Dr. Popovich (who may moonlight as a famous NBA coach on the side or just shares the name) has focused his research efforts on the complex interactions between the immune system and the regrowth and regeneration of the spinal cord after injury. In his lab’s recent work in the journal Molecular and Cellular Neuroscience, Dr. Popovich explores the interaction of Galectin-1 (Gal1) on macrophages and astrocytes at the site of injury in the central nervous system (CNS). As many may already know, a longstanding issue in the field is trying to understand why peripheral nervous tissue (PNS) can regenerate while CNS tissue cannot. Before this paper’s publishing, Gal1 was already known to elicit regeneration in the PNS via its effect on macrophages, but it remained an open question what if any were the effects in the CNS. The Popovich lab took a quantitative approach to answering this question by systematically identifying the expression levels of this protein over a four week time course following spinal cord injury. Interestingly, this work demonstrates a strong upregulation of Gal1 at the injury site as compared to uninjured spinal cord both in macrophages and astrocytes. This suggests a potential target of manipulation going forward in an attempt to tilt the immune-axis in the CNS towards a more conducive environment for regeneration.
Galectin-1 is a switch hitting molecule that changes its activity based on its redox state: when oxidized it is a monomer that acts much like a cytokine, but in its reduced form Gal1 dimerizes and subsequently has a stronger binding affinity with lectins. Of interest to spinal cord injury regeneration is this oxidized monomer form of Gal1, which may assist in axon regrowth. The expression of Gal1 was characterized using the full toolkit of molecular biology (western blots/qRT-PCR) along with immunohistochemistry to determine the localization of Gal1 at various time points. The spinal cord injury was formed using a standard method in which a contusion is applied to the spinal cord of an anesthetized rat.
As stated before, a clear increase in the protein Gal1 monomer at the injury site occurs at the 7 and 14 day time points, which is the height of the inflammatory response in spinal cord injury. mRNA was upregulated at the 3 day time point. Further, protein upregulation was confirmed by quantifying immunoreactivity density. The rest of the paper focuses on the localization and cell specific expression of Gal1. Using fluorescent microscopy, the Popovich lab demonstrates Gal1 up-regulation in macrophages and astrocytes in a series of beautiful images, but furthers the paper by quantifying their images rigorously to provide valuable data on where and when Gal1 is being upregulated in the CNS.
Figure 1:A comparison of Gal1 and OX42 a macrophage/microglia marker in uninjured (left) and injured (right) spinal cord. Notice the greater expression of Gal1 in macrophages in the injured tissue in e’ and e’’.
One of the issues raised near the conclusion of the paper is what the monomeric Gal1 is actually doing to macrophages. Gal1 may be reducing these macrophages overall inflammatory response or causing them to differentiate into a “reparative” phenotype. The final two figures of the paper demonstrate one aspect of Gal1 modulatory effect on macrophages: the protein seems to reduce their levels of phagocytosis. Using ED1 as a marker for phagocytic activity, the paper demonstrates a reduction in the colocalization of Gal1 and ED1 at 7 days. Further these macrophages seem to contain less phagocytosed lipids as imaged using the Oil Red O (ORO) stain. Running a linear regression between the coexpression of Gal1 and ORO showed a negative relationship between ORO and Gal1 expression. In sum these figure suggests that Gal1 promotes less phagocytosis in inflammatory macrophages.
Figure 2:A comparison of Gal1 and ORO (a marker for phagocytosed lipids). Notice the significant negative correlation between Gal1 immuno- positive cells and ORO density. Conversely, r-t represent a sub-population of macrophages that go against the trend and are highly Gal1+ and have high ORO density.
Overall this paper demonstrates a coherent and quantitative approach to the molecular biology of spinal cord injury, and brings clarity to much of the previous work done on Gal1. By stringently observing time points and quantifying not only expression but cellular localization the Popovich lab has provided a wealth of data on Gal1’s role in the immune response to spinal cord injury. It will be interesting to learn how this data is being used to potentially manipulate the immune response in spinal cord injury, perhaps by increasing Gal1’s expression levels at the injury site.
Marc Marino is a first year Neurosciences student currently rotating with Dr. Roberto Malinow. He is currently enjoying the fact that the San Diego Padres look like they were all injected with a bucket of Gal1 (no longer inflamed and terrible).
Gaudet AD, Sweet DR, Polinski NK,Guan Z, Popovich PG. Galectin-1 in injured rat spinal cord: Implications for macrophage phagocytosis and neural repair. Molecular and Cellular Neuroscience. 2015; (64):84-94. doi: 10.1016/j.mcn.2014.12.006