Cloistered Retreat Takes the Pulse of BACE Research

07 Dec 2013

Could β-secretase go the way of its cousin? Once valued as a target by Alzheimer’s drug developers, γ-secretase fizzled when inhibitors targeting the protease sped up cognitive decline and caused skin cancers in clinical trial volunteers. Researchers blamed the disappointing outcomes on interference with crucial biological processes driven by γ-secretase substrates, notably the cell-surface protein Notch (see Aug 2010 research news). With nearly a dozen pharmaceutical companies poised to develop BACE inhibitors, some researchers have been whispering concern that history may repeat itself. That sentiment boiled to the surface at BACE Proteases in Health and Disease, a meeting organized by Stefan Lichtenthaler, Technical University Munich, Germany. At the meeting, held October 6–8 at Kloster Seeon, a 10th-century Benedictine monastery 90 minutes east of the Bavarian capital city, researchers from academia and industry debated the recent science around BACE, including whether known and yet-to-be-discovered substrates might spell doom for potential BACE drugs.

BACE drug programs got off to a slow start because it was difficult to model the large, wide-open active site of the protease and design brain-penetrant drugs to fit it. Just as researchers solved that problem, another emerged. BACE1 knockouts, once thought to be innocuous, turned out to have subtle, potentially troubling side effects, including poor myelination, loss of dendritic spines, axon guidance defects, neural network defects, seizures, and even memory problems. At the same time, screens began turning up dozens of potential BACE substrates in addition to amyloid precursor protein. With BACE inhibitors already in Phase 1 to 3 clinical trials, some researchers began to stress that more work was needed to predict and prevent potentially dangerous side effects of chronic BACE inhibition.

Is Neuregulin the Notch of BACE Inhibitor Development?

The usually silent Benedictine monks might have wrung their hands over the hubbub 70 passionate scientists generated when they were cloistered on this beautiful island retreat for two days. A panel discussion captured the hot-button issues. “Neuregulin is the Notch of BACE inhibitor development,” was how Bart De Strooper, KU Leuven, Belgium, summed up a sinking feeling among academic researchers. That sent shivers down the spines of some industry representatives, who cautioned against throwing BACE out with the neuregulin bathwater.

Scientists at this meeting agreed that neuregulin is a bona fide BACE substrate, but opinion diverged on how that will affect drug development. Dieder Moechars, Janssen Research and Development, emphasized that it may be possible to reduce Aβ production with a partial BACE inhibition, limiting adverse events. Christian Haass, Ludwig-Maximilians University, Munich, agreed that moderate inhibition of BACE might work, but pleaded for a step-by-step approach to avoid the discouraging fallout that followed γ-secretase inhibitor (GSI) trials. GSI side effects were mechanism-based and predictable, Haass said, and the outcome of the clinical trials shook confidence among patients and funding agencies, casting public doubt over the amyloid hypothesis.

Many of the phenotypes that emerge by knocking out BACE can be traced to problems during development, such as poor myelination, lack of pigmentation, and stunted growth. Even so, BACE also regulates crucial processes in adults, Haass said, including maintenance of muscle spindles and dendritic spines. In addition to neuregulin, tens of potential BACE substrates have emerge from various screening approaches (see part two of this story). “There are still other substrates to be identified, and we have no idea what they do in the brain,” Haass said.

On that point, Bruce Albala, Eisai Inc., the fourth member on the panel, expressed a concern among industry researchers. “We need to know which of these phenotypes is more important,” he said. “We are in a business that is increasingly harder to sustain, and a compound that fails because of safety issues helps no one.” Albala said that a consensus paper from the academic community that outlines the most important safety issues would help all industry navigate the BACE drug-development process. “We can’t wait for academia to exhaustively research and vet all the potential substrates to get the go-ahead for industry to begin development,” he added.

