A Guide to Astrocyte Markers

Astrocytes are one of three types of glial cells in the central nervous system (CNS), comprising nearly 50% of non-neuronal CNS cells. The most diverse glial cell phenotypically, they play a key role in maintaining and supporting tissue homeostasis neuronal structure, energy metabolism, trophic factor delivery, synaptic transmission, long-distance communication, and inflammation. These cells proliferate throughout the entire lifespan of an individual, even during aging, which makes them an incredible therapeutic target for multiple CNS diseases. Despite their cryoprotective role, astrocytes can also be cytotoxic, depending on their activation phenotype, and the proteins they express are directly related to their functional state. In this article, we outline the protein markers of astrocytes as documented in recent literature.

Structural Proteins

Astrocytes are complex cells morphologically, consisting of soma and processes, which form intricate branching processes. These branching processes facilitate cell-cell, cell-synapse, and cell-blood vessel communication throughout the CNS. Immature astrocytes express several structural proteins that play a critical role in astrocyte differentiation and formation of branching processes, including the intermediate filament proteins VIM, nestin, and synemin; however, these proteins are also expressed by other CNS cell types, including Bergmann glia. Thrombospondins, secreted glycoproteins that are also expressed in immature astrocytes, support normal synaptic structure.

Actin-related processes play a critical role in forming and supporting the complex branching structures found in mature astrocytes, and encompass several structural proteins that are enriched in astrocytes: CNN1, FMN2, NEBL, PDZ, PDLIM7, and SYNPO2. Additional structural markers of fine, peripheral astrocyte processes include ezrin and radixin.

One of the foundational three astrocyte marker proteins, GFAP plays a critical mechanical role in supporting astrocyte structure and the blood-brain barrier. While GFAP labels mainly the extensive branches of white matter astrocytes, making it the best marker for studying astrocyte morphology, it is expressed by other cell types, including neuronal stem cells.

Transcription Factors

Transcription factors also play a critical role in astrocyte differentiation, structure, and function, and they generally have proven more specific to astrocytes than commonly used structural protein markers. NKX2-1, a NK homeobox gene-coded protein, regulates GFAP expression and stem cell division. Two other NK homeobox gene-coded proteins, NKX3-1 and NKX6-1, are exclusively expressed by olfactory bulb and brainstem astrocytes, respectively. The nuclear transcription factor SOX9 is almost exclusive to astrocytes, and has been used as a reliable marker of astrocytes in the adult brain (outside neurogenic zones).

One of the first transcription factors used as a marker of astrocytes is the nuclear factor 1 (NF1) family member NF1A, which maintains astrocyte-synapse supporting functions in the hippocampus. Other NF1 family members expressed by astrocytes include NF1B and NF1X, which are present in the olfactory bulb, hippocampus, and cortex, and co-localize with ALDH1L1, a marker of astrocyte metabolism, in the brainstem. ALDH1L1, along with GFAP and S100β, is one of the classic astrocyte markers, and is the most selective of the three.

Membrane Proteins

Astrocytes support and maintain ion and energy homeostasis through several transmembrane channels and transporter proteins, many of which are specific to astrocytes. Gap junction channels are formed by a group of proteins called connexins. Cx26, Cx30, Cx40, and Cx43 support exchange with the extracellular milieu in addition to protein interactions, cell adhesion, and intracellular signaling. AQP4 supports water homeostasis, although it is not specific to astrocytes, expressed also in ependymal cells and Bergmann glia.

KIR 4.1 is responsible for potassium uptake from synaptic clefts by astrocytes, and BEST1 facilitates calcium-dependent transport of chloride ions. Glutamate-glutamine shuttle is an astrocyte-specific function, facilitated in part by sodium dependent glutamate transporters EAAT1 and EAAT2 (GLAST and GLT1 in rodents). ASCA-1 is a human and rodent-reactive antibody that specifically detects EAAT1/GLAST and labels all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin. While these markers are abundant in fine astrocyte processes, they are expressed by all other CNS cell types to a variable extent, and they are purported to play a key role in the development of neurological disorders.

