Of the many facets of ApoE’s biology, the most complex one may be its activation of cell surface receptors. ApoE binds to a family of low-density lipoprotein receptors (LDLRs), and scientists are just beginning to parse how those interactions influence cell and neurobiology in adult animals. At "ApoE, Alzheimer’s and Lipoprotein Biology," a symposium held 26 February-2 March 2012 in Keystone, Colorado, the functions of those receptors in neural plasticity emerged as a major theme.

A quintessential example of LDLR signaling is in development. Reelin, an LDLR ligand, is a de-facto competitor of ApoE and, hence, inextricably entwined with its effects on receptors and downstream signaling in neurons. Reelin has long been known to play an essential role in migration of neural cells and pattern formation in the brain. In reeler mice, a naturally occurring mutant strain that completely lacks this protein, Purkinje cells fail to navigate to their proper place in the cerebellum and the mice develop severe ataxia. The cerebral cortex and other laminated structures of the brain develop abnormally as well, and the mice die soon after birth. While reelin was traditionally viewed as a developmental regulator, in recent years scientists have taken a second look at the protein's role in the adult brain.

At Keystone, Gabriella D’Arcangelo, Rutgers University, Piscataway, New Jersey, noted that reelin expression in the brain changes soon after birth. Rather than being produced solely by the Cajal Retzius cells that orchestrate embryonic neurodevelopment, reelin occurs throughout the brain in adult mice. What is its function? To test this, D’Arcangelo has been studying mice heterozygotes for reelin-null genes. She noticed a paucity of dendrites in these animals shortly after birth, but by postnatal day 21, the dendrite complement looked the same as in wild-type animals. The data indicate that maturation of brain connections in reelin-deficient animals may just take a little longer than usual. D’Arcangelo and colleagues also found that fewer spines decorate dendrites in postnatal reelin heterozygotes, and that recombinant reelin from conditioned medium restores spine numbers to cultured hippocampal cells. Their data suggest that well after birth, reelin helps the maturation of synapses.

If reelin is still important in the postnatal period, what about in adults? In 10-month-old reelin heterozygotes, the numbers of both dendrites and spines appear normal, again suggesting that the brain eventually matures even with less-than-normal reelin. However, on closer inspection, D’Arcangelo and colleagues did find that not all is quite right with dendritic spines. For example, while the total amount of the important synaptic scaffold protein post-synaptic density 95 (PSD95) appears the same as in normal mouse brain, considerably less of it makes its way into spines. Similarly, spines contain too little of the NR2A and NR2B glutamate receptor subunits and the PTEN kinase (see Ventruti et al., 2011). These proteins form a complex with PSD95, perhaps explaining why all three are deficient in reelin heterozygotes, said D’Arcangelo. These molecular differences may help explain synaptic plasticity and learning and memory deficits in these animals. They may also relate to Alzheimer’s, D’Arcangelo said, since postmortem analysis indicates less reelin is produced in the brains of people with AD, and spine and synapse losses are characteristics of the disease. Transgenic APP mice make less reelin that do wild-type animals (see Chin et al., 2007 and ARF related news story).

How might reelin loss play into AD pathology? The protein is a major ligand for ApoE receptor 2 (ApoER2) and the very low-density lipoprotein receptor (VLDLR). Signaling through these receptors supports synaptic plasticity and may be antagonized by ApoE4. Working with Edwin Weeber, University of South Florida, Tampa, D’Arcangelo found that reelin heterozygotes underperform in certain learning and memory paradigms and have synaptic deficits, including weaker long-term potentiation. Weeber has been testing if reelin can rescue deficits in adult rodents. At the conference, he wowed the audience with dramatic effects of reelin on learning and memory, not only in reelin heterozygotes, but even in wild-type.

Weeber previously reported that reelin rescues spatial navigation deficits driven by RAP, a protein that binds and blocks all ApoE receptors. Reelin also corrects learning and memory deficits in reeler heterozygotes, which are haploinsufficient (see ARF related conference story).

