This is Part 2 of a 5-part series. See also Part 1, Part 3, Part 4, and Part 5.
Topic 1: ApoE
25 December 2007. Apolipoprotein E (ApoE) was first identified in the 1970s as playing a role in regulating plasma cholesterol levels, and in genetic hyperlipoproteinemias. ApoE is produced in the CNS, where it may be involved in the process of repair following neuronal injury. In 1993, its gene became a focus of AD genetics with the discovery of the increased risk borne by ApoE4 carriers and decreased risk found in ApoE2 carriers. Fourteen years later, hundreds of genetic studies have established incontrovertibly that this 34 kDa glycoprotein has by far the strongest impact of all genetic risk factors for LOAD and for cerebral amyloid angiopathy (CAA) known to date. ApoE4 drives down age of onset of AD, acting most strongly in a person’s fifties and sixties. Two E4 alleles increase risk 10-fold in men, 12-fold in women. ApoE remains understudied, in part because its complex association with lipids make its brain biochemistry challenging. ApoE is probably not directly related to APP or its processing machinery, so isoform-specific differences in its physiological function may hold independent clues to how ApoE changes neuronal function to lead to AD.
Previous research has focused on ApoE’s interaction with the Aβ peptide, coining the term “pathological chaperone.” It is established that ApoE affects assembly, clearance, deposition, and toxicity of Aβ, but no consensus mechanisms have emerged. Overall, human ApoE4 may be less efficient than E2 and E3 at clearing secreted Aβ from extracellular spaces and preventing its adverse effects on synapses, and at the same time it more strongly facilitates Aβ deposition. It is unclear which forms of Aβ ApoE modulates in human brain, but it is clear that different human ApoE isoforms somehow regulate Aβ conformation differently, (see ARF related news story; Holtzman et al., 2000).
This report below focuses on new research since then. Some groups examine how ApoE relates to the best-characterized biomarkers known to date as a function of age, which remains the strongest overall risk factor for LOAD. Basic science opportunities have opened up around ApoE receptors and lipidation, and some established lines of investigation are ready for translational efforts.
ApoE Interaction With Biomarkers
ApoE genotype appears to affect age-related changes in some CSF markers such that people who carry an ApoE4 allele are more likely to develop a pathogenic marker profile that predicts AD. For example, CSF Aβ42 and tau/phospho-tau are candidate predictive markers for AD. Meta-analysis has established that CSF Aβ42 levels are reduced by about half in AD, and a less marked drop occurs already in amnestic MCI/incipient AD. Total tau and p-tau levels in CSF increase two- to threefold in AD. A high tau/Aβ42 ratio is widely thought to put a person at high risk of developing AD within the next 3 years.
Adding ApoE to this background, a cross-sectional study of 184 community volunteers ranging from 21 to 88 years of age who were cognitively normal at enrollment suggests that ApoE influences age-related changes in CSF Aβ42, though not Aβ40 or tau. In people without an ApoE4 allele, CSF Aβ42 levels rose slightly until midlife and then edged down in old age. By contrast, people with an E4 allele had a much steeper decline after age 60 (Peskind et al., 2006). Among ApoE4 carriers, men and women had different trajectories. Men carrying an E4 allele had a slow linear decline in CSF Aβ42 starting in their twenties, while women had an increase until their mid-fifties and then a sharp drop. These time lines mirror age-related changes in testosterone in men and estradiol in women. Furthermore, the ratio of tau/Aβ42 was similar for all participants until age 60. After that, it became highly variable. In some people the ratio stayed stable, but in ApoE4 carriers it tended to become high enough to predict AD, and some converted during the follow-up period (Li et al., 2007; also Fagan et al., 2007). This suggests that ApoE4 accelerates pathogenic Aβ deposition starting already in mid-life. The mechanistic underpinning of interactions between ApoE4, gonadal hormones, and Aβ42 remains unknown, but these data in normal people imply that primary prevention efforts should begin before age 50.
