In today’s Nature Medicine, a team of researchers led by Yakeel Quiroz, Joseph Arboleda-Velasquez, Francisco Lopera, and Eric Reiman describe the case of a Colombian woman who inherited the autosomal-dominant E280A mutation in presenilin 1 but stayed cognitively well for decades past this mutation’s typical age of disease onset. Called Paisa, this mutation causes Aβ overproduction, triggering neurodegeneration and cognitive decline by a person’s 40s. Yet this woman stayed sharp for three decades after that. Now in her 70s, she struggles with short-term memory but still remains independent. Astoundingly, amyloid PET scanning revealed a massive buildup of amyloid plaques in her brain, far higher than those seen in young mutation carriers who are cognitively impaired. Despite all this amyloid, she has very little tau pathology, mostly confined to the medial temporal lobe, and her brain glucose metabolism is almost normal. It’s as if the chain of pathogenic events is broken after amyloid. What explains her remarkable resistance?
- The ApoE Christchurch mutation seems to block secondary tau pathology.
- This protective effect is similar to, but greater than, that of ApoE2.
- Protection correlates with lost binding to HSPGs, believed to propagate tangles.
The researchers believe the answer is a rare mutation in ApoE3 that disrupts the protein’s ability to bind lipoprotein receptors and other proteoglycans. The Colombian woman inherited two copies of this ApoE variant, known as the Christchurch mutation after the city where it was first identified. In vitro experiments showed that ApoE3ch behaves like the ApoE2 allele, also known to be protective. Both isoforms poorly bind heparan sulfate proteoglycans (HSPGs), the extracellular matrix molecules that have been implicated in the propagation and uptake of toxic forms of tau. In a related study, posted November 2 as a preprint on medRχiv, the same authors report that inheritance of two copies of ApoE2 also confers extra protection against AD, particularly tau pathology. Homozygous ApoE2 carriers develop far fewer tangles than ApoE2/3 carriers, or any other ApoE genotype.
“Together, the two studies make the case for ApoE having a profound effect on neuroprotection,” Reiman, at Banner Alzheimer’s Institute in Phoenix, told Alzforum. “We’re hoping this will galvanize interest in understanding ApoE-related mechanisms and developing treatments that target ApoE.”
Others shared his excitement. “This new study demonstrates, for the first time, that functional ApoE is truly required for full pathological and clinical development of AD,” Yadong Huang and Maxine Nelson at the University of California, San Francisco, and Kelly Zalocusky at the Gladstone Institute of Neurological Disease wrote in an accompanying Nature Medicine editorial. David Holtzman at Washington University, St. Louis, noted in an email, “[The data] suggest several mechanisms to further explore regarding how ApoE may be modulating inflammation, tauopathy progression, and neurodegeneration.”
Plaques but No Tangles. A woman with a familial AD mutation and two ApoE3 Christchurch alleles (left scans) had a very high plaque (top, red), but low tangle burden (middle) and healthy brain metabolism (bottom) compared with a typical mutation carrier (right scans). [Courtesy of Arboleda-Velasquez et al., Nature Medicine.]
Mouse studies from Holtzman’s lab first suggested that ApoE4 can worsen tau pathology independently of its effect on amyloid (Sep 2017 news), and human studies are starting to hint at the same thing, seeing more tangles in the brain and more phosphorylated tau in the cerebrospinal fluid of ApoE4 carriers than noncarriers, particularly women (Aug 2018 conference news). A recent tau imaging study found faster cognitive decline in ApoE4 carriers than noncarriers with equal tau burden, suggesting the allele somehow speeds up neurodegeneration (Nov 2019 news).
Lessons From Studying Resilience
The scientists working with the Colombian kindred did not set out to study ApoE. Led by Lopera at the University of Antioquia, Colombia, they studied people who appeared to escape the otherwise deterministic Paisa mutation in hopes of elucidating the AD pathogenesis pathway and finding new therapeutic approaches. Most E280A carriers are diagnosed with MCI at age 44, and dementia by age 49. However, the woman in this case study did not develop symptoms until three decades later, and what symptoms she has now are limited to problems remembering recent events. Her other neurological tests are normal, and her memory troubles have remained stable over two years of testing.
Quiroz and colleagues at Massachusetts General Hospital brought her to Boston to examine her biomarkers to try to find an explanation. Strikingly, amyloid PET imaging revealed a brain loaded with plaques. Her PiB distribution volume ratio (DVR) was 1.96, much higher than the 1.50 typically seen in Paisa carriers with MCI. “Her brain has one of the highest levels of amyloid pathology I’ve seen in the family,” said Quiroz, who leads PET studies with the Paisa population.
