31 Oct 2016

Beta-secretase inhibitors are a leading therapeutic strategy being evaluated for Alzheimer’s disease, but as scientists discussed last month at the 2nd Kloster Seeon meeting on BACE Proteases in Health and Disease, there is still much to learn about the potential pitfalls of blocking these enzymes. Lo and behold, amid new data reinforcing worries of potential sides effects of BACE therapy (see Part 2 and Part 3 of this series), there were also some notable positive signs. Researchers reported how BACE inhibitors prevent dystrophic neurites from forming in animal models of AD, and might even help resolve dystrophies that are already there when treatment starts. Inhibitors also reined in neural hyperactivity in mice and restored long-range electrical synchronies that may be crucial for memory consolidation.

Dispatching Dystrophies
Researchers led by Jochen Herms at Ludwig Maximilians University, Munich, have used cranial windows in mice to monitor what inhibiting BACE does to neuritic dystrophy. The term denotes axons and dendrites swelling up near amyloid plaques. Packed as they are with BACE and APP, dystrophic neurites are potential sites for rampant Aβ production. To visualize the neurites, the researchers crossed APPPS1 transgenic mice with the VGlut1 venus mouse, which expresses a highly fluorescent form of green fluorescent protein in glutamatergic synapses. 

Herms showed exquisite images of dystrophic neurites that emerged beside plaques. These swellings grew over a period of weeks. Electron microscopy showed they were full of vesicles and proteins, including BACE and the lysosomal marker LAMP1. This could be a sign that late endosomes are getting stuck in the neurites and their proteins are not being properly degraded by the lysosomal system, said Herms. After about 12 weeks, the dystrophic neurites fizzled out. This most likely reflects outright destruction of the dystrophy, said Herms, but he also thinks that some neurons may be repairing dystrophies that lie furthest from plaques. 

How would plaques and neurites respond to BACE inhibition? Herms treated the animals with NB-360, Novartis’ forerunner to CNP520, which is being tested in the Generation prevention trial of people who have two copies of the ApoE4 gene (see Aug 2016 conference news). In contrast to untreated mice, which accumulated new plaques and new dystrophies, the NB-360-treated animals made little of either. “The size of the dystrophic parenchyma stayed the same or grew only very slightly,” said Herms.

His talk spurred extensive discussion. Christian Haass, also of Ludwig Maximilians University, was intrigued that dystrophies only seemed to form after plaques had appeared, suggesting the plaques, and not a soluble form of Aβ, are the source of toxicity in this model. Herms agreed. “We have looked at synapses before plaques form and we do not see any pathology, so we have no evidence for soluble oligomeric toxic Aβ,” he said.

Robert Vassar, Northwestern University, Chicago, reminded the audience of David Holtzman’s two-photon work more than 10 years ago that demonstrated that anti-Aβ antibodies could shrink at least the smaller dystrophic neurites (see Jan 2005 news). “The difference here is that you are not removing plaques, so maybe the combination of an antibody and a BACE inhibitor would rescue the dystrophies,” he suggested. Herms said he thought BACE inhibitors by themselves might rescue some of the dystrophic neurites. “Those furthest from plaques may improve,” he said. “This may turn out to be a positive effect of BACE inhibition.” Vassar co-organized the Seeon meeting with Stefan Lichtenthaler from the German Center for Neurodegenerative Diseases in Munich.

Correcting Circuitry
Another upside of BACE inhibition might be that it could restore electrical connectivity in the brain, some scientists believe. Hyperactivity underlies silent seizures in some mouse models of AD, and possibly in people in the early stages of dementia as well (see Sep 2007 news; May 2012 news). Marc Aurel Busche from the Technical University of Munich previously reported that Aβ immunotherapy exacerbated hyperactivity in mice, possibly by unleashing soluble forms of Aβ from plaques (see Nov 2015 news). What would BACE therapy do?

Working in Arthur Konnerth’s lab, Busche has perfected live, two-photon calcium imaging to measure the activation state of neurons. He can simultaneously quantify the activity of individual cells and—thanks to a CCD camera that captures high-resolution images of whole regions of the brain at once—the activity of long-range neural circuits. Busche used these techniques to characterize the effect of NB-360 on APP23xPS45 transgenic mice (see Busche et al., 2008). 

Tracing BACE. A green fluorescent protein tag allowed Tesco and colleagues to track BACE1 in axons. The somatodendritic protem MAP2B is labelled red. [Image courtesy of Selene Lomoio.]

Busche treated six- to seven-month-old mice for six weeks with inhibitor or placebo, then tested them in the Morris water maze. Then he imaged brain calcium by two-photon microscopy before sacrificing the animals to measure brain Aβ. The treated mice had fewer plaques and less soluble Aβ than controls, and navigated more quickly to the hidden platform in the water maze. They had almost no hyperactive neurons. Busche thinks NB-360 reduced hyperactivity by blocking Aβ production and allowing the mouse brain to fully expunge any Aβ oligomers that were loitering around the brain—Charlie Glabe’s OC antibody, which recognizes an epitope found in fibrillar oligomers but not soluble Aβ, failed to bind near plaques in the treated animals, but did in controls. 