Both ApoE and Aβ occur in different conformations, and the particular conformation determines their activity. ApoE subserves numerous functions in the brain, yet isoform-specific and AD-related functions of ApoE4 in human brain remain elusive. In brain, ApoE occurs as lipoprotein particles released by astrocytes. The neuronal and glial surface receptors for these particles include members of the LDL receptor gene family, such as LRP, VLDLR, LDLR, ApoER2, MEGF7, and others. The functional conformation of ApoE receptors in brain cannot be assumed from their conformation on peripheral cells, nor from overexpression by clonal cell lines. Both the conformation of ApoE and the context and conformation of the CNS receptors are thought to produce unique affinity profiles that are not understood. Tools needed in this area include ApoE receptor antibodies that distinguish one receptor family member from another, or that detect conformational differences within a specific receptor.
The receptors mediate ApoE signaling, and some also mediate its endocytic uptake. Both ApoE signaling and uptake require more study. One current hypothesis for how ApoE and Aβ42 interact focuses on isoform-specific effects of ApoE receptor-mediated metabolism of ApoE and Aβ. Studies using a co-culture system of neurons overlaid on glial cells that secrete human knock-in ApoE isoforms have suggested that ApoE4 and Aβ oligomers act synergistically to exert neurotoxicity (Manelli et al., 2006). Further experiments since then suggest that ApoE receptors mediate this toxicity. One hypothesis ripe for investigation is that ApoE4 influences intraneuronal trafficking of oligomeric forms of Aβ, leading to its accumulation in vesicular compartments and toxicity, whereas ApoE2 and 3 preferentially facilitate lysosomal degradation of Aβ. On a broader note, the group agreed that research on Aβ oligomers, on this and other questions, would benefit from a clarification of the nomenclature and various preparations used in the literature (see commentary).
In evolution, LDL receptors developed about a billion years ago, whereas ApoE arose some 600 million years later, suggesting that the ligand evolved separately and made use of an existing set of endocytosing receptors to get its lipid cargo (such as cholesterol and other lipids) into neurons. For their part, neuronal ApoE receptors serve the function of importing glia-secreted lipoprotein particles and relieving neurons from having to synthesize membrane components themselves. But importantly, the ApoE receptors also have other functions that evolved prior to ApoE transport, for example, signaling to the nucleus and to synaptic proteins. One hypothesis holds that ApoE might interfere with pathways normally routed through ApoE receptors by their cognate receptors, and in this way contribute to AD pathogenesis.
Mechanisms remain largely unknown, but fundamental knowledge about ApoE receptor signaling in neuronal function is growing. For example, the ApoE receptors VLDLR and ApoER2 regulate binding and signaling of their cognate ligand reelin. Mice lacking these receptors have neurodevelopmental effects, and humans with mutations in VLDLR suffer mental retardation (Boycott et al., 2005). ApoER2 is present at the postsynaptic density, where it associates with NMDA receptor complexes. Specifically, a particular splice form of ApoER2 in mice is able to enhance LTP and is needed for learning and memory. Activity appears to regulate the requisite ApoER2 splicing. Reelin, which is secreted by interneurons in adult neocortex and hippocampus, clusters VLDLR and ApoER2 receptors and induces src kinase signaling. This signaling leads to a PSD-95-mediated phosphorylation of NMDA receptor subunits and, in a separate pathway, also to inhibition of the tau kinase GSK3β (Beffert et al., 2005). Furthermore, the same ApoE receptors that are needed for LTP are also important for survival of cortical neurons in adult brain (Beffert et al., 2006).