Even so, flortaucipir PET lit up only the medial temporal and occipital lobes, where tangles tend to start early in disease. What’s more, FDG PET revealed that her brain’s glucose metabolism remained robust, better than that in younger carriers with MCI, particularly in the precuneus (see image above). Her hippocampal volume is similar to those of carriers in their 40s, and she has relatively low levels of plasma NfL, a marker of neuronal injury.
Joseph Arboleda-Velasquez from the Schepens Eye Research Institute of Mass. Eye and Ear led genetic analyses. Whole-exome sequencing turned up but one likely explanation for her resilience: the Christchurch R136S mutation. This variant has been previously reported in the cardiovascular literature. It disrupts the LDL receptor binding domain of ApoE, crippling the protein’s ability to traffic fats. The Christchurch mutation was first identified in a man who had an ApoE2/2 genotype and hyperlipoproteinemia Type III (Wardell et al., 1987). This type of dyslipidemia develops in about 10 percent of homozygous ApoE2 carriers. It is marked by high levels of low-density lipoproteins, triglycerides, and cholesterol. Notably, the Colombian woman also has hyperlipoproteinemia Type III, with a blood cholesterol level of 512 mg/dl and triglycerides at 692 mg/dl, three times the normal level. She takes statins and has not experienced any cardiovascular problems to date.
Comparing Isoforms Yields Clues to Mechanism
The dyslipidemia in this woman suggested that ApoE3ch might share some characteristics with ApoE2. To explore this, Arboleda-Velasquez, Michael O’Hare, and colleagues compared ApoE isoforms in vitro. Normal ApoE3 stimulates aggregation of synthetic Aβ42, and ApoE4 even more so. In contrast, the Christchurch variant of ApoE3 behaved like the E2 allele, producing less clumping. Aggregation was lowest in the absence of any ApoE. This hints that the protective effect of the Christchurch mutation was due to a loss of function. Curiously, the finding implied that the Colombian woman might have even more plaques in her 70s without the Christchurch mutation. Even so, her amyloid load was well high enough to cause impairment in most people, hence there had to be another explanation for her cognitive preservation.
What about substrate binding? ApoE binds LDL receptors. ApoE4 does it the best, with E3 next and E2 weakest. ApoE3ch has a binding affinity that is intermediate between ApoE3 and ApoE2 (Lalazar et al., 1988). ApoE2 homozygotes with the Paisa mutation fare less well than does the woman with two Christchurch mutations. This made LDLR binding a poor candidate to be the protective mechanism, Reiman said. Somehow, ApoE3ch provides stronger protection than even a double dose of E2.
On a different measure, however, ApoE3ch was in a class by itself. The same domain on ApoE that binds LDLRs also recognizes HSPGs, a large class of polysaccharide molecules found as receptors on the neuronal cell membrane as well as in the extracellular matrix. One such HSPG is heparin, an anticoagulant secreted by mast cells. When the authors ran ApoE isoforms on a commercial heparin column, ApoE4 clung most tightly, while ApoE3 eluted earlier, and ApoE2 even sooner. ApoE3ch, however, barely bound heparin at all, with most of it coming quickly off the column (see image above). The dramatic effect of the Christchurch mutation in this assay means that how well ApoE binds HSPGs could be a factor in triggering downstream neurodegeneration. The HSPG data reinforce the idea that the Christchurch mutation results in a loss of normal ApoE function, Reiman said.
The Christchurch mutation appears to be recessive. Four other Paisa mutation carriers in the Colombian kindred inherited a single copy of it, and all developed cognitive impairment at the normal age. However, Quiroz noted that the sample was small. She has since identified another 20 or so Paisa mutation carriers with a heterozygous Christchurch mutation, and hopes this larger sample will produce a more definitive answer as to whether a single allele confers any protective effect.
“Additional in vitro and in vivo studies are required for unraveling the mechanisms underlying the protective effect,” Danny Michaelson at the University of Tel Aviv wrote to Alzforum (full comment below).
But How Does This Protect the Brain?
HSPGs have been implicated in the buildup of amyloid plaques, as well as in promoting the microglial response to amyloid (Apr 2016 news; Bussini et al., 2005; Zhang et al., 2012). Other studies link HSPGs to tau pathology. Marc Diamond at the University of Texas Southwestern Medical Center in Dallas and others have reported that these sticky receptors allow tau fibrils to glom onto neurons, facilitating uptake and propagation (Mar 2013 conference news; Apr 2015 news; Rauch et al., 2018; May 2018 news). Perhaps ApoE facilitates tau binding to HSPGs, Reiman speculated. In that case, the Christchurch variant’s weak affinity for HSPG would disrupt the spread of toxic tau through the brain. Interventions that weaken ApoE-HSPG binding might have therapeutic potential, he believes.