Weeber and colleagues use thin tubes, or cannulae, to deliver reelin into brain ventricles. From there, the ligand reaches the hippocampus, where it activates the downstream kinase Dab1 and induces CREB phosphorylation. Weeber showed that in wild-type mice, a single shot of reelin to the ventricles boosts both spine density and long-term potentiation in the hippocampus. The enhancement seems confined to the post-synapse, since paired-pulse facilitation, which relies on presynaptic strengthening, was unchanged. Mice that are missing ApoER2 fail to respond to reelin, singling out that receptor as one that initiates synaptic changes in response to this ligand, said Weeber.

Do these molecular and electrophysiological effects amount to any behavioral changes? Weeber showed that a single shot of reelin to the ventricle substantially improved spatial learning and memory in wild-type mice. The researchers used the Morris water maze. It involves several days of training, during which mice get progressively faster at finding an underwater platform. Typically, differences between control and treated animals emerge after a few days; in this case, however, the treated mice did so much better that the difference was statistically significant on day one. Each day’s training involves four trials in the water bath, said Weeber, and the treated mice already outperformed controls by the third trial.

Researchers at the meeting seemed impressed by how fast and robustly the learning improved. But lest anyone considers popping reelin pills, the long-term effects are not clear. During question time, it emerged that if animals are given reelin daily, they become quite dumb, falling far behind untreated controls in the water maze.

Nevertheless, researchers at the meeting wondered if reelin might rescue deficits in disease models. Weeber said he is currently testing AD mice in the same experimental design and is planning to look at mice expressing human ApoE isoforms. He reported that reelin rescues spatial memory in a model of Angelman syndrome, a developmental disease where loss of a ubiquitination factor essential for regulating synapse architecture causes motor and cognitive deficits.

In his talk, meeting co-organizer Joachim Herz, University of Texas Southwestern Medical Center, Dallas, noted that reelin and Aβ seem to antagonize each other. While the ApoE receptor ligand boosts LTP, and learning and memory, Aβ suppresses the latter and strengthens long-term depression (LTD). Herz previously outlined how ApoE isoforms play into this dynamic. On binding to receptors, ApoE4, a major risk factor for AD, induces their uptake and sequestration inside the cell, thereby limiting reelin signaling at the cell surface (see ARF related conference story). This ultimately retards incorporation of glutamate receptors on the cell surface, said Herz. It also prevents reelin from rescuing against Aβ-induced LTD. ApoE3 and E2, in contrast, do not perturb ApoER2 distribution in the cell or limit reelin signaling.

Could ApoER2 sequestration by ApoE4 be prevented or reversed? Herz and colleagues screened for small molecules that can do just that. They identified several compounds that Herz says are promising and awaiting patent protection. He showed that, in an ApoE4 background, one of the compounds normalized ApoER2 on the cell surface and restored the ability of reelin to rescue Aβ-induced synaptic deficits. How the compound works is not exactly clear, but Herz hinted that it perturbs intramolecular domain interactions that are unique to ApoE4.

How does Aβ antagonize reelin signaling? Researchers are still trying to understand the toxic effect of the peptide, but in his short talk Steve Barger, University of Arkansas for Medical Sciences, Little Rock, reported that both ApoE4 and some forms of Aβ are competitive antagonists of ApoER2 and/or VLDLR ligands, and thereby block signaling. ApoE2 and 3 activate these receptors, Barger said, because they have at least one cysteine and form disulfide dimers that span the divide between individual receptors and thereby stabilize receptor dimers. Because ApoE4 lacks a crucial cysteine residue, it cannot form dimers. Instead, it appears to bind the receptors as monomers, blocking dimeric ligands and suppressing signaling. Barger used a luciferase reconstitution assay, with N- and C-terminals of the enzyme on different ApoE receptors, to demonstrate ApoE receptor dimerization by reelin and ApoE2/3. These agonists also enhanced NMDA receptor activity in a manner sensitive to RAP, or knockdown of ApoER2 expression. Interestingly, while he found that fibrils of Aβ did the same, Aβ oligomers bound to the receptors but did not induce dimerization; instead, they blocked the effects of reelin on dimerization and NMDAR activation. Barger concluded that both ApoE4 and Aβ oligomers suppress reelin signaling in the same manner.

