Part four of a four-part story.
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 colocalizes 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
- Merck BACE Inhibitor Clears a Safety Hurdle, Gets New Trial
- Follow the Leader to Higher BACE Levels
- Kill Two Birds: Muting Trafficking Protein Lowers Aβ and β-CTF
- Divorce Protein Style—APP Fragments Go Their Own Way in Cells
- Going It Alone—APP, BACE1 Take Distinct Routes to Endosome
- LOADing Up—Largest GWAS to Date Confirms Two, Adds Two Risk Genes
- GWAS Mega-Meta Yields More Risk Genes, BIN1 Binds Tau?
- Stress and Aβ—The Apoptosis Connection
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- 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) 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.
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- 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.
- Lilly Halts Phase 2 Trial of BACE Inhibitor Due to Liver Toxicity
- Building a Roadmap for Shared Combination Drug Trials
- BACE Inhibitors Hitch Ride into Endosomes via Membrane Anchor
- Assembly, Traffic Escorts, Fats All Control APP Processing
- Kill Two Birds: Muting Trafficking Protein Lowers Aβ and β-CTF
- Paracrine Signal From BACE1-Clipped Neuregulin Rescues Myelin
- Rounding Second BACE: Another Route to Memory Loss?
- BACE Secrets: Newly Identified Substrates May Regulate Plasticity
- Udayar V, Buggia-Prévot V, Guerreiro RL, Siegel G, Rambabu N, Soohoo AL, Ponnusamy M, Siegenthaler B, Bali J, Simons M, Ries J, Puthenveedu MA, Hardy J, Thinakaran G, Rajendran L. A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of β-amyloid production. Cell Rep. 2013 Dec 26;5(6) PubMed.
- Buggia-Prévot V, Fernandez CG, Udayar V, Vetrivel KS, Elie A, Roseman J, Sasse VA, Lefkow M, Meckler X, Bhattacharyya S, George M, Kar S, Bindokas VP, Parent AT, Rajendran L, Band H, Vassar R, Thinakaran G. A function for EHD family proteins in unidirectional retrograde dendritic transport of BACE1 and Alzheimer's disease Aβ production. Cell Rep. 2013 Dec 26;5(6) PubMed.