Could Juicing Up Trafficking Abolish ApoE4’s Alzheimer’s Risk?

What makes ApoE4 toxic? Joachim Herz at the University of Texas Southwestern Medical Center in Dallas has a new answer to this old question. Because this isoform is more positively charged than ApoE3 or 2, it becomes sticky and insoluble in the mildly acidic environment of early endosomes and gums up the endolysosomal pathway. This, he believes, slows recycling of ApoE and nearby cell-surface receptors, stressing the cell. Making endosomes more acidic by blocking the proton leak channel NHE6 unclogs the system and restores normal trafficking. In a July 9 preprint on bioRxiv, Herz and colleagues report that knocking out NHE6 in adult amyloidosis mice activated microglia, largely prevented plaque deposition, and protected synapses. Notably, in the knockouts, both ApoE4 and ApoE3 were trafficked as readily as was the protective variant ApoE2.

  • ApoE4 stalls early endosomes, slowing receptor recycling.
  • Blocking a proton leak channel acidifies endosomes, restoring normal trafficking.
  • In amyloidosis mice, this curbed plaque and protected synapses.

Herz believes that inhibiting NHE6 via a small molecule might benefit both E3 and E4 carriers. “This is a conceptually unique approach to reduce the risk of AD,” he told Alzforum.

Others agreed the data offer a fresh perspective. “This work provides important mechanistic insights as to how ApoE4 aggregates with itself and with receptors,” said Guojun Bu at the Mayo Clinic in Jacksonville, Florida. Meanwhile, Eric Morrow at Brown University in Providence, Rhode Island, was interested in the therapeutic implications. “This paper provides experimental evidence in support of a novel treatment paradigm for reducing amyloid plaques in Alzheimer’s disease,” Morrow wrote to Alzforum.

Speed the Traffic, Lose the Plaque? Amyloid plaques (white) in APP knock-in mice (left) were nearly abolished by removing the endosomal proton leak channel NHE6 (right). [Courtesy of Pohlkamp et al., bioRxiv.]

Endolysosomal dysfunction is part of the pathogenesis of AD, with many risk genes linked to it (Aug 2019 news; Dec 2019 news; Oct 2020 news). 

Herz explored how ApoE4, the main risk factor for late-onset AD, moves through this system. Compared to ApoE3, E4 has one extra positive charge, with one neutral cysteine changed to a positive arginine. This raises ApoE4’s isoelectric point, or the pH at which it has a net neutral charge. Tellingly, perhaps, for ApoE4 this occurs at pH 6.4—the exact pH of early endosomes. This means that in this environment, ApoE4 becomes uncharged and less soluble, sticking to its receptors and aggregating with itself. That slows down endosome maturation, causing the vesicles to stall.

In cell culture, Herz found, lowering the pH in endosomes by blocking the sodium/hydrogen exchanger NHE6 resolved the issue, allowing ApoE to dissociate from receptors and move through the system normally (Xian et al., 2018). 

Slowing the System. ApoE (upper right corner) is taken up by early endosomes, along with nearby receptors, and quickly recycled. At the early endosomal pH of 6.4, however, ApoE4 becomes sticky, stalling endosomes and delaying receptor recycling. [Courtesy of Pohlkamp et al., bioRXiv.]

Would this work in the brain? To test this, joint first authors Theresa Pohlkamp, Xunde Xian, and Connie Wong generated conditional knockout mice that lost NHE6 when fed tamoxifen. Because NHE6 is essential for proper brain development, the authors triggered its loss once the mice were grown. They isolated neurons from these mice to test their properties. In cultured wild-type neurons, adding ApoE3 or ApoE4 slowed recycling of the ApoE receptor 2, also known as LRP8, as well as that of other receptors such as AMPA and NMDA. In NHE6 knockout neurons, however, ApoE3 and ApoE4 did not harm receptor trafficking.

