Neurons respond awkwardly when astrocytes shower them with too many complements. According to a study in the December 18 Neuron online, astrocytes pump out the complement protein C3 in response to Aβ, and neurons react by shriveling some synapses and ramping up the activity of others. The astrocytes’ toxic overture is turned on by the transcription factor NFκB, well known for its role in inflammation. Led by Hui Zheng at Baylor College of Medicine in Houston, the researchers reported that blocking this pathway eliminated memory problems in an AD mouse model. The work adds to a growing body of evidence implicating neuroinflammation as a key player in the neurodegenerative process.
The complement system consists of roughly 30 proteins. Throughout the body, it targets microbes, sickly cells, and biological flotsam for disposal, and ramps up inflammatory responses to ensure this happens. In the central nervous system (CNS), complement expression surges during development, and again in the context of neurodegenerative disease or brain injury. In early life, complement plays a role in pruning synapses, a process that optimizes neural transmission (see Stevens et al., 2007). Effects on the adult brain are less clear, but complement proteins are expressed by astrocytes in response to inflammatory signals (see Zamanian 2012). The complement protein C3 triggers the clearance of Aβ in AD mouse models; however, some research suggests that ridding the brain of complement protects such mice from cognitive decline even in the face of mounting Aβ (see Fu et al., 2012, and Aug 2013 conference coverage).
First author Hong Lian and colleagues wanted to clarify the role of complement activation and NFκB in neurodegeneration. The transcription factor activates complement expression along with many other inflammatory genes, such those that make cytokines. When silent, NFκB is held in check in the cytoplasm by its inhibitor, IκB. Kinases that phosphorylate the inhibitor target it for destruction, freeing NFκB to move into the nucleus and switch on genes. High levels of activated NFκB have been observed in brain tissue from people with Alzheimer’s, Parkinson’s, and Huntington’s diseases.
Lian started by generating transgenic mice without IκB, leaving any NFκB permanently active. They created mice lacking the inhibitor in neurons only, in the whole brain, or in astrocytes only. In the latter two, the researchers detected about sixfold higher levels of C3 mRNA expression in the brain, resulting in nearly double the amount of C3 protein as in control mice. Mice lacking IκB only in neurons had a normal amount of the protein, suggesting that astrocytes are the cells that make C3 in the brain, and that this is regulated by NFκB.
To study what astrocytic C3 does to neurons, the researchers co-cultured mouse hippocampal neurons with normal astrocytes or with those lacking IκB. After sharing a dish with IκB-deficient astrocytes for two weeks, neurons started losing dendritic spines. As these shrank or disappeared, the dendritic arbors lost some of their branches. The number of synapses declined, as well, as measured by a loss of the synaptic proteins synaptophysin and MAP2, and of the excitatory synaptic protein VGluT1. None of this occurred when C3 was depleted from the co-culture medium, while adding C3 to pure neuron cultures recapitulated the effects.
To see how this played out in vivo, the researchers used a viral system to label neurons and their dendrites with green fluorescent protein. Fluorescent microscopy revealed that in mice lacking IκB in their astrocytes, dendritic spines of all shapes and sizes—from stubby, to mushroom, to delicate long ones—took a hit. Long-term potentiation, a measure of synaptic plasticity, weakened. The mice were forgetful. In contextual fear testing of learning and memory, they froze less often than normal mice when placed in an environment where they had previously experienced a foot-shock.
If astrocytes are sending out toxic C3 signals, how are the neurons receiving them? The researchers suspected the C3a receptor, and indeed, adding a C3aR antagonist to the co-cultures prevented spine loss and C3aR-deficient neurons thrived among IκB-negative astrocytes. Treating the IκB-negative mice with a C3aR antagonist restored dendritic spines, long-term potentiation, and memory. Together, these results suggested that through its receptor on neurons, C3 secreted from astrocytes dampens synaptic plasticity and compromises learning in mice.
Signaling through C3aR has been reported to boost levels of intraneuronal calcium, and the researchers found that this was the case in neurons co-cultured with astrocytes lacking IκB. Those neurons had more AMPA receptors on their cell surfaces and their mini excitatory post synaptic currents (mEPSCs) crested to higher amplitudes. The C3aR antagonist blocked all these effects. These results suggested that the synapses that remained signaled more vigorously.
How would this relationship between astrocytic C3 and neurons play out in the context of AD? To chip away at this question, the researchers first treated normal astroglial cultures with a fibrillar preparation of Aβ42 peptides (as described in Stine et al., 2003). These boosted translocation of NFκB to the nucleus and ramped up C3 expression. The researchers also detected elevated levels of C3 in APP/TTA transgenic mice, which overexpress human APP under control of the tetracycline promoter (see Jankowsky et al., 2005). Surprisingly, treatment with the C3aR antagonist completely reversed memory deficits in the mice: They performed as well as wild-type mice on the Morris water maze test. Postmortem brain samples from AD patients expressed more nuclear NFκB, as well as C3, than control samples.
Lian proposed a model whereby C3 secreted by astrocytes engages the C3a receptor, which triggers an uptick in intraneuronal calcium. This then leads to the other problems, including loss of dendritic spines. How these effects are connected is still unclear.
Chris Norris of the University of Kentucky in Lexington considers the paper impressive, but found it odd that C3 triggered loss of dendritic spines while at the same time stimulating synaptic activity. Lian said the researchers are currently working to tease out how these seemingly contradictory effects are related.
Mark Mattson of the National Institute on Aging in Bethesda, Maryland, who was not involved in the study, said that the pathway may be more detrimental when neurons are also dealing with toxic proteins, such as complement or Aβ. “When cells are under stress, they may be less able to clear calcium,” he said. “Loss of spines in response to complement may have to do with a toxic calcium overload,” he suggested.
Ben Barres of Stanford University in California thought the study was very interesting, but was skeptical that neurons express C3aR (see full comment below). He wrote that the receptor is primarily expressed on microglia—the immune cells of the CNS (see Schafer et al., 2012). Lian and colleagues did not examine the role of microglial cells in the C3 pathway, but said that finding out how they fit into the picture is a prime focus of their current research.—Jessica Shugart
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