The apolipoprotein E gene is the strongest genetic risk factor for late-onset Alzheimer disease. Figuring out how it drives susceptibility remains a tall order. At a one-day symposium in St. Louis, researchers from across the United States and abroad took stock of the latest on potential mechanisms. Receptors, including ApoE receptor 2, topped the hit parade of ApoE accomplices that might mediate actions of the risky isoform.
St. Louis: ApoE Receptors—Hold Sway Over Synaptic Function
The apolipoprotein E gene is the strongest genetic risk factor for late-onset Alzheimer disease. One ApoE4 allele almost quadruples a person’s chances of getting the disease, while having two drives up the risk by 12- to 15-fold. Not surprisingly, some researchers believe that understanding ApoE could be the key to finding much-needed treatments and preventions for AD. But does ApoE get the attention it deserves? The apolipoprotein has been—and unfortunately still is—a Cinderella compared to amyloid-β precursor protein (APP) and the presenilins, which attract the lion’s share of the research dollars and most of the attention at scientific meetings. “ApoE, ApoE Receptors and Neurodegeneration,” a one-day symposium held 7 June 2010 at Washington University, St. Louis, Missouri, bucked the trend by putting ApoE center stage. Though the meeting addressed the relationship between the apolipoprotein and the amyloid-β cascade, it also stressed ApoE’s own intricate biology. The take-home message from the meeting, which was organized by members of the NIA/NIH program project grant (PPG) on ApoE Receptor Biology and Neurodegeneration, was that there may be a lot more to ApoE’s involvement in AD than its dalliances with Aβ.
A case in point is signaling through the ApoE receptor 2 (ApoER2), one of several members of the low-density lipoprotein (LDL) receptor family. Researchers led by Joachim Herz, University of Texas Southwestern Medical Center, Dallas, had previously reported that this receptor plays a key role in neuronal survival, and at the conference, Herz presented the latest twist implicating ApoE in the plot, as well. ApoER2 knockout mice lose almost half their corticospinal neurons by four months of age (see ARF related news story on Beffert et al., 2005). The reasons for this are not entirely clear, but upon binding to its normal ligand reelin, ApoER2 activates downstream cascades such as those triggered by Jun terminal kinases. Reelin also enhances long-term potentiation (LTP) and reverses Aβ-induced reduction of LTP in the hippocampus (see ARF related news story and ARF news story). The ApoER2 ligand even neutralizes LTP suppression by toxic Aβ dimers isolated from human AD patients (see ARF related news story). All told, the data suggest a complex interplay among Aβ, ApoE, and ApoE receptors, Herz said in his keynote talk. One of the unanswered questions—one that is particularly relevant to AD risk—is whether ApoE genotype influences this interplay. In St. Louis, Herz presented newly published data to suggest that it does.
Herz showed that, in keeping with its role in beefing up LTP, reelin enhances NMDA receptor-driven calcium spikes when added to neurons in culture. The spikes still occur if ApoE2 or ApoE3 are added to cells prior to reelin (though in the presence of ApoE3 the spikes do not last long); however, pre-incubating the neurons with ApoE4 completely suppresses the calcium influx. ApoE4 also suppressed LTP in a more physiological setting. For this, Herz and colleagues turned to ApoE targeted replacement (TR) mice developed by Patrick Sullivan at Duke University. These animals have their mouse ApoE gene replaced with one of the three human ApoE isoforms and have become a model of choice in the field. Reelin enhanced LTP in hippocampal tissue slices from ApoE2 or ApoE3 TR mice, just like it does in wild-type tissue, but failed to do so in ApoE4 slices. How might this relate to Aβ, which puts a damper on LTP? Herz said that Aβ-containing extracts from Alzheimer’s Disease Research Center brain tissue samples had little effect on LTP in ApoE2 slices and no effect on reelin-driven LTP. In ApoE3 slices, AD extracts suppressed LTP slightly, and this was overcome with reelin. But in ApoE4 slices, the Aβ preparation suppressed LTP, and reelin was unable to restore it. The experiments, by Herz lab members Ying Chen, Murat Durakoglugli, and Xunde Xian, were described in the June 14 PNAS online, and the paper is freely available for download.
