (PN) cell lysates of the OPC cell line Oli-neu were analyzed after treatment with siRNA silencing NG2 expression (siNG2) or control siRNA (siC). Full-length NG2 levels were reduced as well as PTGDS protein levels, fitting to the reduced mRNA levels of PTGDS found in NG2-KO OPC (B). <b>E</b> Expression of PTGDS mRNA by the OPC cell-line Oli-neu as revealed by <i>in situ</i> hybridization. (A&B: for NG2-KO OPC, 4 independent sorts were analyzed, for WT 3 sorts were analyzed, ΔCT = (CT target)–(CT GAPDH), ΔCT and ΔΔCT values are in log2 scale; for <b>C:</b> 4 animals were analyzed for NG2-KO (KO1-4) and BL6/N (WT1-4) mice; for <b>D</b>: 4 independent transfections were analyzed per siRNA, two tailed student´s t-test was performed.)</p

NG2 intracellular fragments influence PTGDS protein levels.

<p><b>A</b> Expression of NG2_del, NG2_ICD (both red) and Mock (empty Plasmid) constructs in the OPC cell-line Oli-neu, resulted in a reduction of PTGDS protein levels in post nuclear lysates (PN). Overexpression of these constructs leads to a changed ratio of protein levels between the NG2 FL and the small NG2 (intracellular) cleavage fragments the CTF and the ICD (compare to mock, see B). <b>B</b> Cartoon of NG2, NG2 expression constructs (red) and the cleavage sites for α- and γ-secretase leading to the creation of the CTF (12 kD) and ICD (8.5 kD, both blue). (A: 4 independent transfections were analyzed per construct; two tailed student’s t-test was performed.)</p

Expression of PTGDS and Nptx2 in primary OPC.

<p><b>A</b> Expression of cell-type specific markers was analyzed by Western-Blot of total cell lysates of pOPC and the negative (neg.) sort fractions, HEK cells and primary cortical neurons (DIV5 in culture). <b>B-D</b> show the expression of proteins in pOPC over time, related to DIV0. In <b>B</b> a peak of the OPC protein NG2 is shown at DIV1, PLP indicates differentiation into oligodendrocytes starting at DIV2, GFAP shows astrocyte differentiation starting at DIV2. <b>C</b> PTGDS expression peaks together with NG2 at DIV1. <b>D</b> Nptx2 expression increases together with NG2 at DIV1, peaks at DIV2 and returns to basal levels at DIV4. <b>E</b> Expression of PTGDS and Nptx2 mRNA in pOPC at DIV1, as revealed by <i>in situ</i> hybridization, OPC were identified by antibody staining of NG2 protein. (A-D: 3 sorts were analyzed for each time point. Two tailed student’s t-test was applied.)</p