Astrocytes also facilitate GABA transport, mainly at synapses, through GAT-3, an astrocyte-specific GABA transporter. GAT-1 is also expressed by astrocytes, but is less specific than GAT-3, as most GAT-1 is found in neurons. CD49f, a transmembrane protein that interacts with extracellular matrices, is another astrocyte-specific marker that can sort astrocytes from neurons and oligodendrocytes.

Metabolic Markers

Located in the cytoplasm of gray matter astrocytes, S100β is another of the three classical astrocytes markers. Composed of multiple isoforms, S100 proteins are calcium-binding proteins that play a number of physiological roles, including regulating calcium balance, energy homeostasis, and inflammation. S100β is specific to astrocytes.

Astrocytes are the only glial cells that store glucose, which they do by synthesizing glycogen via glycogen synthase (GS). Astrocytes utilize the glucose they’ve stored through glycogen phosphorylase (GP). Astrocytes can even facilitate glucose synthesis in the brain during periods of starvation by gluconeogenesis using glucose 6-phosphatase (G6PC) and fructose 1,6-bisphosphate (FBP). And because astrocytes exhibit a higher glycolytic rate than neurons under normal conditions, PFKFB3 and PFK are excellent astrocyte-enriched markers. Additional proteins involved in glycolysis that are either enriched in or specific to astrocytes include PKM2, PDK4, LDH5, and MCT4. PC and MCT1 are also expressed by astrocytes during glycolysis, but they are less specific markers as they are additionally expressed by other glial cells.

Fatty acid oxidation and glycogenic amino acid catabolism occur at much higher rates in astrocytes than in neurons. ABHD5, GK, GPAT3, and IDI1 all contribute to membrane lipid metabolism, while CTP1a and CTP2 are astrocyte-specific markers that transport long-chain fatty acids into mitochondria. FABP7, which binds both saturated and unsaturated long-chain fatty acids, is enriched in astrocytes filled with cytoplasmic granules believed to originate from damaged mitochondria, but is also expressed in neural stem cells throughout development. Acetyl-coA from fatty acid oxidation is further transformed into acetoacetate in astrocytes via HMGCS2. GLDC, GPT2, HIBADH, SERHL, SDSL, PRODH, and 3PGDH are proteins involved in amino acid catabolism and are enriched in astrocytes. Fatty acid oxidation in astrocytes is regulated by T3, which is produced from T4 by DIO2 and aided by HADHA, most of which co-localizes with GFAP. T4 is produced by upstream astrocytic thyroid hormone metabolism, which is mediated by SLCO1C1/OATP1C1.

Cholesterol, required for axonal growth and synaptogenesis, is synthesized by astrocytes via SCAP and HMGCR sequentially. APOE and ABCA7 are cholesterol transporters, and their transcription is controlled by LXR/RXR. Astrocytes also facilitate glutamate uptake and recycling at synapses via astrocyte-specific enzyme GLUL and the amino acid transporters SNAT3 and SNAT5. Some of this glutamate is transported to the mitochondria by GC-2, which is enriched in astrocytes but also expressed by oligodendrocyte progenitor cells, and is then deaminated by GDH.

Table of Astrocyte Markers

The table below lists human and rodent protein markers of astrocytes as described by recent literature. The proteins listed include both general and specific astrocyte markers, and comprise the major groups of astrocyte markers: structural proteins, transcription factors, transmembrane proteins, and metabolic markers. Accompanying each marker are links to relevant antibodies and ELISA kits, as these immunodetection tools are routinely used in cell characterization studies via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and can serve as a useful reference for astrocyte immunophenotyping.

Note: *Some markers are protein isoforms, multi-subunit protein complexes, or protein families composed of several distinct genes. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

References

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