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After traumatic brain injury oligodendrocytes regain a plastic phenotype and can become astrocytes

Xianshu Bai, Na Zhao, Wenhui Huang, Laura C. Caudal, Renping Zhao, Johannes Hirrlinger, Wolfgang Walz, Frank Kirchhoff, Anja Scheller

Preprint posted on June 18, 2021 https://www.biorxiv.org/content/10.1101/2021.06.18.448919v1

A cytobridge in the brain- AO cells are half-way there!

Selected by Ranabir Chakraborty

Categories: cell biology, neuroscience

Background:

The degree of neuron insulation by lipid-rich membranes (myelination) is a major factor in determining the rate of propagation of action potentials along neurons. Myelination ensures rapid conduction of electrical impulses which is necessary for efficient information processing in the brain. Oligodendrocytes, generated from Oligodendrocyte Progenitor Cells (OPCs), are the myelin-forming cells in the brain that can generate up to 50 myelinating processes, each capable of independently wrapping around axons (1). While oligodendrocytes are terminally differentiated cells, a plastic phenotype within the lineage was first demonstrated by Raff and colleagues in 1983, who showed that O-2A precursor cells have a microenvironment-dependent ability to generate either oligodendrocytes or type 2 astrocytes (2). Nerve/Glia Antigen 2 (NG2) expressing glial precursor cells (of oligodendroglial lineage) also demonstrate such plasticity in an age-dependent manner in vivo (3). Besides precursor cells, plasticity of mature oligodendrocytes has also been demonstrated in the goldfish optic tract. Upon axonal injury, these cells revert to a precursor-like bipolar morphology, and eventually remyelinate the axons (4). However, the plasticity of mature oligodendrocytes in the adult mammalian brain remains elusive. In this preprint, Bai et al. demonstrate a novel type of cell generated from the oligodendroglial lineage, including mature oligodendrocytes, in an acutely injured brain that can potentially become astrocytes.

Key results:

The authors made use of the Cre/loxP transgenic system to investigate the potential contribution of oligodendroglial lineage cells (OPCs and/or mature oligodendrocytes) towards generation of astrocytes upon acute stab-wound injury (SWI). Using double transgenic mice expressing Cre recombinase in oligodendroglial cells (NG2-CreERT2; R26flSTOPfltdTomato reporter), it was observed that stab wounds after tamoxifen-induced recombination cause an increase in the expression of Glial Fibrillary Acidic Protein (GFAP), a marker for astrocytes, in about 25% of recombined (oligodendroglial) cells. Of these, about 28.5% of cells were newly generated astrocytes that not only extended elaborate processes, but also expressed another astrocytic marker, Glutamine synthetase (GS). Increasing the duration of tamoxifen-induced recombination before SWI substantially increased the number of newly generated astrocytes from recombined cells, suggesting these cells are distinct from astrocytes already present in the brain.

In order to understand the origin of astrocytes, the authors used a split Cre system, where the presence of a functional recombinase enzyme depends on the complementation of N- and C- terminals of Cre (NCre and CCre) expressed under different promoters. Transdifferentiation, or direct conversion of one terminally differentiated cell type to another without the involvement of a dedifferentiated state, is brought about by a downregulation, and simultaneous upregulation of former (cell type that transdifferentiates) and latter (new cell type that is formed) cells respectively. As such, the authors utilized transgenic mice differentially expressing NCre and CCre in astrocytes (GFAP-NCre) and oligodendrocytes (PLP-CCre) respectively. SWI induces Cre complementation, with recombined cells immunopositive for both astrocyte and oligodendrocyte markers. This points towards the potential of mature oligodendrocytes to express astrocytic genes. These astrocytes also respond to injury by undergoing astrogliosis, thereby validating their functionality in the brain.

Using a double transgenic mice line (PLP-DsRed1/GFAP-EGFPGFEA), the authors were able to detect a subset of cells, referred to as AO cells, that expressed both fluorophores in the injured brain, suggesting co-expression of Astrocytic and Oligodendrocytic genes (hence the name). These cells also expressed several oligodendrocyte-specific proteins, but not astrocyte or other glial markers, indicating an oligodendrocytic lineage. During their conversion to astrocytes, AO cells show upregulation of the GFAP promoter, and downregulation of the PLP promoter, leading the authors to classify them as a “transitioning cell”.

The lack of a definitive cytological state was also observed in the electrophysiological properties of AO cells (in PLP-DsRed1/GFAP-EGFP mice), which were found to be quite distinct in comparison to astrocytes or oligodendrocytes. Although these cells exhibited mostly K+ currents (whole-cell patch-clamp recordings at -80 mV holding voltage) which are typical of glia, the type of these currents (voltage-gated, non-rectifying; outwardly or inwardly rectifying K+ currents) varied a great deal between individual AO cells, indicating a transitional cell state without a definitive physiological property.

To go beyond the fixed time-point based experimental approaches, the authors used a 2-photon microscope to visualize the transition to astrocytes. In PLP-DsRed1/GFAP-EGFP mice, AO cells were detected as early as 3 days post injury (dpi) (Figure 1). Upon tracing a single oligodendrocyte (DsRed1+EGFP-), AO cell identity (DsRed1+EGFP+) could be seen by 5 dpi, which eventually converted to an astrocyte by 6 dpi (DsRed1 EGFP+). However, not all AO cell transitioned to astrocytes; some reverted to the oligodendrocyte fate, suggesting a potential heterogeneity in committing to becoming an astrocyte.

