Picture a bit of bacterial membrane floating about the brain. When a patrolling microglia cell notices that, it prods neighboring microglia to prick up their ears, too. Researchers now report that their clarion call consists of a protein called galectin-3, which binds toll-like receptor 4 (TLR4) on other microglia. In the March 10 Cell Reports, first author Miguel Burguillos of Queen Mary University in London, and colleagues also report that galectin-3 and TLR4 abound in the brains of people who suffered ischemia, suggesting the same alert system could be active in humans after loss of blood flow to the brain. As for neurodegenerative conditions, galectin-3 is known to flood the spinal cords of people with amyotrophic lateral sclerosis. Researchers are aware of a powerful neuroinflammatory component to several neurodegenerative diseases, but have not yet analyzed galectin-3 in detail (see Feb 2015 news; news story; Jul 2014 news).
Galectins: Sweet-Toothed, Two-Faced
A family of sugar-binding proteins, galectins interact with carbohydrates on other molecules, such as glycoproteins. They operate inside cells and in the extracellular space, and participate in a variety of processes, including proliferation, neurite growth, and apoptosis. Galectins can take on opposing roles depending on their location and circumstances. For example, galectin-1 modulates binding of cells to the extracellular matrix; promoting this binding in melanoma cells but inhibiting it in muscle (reviewed in Cooper and Barondes, 1999).
Scientists have long been aware that galectin-3 participates—somehow—in inflammation, Burguillos said. In fact, researchers use it as a marker for activated microglia and macrophages, though in that context it is often known by another name, Mac-2. Galectin-3 can be found in the nucleus, cytosol, and cellular membranes, and the cell exports it in response to inflammatory stimuli such as lipopolysaccharide, a component of bacterial membranes, or interferon-γ (Li et al., 2008; Jeon et al., 2010).
Galectin-3 seems to either promote or prevent inflammation, depending on its situation. For example, in a mouse model of ALS, knocking out the gene enhanced neuroinflammation and sped up the disease, suggesting galectin-3 must be anti-inflammatory (Lerman et al., 2012). However, in a project on mice with experimental autoimmune encephalomyelitis, loss of galectin-3 alleviated disease, suggesting pro-inflammatory ability (Jiang et al., 2009).
Burguillos focused on the secreted, extracellular form of galectin-3 released in response to LPS or ischemia. He worked in the laboratories of co-senior authors Bertrand Joseph of the Karolinska Institute in Stockholm and Tomas Deierborg of Lund University in Sweden. In collaboration with co-senior author Jose Venero of the University of Seville, Spain, they found a pro-inflammatory role for galectin-3 in this context.
Previous studies indicated that galectin-3 could bind to toll-like receptors as well as to LPS, which instigates inflammation via TLR4 (Mey et al., 1996). Therefore, the authors asked if galectin-3 itself might also interact with TLR4. In the mouse microglial BV2 cell line, extracellular galectin-3 co-localized with TLR4, and the two proteins co-immunoprecipitated. Burguillos measured expression of immune-related genes in the galectin-3-treated cells, and observed a pro-inflammatory profile.
Burguillos reasoned that if galectin-3 mediates inflammation, then blocking the protein should dampen microglial responses to LPS. In BV2 cultures, neutralizing galectin-3 with either interfering RNA or antibodies reduced production of nitric oxide synthase, a marker for inflammation. Experiments with primary microglia corroborated these results.
All told, the findings suggest that secreted galectin-3 can amplify inflammation. Would this hold up in vivo? Burguillos examined galectin-3 activity in two different mouse models of neuroinflammation. First, he created a situation somewhat akin to Parkinson’s disease by injecting LPS into the substantia nigra. Galectin-3 and TLR4 co-localized in cells expressing the microglial marker Iba1, macrophages gathered near the injection site, and half the dopaminergic neurons died. In galectin-3 knockout mice, fewer macrophages were recruited and the neurons survived.
Second, Burguillos modeled ischemia. Occluding the carotid arteries for 13 minutes caused a rise in populations of Iba1-positive microglia and death of pyramidal neurons in the hippocampus of wild-type mice. Again, galectin-3 knockouts were protected.
Finally, Burguillos examined brain tissue samples from five people who had died of cardiac arrest, which would limit blood flow to the brain. These samples contained more galectin-3 and TLR4 than brains from age-matched controls.
The authors conclude that galectin-3 release starts a chain reaction of inflammation by activating other microglia. They suspect it acts only on nearby cells—microglia and perhaps astrocytes or monocytes—in a paracrine manner. Cells take up galectin-3 quickly, Burguillos pointed out, so it would probably not reach far-flung microglia. The human data suggest, but by no means prove, that a similar process occurs in people, he said.
Though Burguillos studied ischemia, he speculated that galectin-3 might be involved in neuroinflammation in other conditions where TLR4 has been implicated, including Alzheimer’s, Parkinson’s, and ALS (Chen et al., 2012; Noelker et al., 2013; Casula et al., 2011). What exactly do the new data mean for those conditions? “It is hard to decide,” said Stanley Appel of the Methodist Neurological Institute in Houston, citing the disparate pro- or anti-inflammatory roles of galectin-3 in different disease models. Appel speculated that galectin-3 might affect inflammation via receptors other than TLR4 in different conditions. Burguillos pointed out that he found a pro-inflammatory role for extracellular galectin-3, but the intracellular version could conceivably do the opposite, and this galectin-3 might contribute to the biology of other diseases.
In the case of ALS, galectin-3’s role remains in question, but it does seem to be involved. Scientists have reported its upregulation in the microglia and muscles of SOD1 rats, cerebrospinal fluid of people with ALS, and spinal cords of people who died of the disease, leading to the suggestion it could be a biomarker (Nikodemova et al., 2014; Gonzalez de Aguilar et al., 2008; Zhou et al., 2010).
“Galectin-3 may play a role in ‘propagating’ proinflammatory microglial responses,” commented Mariko Bennett of the Stanford University School of Medicine, who was not involved in the work, in an email to Alzforum (see full comment below). “While further work is needed to demonstrate the paracrine nature of these effects … perhaps this system represents a mechanism by which small amounts of injury or inflammation can lead to widespread, prolonged, and possibly neurotoxic microglia activation in neurodegenerative disease.”
However, Oleg Butovsky of Brigham and Women’s Hospital in Boston, who also was not involved in the study, questioned the cell culture experiments. He cautioned that that the Iba1 marker the authors used to identify microglia also occurs in peripheral monocytes, which usually express more galectin-3 than microglia do, and that neither BV2 cells nor microglia from juvenile mice express the same pattern of genes as adult microglia. He said he would like to see the experiments repeated in microglia from adult mice (Butovsky et al., 2014). Burguillos concurred that cultures from adult mice would strengthen his argument.—Amber Dance
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