The work suggests that the ApoE4 isoform might increase susceptibility to AD by undermining the normal processes of LTP and by exacerbating Aβ’s suppression of it. As for a mechanism, Herz believes that ApoE4 blocks beneficial reelin signaling because it sequesters ApoER2 receptors inside cells. When Chen and colleagues looked at steady-state levels of ApoER2 receptors in primary cortical neurons, they found that the receptors were internalized and became trapped in cells treated with ApoE4. However, receptors traffic normally and return to the cell surface in the presence of ApoE2/3. The upshot is that ApoE4 dampens ApoER2 signaling, prevents NMDA receptor activation, and blocks LTP. But some big questions remain, said Herz. These include how ApoE4 alters ApoER2 trafficking and whether the mechanism is amenable to drug treatment. He also thought it will be important to find out how Aβ and ApoE dovetail with respect to synaptic regulation.
Given these findings, one potential therapy for AD might be to activate ApoER2 receptors. Edwin Weeber, University of South Florida, Tampa, has examined this idea using reelin heterozygote mice, whose reelin level is reduced by half. These mice show defects in LTP and perform poorly in a contextual fear learning and memory paradigm. Injecting reelin into their brain ventricles restores reelin and rescues learning and memory.
But can reelin have any effect in wild-type mice, and how might it work? To test this idea, Weeber turned to an in-vivo model of receptor blockade. Injecting into the brain of mice a daily shot of RAP, or receptor associated protein—which binds to all the apolipoprotein receptors—cripples the animal’s performance in water maze tests of spatial memory. But a single shot of reelin into the brain ventricles counteracted RAP; it enhanced associative learning and improved spatial learning performance for days, significantly reducing escape latency in the water maze, Weeber said.
Weeber also reported improved LTP when adding reelin to hippocampal slices from Tg2576 AD mice. He has not tried to give reelin to AD mice in vivo yet. Nonetheless, Weeber thinks that the ApoER2 receptor system could be of interest as a therapeutic target for AD and other neurodegenerative diseases.
Hyang-Sook Hoe, Georgetown University, Washington, DC, also studies ApoER2 receptors—in synapse and spine formation. Using a cell co-culture system, she found increased clustering between COS7 cells and primary hippocampal neurons—as the two form synapses with each other—if the former overexpress ApoER2. The neurons also expressed a third more synaptophysin, a marker of synaptic vesicles, suggesting that ApoER2 stimulates synaptic differentiation. In addition, she tested if the apolipoprotein receptor has post-synaptic effects by looking at dendritic spine formation. Overexpressing ApoER2 in the COS7 cells boosted neuronal spine density in the primary neurons, whereas knocking down the receptor had the opposite effect. These results suggest that ApoER2 can profoundly affect synaptic development. Fitting this idea, Hoe found that ApoER2 knockout mice have about a third fewer dendrites than wild-type animals.
Delving into the mechanism, Hoe focused on PSD-95 and X-11, post-synaptic proteins that both bind to ApoER2. In St. Louis, she reported that in the same heterologous co-culture system, PSD95 and ApoER2 synergistically enhance presynaptic differentiation and also have an additive effect on dendritic spine formation in hippocampal neurons. X11 had the opposite effect. The results point to a model where PSD-95 would bind to ApoER2 to enhance spine formation and X11 binds to ApoER2 to suppress it.
What do these findings have to do with ApoE—and perhaps AD? For this question, Hoe also availed herself of ApoE TR mice, counting the spine density in cortical layers II/III of their brains. She found a dearth of dendritic spines in ApoE4 TR mice compared to wild-type. ApoE4 TR mice as young as four weeks old lose 25 percent of their shaft spines; by one year of age the loss amounts to 55 percent. Hippocampal spines appeared normal, however. It is not clear how this loss relates to the ApoE4-dependent LTP suppression and aberrant ApoER2 trafficking described by Herz, or to Aβ in any way. Going forward, Hoe said she plans to determine whether ApoE isoforms differentially influence the interaction between ApoER2 and PSD-95 or X-11.