One hypothesis worth testing is that ApoE might interfere with reelin’s effect on the NMDA receptor. Direct tie-ins with AD pathways do not exist yet, but regulation of NMDA receptor subunits is becoming a point of convergence. Separate research has shown that Aβ dampens the excitability of excitatory synapses by downregulating NMDA receptor components, leading to an LTD-like state. Opposing that, ApoE tends to tune up the excitability of the same neurons by signaling through ApoE receptors and PSD-95 to NMDA receptors. In the same vein, Aβ tends to downregulate dendritic spines, whereas ApoE upregulates them. Rigorous isoform-specific effects, or relationships among Aβ, reelin, and its competitive ligand ApoE, in the same model system of neuronal excitability have not been found yet. However, it is known that hAPP-transgenic mice contain fewer than normal reelin-positive pyramidal neurons in their entorhinal cortex (Chin et al., 2007). The study of ApoE receptors in synaptic plasticity and neuronal survival offers opportunities for understanding how ApoE influences AD risk with approaches outside of the classic AD pathologies. Note, however, that since the Bar Harbor conference was held, a study has linked APP processing to ApoE receptors by reporting evidence that the intracellular APP stub AICD suppresses LRP1 transcription and indirectly affects ApoE and cholesterol levels (Liu et al., 2007).
ApoE occurs in the body not as a free protein but packaged with cholesterol and phospholipids in HDL-like particles. In extracellular spaces, ApoE particles influence the transport, clearance, and conformation of Aβ in ways still unknown. Therefore, understanding the protein’s lipidation may yield clues to AD pathogenesis and therapeutic approaches. Research into whether the composition of these lipoprotein particles affects AD pathology is beginning, but the field needs more exploration.
Cardiovascular research has shown that lipidation of HDL occurs in part through the transmembrane protein ABCA1, which transports cholesterol and phospholipids onto nascent apolipoprotein particles. ABCA1 protein occurs in the cell membrane of CNS neurons and glia, and the gene has come up in genetic association studies of AD risk (see Alzgene). Crosses of human APP-transgenic mouse strains with ABCA1 knockout mice indeed have low ApoE and low HDL levels in blood, CSF, and brain. Analysis of these mice showed that when ABCA1 does not lipidate astrocyte-secreted ApoE normally, the HDL particles are rapidly degraded, suggesting that ABCA1 affects HDL levels in the central nervous system (see ARF related news story). But surprisingly, subsequent crosses of different lines of APP-transgenic mice to ABCA1 knockout mice showed more severe amyloid pathology in the brain parenchyma and blood vessels than did the APP transgenics alone. This would suggest that poorly lipidated HDL particles are fibrillogenic (see ARF related news story). By that logic, elevated ABCA1 expression would be predicted to increase ApoE lipidation and decrease Aβ deposition, and ongoing research suggests this is indeed the case. In PDAPP mice overexpressing ABCA1, ApoE-containing HDL particles in the CNS are larger and contain more lipid, and the mice have fewer fibrillar amyloid deposits in cortex and hippocampus. This work indicates that the lipidation of ApoE influences where and in which conformation Aβ aggregates. This area of research has stimulated translational interest in agonists of the transcription factor LXR. These agonists can induce ABCA1, but at present remain unsuitable for chronic treatment in humans (see Koldamova and Lefterov, 2007; Cao et al., 2007; Riddell et al., 2007; Lefterov et al., 2007; Narlawar et al., 2007).
Next, this line of investigation could ask whether, and how, changes in ABCA1 levels influence ApoE clearance in CNS or Aβ metabolism. Mouse studies could assess whether ABCA1 influences Aβ-related learning and memory abnormalities. Mechanistic studies could tackle how ABCA1 mediates ApoE lipidation and how that changes ApoE-Aβ interactions. Whether ABCA1 affects the size of lipoprotein particles differently depending on if astrocytes express ApoE2, 3, or 4 is also unknown.