Patrick May, Eli Lilly and Company, agreed. May further stressed that inconsistency between the preclinical models presents a problem for pharma. Phenotypes often are not reproducible from lab to lab. “Furthermore, it is challenging to take controversial preclinical data and apply it to clinical studies,” he said. Ryan Watts, Genentech, echoed that sentiment. “For example, we don’t see the ocular phenotype, and the models may not always predict what will happen in humans treated with BACE inhibitors,” he said (see Cai et al., 2012). Mark Albers, Harvard Medical School, Boston, said it is unfair to look at every drug through the prism of side effects in animals. Albers noted that given what researchers now know about statins, one of the most successful drug classes ever, they might have never brought them to market because statins affect many more targets that the intended one, cholesterol biosynthesis. “Attempts to predict what will happen in humans are often inaccurate,” he said.

On phenotypes, Lichtenthaler concurred that the field needs to reproduce results in different models. “We really need to think about whether we are looking at effects that are due to BACE and its substrates, or due to the model itself,” he said. That some phenotypes cannot be reproduced does not automatically mean the results are wrong; rather, it could reflect modifier effects unique to specific genetic backgrounds. Lichtenthaler cautioned against over-interpreting knockout data. “A knockout is not the same thing as a drug-treated animal,” he said. Genetic knockout can remove a gene entirely from birth, allowing compensatory mechanisms to develop in some instances, whereas drug treatment reduces the target partially during aging.

For their part, pharmaceutical companies seem to be taking basic research discoveries seriously. Matthew Kennedy of Merck described how his company compared knockout phenotypes reported in the literature with their in-house models and with BACE inhibitor-treated animals. He noted that they have never seen seizures, impaired cognition, or retinal neurodegeneration with their inhibitors. Merck’s researchers have, however, noticed hypomyelination, reduced prepulse inhibition, and slower nerve conduction with some compounds. Merck’s lead compound has undergone Phase 1 testing (see Jul 2012 news story) and enrollment for a Phase 2/3 trial is underway (see March 2013 news story). Haass said he was impressed that companies are working on these phenotypes step by step.

De Strooper deplored a general lack of feedback from pharma once drugs enter trials. “There is a black hole. That compromises communication between companies and academia,” he said. De Strooper challenged industry to share samples and publish data, especially when things go wrong. Others objected to singling out industry for failing to publish negative data when the problem is rife among academic labs, as well (see May 2013 news story; Sep 2004 news story). While pharma does not share with everyone, they do share samples with collaborators. Lilly’s May cited the analysis of CSF samples from Lilly trials of ß- and γ-secretases by Erik Portelius and colleagues at the University of Gothenburg, Sweden (see February 2012 news story; Portelius et al., 2012).

One easy point of consensus was that developing drugs for AD has become so expensive that a new approach may be needed. “The way we develop drugs right now suits antibiotics and other drugs with short duration of action, but for drugs that will have to be tested and used long-term it hardly seems feasible,” said De Strooper. “We need to figure out how our respective societies as a whole can facilitate trials by taking on part of the burden.”—Tom Fagan

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Cai J, Qi X, Kociok N, Skosyrski S, Emilio A, Ruan Q, Han S, Liu L, Chen Z, Bowes Rickman C, Golde T, Grant MB, Saftig P, Serneels L, De Strooper B, Joussen AM, Boulton ME. β-Secretase (BACE1) inhibition causes retinal pathology by vascular dysregulation and accumulation of age pigment. EMBO Mol Med. 2012 Sep;4(9):980-91. PubMed.
Portelius E, Zetterberg H, Dean RA, Marcil A, Bourgeois P, Nutu M, Andreasson U, Siemers E, Mawuenyega KG, Sigurdson WC, May PC, Paul SM, Holtzman DM, Blennow K, Bateman RJ. Amyloid-β1-15/16 as a Marker for γ-Secretase Inhibition in Alzheimer's Disease. J Alzheimers Dis. 2012 Apr 18; PubMed.

Other Citations

BACE—Substrates, Functions, Developmental Phenotypes

11 Dec 2013

BACE1 is the major β-secretase for amyloid precursor protein, making it a prime target for drug developers. However, because this protease cleaves many substrates, scientists worry about adverse effects of BACE inhibition (see part one). The BACE1 homolog BACE2 is thought not to play a role in AD, but it, too, cleaves many substrates in tissues outside the brain and most inhibitors known to date block both BACE1 and 2. What are those substrates and what functions might BACE inhibitors compromise? Those questions dominated BACE Proteases in Health and Disease, a meeting hosted by Stefan Lichtenthaler, Technical University of Munich, on October 6–8, 2013. The meeting attracted a Who's Who of BACE researchers from academia and industry.