In summary, several speakers showed that ApoER2 can profoundly affect synapse formation and function, suggesting one rationale for how different ApoE isoforms might render a person susceptible to cognitive decline.—Tom Fagan
This is Part 1 of a three-part series. See also Part 2 and Part 3.
ApoE stands out for a frustrating mark of distinction. Despite its established status as the strongest genetic risk factor for late-onset Alzheimer disease, scientists digging for the why and the how have unearthed a myriad of potential interactions but no consensus mechanism. Indeed, 17 years after the gene discovery, figuring out how ApoE4 drives up a person’s susceptibility remains a tall order. At a one-day symposium called “ApoE, ApoE Receptors and Neurodegeneration,” held 7 June 2010 at Washington University, St. Louis, Missouri, researchers from across the U.S. and abroad took stock of the latest on potential mechanisms. Receptors, including ApoE receptor 2 (see Part 1 of this three-part series), topped the hit parade of ApoE accomplices that might mediate nefarious actions of the risky isoform, but participants paid due consideration to ApoE’s influence on lipid and Aβ metabolism as well (see also Part 3).
In the brain, astrocytes produce and release ApoE, which finds its way to receptors on the surface of neurons. Those receptors include the family of low-density lipoprotein receptors. It is not proven that these receptors dovetail with ApoE’s involvement in AD pathology, but one of them, the low-density lipoprotein-related protein 1 (LRP1), does bind Aβ and even APP among a variety of ligands. In his presentation, Guojun Bu of WashU, showed how his lab members used the Cre/Lox system, under the Cam kinase promoter, to conditionally delete LRP1 from the forebrain of adult mice. Bu reported that getting rid of LRP1 boosts brain ApoE levels; it also suppresses the synaptic markers synaptophysin and PSD-95, as well as the number of dendritic spines in the forebrain. On top of this, the animals exhibit glial activation, neural inflammation, and deficits in their memory and motor function. These data come on the heels of similar in vitro results, when Bu knocked LRP1 down with a lentiviral approach. In these cultures, neurites degenerated and neurons died off (see, e.g., Fuentealba et al., 2009).
How does a loss of LRP lead to the effects, which, after all, model aspects of AD? Bu said the most likely mechanism works through perturbation of brain lipid metabolism. The Bu team found that LRP1 deletion in neurons correlates with a dearth of brain cholesterol, sulfatides, and cerebrosides, among other lipids. After these early events, dendritic spines degenerate and synapses disappear as animals age, and eventually neurons degenerate as these animals grow very old. Bu also noted that LRP1 knockouts lose NMDAR1 and GluR1 glutamate receptor subunits. That could be related to the synapse loss, or it could be an independent effect, he said. Is this relevant to AD? On this, Bu noted previous work from Eddie Koo’s lab that suggested LRP levels in the AD brain plummet by about 50 percent (see Kang et al., 2000) and said he is trying to confirm that data. But in addition, LRP1 levels are lowest in the brains of mice that have targeted replacement (TR) of their endogenous ApoE gene with that for human ApoE4, indicating that LRP1 not only regulates ApoE levels, but vice versa.
The finding suggests that ApoE4 might increase susceptibility to AD by suppressing LRP1. In keeping with this, Bu noted findings from two other meeting presenters. Joachim Herz, University of Texas Southwestern Medical Center, Dallas, reported that ApoE4 can ablate ApoE receptor 2 (ApoER2) from the cell surface by sequestering it intracellularly, while Hyang-Sook Hoe and colleagues at Georgetown University, Washington, DC, found reduced spine density in ApoE4 TR mice (see Part 1 of this series). Whether LRP1 levels fall and synapses fade away in ApoE4 mice because the apolipoprotein sequesters LRP1 in the same way as it does ApoER2 is not clear.
