Keystone: Does ApoE Fragmentation Drive Pathology?

Series - Keystone: Slalom No Match for Ins and Outs of ApoE Research:

Fragmentation can bog down your hard drive, and some scientists think it also wreaks havoc on processing in the brain. At “ApoE, Alzheimer’s and Lipoprotein Biology,” a Keystone symposium held 26 February-2 March 2012 in Keystone, Colorado, researchers discussed whether cleavage of the notorious lipoprotein explains why it is the strongest genetic risk factor for Alzheimer’s disease. Fragments of ApoE have been reported to poison neurons, and ApoE4, the isoform that confers AD risk, gives itself up to proteolysis more readily than does ApoE2 or E3. Stopping that proteolysis, then, might seem a therapeutic approach, and several presentations at the symposium focused on that idea. But as the AD field has come to acknowledge over the years, nothing about ApoE is straightforward.

Fragmentation represents but one way in which ApoE4 may increase risk for AD. Many researchers in the field believe it may be secondary to ApoE’s more widely accepted meddling with Aβ (see Part 1 of this series) and synaptic plasticity (see Part 2 and Part 3). At Keystone, a hypothetical scenario emerged to tie these nefarious properties together. The hypothesis has Aβ and plasticity effects driving early pathology; proteolytic fragments would emerge later as cells begin to ramp up ApoE production in an attempt to repair ongoing neuronal damage. (ApoE, being the major lipid and cholesterol carrier in the brain, plays a crucial role in the repair of cell membranes.)

Robert Mahley, from the Gladstone Institute for Neurological Disease, San Francisco, California, said in his keynote address that ApoE4 is a risk factor for other neurodegenerative diseases as well, such as Parkinson’s, multiple sclerosis, and traumatic brain injury. (ApoE is listed in PDGene and MSGene, though not among the Top 10.) Mahley suggested that ApoE might play a role beyond AD because it sets the stage for multiple “second hits” that promote different downstream pathologies. Blocking ApoE proteolysis, then, might stem deterioration of neurons quite broadly in neurodegenerative and brain injury conditions.

Why does ApoE4 more readily undergo fragmentation than the other isoforms? Together with Gladstone colleagues Karl Weisgraber and Yadong Huang, Mahley reported years ago that ApoE4 assumes a different tertiary structure from ApoE2 and E3, rendering it vulnerable to proteases (see ARF related news story). In ApoE4, substitution of an arginine for a cysteine at position 112 frees up other amino acids in the N- and C-termini of ApoE4 to interact, and this domain interaction exposes amino acids to proteases that are hidden in ApoE2 and 3. One of the criticisms researchers have voiced of this scenario is that lipidation of the protein protects it from proteolysis, and ApoE is mostly lipidated.

Nevertheless, if proteolytic susceptibility is ApoE4’s Achilles' heel, Mahley and colleagues would like to save it from protease arrows. For about a decade, the scientists have worked on developing “structural correctors.” As outlined in a paper published last month (see Chen et al., 2012), a high-throughput, fluorescence resonance energy transfer (FRET) assay identified small molecules that disrupt ApoE4 domain interactions in cultures of neuronal cells. These molecules reduce ApoE fragmentation in neuronal cells and prevent ApoE4-induced mitochondrial toxicity. They also unblock stalled endoplasmic reticulum/Golgi trafficking of ApoE4 to levels seen in cultured neurons expressing ApoE3. Intracellular sequestration of cell-surface receptors by ApoE4 attenuates receptor signaling and weakens synaptic signaling (see Part 3 of this series).

In the NSE-ApoE4 mouse model, structural correctors given daily for 10 days reduced ApoE4 fragments in the whole brain, including the hippocampus, by about a fifth, said Mahley. The treatment also boosted levels of the mitochondrial enzyme cytochrome c oxidase 1 by half. Its loss indicates damage to the organelles, and its levels are lower in NSE-ApoE4 mice than controls. Researchers at the meeting were intrigued by the cytochrome c oxidase rescue, and felt that other groups may want to replicate the effect. In response to questions about clinical development, Mahley said the molecules are being modified as potential therapeutics, and suggested they may be ready for testing in the clinic in two years.

While Mahley and colleagues have identified the most toxic of the known ApoE4 fragments—they are those containing the C-terminal lipid-binding domain—which protease generates them remains a mystery despite years of effort to identify it. The main proteolytic sites are methionine 272 and leucine 268, and the protease(s) responsible seem unique to neurons, noted Mahley. Fragments are not found in other brain cells or in peripheral cells.

ApoE fragments are found in the human brain, as Yadong Huang, also from the Gladstone Institute, noted in his talk. His group examined tissue samples from 41 human volunteers—25 AD patients and 16 controls of different ApoE genotypes. Huang reported that there are very few fragments in the brain tissue from controls, twice as many in AD cases, and that homozygote ApoE4 carriers had more fragmentation than heterozygote carriers. ApoE4 carriers had more fragments regardless of whether they had dementia, and AD patients homozygous for ApoE3 showed more fragmentation than did homozygous ApoE3 controls. This is an important point, Huang said during questions time. “ApoE3 can get proteolytically cleaved,” he said, “and in some AD cases, perhaps the protease responsible is elevated.” Huang said that he also sees ApoE fragments in plaques and in tangles.