Figure 1: Conversion of oligodendrocytes to astrocytes via transitional phases upon SWI. In vivo 2-photon microscope facilitates the visualization of a newly formed astrocyte (arrowhead, c) from oligodendrocyte (open triangle, a) via AO cell (asterisk, b). Modified from Bai et al., Figure 5a-c (made available under a CC-BY-NC-ND 4.0 international license).

Although acute SWI is an efficient approach for the detection of AO cells, two physiological injury models (Pial Vessel Disruption, PVD and transient Middle Cerebral Artery Occlusion, MCAO) were assessed for their fidelity in identifying cell fate transition. Under both of these injury conditions, AO cells were observed near the lesion sites, providing substantial evidence for the physiological relevance of this process. Since these injuries also affected the Blood-Brain Barrier (BBB), which would lead to an elevated level of cytokines in the affected region, the authors hypothesized inflammatory molecules to be a key player in the generation of AO cells. Not only was Interleukin-6 (IL-6) level increased after SWI, cortical injection of IL-6 also induced the formation of AO cells, which could be reduced by Leukaemia Inhibitory Factor (LIF). Such regulation by cytokines provides a possible mechanism that could act in concert with each other, or other molecular players yet unknown, to generate astrocytes from oligodendrocytes.

The reason behind choosing this preprint:

The generation of different cell types during development requires fate determination of stem/progenitor cells, which is provided by several intrinsic and extrinsic cues. Fate switch, however, remains an underexplored avenue. In an organ that mostly comprises post-mitotic cells (neurons), the plastic potential of non-neuronal cells becomes an important aspect of the efficient maintenance of homeostasis. Bai and colleagues quite elegantly demonstrate the conversion of mature oligodendrocytes and their progenitors into astrocytes upon injury. This paper also brings to attention another class of heterogeneous astrocytes (AO cells) that might have a distinct response to injury. Consequently, oligodendrocyte plasticity could emerge as a potential therapeutic target for the treatment of brain trauma/injuries.

Questions for the authors:

• Given the variations in electrophysiological properties of individual AO cells, are the genes coding for different ion channels differentially expressed?
• What factors determine if an AO cell will be converted to an astrocyte, or back to an oligodendrocyte?
• Do the astrocytes generated from AO cells become functionally integrated into the glio-vascular network?

 

References:

1. Baumann N, Pham-Dinh D. Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiological reviews. 2001.
2. Raff MC, Miller RH, Noble M. A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature. 1983;303(5916):390-6.
3. Zhu X, Hill RA, Dietrich D, Komitova M, Suzuki R, Nishiyama A. Age-dependent fate and lineage restriction of single NG2 cells. Development. 2011;138(4):745-53.
4. Ankerhold R, Stuermer CA. Fate of oligodendrocytes during retinal axon degeneration and regeneration in the goldfish visual pathway. Journal of neurobiology. 1999;41(4):572-84.

 

 

Posted on: 6th August 2021

doi: https://doi.org/10.1242/prelights.30269

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Author's response

Anja Scheller and Xianshu Bai shared

Given the variations in electrophysiological properties of individual AO cells, are the genes coding for different ion channels differentially expressed?

This is highly likely to be the case for the AO cells. As we observed, AO cells have several distinct electrophysiological properties, being similar to oligodendrocytes and to astrocytes during their transition. Oligodendrocytes and astrocytes are two different macroglial cell types sharing common ion channel expression, but the expression level differs. Therefore, it is highly likely that AO cells at different transition level express variable levels of ion channels. However, single-cell RNA sequencing will delineate the differences and provide more information about ion channel expression of these cells.

What factors determine if an AO cell will be converted to an astrocyte, or back to an oligodendrocyte?

From our Split-Cre data, we observed more astrocytes derived from AO cells at the cortical layer 1-3, while AO cell derived oligodendrocytes are more located at the deeper layer of the cortex. This observation indicates that sub-region enriched micro-environmental factors navigate AO cell differentiation. From our unpublished data, we found that IL-6 mRNA was more pronounced at the upper layers. Many colleagues have shown that IL-6 facilitates astroglial generation from neural progenitor cells, therefore we strongly believe that the cytokine released in the different environments influences the differentiation to either astro- or oligodendrocytes.

Do the astrocytes generated from AO cells become functionally integrated into the glio-vascular network?

This is an excellent question. Four weeks post cortical stab wound injury in NG2-CreERT2 x floxed tdTomato mice, we observed only few cells positive for tdTomato co-expressing GFAP and coincidently wrapping blood vessel with their processes. However, since OPCs can also generate astrocytes after acute injuries, we cannot conclude that AO cell derived astrocytes are functionally integrated into the glio-vascular network. Therefore, at this time point, we cannot answer this important question, but we showed in our manuscript that AO cell derived astrocytes can proliferate and have the morphology of typical astrocytes. We regard this as strong evidence for full integration of AO cell derived astrocytes in cortical circuits including the neurovascular unit, i.e., functioning like all the other astrocytes.

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