Finally, Busche reported that the BACE inhibitor cleared up disturbances in slow-wave oscillations. These patterns of long-range, synchronized firing spread over the entire brain when an animal is asleep or anesthetized. Scientists believe they help consolidate memories. In APP/PS mice, this synchrony breaks down as oscillations between the frontal and occipital cortices become uncoupled (see Busche et al., 2015). Camera-based calcium imaging of whole brain hemispheres revealed that NB-360 restored this coupling and that this restoration correlated with improved learning and memory.

Overall, Busche said he thinks that Aβ leads to hyperactivity, which breaks long-range circuits, which in turn leads to memory impairment. He suggested using functional MRI or EEG recordings to identify people with long-range circuity problems as potential candidates for BACE inhibition.

Researchers at Seeon were intrigued by the opposite effects the BACE inhibitor and immunotherapy appear to have on hyperactivity. Some asked if the type of immunotherapy used might be critical, since some antibodies might solubilize toxic forms of Aβ, while others corral them. “From a therapeutic perspective, this is extremely important question to answer,” agreed Busche.

At a more basic level, researchers still have no idea what kick-starts the dystrophy that might be a driving force behind many of these electrical disturbances in the brain. Hints came from Giuseppina Tesco, who studies BACE1 trafficking in her lab at Tufts University, Boston. In prior work, she found that transport of the protease depends on the GGA3 protein, which helps recycle other proteins from the cell membrane (see Jun 2007 news; Dec 2013 conference news). Most of the work on GGA3 and BACE has been done in non-polarized cells, but now Selene Lomoio in Tesco’s lab did this in primary neurons.

Lomoio capitalizes on the super-high resolution of total internal reflection fluorescence microscopy to track the movement of molecules in living cells. Tesco showed live images of both GGA3 and BACE1 scooting along axons (see movie below). Both travelled antero- and retrogradely at about the same speed, though BACE sped along faster than GGA3, suggesting that other carriers might contribute to trafficking of the protease. Nonetheless, in GGA3 knockout neurons, most BACE vesicles just stopped, even though other proteins, such as synaptophysin, motored along at normal speed. Furthermore, in GGA3 KOs, BACE accumulated in swollen axons that looked very like dystrophic neurites. Tesco said the swellings most likely reflect defective trafficking of molecules to the lysosome for degradation.

Others were impressed by the quality of Tesco’s data and peppered her with questions about what it might imply about dystrophic neurites. Tesco does not know if GGA3 levels drop in dystrophic neurites in AD—unfortunately, GGA3 antibodies are not good enough for immunostaining. Others wondered if any toxic Aβ species block GGA trafficking or if loss of GGA3 leads to an increase in production of Aβ from APP. Tesco agreed all these were testable hypotheses. “This experimental approach could yield mechanistic insight into the accumulation of BACE1 in dystrophic neurites in AD,” she said.

Tesco’s findings gibe with work from Vassar’s lab at Northwestern University, Chicago. He reported that BACE1 accumulates in dystrophic neurites in AD, where it increases cleavage of APP and releases Aβ (see Sadleir et al., 2016).—Tom Fagan

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References

News Citations

1. What Exactly Does BACE Do in Adults? 26 Oct 2016
2. BACE Inhibition and the Synapse—Insights from Seeon 28 Oct 2016
3. New Ways to Target Aβ and BACE Show Promising Phase 1 Data 23 Aug 2016
4. Window to the Brain Shows Dystrophic Neurites Shrinking 31 Jan 2005
5. Do "Silent" Seizures Cause Network Dysfunction in AD? 7 Sep 2007
6. Epilepsy Drug Calms the Hippocampus, Aids Memory 9 May 2012
7. Aβ Immunotherapy Revs Up Neurons in Mice—What About in People? 13 Nov 2015
8. Stress and Aβ—The Apoptosis Connection 11 Jun 2007
9. Meeting Explores Complex Biology of BACE Regulation 26 Dec 2013

Antibody Citations

1. Amyloid fibrils and fibrillar oligomers

Paper Citations

1. Busche MA, Eichhoff G, Adelsberger H, Abramowski D, Wiederhold KH, Haass C, Staufenbiel M, Konnerth A, Garaschuk O. Clusters of hyperactive neurons near amyloid plaques in a mouse model of Alzheimer's disease. Science. 2008 Sep 19;321(5896):1686-9. PubMed.
2. Busche MA, Kekuš M, Adelsberger H, Noda T, Förstl H, Nelken I, Konnerth A. Rescue of long-range circuit dysfunction in Alzheimer's disease models. Nat Neurosci. 2015 Nov;18(11):1623-30. Epub 2015 Oct 12 PubMed.
3. Sadleir KR, Kandalepas PC, Buggia-Prévot V, Nicholson DA, Thinakaran G, Vassar R. Presynaptic dystrophic neurites surrounding amyloid plaques are sites of microtubule disruption, BACE1 elevation, and increased Aβ generation in Alzheimer's disease. Acta Neuropathol. 2016 Aug;132(2):235-56. Epub 2016 Mar 18 PubMed.

Further Reading

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