In general, the AD field at large has generated little independent evidence to corroborate findings on ApoE fragmentation. Researchers debate whether, and under what circumstances, neurons express ApoE, and data on neuronal ApoE fragmentation have not caught on in a broad way. However, researchers in Bradley Hyman’s lab at Massachusetts General Hospital, Charlestown, did report seeing more ApoE N-terminal fragments in plaques in Alzheimer’s cases than controls (see Jones et al., 2011).

The human brain appears to have three major ApoE fragments of 29, 14-20, and 12 kDa, respectively, Huang said. His group has since looked in cerebrospinal fluid and found these three fragments among a small number of cases. Huang is now working with collaborators to test a larger number of CSF samples. If the fragments reliably turn up in the CSF, then they may form the basis of a future diagnostic test for neuronal damage, he suggested. At present, CSF tau and phospho-tau are the leading CSF markers for neuronal damage.

Given that ApoE fragmentation only seems to occur in neurons (see Brecht et al., 2004), are all neurons equally at risk? Huang’s group reported at Keystone that hilar GABAergic interneurons seem particularly vulnerable. In a mouse model expressing a truncated ApoE found in human brain, these neurons are decimated by the time the animals reach 12 months of age (see Andrews-Zwilling et al., 2010), and spatial memory deficits in the mice correlate with the loss. While neuron loss is a hallmark of AD, many established animal models do not recapitulate it. Some researchers at the symposium were intrigued that it occurs in this model. Tau pathology emerges in these neurons as well, noted Huang, and genetically removing the microtubule-binding protein protected against both neuron loss and learning deficits.

To test what ApoE fragments and tau might have to do with the health of GABAergic neurons in people, Huang is now studying induced pluripotent stem (iPS) cells generated from non-demented older individuals. When generating neuronal cultures from these iPS cells, he saw fewer GABAergic neurons from ApoE4/4 than ApoE3/3 donors, indicating ApoE4 stem cells have a hard time making this particular type of cell; total neuron production was normal. Looking more closely at the E4 cells, the scientists found more ApoE fragmentation, and more tau phosphorylation as judged by binding of the AT8 antibody. Again, the structural correctors rescued both phenotypes and boosted expression of the GABAergic neuron marker GAD67, Huang said.

Huang believes that, rather than block differentiation of GABAergic neurons, ApoE4 brings on their premature death. Cell culture stresses the cells, he said, and that makes them produce a lot of ApoE. In this sense, the culture may mimic what is going on in the brains of people with AD or other neurodegenerative diseases. Huang plans to use this system to study why tau gets phosphorylated. So far, it appears there was no change in the kinases typically suspected of modifying tau, including GSK-3β and Cdk5; instead, Huang thinks tau phosphorylation may be kick-started through the reelin/ApoE receptor signaling pathway.—Tom Fagan.

This is Part 4 of a five-part story. See also Part 1, Part 2, Part 3, Part 5. Download a PDF of the entire series.

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References

News Citations

  1. Keystone: Symposium Emphasizes Key Aspects of ApoE Biology
  2. Keystone: Probing the Function of Lipoprotein and Related Receptors
  3. Keystone: ApoE Receptors and Ligands in Memory and AD
  4. ApoE Catalyst Conference Explores Drug Development Opportunities
  5. Keystone: Therapies Around ApoE—Has Their Time Come?

Paper Citations

  1. Chen HK, Liu Z, Meyer-Franke A, Brodbeck J, Miranda RD, McGuire JG, Pleiss MA, Ji ZS, Balestra ME, Walker DW, Xu Q, Jeong DE, Budamagunta MS, Voss JC, Freedman SB, Weisgraber KH, Huang Y, Mahley RW. Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons. J Biol Chem. 2012 Feb 17;287(8):5253-66. PubMed.
  2. Jones PB, Adams KW, Rozkalne A, Spires-Jones TL, Hshieh TT, Hashimoto T, von Armin CA, Mielke M, Bacskai BJ, Hyman BT. Apolipoprotein E: isoform specific differences in tertiary structure and interaction with amyloid-β in human Alzheimer brain. PLoS One. 2011;6(1):e14586. PubMed.
  3. Brecht WJ, Harris FM, Chang S, Tesseur I, Yu GQ, Xu Q, Dee Fish J, Wyss-Coray T, Buttini M, Mucke L, Mahley RW, Huang Y. Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice. J Neurosci. 2004 Mar 10;24(10):2527-34. PubMed.
  4. Andrews-Zwilling Y, Bien-Ly N, Xu Q, Li G, Bernardo A, Yoon SY, Zwilling D, Yan TX, Chen L, Huang Y. Apolipoprotein E4 causes age- and Tau-dependent impairment of GABAergic interneurons, leading to learning and memory deficits in mice. J Neurosci. 2010 Oct 13;30(41):13707-17. PubMed.

Other Citations

  1. NSE-ApoE4 mouse model

External Citations

  1. PDGene
  2. MSGene

Further Reading

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