“This theory warrants experimental testing in cells and animals and, if successful, further clinical studies in humans,” Huang, Nelson, and Zalocusky noted in their editorial. Diamond wrote to Alzforum, “These results imply that we should ask more questions about a direct relationship between ApoE and tau. There are definitely reports of their interaction, so this could be an interesting avenue.”
For his part, Holtzman was most intrigued by the inflammation angle. “One wonders whether the effect of this ApoE variant is somehow decreasing the ability of ApoE to initiate a microglial-mediated inflammatory response to tau pathology,” he wrote (full comment below). His lab recently reported that ApoE4 exerts its toxic effects through microglia in tauopathy mouse models (Oct 2019 news).
Jacob Raber at Oregon Health and Science University in Portland suggested using unbiased omics approaches to explore the mechanisms underlying these isoform effects. “Together with other reports, these data support that tau and ApoE might be especially good therapeutic targets in AD,” he wrote (full comment below).
Could ApoE-HSPG binding be targeted therapeutically? The authors raised an antibody, 1343A, against ApoE’s LDLR binding region. When they incubated regular ApoE3 with it prior to adding it to the heparin column, the protein behaved like the ApoE3ch variant, eluting rapidly without apparent contact with this experimental HSPG stand-in. Quiroz said the scientists are further characterizing this antibody to see if it is suitable for clinical development.
A small-molecule approach would be cheaper, and more practical for repeat administration, than an antibody. Researchers led by Carl Frieden at WashU have previously described one such molecule, EZ482, that binds ApoE and alters its shape in such a way as to block HSPG binding (Mondal et al., 2016). Frieden told Alzforum that his group did not study EZ482’s effects in vivo, or develop it further. He is starting a project to look for additional compounds that alter ApoE binding. Meanwhile, researchers at the Gladstone Institute previously identified compounds similar to EZ482 that corrected some of ApoE4’s toxic effects in cell culture (Chen et al., 2012; Wang et al., 2018).
Reiman’s group is also interested in finding small molecules that block ApoE binding to HSPGs. However, he believes silencing ApoE with antisense oligonucleotides or RNA interference might be a simpler approach, and perhaps the first thing to test in clinical trials.
Joachim Herz at the University of Texas Southwestern Medical Center in Dallas noted that weakening the association of ApoE with HSPG receptors at the cell surface would effectively decrease the ApoE concentration in endosomes. Herz believes that ApoE4 wreaks much of its mischief there, because the apolipoprotein unfolds inside these acidic compartments and blocks recycling of neuronal receptors such as AMPA and NMDA. He found that a small molecule that inhibits the endosomal proton leak channel NHE6 sped up receptor recycling, weakening ApoE4 toxicity (Xian et al., 2018). About Arboleda et al., Herz wrote, “[These data] nicely provide circumstantial support to our model that ApoE4 aggregation in the early endosome is a key event in AD pathogenesis that can be effectively reduced or abrogated with small-molecule inhibitors of NHE6.”
“Undoubtedly, this work opens exciting new avenues in AD research and reveals promising possibilities for potential AD therapeutics,” Yadong, Nelson, and Zalocusky noted.
ApoE2 Strengthens Link to Tau Toxicity
The separate ApoE2 homozygote study complements the ApoE3ch findings. Using data from 4,108 autopsy-confirmed AD cases and 989 controls assembled by the Alzheimer’s Disease Genetics Consortium, Reiman and colleagues found that the 24 ApoE2 homozygotes in this sample ran but a third the risk of AD as did ApoE2/3 heterozygotes. In cases where AD did develop, it happened much later in life. Alzforum covered some of these data at AAIC last July in Los Angeles (Aug 2019 conference news).
The manuscript details a new discovery beyond this meeting story. Similar to the Christchurch mutation, ApoE2 in this pathology-confirmed sample seemed to ameliorate tau pathology. ApoE2 homozygotes died with an average Braak tangle stage of 2.3, heterozygotes, 3.2, compared with 4 or higher for the other ApoE genotypes. The results remained significant even after adjusting for plaque burden.
Notably, the protection from ApoE2 appeared unique to Alzheimer’s. There was no difference in the risk for cerebral amyloid angiopathy, Lewy body disease, hippocampal sclerosis, or vascular brain injury in this large sample. This once again suggests a specific effect of ApoE on the secondary tauopathy that develops in Alzheimer’s disease in the presence of amyloid, Reiman said. He added that the results seem specific for secondary tauopathies, because some evidence suggests ApoE2 homozygotes are more susceptible to primary tauopathies such as progressive supranuclear palsy (Zhao et al., 2018). “This result points towards additional mechanisms through which the ApoE2 allele confers its protective effect, beyond decreasing amyloid deposition,” Gemma Salvadó at the Barcelonaβeta Brain Research Center, Spain, wrote to Alzforum (full comment below).—Madolyn Bowman Rogers
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