Just because astrocytes shield neurons from threats doesn’t mean they do so voluntarily. Injured motor neurons prompt astrocytes with “help me” signals, according to a paper in the October 27 Nature Communications. Researchers led by András Lakatos of the University of Cambridge, England, and Rickie Patani of University College London reported that the ephrin type-B receptor (EphB1) on neurons engages a ligand on astrocytes, which then ramps up expression of neuroprotective and anti-inflammatory genes in the glia. This lifeline appeared to be impaired in a mouse model of amyotrophic lateral sclerosis (ALS), and in astrocytes derived from ALS patient stem cells. The findings could help explain how neurons become vulnerable to damage in ALS and possibly other neurodegenerative diseases.

  • EphB1 receptors on injured motor neurons activate STAT3 in astrocytes via the glial Ephrin-B1 ligand.
  • This “reverse signaling” promotes neuroprotective, anti-inflammatory transcription.
  • The neuroprotective pathway falters in an ALS mouse model and in astrocytes from ALS patients.

Astrocytes can either nurture neurons—tending to their synapses and bathing them in nutrients—or pummel them with damaging inflammation that hastens their death. How the glial cells sway in one direction or the other is a matter of intense research. Some studies report that feisty microglia transform astrocytes into agents of damage, while other contend that signals from damaged neurons steer the trajectory of the glial responses (Jan 2017 news; Apr 2017 newsHooten et al., 2015; Murdock et al., 2015). Understanding the drivers of astrocytic behavior may be of critical importance in the context of ALS, in which neuroinflammation is linked with rapidly progressing disease (Mar 2017 news). 

First author Giulia Tyzack and colleagues asked if signals delivered by injured motor neurons might rally astrocytes to protect them. Specifically, they investigated whether EphB1—a receptor expressed on neurons in response to injury—might send signals to astrocytes by binding its ligand, ephrin-B1, on the astrocyte surface (Wang et al., 2005). Ephrin receptors and their ligands are known to work bidirectionally, transducing signaling cascades into both the ligand and receptor bearing cells upon engagement (Pasquale, 2008Klein, 2009). To provoke such a response, Tyzack and colleagues snipped the axons of facial motor neurons in mice. They found that EphB1 expression nearly tripled in the damaged cells within a day and continued to rise for another week. Astrocytes in the vicinity of the damaged motor neurons expressed ephrin-B1 at nearly twice the level of cells distant from the injury, and had triple the expression of phosphorylated STAT3, a hallmark of astrocyte activation (Herrmann et al., 2008; Anderson et al., 2016). 

Neuronal SOS?

Following damage to axons, motor neurons in sections of the facial motor nucleus (top) expressed the EphB1 receptor, while nearby astrocytes (bottom panel) revved up expression of its ligand, ephrin-B1. [Courtesy of Tyzack et al., Nature Communications, 2017.]

The researchers next investigated the nature of the activation of astrocytes by EphB1. When they treated cortical mouse astrocytes with recombinant EphB1 in a clustered state, mimicking the conformation of the receptor on the neuronal surface, astrocytes responded by phosphorylating STAT3 and shuttling it to the nucleus. This also occurs in response to IL-6, a cytokine known to ramp up inflammatory responses in astrocytes. Both EphB1 and IL-6 also boosted GFAP expression on astrocytes, and triggered the cells to transform into a reactive shape. 

However, the two proteins evoked dramatically different gene-expression profiles in the astrocytes, with many differentially expressed genes being involved in immune response. IL-6 promoted higher expression of pro-inflammatory genes such as Cebpd and Ptx3, while Eph1B boosted higher expression of immune regulators and homeostatic genes. The findings suggest that EphB1 shifts STAT3-mediated processes from potentially damaging pro-inflammatory responses toward neuroprotective ones.

To discern whether this transcriptional shift had functional consequences, the researchers triggered excitotoxicity in mouse spinal cord neurons by treating them with glutamate. Cleaved caspase 3, a marker of apoptosis, shot up by nearly 60 percent in response. However, if they first bathed the cells in medium from astrocytes that had been treated with EphB1, then activated caspase only increased by 35 percent. Medium from IL-6-treated astrocytes offered no protection.

How would this protective pathway function in ALS? The researchers addressed this by looking for markers of the pathway in SOD1-G93A mice. In lumbar spinal cord sections of symptomatic mice, the researchers found negligible expression of EphB1 in neurons, while nearby astrocytes only weakly expressed nuclear STAT3. The same was true in healthy wild-type mice. To see if they could awaken the protective signals, the researchers snipped the sciatic nerve. In response, levels of all three markers rose subtly in both ALS and wild-type mice at day 1. However, while they continued to rise in wild-type mice over the following week, they plateaued in ALS mice. The findings suggested that not only does the protective pathway appear silent in ALS mice, it also fails to switch on in response to acute injury.