Long a backwater to γ-secretase, recent high-profile discoveries and the entry of pharmaceutical companies have invigorated BACE research. While BACE1 knockout mice initially were reported to be mostly normal, subtle phenotypes soon emerged. Axonal guidance defects, retinal pathology, loss of dendritic spines and muscle spindles, seizures, hyperactivity, and memory defects are among the faults that have been reported in these mice. Some of this is developmental, but not all (see part three).

Researchers continue to identify potential BACE substrates, with the number now reaching dozens. Robert Vassar, Northwestern University, Chicago, one of the original discoverers of the secretase in 1999, summed up the million-dollar question facing the field: How much BACE inhibition is needed to benefit Alzheimer's disease, and will that amount be safe? Vassar cited the recent discovery of the A673T mutation that protects APP against BACE cleavage, and their carriers against AD (see July 2012 news story). "That suggests about 20 percent decrease of Aβ over production a lifetime might be sufficient to prevent AD," said Vassar, suggesting that 50 percent inhibition of BACE might be warranted for AD prevention, and more if amyloid has already accumulated. "To address the potential side effects, we need to sort out which of the symptoms in knockout mice are due to developmental versus later blockage of BACE," he said.

Substrates and Developmental Phenotypes
To understand what BACE1 does, Vassar’s group has localized the protease in the adult brain using immunohistochemistry and electron microscopy. They concluded that BACE1 abounds in presynaptic terminals, where it seems to be required for axon guidance (see Kandalepas et al., 2013). In BACE1 knockouts, olfactory sensory neuron axons fail to find their way to glomeruli in the olfactory bulb; also, infrapyramidal bundle mossy fibers projecting from the dentate gyrus turn porematurely toward their terminal area in the CA3 region of the hippocampus. In rodents, shorter infrapyramidal bundles compromise memory, noted Vassar.

Work from the lab of Mark Albers, Harvard Medical School, Boston, adds a twist to this role of BACE. Albers and colleagues reported that enhanced BACE1 cleavage of human APP contributes to miswiring in olfactory glomeruli (see Aug 2012 news story). In Munich, Albers reported that in the absence of APP or APP-like protein 2 (APLP2), another BACE substrate, olfactory neuron axons fail to target to the correct glomeruli. This mimics the axonal guidance deficits seen in BACE1 knockouts, suggesting that failure to process APP/APLP2 drives that phenotype.

Vassar believes that the unprocessed neural cell adhesion molecule CHL1 could underlie defects in BACE knockouts. CHL1 knockouts have almost identical phenotypes to BACE1 nulls, including axon guidance defects (see Hitt et al., 2012). Other researchers at the meeting also fingered CHL1 as a BACE substrate. Using proteomics approaches, researchers at Bart De Strooper’s lab at KU Leuven, Belgium, and Peer-Hendrik Kuhn and colleagues at Lichtenthaler’s lab identified CHLI and the related L1 as BACE1 substrates (see Jun 2012 news story). De Strooper said that BACE inhibitors cause full-length L1 and CHLI to accumulate in synaptosomes. He also noted that without CHL1 signaling, somatosensory thalamic neurons fail to project to their proper location in the brain, again suggesting guidance defects.

Bastian Dislich, Technical University of Munich, uncovered CHL1 with a mass spectroscopy-based proteomic approach that Vassar called a tour de force. Dislich raised three generations of BACE1 knockouts on chow laced with lysine containing heavy isotopes. He took brain extracts from those animals at postnatal day three, and mixed them with extracts from wild-type, age-matched mice that had been raised on regular food. By examining the ratio of heavy to light species in the mass spec, he identified peptides that were more abundant in the BACE1 knockouts. They would be likely substrates for BACE1. The strategy identified APP, APLP2, and a number of other membrane-bound proteins, including CHL1. Dislich said that about 30 percent of those hits function in axon guidance or neurite outgrowth. He plans to check these with Western blots to see whether the full-length proteins, or the ectodomains that would be cleaved by BACE1, accumulate.

