SOX9 Is an Astrocyte-Specific Nuclear Marker in the Adult Brain Outside the Neurogenic Regions

Abstract

Astrocytes have in recent years become the focus of intense experimental interest, yet markers for their definitive identification remain both scarce and imperfect. Astrocytes may be recognized as such by their expression of glial fibrillary acidic protein, glutamine synthetase, glutamate transporter 1 (GLT1), aquaporin-4, aldehyde dehydrogenase 1 family member L1, and other proteins. However, these proteins may all be regulated both developmentally and functionally, restricting their utility. To identify a nuclear marker pathognomonic of astrocytic phenotype, we assessed differential RNA expression by FACS-purified adult astrocytes and, on that basis, evaluated the expression of the transcription factor SOX9 in both mouse and human brain. We found that SOX9 is almost exclusively expressed by astrocytes in the adult brain except for ependymal cells and in the neurogenic regions, where SOX9 is also expressed by neural progenitor cells. Transcriptome comparisons of SOX9ϩ cells with GLT1ϩ cells showed that the two populations of cells exhibit largely overlapping gene expression. Expression of SOX9 did not decrease during aging and was instead upregulated by reactive astrocytes in a number of settings, including a murine model of amyotrophic lateral sclerosis (SOD1G93A), middle cerebral artery occlusion, and multiple ministrokes. We quantified the relative number of astrocytes using the isotropic fractionator technique in combination with SOX9 immunolabeling. The analysis showed that SOX9ϩ astrocytes constitute ϳ10 -20% of the total cell number in most CNS regions, a smaller fraction of total cell number than previously estimated in the normal adult brain. Significance StatementAstrocytes are traditionally identified immunohistochemically by antibodies that target cell-specific antigens in the cytosol or plasma membrane. We show here that SOX9 is an astrocyte-specific nuclear marker in all major areas of the CNS outside of the neurogenic regions. Based on SOX9 immunolabeling, we document that astrocytes constitute a smaller fraction of total cell number than previously estimated in the normal adult mouse brain.

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

“…Previous studies in rodents showed that Id3 is only expressed in developing astrocytes (Lamantia, Tremblay, & Majewska, 2014; Molofsky et al, 2013). Sox9 is initially (E8.5–E18.5) expressed in all glial (precursor) cells, and is subsequently downregulated in myelinating oligodendrocytes (Stolt et al, 2003) and becomes astrocyte‐specific (Sun et al, 2017). Also NFIA is initially (E11.5) expressed in all developing glial cells, but downregulates Olig2 and becomes exclusive to astrocytes at E13.5–E16.5 (Deneen et al, 2006; Molofsky et al, 2012).…”

Oligodendrocytes Support Neuronal Glutamatergic Transmission via Expression of Glutamine Synthetase

“…Following synaptic release, glutamate uptake and degradation are tightly regulated to achieve temporal and spatial signaling specificity and prevent cellular excitotoxicity (Kim et al, 2011; Sattler and Rothstein, 2006; Sheldon and Robinson, 2007). Currently, astrocytes are considered the sole glial cell type that contributes to glutamate uptake and degradation in the CNS (Jayakumar and Norenberg, 2016; Liang et al, 2006; Ortinski et al, 2010; Papageorgiou et al, 2018; Schousboe et al, 2013; Schousboe, 2019; Sun et al, 2017; Tani et al, 2014; Trabelsi et al, 2017; Yuan et al, 2017), as they express high levels of glutamate transporters and glutamine synthetase (GS), an enzyme that converts glutamate into glutamine. In keeping with this view, GS is frequently used as an astrocyte-specific marker (Armbruster et al, 2016; Habbas et al, 2015; Okuda et al, 2014; Papageorgiou et al, 2018; Theofilas et al, 2017; Tong et al, 2014).…”

“…However, GS expression has also been reported in oligodendrocytes (Bernstein et al, 2014; Takasaki et al, 2010), glial cells known for producing myelin and ensheathing axons in the CNS (Pan and Chan, 2017). Although these results remain controversial (Anlauf and Derouiche, 2013; Jayakumar and Norenberg, 2016; Sun et al, 2017), it is of great physiological and clinical importance to identify all potential cellular and molecular components involved in the life cycle of glutamate. GS deletion from the brain results in neonatal death (He et al, 2010), and mutations in the GS gene produce severe neuropathology in humans (Häberle et al, 2012; Spodenkiewicz et al, 2016).…”

Neuroprotective potential of astroglia

“…Such studies help to identify potential candidates for new and possibly better molecular markers for astrocytes. An interesting addition to the pool of astrocytic markers is SOX9, a transcriptional factor that has been reported as selective for astrocytes in most brain areas (Sun et al, ). This protein is characteristically accumulated in the nuclei of the cells, which sometimes is very useful when, for example, one needs to count astrocytes, which is not a trivial task using antibodies against GFAP that predominantly stain processes, creating a mesh‐like staining pattern.…”