Next, the authors crossed NHE6 knockout with APOE4 knock-in mice. In hippocampal slice cultures of the latter, long-term potentiation is impaired; removing NHE6 restored synaptic plasticity to normal.

To examine how NHE6 affects amyloid pathology, the authors crossed the conditional NHE6 knockouts with APPNL-F mice. These carry human APP with the Swedish and Iberian mutations, but express the protein at endogenous levels. They develop plaques and gliosis by 6 months, and lose synapses by 9 months, but have no detectable memory loss. In these mice, getting rid of NHE6 profoundly affected amyloid, cutting plaque and insoluble Aβ by 80 percent at one year of age. Curiously, the benefits were the same whether the mice carried mouse ApoE or humanized ApoE4.

How might endosomal trafficking affect plaque? Possibly via glia, Herz thinks. Removing NHE6 generally amped up activation of both microglia and astrocytes, as seen by higher Iba1 and GFAP staining, respectively. However, the number of microglia milling around plaques was unchanged, and they ingested equal amounts of amyloid. To Herz, this means that activated microglia process, and dispose of, amyloid more efficiently. He points to recent work by David Holtzman and colleagues at Washington University, St. Louis, which suggests that microglia with heightened ApoE recycling ramp up catabolic pathways that break down lipids and proteins (Jul 2021 news). 

Because NHE6 knockouts lose the protein in all cells, it is unclear which cell type drives the beneficial ApoE recycling. In ongoing work, Wong is generating conditional knockouts for microglia, astrocytes, or neurons. Together with Pohlkamp, she is also making mice with inducible NHE6 that can be dialed up or down, to define the effects of manipulating this protein with more nuance.

Could inhibiting NHE6 prevent AD in people? Herz believes so, but technical hurdles abound. While many NHE inhibitors exist, most target isoforms other than NHE6. They are highly charged, hence do not cross the blood-brain barrier. To treat AD, scientists would need to develop inhibitors with a different biochemistry, Herz said.

Bu cautioned that changing endosomal pH would affect trafficking of other proteins as well, potentially causing side effects. In people, loss-of-function mutations in NHE6 cause the neurodevelopmental disorder Christianson syndrome. Even in NHE6 knockout mice, which lost the protein only as adults, its absence triggered degeneration of Purkinje cells in the cerebellum. Morrow, who studies Christianson syndrome, noted that diffuse tau deposits have been found in the brains of some patients. “Further careful study of the functions of NHE6 in the nervous system and the effects of mutations in NHE6 is warranted,” he wrote.

Herz believes that a partial knockdown of NHE6 might negate the risk from ApoE4 without triggering degeneration. In his studies, even a fraction of a pH unit change in endosomes was enough to restore trafficking. “We think there’s considerable therapeutic width,” he told Alzforum.—Madolyn Bowman Rogers

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References

News Citations

  1. Familial AD Mutations, β-CTF, Spell Trouble for Endosomes
  2. Lysosomal Diseases: Stepping Stones to Gene Therapy for Alzheimer’s?
  3. In Astrocytes, ApoE4 Bungles Endocytosis, PICALM Picks Up the Slack
  4. Taming ApoE Via the LDL Receptor Calms Microglia, Slows Degeneration

Research Models Citations

  1. APOE4 Targeted Replacement

Paper Citations

  1. Xian X, Pohlkamp T, Durakoglugil MS, Wong CH, Beck JK, Lane-Donovan C, Plattner F, Herz J. Reversal of ApoE4-induced recycling block as a novel prevention approach for Alzheimer's disease. Elife. 2018 Oct 30;7 PubMed.

Other Citations

  1. APPNL-F

Further Reading

Primary Papers

  1. Pohlkamp T, Xian X, Wong CH, Durakoglugil MS, Werthmann GC, Saido TC, Evers BM, White CL 3rd, Connor J, Hammer RE, Herz J. NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load. Elife. 2021 Oct 7;10 PubMed.

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