Kuhn identified contactin-2, another cell-adhesion molecule, and seizure protein 6, which has ties to epilepsy. In BACE knockouts and in cells treated with BACE inhibitors, ectodomains of both of these proteins are poorly shed from cell surfaces. In Munich, Kuhn reported that BACE processes another cell membrane protein, delta/notch-like epidermal growth factor receptor, or DNER for short (Eiraku et al., 2002). DNER ectodomain shedding seems essential for proper brain development. Kuhn said that by processing DNER, BACE1 drives the differentiation of nestin-positive radial glial cells into neurons. In BACE1 knockouts and under BACE1 inhibition, these glia instead persist at postnatal day seven, disrupting the patterning of the neuronal cortex.

Riqiang Yan, Lerner Research Institute, Cleveland, who also discovered BACE at the same time as Vassar and others, described uncannily similar machinations are afoot with cell differentiation in the hippocampus of BACE1 knockouts. There he found increased astrogenesis at the expense of neurogenesis. “The knockout shifts the fate of neuronal stem cells,” he said. What drives that shift? In this case, enhanced Notch signaling seems to be the culprit. Yan reported that BACE1 cleaves the notch ligand Jagged1 from cell surfaces. In the absence of the protease, Jagged1, which abounds on granule cells of the hippocampus, accumulates and activates Notch on adjacent cells.

Scientists continue to uncover novel aspects to cleavage of neuregulin, a classic BACE substrate, as well. Christian Haass, Ludwig-Maximilians University, Munich, reviewed work his group published earlier this year showing that BACE1 cleavage helps release a soluble epidermal-like growth factor domain from neuregulin in neurons, which then signals through cell surface receptors on adjacent myelinating cells (see May 2013 news story). The soluble EGF domain can by itself rescue myelination defects in BACE1 knockout zebrafish, said Haass. His group found that this signaling pathway seems crucial for peripheral myelination only. Myelination in the central nervous system remain unaffected in BACE1 knockouts—at least in fish and mice.

Researchers at the meeting seemed in general agreement that CHL1 represents a bona fide BACE substrate, but many said it was too early to determine if other hits that have turned up in screens will prove to be true substrates. There could be indirect effects, for example, blocking cleavage of a real substrate could limit downstream processing of other proteins by other proteases. “Just because BACE seems to cleave a protein does not make that protein a substrate,” said De Strooper. The litmus test must be strict and must require that BACE1 co-localize with the protein and cleave it in a physiological setting, he said.

Most also agreed that it remains to be seen whether developmental phenotypes seen in BACE knockouts will have any bearing on what might happen in people treated with a BACE inhibitor. While some warned that developmental pathways often reactivate later in life, particularly during disease, most people at the meeting seemed to think that these are less relevant to BACE therapeutics than adult phenotypes. “We need a much better understanding of adult phenotypes as a way to predict side effects of BACE inhibitors,” concluded Lichtenthaler (see part three).—Tom Fagan

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Blocking BACE—Do Adult Mouse Phenotypes Predict Side Effects?

13 Dec 2013

Despite Eli Lilly terminating development of its BACE inhibitor LY2886721 (see Jun 2013 news story), the protease remains a popular target among pharmaceutical companies looking for the next Alzheimer's disease therapy. While BACE inhibitors may reduce production of Aß and slow or halt progression of AD, could they also wreak havoc on the aging brain? Leaders in the field debated that point at BACE Proteases in Health and Disease, a three-day meeting organized by Stefan Lichtenthaler, Technical University of Munich, October 6–8 (see part one). While many agreed that developmental phenotypes of BACE deficiency may be less relevant to therapeutic success (see part two), most researchers at the meeting expressed uneasiness that blocking normal BACE functions in adults might have unexpected consequences.

