Does ApoE4, the strongest genetic risk factor for sporadic Alzheimer’s, prevent synaptic pruning? Counterintuitively, that is what scientists conclude in the September 6 Proceedings of the National Academy of Sciences. The researchers, led by Ben Barres of Stanford University in Palo Alto, California, in collaboration with David Holtzman and colleagues at Washington University in St. Louis, report that the risk allele hinders phagocytic activity in astrocytes, preventing them from nibbling on synapses. How could that promote dementia? They also report that human ApoE4 leads to a glut of the complement protein C1q in the brains of transgenic mice. The researchers speculated that C1q decorates uncleared, deteriorating synapses, which could drive neuroinflammatory responses in the brain as the animals age. Researchers believe C1q tags synapses for destruction by microglia in AD and potentially other neurodegenerative diseases (see Nov 2015 conference news).
“This work is highly exciting,” said Guojun Bu of the Mayo Clinic in Jacksonville, Florida. “It introduces a new dimension to the diverse functions of ApoE as a risk factor for AD,” he added. Bu was not involved in the study.
Apolipoprotein E exists in three different flavors—ApoE2, ApoE3, and ApoE4. While ApoE2 reduces AD risk, ApoE4 elevates it. Researchers have long sought to understand how the apolipoprotein, the only one in the brain, exerts its influence over AD. Previous findings indicate that ApoE affects the uptake of Aβ by astrocytes, and that ApoE4 somehow inhibits this process (see Holtzman et al., 2000; Koistinaho et al., 2004; Apr 2013 news). However, transgenic knock-in mice expressing human ApoE4 display behavioral and synaptic deficits independent of Aβ pathology (see Bour et al., 2008; Dec 2009 news). Furthermore, Barres and colleagues had previously reported that astrocytes, which express most of the ApoE in the brain, play a key role in refining neural circuitry by pruning synapses (see Dec 2013 conference news). This prompted the researchers to investigate the relationship between ApoE genotype and this synapse-eating activity of astrocytes.
First author Won-Suk Chung—who has since moved from Barres' lab to the Korea Advanced Institute of Science and Technology in Daejeon—started by asking whether ApoE alleles could differentially influence the phagocytic activity of astrocytes. Chung and colleagues designed a cell culture system to address this. The researchers grew astrocytes from ApoE knockout mice, then added culture medium from astrocytes derived from ApoE2, E3, or E4 transgenic knock-in mice. This culture medium contained ApoE as well as any other factors the astrocytes were pumping out. Chung then added phagocytic prey—fluorescent synaptosomes—to the mix, and monitored phagocytosis. He found that cultures bathed in medium from ApoE2-expressing astrocytes engulfed the most synaptosomes, while cultures bathed in ApoE4-laced medium took up the least. The same was true when the researchers used synaptosomes labeled with pHrodo, a dye that emits fluorescence only after arrival in the acidic belly of the astrocyte (i.e., acidic organelles such as the lysosome).
To determine if it was the ApoE in the medium that influenced phagocytosis, the researchers next added pure lipidated ApoE to ApoE knockout astrocyte cultures. The ApoE effect held up, but only if the researchers also added an opsonin. Opsonins bridge the gap between phagocytic receptors on cells and the cargo they engulf. This result indicted that ApoE proteins do not directly alter astrocyte phagocytosis, but instead boost or inhibit phagocytic pathways with the help of other proteins.
To extend their findings to a relevant in vivo model, the researchers investigated how the different ApoE alleles affected synapse pruning during early postnatal development, a task that falls to astrocytes as well as microglia. To quantify this, they focused on commonly studied neural connections in the eye that undergo massive pruning during development, namely the extension of retinal ganglion cell (RGC) axons into presynaptic terminals in the dorsal lateral geniculate nucleus (dGLN). In various ApoE knock-in strains, the researchers injected fluorescent molecules into the eye to label the RGC axonal projections, then monitored fluorescent uptake into astrocytes gathered around the synaptic terminals in the dGLN. As with their cell culture experiments, they found that astrocytes in ApoE2 knock-in mice had gorged on the most synapses, while those in ApoE4 animals had taken up the least. The intermediate level of phagocytosis in ApoE3 knock-in animals was on par with that in ApoE wild-type or knock-out mice, suggesting that ApoE2 and ApoE4 alleles had dominant phagocytosis enhancing and inhibiting effects, respectively.
The researchers hypothesized that the reduced level of synaptic clearance in ApoE4 carriers would lead to an accumulation of nonfunctional or “senescent” synapses, which could interfere with neural circuitry, trigger neurodegeneration, and partly explain increased susceptibility to AD. The researchers had previously reported that senescent synapses build up with age, and that they are decorated with the complement protein C1q (see Dec 2007 news; Stephan et al., 2013). Therefore, they relied on C1q levels as a proxy for the number of such synapses in the hippocampi of different ApoE knock-in mouse strains. In keeping with their hypothesis, the researchers found low levels of C1q in the hippocampi of nine-month-old ApoE2 mice compared to ApoE3 or ApoE4 mice. By 18 months, ApoE4 knock-in mice had significantly more hippocampal C1q compared to the other strains.
Barres explained that the presence of synaptic C1q alone is insufficient to trigger the complement cascade that ultimately beckons hungry microglia to dine on the synapses. Rather, he proposed that some type of second hit triggers the cascade. While microglial pruning of synapses is beneficial during development, studies have suggested that in the context of AD, this pruning kicks into overdrive and hastens neurodegeneration instead (see Apr 2016 news). The connection between astrocytic and microglial pruning functions is somewhat blurry, but the current findings help bring it into focus, Barres said. “This is the first paper to link ApoE allele to buildup of synaptic C1q—and thus to synapse degeneration by the complement cascade,” Barres said. “The pieces are starting to come together.”
“These data provide a new mechanism for the APOE4-related neurodegeneration and APOE2-related neuroprotection, namely phagocytic capacity,” commented Mary Jo LaDu, Ana Valencia Olvera, Deebika Balu, and Maria Evangelina Avila-Munoz of the University of Illinois in Chicago in a joint comment to Alzforum. However, the commentators pointed out that some of the results contradict previous findings suggesting that C1q promotes neuronal survival and protects against synapse loss. “Critical to these questions is the interactions between astrocytes and microglia,” they added (see full comment below).
Other unanswered questions include how ApoE influences astrocyte phagocytosis. Barres and colleagues hypothesized it might interfere with the binding of synapses to phagocytic receptors on the astrocyte surface—either enhancing binding in the case of ApoE2, or inhibiting it in the case of ApoE4. Bu thinks ApoE may be uniquely positioned to do this. “ApoE binds to a variety of cell surface receptors including transport receptors LRP1 and LDLR, as well as signaling receptors ApoER2/LRP8 and VLDLR,” he said. “Whether one or more of ApoE receptors or an alternative receptor involved in regulating phagocytosis mediate the ApoE isoform-dependent effects requires further investigation.”—Jessica Shugart
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