01 May 2019

Despite years of study, ApoE still has secrets to give up. At the 14th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 27–31 in Lisbon, Portugal, researchers indicted ApoE4 on several fronts. They charged it with holding back the microglial response to amyloidosis. They said it bungled disposal of damaged mitochondria, perhaps perturbing cellular energy metabolism. They implicated it in axonal remodeling, and said this may explain why the highly arborized neurons of the entorhinal cortex are particularly vulnerable to Alzheimer’s. Finally, as peripheral ApoE is beginning to attract more attention, scientists are accounting for the different pools of this lipoprotein that course through the human body. Others elaborated on how ApoE4 damages blood vessels in the brain and correlates with neuroinflammation. Quite the rap sheet.

The APOE4 allele locks microglia in a homeostatic state.

It impairs mitophagy and axonal plasticity.

Peripheral APOE4 causes vascular problems in brain.

First, microglia, the “phenom” in AD research these days. Many genetic risk factors act through their effect on these cells (see Part 4 of this series), and some appear to be in cahoots with each other. Oleg Butovsky of Brigham and Women’s Hospital, Boston, previously reported that TREM2 and ApoE cooperate to rouse microglia into a “microglial neurodegenerative” (MGnD) state, in which they clean up dead neurons and other debris (Feb 2015 conference news; Sep 2017 news).

Microglial Replacement. ApoE4 microglia freeze into a homeostatic state; could replacing them with ApoE3 monocytes from blood restore normal function? [Courtesy of Oleg Butovsky.]

How does the ApoE4 allele affect this activation? In Lisbon, Butovsky said it has the same effect as knocking out ApoE, i.e., it traps microglia in a homeostatic state. His team isolated microglia from adult mice that carry humanized ApoE3 or ApoE4. In the former, microglia activated as the animal aged, but in the latter, homeostatic gene expression stayed high. Then the scientists challenged microglia by injecting dying neurons into mouse brain. Normally, this spurs microglia to enter the MGnD state. In E4 mice, however, it did not. They managed to turn down their homeostatic genes, but not to ramp up MGnD genes, like a teenager who can’t work up the motivation to get off the sofa and clean his room. Butovsky concluded that ApoE4 microglia are unable to respond properly to cellular damage in the brain.

In addition, aberrant behavior of ApoE4 microglia during development may render the brain vulnerable to Alzheimer’s, Butovsky believes. Charlotte Madore in his group found that microglia in ApoE3 mice assumed an MGnD phenotype during the first two weeks of life, when the cells are busy pruning inactive synapses. Then microglia settle into a calmer mode, turning up homeostatic genes. In newborn ApoE4 mice, however, microglia maintained high expression of homeostatic genes throughout infancy and into adulthood. Madore thinks this may prevent them from appropriately sculpting brain circuitry, and could lead to behavioral abnormalities later in life. Similarly, genetic ablation of TREM2 makes microglia “superhomeostatic” and prevents them from migrating toward tissue damage (May 2017 news). TREM2 knockout mice have abnormal social behavior as adults (Filipello et al., 2018; Calcagno et al., 2018).

Madore is investigating if these microglial differences interact with early life stress. Butovsky, working with Arie Kaffman at Yale University in New Haven, Connecticut, previously reported that early stress can cause lasting changes in microglia gene expression (Delpech et al., 2016). Madore found that stressors such as lack of bedding material cause mouse microglia to ramp up MGnD gene expression. If E4 mice cannot mount this response, perhaps that affects the brain in ways that leave mice susceptible to degeneration later, she speculated. In people, childhood epilepsy in ApoE4 carriers has been linked to an increased risk of AD late in life, hinting that early stressors could interact with genetic factors to predispose someone to the disease, Madore believes (Joutsa et al., 2017).

If ApoE4 microglia make the brain less able to combat AD, perhaps replacing them with healthier cells could be therapeutic, Butovsky suggested. In other studies, depleting brain microglia stimulated blood monocytes to enter the brain and repopulate the microglial niche (Lund et al., 2018; Cronk et al., 2018; Bennett et al., 2018). Microglia numbers can be reduced by preventing their proliferation with CSF1R blockers. Such drugs are under development for cancer, and treatment with one lessened pathology in a mouse model of AD (Sosna et al., 2018; Cannarile et al., 2017). Butovsky suggested infusing healthy ApoE3 monocytes into the bloodstream before CSF1R inhibitor treatment, to allow these cells to move into the brain and take over immune surveillance duties there. He is testing this idea in mouse models, collaborating with Li-Huei Tsai at MIT and Mathew Blurton-Jones at the University of California, Irvine.

ApoE affects other cellular processes that could play a role in AD, as well. Shira Simonovitch of Tel Aviv University, Israel, working with Daniel Michaelson and Ronit Pinkas-Kramarski there, previously reported that ApoE4 suppresses autophagy (Simonovitch et al., 2016). In Lisbon, Simonovitch extended this finding to mitochondrial disposal. The cell’s energy powerhouses are dynamic organelles that fuse and divide in response to stressors. Fusion boosts their health, while fission allows malfunctioning mitochondria to be chewed up by autophagosomes, a process known as mitophagy.

Simonovitch compared this in transgenic mice carrying human ApoE3 or ApoE4. In hippocampal sections from the E4 mice, Simonovitch counted three times as many elongated mitochondria as in E3 hippocampus. This hints at a block in fission, and staining for specific proteins revealed a dearth of the fission protein Drp-1 and an excess of the fusion protein Mfn1.