For now, those functions center around Aß precursor protein, neuregulin, and the melanin scaffold PMEL, all established BACE substrates. Jochen Herms, Ludwig Maximilians University, Munich, previously found that inhibiting γ-secretase alters dendritic spine density in wild-type mice (see Bittner et al., 2009). At Seeon, Herms reported that BACE might be involved in the formation of new dendritic spines. Using two-photon microscopy, Herms and colleagues watched spine dynamics in living mice whose neurons express enhanced green fluorescent protein (eGFP). They saw that over a period of 16 days, 3-month-old eGFP mice treated with BACE inhibitors developed by Merck (SCH-1682496) and Eli Lilly (LY2811376) formed half as many new dendritic spines as controls, and lost 10 percent of their total spine number. The effects seemed reversible, because spine numbers slowly recovered during two weeks off the drug. Herms concluded that blocking BACE1 affects spine plasticity more profoundly than spine density.

BACE knockout animals had fewer hippocampal dendritic spines at 16 days of age, but by 32 days were undistinguishable from wild-type, suggesting that the animals may compensate for the absence of BACE1. BACE inhibitors caused no further loss of spines in BACE1 knockouts, but they did reduce spine numbers in wild-type animals. The data confirm that the compounds reduce spine numbers by binding to BACE, rather than some other target, said Herms. Do these spine losses translate into functional deficits? Herms found that BACE inhibitors reduced spontaneous and evoked neurotransmission in mice. In response to questions, Herms said he was unsure how the spine suppression might affect people with Alzheimer’s.

Another defect that raised concern was one reported on last July (see Jul 2013 news story). Carmen Birchmeier, Max Delbrück Center for Molecular Medicine, Berlin, and colleagues found that knocking out BACE1, or blocking it pharmacologically in adult mice, interferes with muscle spindles. Comprising muscle fibers and sensory neurons, spindles intersperse among the skeletal muscles and convey information about muscle tension to the brain, allowing it to coordinate movement. Birchmeier and colleagues found that BACE cleavage of the Ig-containing ß1 isoform of neuregulin supports spindle formation, and that in BACE1-negative mice spindles lose their shape and motor control is compromised. Researchers at the meeting were curious whether this effect can be reversed and what type of dose response underlies it. Birchmeier said she has yet to look at multiple doses; she used the same dose of LY2811376 that Eli Lilly used to reduce Aß production in mice. Some of the spindle deficits seem to be long-lasting, suggesting there could be some irreversible damage, Birchmeier cautioned that more work needs to be done before this is known.

Researchers also debated the importance of pigmentation loss in adult BACE knockouts. Guillaume van Niel, Institut Curie, Paris, and colleagues reported last July that BACE2 processes the melanocyte protein PMEL, which forms a functional amyloid that binds melanin (see Rochin et al., 2013). Pigmentation failure gives BACE2-deficient animals a silvery coat, while BACE1 knockouts animals are the same color as wild-type, van Niel said. Philip Wong, Johns Hopkins University, Baltimore, reported slightly different results. In his hands the pigmentation phenotype in BACE2 knockouts was less severe. Wong said that to the naked eye the knockouts looked no different than wild-type, and only close examination revealed a subtle loss of pigmentation. "I think the key difference is the design of the animals," said Wong. Van Niel and colleagues studied a mouse engineered by researchers at Bart De Strooper's lab at KU Leuven, Belgium. That strain makes a catalytically inactive BACE2, whereas Wong's group knocked out the full gene and their mouse makes no BACE2 at all. "I suspect that because BACE2 is still there, albeit inactive, it acts as a dominant negative and prevents compensation by BACE1 in the mouse from De Strooper's lab," said Wong. The dominant negative may better mimic what happens when people are treated with non-selective BACE inhibitors, agreed Wong, because in that case the proteins are still present, though inactive.

Attendees debated what pigmentation effects might look like in people, and whether they might jeopardize BACE therapeutics. Most agreed the likely effect would be lightened skin color. "I would trade that for Alzheimer's anytime," said Wong. Van Niel suggested that potential sensitivity to melanoma, a more serious side effect, warrants further investigation.