The researchers next asked whether the EphB1 pathway was more or less active in astrocytes derived from ALS patients compared to controls. They generated astrocytes from human induced pluripotent stem cells (hiPSCs) derived from three healthy controls and two ALS patients who carried SOD1-D90A mutations. They also used an isogenic control from one of the patient cell lines, in which the disease mutation had been corrected using transcription activator-like effector nucleases (TALEN). 

Compared with astrocytes from healthy controls or with the corrected patient cells, SOD1-D90A mutated astrocytes upregulated more than 1,900 transcripts and downregulated more than 2,500. This pattern had hints of an inflammatory signature, including five genes known to be upregulated in response to IL-6 in mice, and five downregulated transcripts that had gone up in response to EphB1. Among these 10, expression of PHLDA3, a tumor suppressor with links to cell death, nearly doubled that in normal cells, at both the RNA and protein level, while expression of HSPB8, a protective heat-shock protein, fell 10-fold. These findings suggested that astrocytes from ALS patients had a more pro-inflammatory profile than control cells, and that the protective EphB1 pathway might be muted.

Together, the findings indicate that activation of astrocytes can have multiple outcomes. “The field has been biased in thinking that astrocyte reactivity equals neurotoxicity, but in fact it can be neuroprotective as well,” commented Serge Przedborski of Columbia University in New York. “This duality is something we’ve overlooked for years.” Przedborski and others have long demonstrated that astrocytes gain toxic functions in ALS models. In light of the current findings, researchers should now also consider the potential effects of astrocytes losing neuroprotective functions, he said.

Lakatos and Patani added that efforts to treat ALS by blocking activation of astrocytes should be considered with caution. “Experimental therapies aimed at reducing toxic pro-inflammatory processes should be complemented with approaches that spare or even augment the protective or anti-inflammatory STAT3 downstream signaling,” they wrote to Alzforum. “In the next few years we want to explore key effectors downstream of the EphB1-induced pathway in astrocytes in order to assess their neuroprotective potential.”

The authors acknowledged that it is still unclear how Ephrin B1 signaling provokes protective responses, or what factors actually protect neurons. Przedborski added that given the complex web of communication between neurons and astrocytes, it is likely that factors beyond Ephrin B1 are involved.

Kerry O’Banion of the University of Rochester in New York commented that the findings raise many questions. “More work is required to better understand the potential mechanism of astrocyte neuroprotection uncovered by these studies, and whether the identified deficit of signaling seen with SOD-mutant astrocytes extends to other neurodegenerative disease or, for example, to aging astrocytes.”—Jessica Shugart


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News Citations

  1. Microglia Give Astrocytes License to Kill
  2. ApoE and Tau: Unholy Alliance Spawns Neurodegeneration
  3. Can Immune Gene Expression Predict Pace of Motor Neuron Destruction?

Research Models Citations

  1. SOD1-G93A (hybrid) (G1H)

Paper Citations

  1. . Protective and Toxic Neuroinflammation in Amyotrophic Lateral Sclerosis. Neurotherapeutics. 2015 Apr;12(2):364-75. PubMed.
  2. . The dual roles of immunity in ALS: injury overrides protection. Neurobiol Dis. 2015 May;77:1-12. Epub 2015 Feb 26 PubMed.
  3. . Induction of ephrin-B1 and EphB receptors during denervation-induced plasticity in the adult mouse hippocampus. Eur J Neurosci. 2005 May;21(9):2336-46. PubMed.
  4. . Eph-ephrin bidirectional signaling in physiology and disease. Cell. 2008 Apr 4;133(1):38-52. PubMed.
  5. . Bidirectional modulation of synaptic functions by Eph/ephrin signaling. Nat Neurosci. 2009 Jan;12(1):15-20. Epub 2008 Nov 23 PubMed.
  6. . STAT3 is a critical regulator of astrogliosis and scar formation after spinal cord injury. J Neurosci. 2008 Jul 9;28(28):7231-43. PubMed.
  7. . Astrocyte scar formation aids central nervous system axon regeneration. Nature. 2016 Apr 14;532(7598):195-200. Epub 2016 Mar 30 PubMed.

Further Reading

Primary Papers

  1. . A neuroprotective astrocyte state is induced by neuronal signal EphB1 but fails in ALS models. Nat Commun. 2017 Oct 27;8(1):1164. PubMed.