If reduction of LRP1 might explain, at least partly, why ApoE4 increases risk of AD, then would targeting the receptor help? Bill Rebeck of Georgetown University, Washington, DC, showed that it might, though not by improving synaptic biology. Rebeck showed that an ApoE fragment comprising the region of the protein that binds apolipoprotein receptors (amino acid 141-149) protects in vitro against glial activation/inflammation, which also occurs in the AD brain.
Lipopolysaccharide (LPS) is commonly used to induce an inflammatory glial response, and it elevates nitric oxide (NO) production and suppresses ApoE. In St. Louis, Rebeck reported that adding the ApoE141-149 peptide counteracted both these effects as well as activation of Jun-terminal kinase (JNK), one of two glial kinases LPS activates (the other being ERK). The peptide had no effect in LRP1-negative microglia. Rebeck concluded that LPS works by activating JNK in an LRP1-dependent manner. If that is true, then blocking JNK activation should relieve LPS-induced ApoE suppression. In fact, Rebeck showed that the JNK inhibitor SP600125 increases ApoE levels when injected into mouse brain. He suggested that blocking JNK could be a strategy for raising ApoE levels in people with AD. In the case of ApoE2/3 carriers, this might be beneficial, since those two isoforms enhance synaptic plasticity, which is essential for learning and memory (see Part 1).
The ApoE141-149 peptide may have other uses as well. Rebeck showed that activation of another receptor, ApoE receptor 2, puts the brakes on APP processing, as measured with a luciferase reporter system that quantifies production of the APP intracellular domain (AICD). Dimers and trimers of the 141-149 peptide worked similarly, suppressing AICD, trapping APP on the cell surface, and holding down Aβ production in PS70 cells as well as in primary neuronal cultures. Rebeck reported that a single injection of the peptide dimer into the hippocampus of wild-type mice increases α-secretase cleavage of APP and reduces Aβ levels. Rebeck said the findings, some recently reported (see Minami et al., 2010), could spur the development of small molecules that mimic the effect of the peptide and protect the brain against amyloidogenic processing of APP.
Receptor activation may be but one side of the ApoE coin. In the extracellular space, the apolipoprotein can have other effects, such as on Aβ oligomerization and clearance (see Part 3 of this series) or on cholesterol metabolism. At the conference, Cheryl Wellington, University of British Columbia, Vancouver, addressed the latter. She focuses on the cholesterol transporter protein ABCA1 as a potential therapeutic target. ABCA1 transfers cholesterol from the cell surface to ApoE, forming large lipoprotein particles. Wellington and colleagues showed that ABCA1 deficiency leads to a marked loss of ApoE and an increase in amyloid in mouse brain (see ARF related news story on Hirsch-Reinshagen et al., 2005). In contrast, transgenic mice overexpressing ABCA1 build up less amyloid (see ARF related news story). The data point to decreased clearance of Aβ when ApoE is poorly lipidated, said Wellington, and she suggested this could be used to develop a therapeutic.
One class of therapeutics that induces ABCA1 expression is the liver X receptor (LXR) agonists. Wellington shared unpublished data on the effects of the LXR agonist GW3965 on APP/PS1 transgenic mice. Her group tried the agonist both prophylactically, before the animals develop plaques, and as a treatment for mice with advanced AD-like pathology. At the lower of two doses, the agonist slightly raised ABCA1 levels in the brain, and at the higher dose, ApoE levels climbed as well. But the increase in ABCA1 did not have an effect on Aβ deposition, which only trended lower in animals treated with either dose of GW3965. Cognitive test results were more encouraging. Mice given the agonist prophylactically performed like wild-type mice in a novel object recognition task, in which untreated APP/PS1 controls performed poorly. GW3965 also restored memory when given to older mice as a treatment. GW3965 was developed by GlaxoSmithKline (see Collins et al., 2002) and is sold as a research tool by Tocris Bioscience under license.