On the other side of the coin, could there be some benefits to BACE inhibition besides reducing amyloidogenic processing of APP? Markus Stoffel, ETH Zurich, Switzerland, summarized recent findings from his lab that suggest BACE2 limits proliferation of insulin-producing ß cells in the Islet of Langerhans in the pancreas. BACE2 cleaves a transmembrane protein called TMEM27. Mice modeling maturity-onset diabetes of the young—a rare, inherited form of type 2 diabetes—downregulate this protein, while those overexpressing TMEM27 develop bigger ß cells, produce more insulin, and better control plasma glucose levels. Stoffel's group found that TMEM27 sheds an extracellular domain that likely stimulates tissue growth. In an RNAi screen to identify the sheddase that releases this domain, Stoffel found BACE2. In addition, treating obese mice with a BACE inhibitor stimulated insulin release from the pancreas and improved glucose tolerance.

Researchers agreed that the roles both BACE isoforms play in adult mice and in people are far from fully understood. Answers to some outstanding questions may come from conditionally knocking out BACE1 in adult animals. At least five different groups said that they are presently doing so. "That was fantastic to hear," said Lichtenthaler. Two years ago it seemed that nobody was having success at creating such animals. “They will be important to distinguish adult from developmental phenotypes and help us identify substrates and pathways that rely on BACE in the adult," said Lichtenthaler.—Tom Fagan

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Bittner T, Fuhrmann M, Burgold S, Jung CK, Volbracht C, Steiner H, Mitteregger G, Kretzschmar HA, Haass C, Herms J. Gamma-secretase inhibition reduces spine density in vivo via an amyloid precursor protein-dependent pathway. J Neurosci. 2009 Aug 19;29(33):10405-9. PubMed.
Rochin L, Hurbain I, Serneels L, Fort C, Watt B, Leblanc P, Marks MS, De Strooper B, Raposo G, van Niel G. BACE2 processes PMEL to form the melanosome amyloid matrix in pigment cells. Proc Natl Acad Sci U S A. 2013 Jun 25;110(26):10658-63. PubMed.

Meeting Explores Complex Biology of BACE Regulation

26 Dec 2013

Beta-secretase continues to be an attractive target for those looking for the next Alzheimer's disease therapy. Merck is starting a large trial of its BACE inhibitor MK-8931 in prodromal AD (see Dec 2013 news story), and almost a dozen other companies have BACE inhibitors in various stages of testing. Against this backdrop of therapeutic research, scientists puzzle over some of the basic biology surrounding this protease, including why it seems to be more active in people with sporadic AD, where in the cell it cleaves Aβ precursor protein, and how it gets there. BACE regulation was one of the major themes at BACE Proteases in Health and Disease, a meeting hosted by Stefan Lichtenthaler, Technical University of Munich, October 6-8.

Scientists have known for some time that the AD brain expresses normal levels of BACE messenger RNA, quashing the idea that overactive BACE genes underlie the increased protease activity (see Mar 2004 news story). Instead, researchers have looked to degradation or relocation to APP-rich compartments as explanations for increased BACE processing of APP. Both of these rely on protein trafficking, which has become a major theme in the study of BACE and of γ-secretase and Aβ precursor protein as well (see Dec 2012 news story; Mar 2009 news story).

At the meeting, researchers discussed how production and trafficking of the protein within neurons might contribute to amyloidogenic processing of APP. Gopal Thinakaran, University of Chicago, detailed complex mechanisms for BACE transport in neurons. The journey includes transcytosis, where the protein in the cell membrane is taken up at the dendrites on one end of the cell and brought to the tip of the axons at the other end. Thinakaran's group used a dual labeling system to follow the path BACE took in cultured hippocampal neurons. The researchers engineered the protease to sport both a yellow fluorescent protein tag that traced distribution of total BACE and a motif that bound α-bungarotoxin to selectively follow BACE taken up by endocytosis. Researchers have developed a fluorescently labelled version of this toxin to image trafficking of receptors taken up into cells (see Sekine-Aizawa et al., 2004).

In a series of videos, Thinakaran showed how BACE internalized from the cell surface travels in both anterograde and retrograde directions in axons, but only retrogradely, toward the soma, in dendrites. Thinakaran said this was surprising because cargo normally moves in both directions. "No one has ever shown that an internalized protein only moves retrogradely in dendrites," he said.