Reelin perturbations have been recorded in Alzheimer’s disease, though human genetic data at present are sparse (see reelin on AlzGene). Could these have ramifications unrelated to synaptic signaling? In addition to being downregulated in the AD brain, reelin has been reported to associate with amyloid plaques in AD and in transgenic mouse models. In Keystone, Irene Knuesel, University of Zurich, Switzerland, wondered how it ends up there. Does it have to bind to Aβ, or can it accumulate on its own? Previous work suggested that reelin with the C-terminus aggregates, said Knuesel (see de Bergeyck et al., 1997), but the proteases that generate those fragments and how they relate to AD pathology are completely unknown. Reelin is itself a protease. Knuesel described how neuroinflammation may exacerbate Alzheimer’s pathology by triggering proteolysis and aggregation of reelin.

Knuesel and colleagues searched for reelin proteases in p19 carcinoma cells. While undifferentiated, these multipotent cells express no reelin protease, but when pushed toward neurogenesis with retinoic acid, they cleave reelin into several fragments consistent with N- and C-terminal proteolysis, said Knuesel (see Ducharme et al., 2010). Knuesel and colleagues used these cells to identify ADAM metallopeptidase with thrombospondin type 1 motif, 4 (ADAMTS-4) and tissue plasminogen activator (tPA) as the enzymes that cut reelin near the C-terminus.

To test if reelin proteolysis dovetails with AD pathology, Knuesel injected the virus simulator polyI:C into pregnant mice to induce neuroinflammation in their offspring. In the process, reelin became cut, and its proteolytic fragments accumulated in axon terminals and neurites, said Knuesel. When she treated prenatally exposed mice with a second shot of polyI:C in adulthood, they formed intraneuronal aggregates that Knuesel thinks may contain Aβ precursor protein. Some researchers wonder if these are Hirano bodies, which contain actin and are sometimes associated with AD. In a transgenic mouse model of AD, polyI:C elevated soluble Aβ and tau phosphorylation, and led to a dramatic increase in amyloid plaques. All told, Knuesel said the believes that proteolytic fragments of the ligand may form seeds for aggregation of proteins, including reelin itself and Aβ.—Tom Fagan.

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


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News Citations

  1. Brain Reelin from Missing Cells in Early AD?
  2. St. Louis: ApoE Receptors—Hold Sway Over Synaptic Function
  3. Keystone: Symposium Emphasizes Key Aspects of ApoE Biology
  4. Keystone: Probing the Function of Lipoprotein and Related Receptors
  5. Keystone: Does ApoE Fragmentation Drive Pathology?
  6. Keystone: Therapies Around ApoE—Has Their Time Come?

Paper Citations

  1. . Reelin deficiency causes specific defects in the molecular composition of the synapses in the adult brain. Neuroscience. 2011 Aug 25;189:32-42. PubMed.
  2. . Reelin depletion in the entorhinal cortex of human amyloid precursor protein transgenic mice and humans with Alzheimer's disease. J Neurosci. 2007 Mar 14;27(11):2727-33. PubMed.
  3. . A truncated Reelin protein is produced but not secreted in the 'Orleans' reeler mutation (Reln[rl-Orl]). Brain Res Mol Brain Res. 1997 Oct 15;50(1-2):85-90. PubMed.
  4. . Ceruloplasmin-induced aggregation of P19 neurons involves a serine protease activity and is accompanied by reelin cleavage. Neuroscience. 2010 May 19;167(3):633-43. PubMed.

Other Citations

  1. Download a PDF of the entire series.

External Citations

  1. reelin on AlzGene

Further Reading