Wellington pointed to several positive signs from this study. The effects of the agonist depended on ABCA1, that is, APP/PS1/ABCA1-negative mice did not respond. This suggests that the agonist acts through ABCA1 and not one of the many other proteins that are affected by LXR activity. This has long been a point of uncertainty with this approach. Secondly, the improvement in cognition in the older mice may bode well for using a similar strategy in people who already have AD symptoms. And lastly, that the mice appeared to become sharper without a dramatic change in Aβ accumulation suggests that ABCA1 affects a particular, crucial pool of Aβ, said Wellington. Whether these might be soluble oligomers that are now believed to be the most toxic Aβ species remains to be seen.
The importance of ApoE’s role in lipid metabolism, as it relates to AD risk, was reinforced by Patrick Sullivan, Duke University, Durham, North Carolina, who first developed the ApoE targeted replacement (TR) mice. Sullivan initially stressed that, although ApoE is a risk factor for AD, in some parts of the globe where ApoE4 genotypes are quite common, Alzheimer disease is not. Sullivan argued that environmental factors deserve more attention in AD, noting that at most there is a 60 percent concordance rate for the disease in monozygotic twins. “The environment obviously does matter,” he said.
One environmental factor that has drawn scrutiny is the Westernized diet. It could affect the lipidation status of ApoE and other lipoproteins, and in this way provide one explanation for ApoE’s link to the disease. Sullivan has looked for changes in lipid metabolism in ApoE TR mice. He found no difference in hippocampal cholesterol levels between ApoE3 and ApoE4 TR animals, but did see a difference in the fatty acid moieties attached to the glycerol backbone of phosphatidylethanolamines (PEs). In ApoE4 animals, PEs are less endowed with the long-chain fatty acids eicosapentaenoic acid and docosohexanoic acid (DHA). This may be particularly important to neurons, because the highest concentration of DHA in the entire body occurs at synapses, said Sullivan. DHA was tested in a small clinical trial for AD that showed some encouraging results (see ARF related news story), but a larger trial run by the ADCS proved negative (see ARF related news story).
Sullivan is interested in creating animal models of late-onset Alzheimer disease where the only genetic manipulation is the ApoE gene and where adding non-genetic risk factors would induce AD pathology. “We could then use metabolomics, proteomics, and lipidomics to tease out what pathways might be altered,” he said.
Perhaps the most intensely debated talk at the conference was one of the “hot topic” posters chosen for oral presentation. Katie Youmans from Mary Jo LaDu’s laboratory at the University of Illinois has coupled ApoE targeted replacement with the 5xFAD model developed by Bob Vassar’s lab. The idea was to get a mouse that features rapid acceleration of AD pathology. In these mice, deposition began in the subiculum/deep cortex, unlike in many other mouse models where it first begins in the hippocampus and superficial layers of the cortex. In this regard, the ApoE TR/5xFAD mice better recapitulate the pattern seen in the human brain, said LaDu.
Unexpectedly, Aβ deposits were actually more extensive in ApoE2 TR/5xFAD mice than in their ApoE4/5xFAD counterparts. Youmans told ARF that, though the deposits have the characteristic appearance of plaques (dense cores surrounded by a halo, e.g.), she has yet to fully characterize them. Some researchers, including Sullivan, were skeptical of these data, which seem to contradict most other findings that ApoE2 is protective. But when Youmans looked further, she found that in the ApoE2 crosses, most of the Aβ was extracellular, whereas in the ApoE4 crosses it was intraneuronal. In addition, by four months, the ApoE4 crosses had lost the synaptic marker synaptophysin, but the ApoE2 crosses actually had more of it, despite their greater abundance of extracellular and insoluble Aβ. “We basically figured out why ApoE2 is protective, and we debunked the amyloid hypothesis at the same time,” said LaDu. She clarified, saying that her data suggest amyloid deposits are not the driving force behind AD. Instead, soluble intraneuronal species of Aβ are, and ApoE4 drives their accumulation. This is in keeping with recent advances in the field that point to soluble Aβ as the most toxic species. Whether LaDu’s preliminary data will hold up remains to be seen, but she stressed that they had already repeated the analysis three times and come up with the same findings.—Tom Fagan.