Thinakaran was unsure how to explain this phenomenon. Since cargo travels along microtubules, and these orient in both directions in dendrites, the findings suggest that BACE must somehow select and travel along only those microtubules oriented toward the soma. The protease must navigate properly through branch points, as well, said Thinakaran.

What regulates this process? Thinkakaran's group looked for answers in endosomes. These small membrane compartments originate from the cell membrane, where mature BACE resides, and travel to the cytosol. Thinakaran reported that BACE co-localizes with C-terminal Eps15 homology domain proteins (EHDs), which regulate endocytosis and protein recycling. When Thinakaran's group knocked down EHD1 or 3, transport of total BACE in dendrites progressed as usual, but retrograde transport of internalized BACE slowed to a crawl, and a greater proportion of BACE remained stationary. As a consequence, BACE1 levels in axons decreased considerably.

Thinakaran said these observations could be relevant to Alzheimer's. His group found extensive colocalization of EHD1 and 3 with Aβ and with APP β-C-terminal fragments, which are products of BACE cleavage, in the brains of young APP/PS1 mice well before plaques began to deposit. In addition, knocking down EHDs in neurons from these mice caused a significant reduction in Aβ production. The work suggests that this type of transport helps drive BACE processing of APP.

Working independently, Lawrence Rajendran and his colleagues at the University of Zurich found that Rab11, a GTPase that binds to EHD proteins, also contributes to BACE processing. Rab-GTPases play an important role in regulating membrane trafficking. Rajendran's group developed a multiplex system to assay BACE activity by simultaneously, and quantitatively, measuring Aβand sAPPβ. They combined this with an unbiased RNA interference screen of Rab-GTPases to see if any of them modulated BACE activity. They complemented this screen with one that overexpressed Rab-GAPs, which inactivate specific Rab-GTPases. Rab11 emerged as one of the strongest modulators of BACE. Rajendran’s group then found that silencing Rab11 in various cell lines, including primary neurons from either wild-type mice or APP transgenic mice, reduced β-cleavage of APP and Aβ production.

How would inactivating Rab11 limit BACE cleavage of APP? Rajendran showed that without functional Rab11, neurons take up BACE from the cell membrane into endosomes, but it fails to recycle back to the cell surface. Previously, researchers had shown that internalization to endosomes was sufficient for BACE processing of APP (see Aug 2011 news story), but these new findings suggest that recycling back to the cell surface might be crucial as well. Thinakaran's and Rajendran's work appeared in back-to-back papers in the December 26 Cell Reports.

Researchers at the meeting seemed intrigued by these developments, but noted many outstanding questions. Some asked how EHDs and Rab11, which are known to be involved in bidirectional transport, help carry internalized BACE unidirectionally in dendrites. Thinakaran said he believes that BACE itself may have some role to play. Others wondered what kick-starts BACE endocytosis. Thinakaran said that this could be a stochastic process in resting neurons, but suggested that it might be regulated by synaptic activity.

At the other end of the BACE regulation spectrum, Taisuke Tomita, University of Tokyo, and Giuseppina Tesco, Tufts University, Medford, Massachusetts, described how sluggish degradation of the protease might contribute to disease. Tomita has examined the effect of Bin1 on BACE trafficking to the lysosome. Genome-wide association studies have identified genetic variants near the Bin1 gene as risk factors for AD (see May 2010 news story). While there are hints that Bin1 might bind tau, researchers are still trying to identify the functional variants that confer risk (see Aug 2012 news story). Because Bin1 binds proteins involved in endocytosis, Tomita decided to check on links to BACE.

Tomita reported that when he silenced Bin1 in N2a neuroblastoma cells, endogenous BACE rose—and so did production of Aβ40 and Aβ42. Tomita also used cultured neurons from "floxed" mice that have LoxP sites surrounding the Bin1 gene (see Chang et al., 2007). By infecting these neurons with lentivirus expressing the Cre recombinase, the researchers ablated Bin1. Loss of Bin1 led to high levels of BACE and more sAPPβ and Aβ than seen in control cells. Rajendran's group had previously reported that silencing Bin1 increases sAPPβ and Aβ levels as well (Bali et al., 2012). Tomita further reported that when he knocked down Bin1 in cells with fluorescently tagged BACE1, endocytosis of the protease appeared normal but the protease got trapped in endosomes. Hence Tomita believes Bin1 supports BACE1 trafficking from endosomes to the lysosome.