The scientists also saw that mitochondria in E4 brains had fewer crista, the folds of the inner membrane that produce energy, and western blots indicated changes in several known mitochondrial proteins. In particular, E4 mitochondria had a shortage of the cleaved form of pink1, which is associated with healthy mitochondrial resting potential. The data hint at an energy deficit in the ApoE4 brain; the increased mitochondrial fusion may be an attempt to compensate, Simonovitch suggested.

How about humans? The researchers have no way of measuring this directly in living people, but they compared postmortem samples from the brains of ApoE4/4 and ApoE3/3 AD patients. They found half as much Drp-1 in the former as the latter by western blot. Impairments in mitophagy have been linked to Parkinson’s and other neurodegenerative conditions (Aug 2013 news; Oct 2014 news; Sep 2016 news). Mitochondrial damage occurs in AD models, and removing these damaged organelles lessens pathology and improves memory in mouse and worm models of AD (Nov 2009 news; Feb 2010 news; Feb 2019 news).

Axonal Vulnerability? Entorhinal cortex neurons possess enormous axonal arbors (bottom right) that may leave them susceptible to tau pathology. [Courtesy of Tamamaki and Nojyo, 1993.]

ApoE4 also promotes tau pathology (Sep 2017 news). Neurons in the layer II entorhinal cortex (EC) and hippocampal CA1 region are particularly vulnerable to tau tangles, and Jean-Pierre Roussarie and colleagues at Rockefeller University wondered if ApoE isoforms might influence this. Roussarie works in the laboratory of Nobel laureate Paul Greengard, who passed away April 13 at age 93 (see obituary). Roussarie profiled seven different neuronal types in wild-type mice at three different ages to identify pathways most altered in vulnerable neurons compared with those that resist tau pathology. Together with Olga Troyanskaya’s group at Princeton University, Roussarie and colleagues then combined these expression profiles with a compendium of human genomics and GWAS data for genes associated with tau pathology (Greene et al., 2015; Beecham et al., 2014).

This pinpointed a tau vulnerability gene module that was highly connected in EC neurons, and whose gene members were suppressed during aging and in APPswe/PS1dE9 mice. Genes in this cluster regulate axon extension, microtubule assembly and transport, and neuronal signaling (Brichta et al., 2015; Roussarie et al., 2018). Importantly, gene-expression profiles from the different neuron types were comparable to those in postmortem human brain samples from the same regions, suggesting the results may translate to people, Roussarie said. This work is described in a paper posted to bioRχiv (Roussarie et al., 2018).

How about ApoE? Roussarie compared gene expression in healthy, year-old female ApoE2 and ApoE4 mice, and found 57 neuronal genes that were differently expressed in the E4s, about half turned up and the other half down. These genes overlapped extensively with the tau vulnerability module of axonal plasticity genes, as well as with genes associated with tau tangles in another GWAS (Chibnik et al., 2017). In other words, ApoE4 affects the same set of genes as do aging and AD pathology. Within EC neurons, the effects of ApoE4, aging, and tau pathology all converge on axonal plasticity, Roussarie concluded. He noted that EC neurons extend extremely complex axonal arbors, and speculated that this may leave them vulnerable to perturbations in this process.

While most studies of ApoE focus on the central nervous system, the protein is also abundant in the periphery. In Lisbon, Randy Bateman of Washington University in St. Louis reminded the audience that CNS and PNS versions are processed differently. In the CNS, all three isoforms turn over at the same rate, while in blood, they vary greatly. There, ApoE4 is cleared twice as fast as ApoE3. In line with this, people with one E3 and one E4 allele have more E3 than E4 in their plasma, while the opposite is true in brain (Wildsmith et al., 2012; Baker-Nigh et al., 2016).

To examine brain and plasma ApoE more closely, since then Bateman and colleagues have developed an assay to isolate ApoE from body fluids via monoclonal antibodies and analyze the protein by mass spectrometry. This taught them that, in cerebrospinal fluid, ApoE’s C-terminal domain was either missing or modified, Bateman reported in Lisbon. In addition, plasma and CSF ApoE differed at threonine 194. Though the researchers are still determining what this modification is, there are previously reported candidates at this location (Wernette-Hammond et al., 1989; Halim et al., 2013). These differences may help researchers design therapies to specifically target pathological ApoE, Bateman suggested.

Other research implicates peripheral ApoE in AD. Guojun Bu of the Mayo Clinic in Jacksonville, Florida, previously reported that peripheral ApoE4 makes brain blood vessels leaky and slows cerebral blood flow. This correlated with worse memory and more amyloid plaques in brain, even in animals without any CNS ApoE (Jul 2018 conference news). In Lisbon, Bu added to these data, reporting that blood vessels in mice with peripheral ApoE4 branch less than those in E3s and dilate less frequently, suggesting problems with regulating blood flow. E4s also deposited less collagen around blood vessels and had more astrogliosis in their brains.

To confirm that the peripheral ApoE4 effects also occur in wild-type animals that have CNS ApoE, Bu’s team injected plasma from young ApoE4 mice into aged wild-type mice, and found that it promoted leakage of the blood-brain barrier. ApoE3 plasma had the opposite effect, stemming leaks. “ApoE4’s harm likely comes from a combination of central and peripheral effects,” Bu concluded.—Madolyn Bowman Rogers