This is Part 2 of a three-part series. See also Part 1 and Part 3.
The role of ApoE receptors was high on the agenda at “ApoE, ApoE Receptors and Neurodegeneration,” a one-day symposium held 7 June 2010 at Washington University, St. Louis, Missouri. These cell-surface proteins enable a flurry of ApoE activity, much of it totally independent of amyloid-β pathology, from enhancing synaptic modeling and plasticity to activating microglia (see Part 1 and Part 2 of this series). But whenever AD is discussed, it is not long before Aβ comes up, and the same was true at this meeting as well, which featured a useful summary of where the science of amyloid and ApoE currently stand.
It is well established that apolipoprotein E binds to Aβ in vitro, co-localizes with it in vivo, and profoundly affects Aβ structure, levels, and toxicity in mouse models. Cognitively normal people are more likely to amass amyloid-β in the brain as they age if they carry ApoE4, whereas ApoE2 carriers show not such propensity (see Reiman et al., 2009; Morris et al., 2010). And yet, how ApoE affects Aβ accumulation in real life and over time is far from trivial a question to answer. Over the last few years, David Holtzman and colleagues at Washington University, St. Louis, have tackled this issue by developing a microdialysis procedure to study the comings and goings of Aβ in the brains of AD model mice. With this system they found that ApoE extends the half-life of Aβ in the extracellular space in PDAPP mouse models. Joe Castellano, a graduate student in the Holtzman lab, found that in 18-month-old mice, the half-life of Aβ is around an hour in an ApoE4 background, about half that in mice expressing ApoE2, and 40 minutes in mice expressing ApoE3. This means that ApoE stays around about twice as long in mice expressing the AD risk allele. Amyloid pathology is worse in the E4 mice, suggesting that the longer half-life of Aβ in these animals could result from the peptide simply sticking to plaques. However, the half-life of Aβ is also about 60 minutes in young ApoE4 mice that have no plaques yet. “This is pretty clear evidence that differential Aβ deposition in these animals occurs because of soluble Aβ clearance problems,” said Holtzman. He thought this was an important finding to keep in mind, but added that much work needs to be done to figure out the cellular mechanisms. One avenue of research currently underway in Holtzman’s lab focuses on the nexus among ApoE, its receptors, and Aβ clearance (see ARF related news story).
Daniel Michaelson, Tel Aviv University, Israel, also presented evidence that ApoE4 extends Aβ’s half-life. In a “hot topic” poster abstract chosen for oral presentation, he described how the neprilysin inhibitor thiorphan can help reveal ApoE-dependent Aβ dynamics and effects on neurodegeneration. In ApoE targeted replacement (TR) mice, which carry human ApoE isoforms in lieu of their own, both total and oligomeric Aβ accumulated in hippocampal neurons when neprilysin was inhibited in an ApoE4 background, but not in an ApoE3 background. This fits with Holtzman’s finding that Aβ stays around longer in the presence of ApoE4. Michaelson, collaborating with Eliezer Masliah at the University of California, San Diego, turned to the electron microscope to view the consequences of this long-lived Aβ for cells. With immuno-EM, Michaelson saw that Aβ and oligomers of Aβ were found in mitochondria, but mostly co-localized with ApoE in enlarged lysosomes. Michaelson suggested that Aβ becomes toxic to cells by targeting both the mitochondria and the lysosomal system, resulting in increased apoptosis and cell death. He believes that the LDL receptor-related protein 1 (LRP1) may mediate the effects of ApoE because LRP1 levels rise in ApoE4 TR mice treated with neprilysin. (For more on LRP1, see Part 2 of this series.)