Lipid metabolism can become disturbed with aging. For example, it is possible that physiologically regulated molecules such as ABCA1 and ApoE shift the brain’s lipid environment with age. Mature amyloid fibrils are increasingly viewed as a less toxic form of Aβ aggregate than soluble oligomers. Rather than being stable, plaques are increasingly thought to have an “off-rate,” whereby a certain fraction of fibrils dissolve to release oligomers into the surrounding neuropil. Since hydrophobic forces drive Aβ aggregation, the question arises if elevated brain triglycerides might alter the equilibrium between plaques and oligomers/protofibrils. Biochemical experiments published this month in an open-access article suggest that adding lipids to mature Aβ fibrils releases “reverse oligomers” that are much more toxic to cultured neurons than are the mature fibrils. Different types of lipid can achieve this effect, and they do so with different kinds of amyloid made of Aβ, prion, or tau proteins. Typically, the lipids release oligomers of the A11 conformation (Kayed et al., 2003). Furthermore, stereotactic injection suggests that these “reverse oligomers” are toxic in vivo and impair memory in mice. If this initial data is replicated and expanded, it would suggest that plaques can turn into sources of highly toxic oligomers when their lipid environment changes, perhaps as a function of age. It would suggest that plaques are not inert end stages of pathology but can act as reservoirs of toxicity (Martins et al., 2007).
Other ApoE Mechanisms
A long-standing debate in the field revolves around the question of whether human neurons crank up production of ApoE in response to injury or in the course of aging. Many investigators have found that ApoE expression is largely exclusive to astrocytes, but a knock-in model has reported ApoE expression in hippocampal neurons after injury with kainic acid (e.g., Xu et al., 2006). Related research has shown that fragmentation of ApoE occurs in transgenic mice expressing ApoE in neurons, and human AD brain contains ApoE fragments in an isoform- and region-specific manner, as well. Cell culture studies indicate that the fragments impair the cytoskeleton and mitochondrial function. The enzyme responsible for this ApoE cleavage is thought to be a serine-like protease with chymotrypsin-like properties. The protease has so far eluded research efforts to identify it. Proteases are particularly amenable to drug development, and the relevance and site of action of the ApoE-cleaving activity will become clear once it is found (for detailed coverage, see ARF conference report. The relative contributions to neuronal toxicity of oligomeric Aβ and ApoE fragments are unclear. There was consensus that AD may well develop for different reasons in different people. A person with two ApoE4 alleles, especially if they sustained repeated mild concussions in contact sports or accidents, may develop AD partly due to ApoE toxicity, whereas a person with an presenilin mutation will develop AD primarily as a consequence of Aβ overproduction.
Translational ApoE Research
Given ApoE’s overriding genetic influence on AD risk and onset, much more effort is warranted in bridging the gap between basic research and the clinic (Refolo and Fillit, 2004). Besides LXR agonists (see above), therapeutic approaches under study in academia and industry include the following:
A research program at Gladstone Institute at the University of California, San Francisco, has tested small molecules for their ability to disrupt an intramolecular interaction between structural domains in ApoE4 alone, which results from a single amino acid difference between ApoE3 and E4 at residue 112 (Mahley et al., 2006). Such “structure correctors” would make the ApoE4 protein E3-like in its biochemical interactions with protein and lipid binding partners. The institute last November created the Gladstone Center for Translational Research. The center will pursue this and other ApoE-related therapeutic targets in a collaboration with the pharmaceutical company Merck.
A modified Aβ12-28 peptide has been shown to abolish ApoE4’s promotion of Aβ fibril formation by virtue of blocking interaction between ApoE4 and Aβ (Sadowski et al., 2006).
The wide-open field of ApoE receptor signaling and endocytic pathways may yield therapeutic approaches in the future. ApoE receptors are specific for ligands, and drugs could interfere at this level to block particular responses. For example, ApoE mimetic peptides are under study for their neuroprotective and anti-inflammatory effects in brain injury models (Laskowitz et al., 2007).
In concluding the ApoE sessions, the workshop group came to consensus that ApoE exerts multiple different effects. An effect through Aβ is without question, but a separate effect on neurobiology that is distinct from Aβ likely occurs, as well.—Gabrielle Strobel.
See also Part 1, Part 3, Part 4, and Part 5.