Tesco was the first to show that the lysosome degrades BACE1 after it has been internalized. She had previously reported that whether internalized BACE ended up in lysosomes or re-sorted back to the cell surface depended on Golgi-localized γ ear-containing ARF-binding 3 (GGA3). This protein recognizes a specific di-leucine motif on BACE (see Jun 2007 news story on Tesco et al., 2007). At the meeting, Tesco discussed how this process might be impaired during disease. She reported that after a traumatic brain injury (TBI), GGA3 plummets in the mouse brain while BACE rises. Unexpectedly, however, she also observed this rise in brain BACE after head trauma in GGA3 knockouts. This suggested that some other mechanism controls BACE, as well. Tesco and colleagues traced this effect to the GGA3 homolog GGA1. She reported that after TBI, both GGA3 and GGA1 are quickly lost, but GGA1 bounces back within about seven days while GGA3 levels remain low. She concluded that over the long term, BACE increases may be due solely to loss of GGA3.

Is this relevant to Alzheimer's? Since previous work suggested that GGA3 drops by half in the AD brain, Tesco's group has crossed GGA3 knockouts with 5xFAD mice to see if GGA3 haploinsufficiency affects pathology. Preliminary data suggests that BACE rises in the GGA3 heterozygotes/5xFAD mice. The researchers have yet to look at the effects on Aβ and plaques.

All told, the researchers at this meeting grappled with how trafficking of BACE from the cell surface to the lysosome for degradation, or retrogradely through dendrites and back to the cell surface, could be steps that regulate overall BACE activity in the neuron. "It is crucial that we understand this process," Thinakaran told Alzforum. "We know from Bob Vassar's work that BACE predominantly localizes to presynaptic terminals and turns up in dystrophic neurites near Aβ plaques," he said (see Zhao et al., 2007). "If we could reduce BACE transport to axons, we may find a novel approach to a therapeutic."—Tom Fagan

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Sekine-Aizawa Y, Huganir RL. Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors. Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17114-9. Epub 2004 Nov 24 PubMed.
Chang MY, Boulden J, Katz JB, Wang L, Meyer TJ, Soler AP, Muller AJ, Prendergast GC. Bin1 ablation increases susceptibility to cancer during aging, particularly lung cancer. Cancer Res. 2007 Aug 15;67(16):7605-12. PubMed.
Bali J, Gheinani AH, Zurbriggen S, Rajendran L. Role of genes linked to sporadic Alzheimer's disease risk in the production of β-amyloid peptides. Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15307-11. PubMed.
Tesco G, Koh YH, Kang EL, Cameron AN, Das S, Sena-Esteves M, Hiltunen M, Yang SH, Zhong Z, Shen Y, Simpkins JW, Tanzi RE. Depletion of GGA3 stabilizes BACE and enhances beta-secretase activity. Neuron. 2007 Jun 7;54(5):721-37. PubMed.
Zhao J, Fu Y, Yasvoina M, Shao P, Hitt B, O'Connor T, Logan S, Maus E, Citron M, Berry R, Binder L, Vassar R. Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer's disease pathogenesis. J Neurosci. 2007 Apr 4;27(14):3639-49. PubMed.

BACE Proteases in Health and Disease

Tools

Cloistered Retreat Takes the Pulse of BACE Research

Comments

Make a Comment

References

News Citations

Paper Citations

Other Citations

Further Reading

News

BACE—Substrates, Functions, Developmental Phenotypes

Comments

Make a Comment

References

News Citations

Paper Citations

Further Reading

Blocking BACE—Do Adult Mouse Phenotypes Predict Side Effects?

Comments

Make a Comment

References

News Citations

Paper Citations

Further Reading

News

Meeting Explores Complex Biology of BACE Regulation

Comments

Make a Comment

References

News Citations

Paper Citations

Other Citations

Further Reading

News