For his part, Holtzman agreed that apolipoprotein receptors might be involved in ApoE/Aβ dynamics. Knocking out one of them, the LDL receptor (LDLR), boosts ApoE in the cerebrospinal and interstitial fluids in the brains of PDAPP mice (see Fryer et al., 2005), while overexpressing LDLR in the same transgenic mice ablates the apolipoprotein, probably due to clearance mediated by the LDLR, noted Holtzman (see also ARF related news story on Kim et al., 2009). Overexpression of the LDLR also curtails plaque pathology and limits soluble Aβ as measured by microdialysis. His lab has shown that boosting the LDL receptor can reduce both levels of ApoE in the brain and plaque pathology, Holtzman said. In the poster session, Jacob Basak from the lab showed that astrocytes from LDLR overexpressing mice clear Aβ added to the culture medium much faster than do wild-type astrocytes. That data suggest that enhanced clearance of Aβ by glial cells may explain why LDLR overexpressing mice have less amyloid pathology, though it is still not clear if ApoE is part of the mechanism.
One way ApoE might extend the half-life of Aβ is by affecting its aggregation. In his hot-topic presentation, Tadafumi Hashimoto, who works in Brad Hyman’s lab at Massachusetts General Hospital, Charlestown, described a “split luciferase” assay system to measure Aβ interactions. This system expresses the N-terminal half of the luciferase on one Aβ construct, and the C-terminal half on another. When Aβ molecules aggregate, luciferase is reconstituted and emits light. Hashimoto found that ApoE4 enhances formation of high-molecular-weight Aβ aggregates in HEK293 cells, whereas ApoE2 facilitates formation of lower-molecular-weight species. Furthermore, the lipidation status of ApoE is crucial for its effect on Aβ aggregation. While recombinant ApoE had no effect on in vitro Aβ aggregation, affinity-purified, lipidated ApoE enhanced the formation of Aβ oligomers. The work also suggests that ApoE might extend Aβ’s half-life by promoting aggregation.
One other potential nexus between ApoE and Aβ clearance is the blood-brain barrier (BBB). The barrier may be compromised in AD patients and is known to leak in ApoE knockout mice, but just how ApoE isoforms affect the BBB is unknown, said Kazuchika Nishitsuji, National Center for Geriatrics and Gerontology in Aichi, Japan. In his hot-topic talk, Nishitsuji described how he followed the distribution of the large-molecular-weight dye Evans Blue in the brain after injecting it into the peritoneal cavity as a way of assessing the BBB. He reported that leakage was greater in ApoE4 knock-in animals than in ApoE3 animals. In a cell model of the BBB that co-cultures astrocytes, endothelial cells, and pericytes, electrical resistance across endothelial cells was lower when astrocytes expressed ApoE4 rather than ApoE3. The lower resistance suggests that the endothelial cells were more permeable. How ApoE4 makes these glia more permeable is not clear, but Nishitsuji noted that phosphorylation of occludin was up. Occludin is one of the proteins found in endothelial cell tight junctions, which help hold the BBB together. It also remains to be seen how ApoE4 isoforms might affect endothelial cells and the BBB in an AD mouse model.
All told, this meeting demonstrated that the biology that surrounds ApoE, ApoE receptors, and AD pathology is complex. Many people might like to know how this knowledge could be put to practical use, as in a therapeutic that could prevent or slow Aβ pathology in ApoE4-positive patients. There was little to get excited about on that front. It was mentioned that statins increase LDLR in the periphery and have been investigated as potential AD therapeutics with little success, perhaps because they do not readily penetrate the brain. Holtzman noted one new therapeutic aimed at boosting LDLR levels—an antibody to proprotein convertase subtilisin/kexin type 9, or PCSK9, which degrades LDLR. Amgen is currently putting that human monoclonal antibody through its paces in a Phase 1 clinical trial in hopes to use the antibody to treat high plasma cholesterol. But it is too soon to tell whether the antibody, or better yet, a small molecule inhibitor of the protease, could be beneficial in AD.—Tom Fagan.
This is Part 3 of a three-part series. See also Part 1 and Part 2.
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