Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neuronsReport as inadecuate




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Molecular Neurodegeneration

, 11:67

First Online: 05 October 2016Received: 02 July 2016Accepted: 28 September 2016

Abstract

BackgroundThe protease BACE1 beta-site APP cleaving enzyme is a major drug target in Alzheimer’s disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 SEZ6 and its homolog SEZ6L.

Methods and resultsWe generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins sSEZ6, sSEZ6L were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid CSF and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice.

ConclusionsThis study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice.

KeywordsAlzheimer’s disease BACE1 BACE2 Secretase Neuroproteomics Biomarker SEZ6 SEZ6L AbbreviationsADAlzheimer’s disease

APPAmyloid precursor protein

AβAmyloid β peptide

BACE1β-site APP cleaving enzyme

BSABovine Serum Albumin

CSFCerebrospinal fluid

DIVDays in vitro

Endo HEndoglycosidase H

HEK293THuman embryonic kidney 293

HEPHHephaestin

IL6STInterleukin-6 receptor subunit beta

KOKnock out

LDLRLDL-receptor

LFQLabel-free quantification

MSDMesoscale discovery

PBSPhosphate buffered saline

PNGase FPeptide-N-Glycosidase

SDCSodium deoxycholate

SEZ6Seizure protein 6

SEZ6LSEZ6-like

SEZ6L2SEZ6-like 2

SLICLigase independent cloning

sSEZ6, sSEZ6LSoluble SEZ6 and SEZ6L

WTWild type

Electronic supplementary materialThe online version of this article doi:10.1186-s13024-016-0134-z contains supplementary material, which is available to authorized users.

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Author: Martina Pigoni - Johanna Wanngren - Peer-Hendrik Kuhn - Kathryn M. Munro - Jenny M. Gunnersen - Hiroshi Takeshima - Regina

Source: https://link.springer.com/article/10.1186/s13024-016-0134-z







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