Aims

Amyloid-beta species, e.g. Abeta40 and Abeta42, can further undergo amyloidolytic processing by BACE1 in vitro and in vivo (Liebsch et al., 2019), resulting in the production of the metastable intermediate, Abeta34. Besides being classified as a non-amyloidogenic Abeta species, Abeta34 is an important new marker of amyloid clearance. However, little is known about how Abeta34 is produced and removed from the cell. Therefore, we aim to elucidate the molecular details of Abeta34 metabolism.

Methods

To locate Abeta34 in the cell and to elucidate Abeta-degrading enzymes involved in its further degradation, we performed (i) siRNA-mediated knockdown of candidate enzymes and (ii) transient overexpression of such proteases in SH-SY5Y neuroblastoma cells. Expression levels of proteins were verified by Western blotting and Abeta species were analyzed by ELISA and MALDI mass spectrometry.

Results

Endosomes and lysosomes are the sites where BACE1 is located and active. Therefore, these sites provide optimal conditions for Abeta34 production by BACE1, and so for its degradation by the candidate enzymes, namely Endothelin Converting Enzymes, Insulin Degrading Enzyme, Cathepsin B and Cathepsin D. We found that BACE1 generates Abeta34 in intracellular compartments mainly from longer Abeta species and identified the proteases that have a major role in Abeta34 degradation.

Conclusions

It is important to elucidate the cellular pathways that lead to Abeta34 production and degradation because there is a fundamental need to better understand how current inhibitors impact the balance between BACE1’s amyloidogenic (e.g. APP→Abeta40 and Abeta42) and amyloidolytic activities (e.g. Abeta40 or Abeta42→Abeta34).

Abstract Body

Objectives:

The beta-site APP cleaving enzyme (BACE1) is known for its “amyloidogenic” activity which leads to the production of Abeta40 and Abeta42 peptides. We recently discovered a novel “amyloidolytic” activity for BACE1 whereby it degrades longer Abeta peptides into a common, non-toxic Abeta34 intermediate (Liebsch et al., 2019). Our current work examined whether altered BACE1 activity shifts the equilibrium between Abeta production (Abeta40, Abeta42) and clearance (Abeta34), and how the generation of certain N-terminally truncated Abeta peptides is influenced.

Methods:

Pericytes are vascular mural cells within the neurovascular unit, essential for the blood brain barrier and able to metabolize amyloid peptides (Kirabali et al., 2019). Thus, we compared pericytes with neuroblastoma cells (SH-SY5Y) and organotypic brain slice cultures to analyze the production and clearance of Abeta species by immunoprecipitation – mass spectrometry (IP-MS) and our ultra-sensitive multiplexing assay (custom MSD).

Results:

In the presence of surplus BACE1, longer Abeta peptides were processed via the “amyloidolytic” pathway, yielding elevated levels of Abeta34. N-terminally truncated peptides (Abeta5-x) were detected when BACE1 activity was reduced; the truncated species can aggregate and gain toxic properties in combination with other Abeta peptides.

Conclusions:

Our new findings caution that clinical trials that have utilized BACE1 inhibitors may have inadvertently attenuated (i) the favourable degradation of toxic Abeta species into non-toxic Abeta34 and (ii) increased the generation of N-terminally truncated Aβ peptides. The influence of truncated Abeta5-x species on amyloid seeding, aggregation, and toxicity is not well understood and represents an exciting new area of APP biology.

Aims

Amyloid-beta species, e.g. Abeta40 and Abeta42, can further undergo amyloidolytic processing by BACE1 in vitro and in vivo (Liebsch et al., 2019), resulting in the production of the metastable intermediate, Abeta34. Besides being classified as a non-amyloidogenic Abeta species, Abeta34 is an important new marker of amyloid clearance. However, little is known about how Abeta34 is produced and removed from the cell. Therefore, we aim to elucidate the molecular details of Abeta34 metabolism.

Methods

To locate Abeta34 in the cell and to elucidate Abeta-degrading enzymes involved in its further degradation, we performed (i) siRNA-mediated knockdown of candidate enzymes and (ii) transient overexpression of such proteases in SH-SY5Y neuroblastoma cells. Expression levels of proteins were verified by Western blotting and Abeta species were analyzed by ELISA and MALDI mass spectrometry.

Results

Endosomes and lysosomes are the sites where BACE1 is located and active. Therefore, these sites provide optimal conditions for Abeta34 production by BACE1, and so for its degradation by the candidate enzymes, namely Endothelin Converting Enzymes, Insulin Degrading Enzyme, Cathepsin B and Cathepsin D. We found that BACE1 generates Abeta34 in intracellular compartments mainly from longer Abeta species and identified the proteases that have a major role in Abeta34 degradation.

Conclusions

It is important to elucidate the cellular pathways that lead to Abeta34 production and degradation because there is a fundamental need to better understand how current inhibitors impact the balance between BACE1’s amyloidogenic (e.g. APP→Abeta40 and Abeta42) and amyloidolytic activities (e.g. Abeta40 or Abeta42→Abeta34).