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. 2016 Oct 5;11(1):67.
doi: 10.1186/s13024-016-0134-z.

Seizure Protein 6 and Its Homolog Seizure 6-like Protein Are Physiological Substrates of BACE1 in Neurons

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Free PMC article

Seizure Protein 6 and Its Homolog Seizure 6-like Protein Are Physiological Substrates of BACE1 in Neurons

Martina Pigoni et al. Mol Neurodegener. .
Free PMC article

Abstract

Background: The 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 results: We 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.

Conclusions: This 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.

Keywords: Alzheimer’s disease; BACE1; BACE2; Biomarker; Neuroproteomics; SEZ6; SEZ6L; Secretase.

Figures

Fig. 1
Fig. 1
Specificity of SEZ6 and SEZ6L monoclonal antibodies. a Membranes from mouse brains were probed with the indicated antibodies against SEZ6, SEZ6L, SEZ6L2 or calnexin. Brains were collected from wild type (WT), SEZ6-/- (SEZ6 KO), SEZ6L2-/- (SEZ6L2 KO) or triple knock-out (TKO) mice lacking SEZ6, SEZ6L and SEZ6L2. b Lysates from primary neurons were treated with peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) and blotted for SEZ6 and SEZ6L. For SEZ6, a polyclonal antibody was used in the deglycosylation experiment. * indicates mature SEZ6, ** indicates immature SEZ6. c, d Immunohistochemistry of TKO and WT brains using antibody against SEZ6 (c) or SEZ6L (d)
Fig. 2
Fig. 2
BACE1 is required for SEZ6 and SEZ6L shedding in primary neurons and mouse brain. a Schematic diagram of SEZ6 and SEZ6L domain structure and proposed proteolytic processing. b Detection of soluble SEZ6 and SEZ6L ectodomains (sSEZ6 and sSEZ6L) and full-length SEZ6 and SEZ6L in neuronal supernatant and lysate upon C3 treatment. c Detection of sSEZ6 and sSEZ6L and full-length SEZ6 and SEZ6L in BACE1 KO and WT brains. Brains were separated into soluble fraction (DEA) and membranes (membrane). Note that in this figure, a different molecular weight marker has been used compared to Fig. 1. The 148 kDa band corresponds to the band detected at 170 kDa in Fig. 1. The upper band in panel 2C (*) is due to unspecific signal. Densitometric quantitations of the Western blots are shown, (*; p < 0.05, **; p < 0.01, two-tailed Mann-Whitney test n = 6)
Fig. 3
Fig. 3
BACE2 but not BACE1 cleaves SEZ6 and SEZ6L in a pancreatic β-cell line. a sSEZ6 and sSEZ6L were detected in the supernatant and full-length SEZ6 and SEZ6L in the lysate of the pancreatic β-cell line MIN6 upon BACE1 and BACE2 knock-down by siRNA (siB1, siB2). As a control, cells were treated with non-silencing control siRNA (siCon). Densitometric quantitations of the Western blots are shown, (*; p < 0.05, two-tailed Mann-Whitney test n = 4). b BACE1 (green lines) and BACE2 (blue lines) activity were quantified in enzymatic (solid lines) and cellular (dotted lines) models after pharmacological inhibition with nonselective (inhibiting both BACE1 and BACE2, compounds B and C) and BACE1-selective inhibitors (compound A). Soluble Aβ42 as well as sSEZ6 and sSEZ6L were detected in the supernatant of the neuroblastoma cell line SK-N-BE(2) or in the MIN6 respectively, as indicated. Data were standardized to low and high controls within each assay. Data represent biological duplicates with two or more technical replicates
Fig. 4
Fig. 4
BACE1 controls neuronal cell surface levels of SEZ6 and SEZ6L. Primary, murine neurons were treated with the BACE inhibitor C3 or DMSO as a control. Proteins at the surface were labeled with biotin and enriched using streptavidin pull-down. Biotinylated SEZ6 and SEZ6L were detected by immunoblot. As a control, both proteins were also detected in whole cell lysates. Note, that only the mature 170 kDa form of SEZ6 was biotinylated at the cell surface. As a control, the ADAM10 substrate LDL receptor (LDLR) did not show a change in surface levels upon C3-treatment. As a further control, the cytosolic protein actin was only detected in whole lysates, but not among the surface biotinylated proteins
Fig. 5
Fig. 5
Cleavage site determination of SEZ6. a Comparison of BACE1 cleavage sites in the known APP Swedish mutant, in SEZ6 and SEZ6L. Additionally, the peptide (SEZ6 pep) used for the in vitro assay is aligned. Numbers next to the N- and C-terminal amino acids of the peptide indicate the amino acid number within the sequence of the full-length protein. Amino acids at the cleavage site are shown in green. Amino acids of the transmembrane domains are in red. Domains of SEZ6 and SEZ6L are shown with indicated symbols. The most C-terminal tryptic peptide of the secreted SEZ6 ectodomain detected in our previous study is underlined in black. b Extracted ion chromatogram of full-length peptide incubated with BACE1, BACE1 plus C3 or without BACE1 showing the peaks of the two cleavage products as well as the full-length peptide. Identification of the full-length peptide (c), the N-terminal (d) and the C-terminal cleavage product (e) by fragment ion spectra. The mapped y and b fragment ions are indicated in the sequences as well as in fragment ion spectra. Neutral loss fragment ions are indicated in light blue for b and orange for y ions
Fig. 6
Fig. 6
SEZ6 is a substrate for γ-secretase. a HEK293T cells were stably transfected with empty vector (Empty) or SEZ6 expression construct with an N-terminal HA-tag and a C-terminal FLAG epitope tag. Cells were treated with C3 or DMSO as a control. sSEZ6 was detected in the cell supernatant and full-length SEZ6 in the lysate. Calnexin was used as a loading control. b Cells were treated with DMSO, C3 or the γ-secretase inhibitor DAPT. The C-terminal SEZ6 fragment was detected by immunoblot using an anti-FLAG-tag antibody
Fig. 7
Fig. 7
Proteomic analysis of CSF from BACE DKO and WT mice. a Volcano plot of proteomic analysis of BACE1 and BACE2 double knockout (BACE DKO) and WT mouse CSF. The minus log10 transformed t-test p-values are plotted against the log2 transformed label-free quantification intensity ratios of BACE DKO and WT CSF for every relatively quantified protein. Proteins with a t-test p-value < 0.05 are shown as red circles. Already known BACE substrate candidates with a p-value < 0.05 are marked with gray filling. Proteins that remain significant after Benjamini-Hochberg false discovery rate correction (FDR < 0.05) have bold letters (SEZ6 and SEZ6L). b Detection of sSEZ6 and sSEZ6L in mouse CSF. Densitometric quantitation of the Western blot is shown, (**; p < 0.01, one-way ANOVA followed by two-tailed Student’s t-Test, n = 3). The dotted line indicates that the samples were loaded onto the same blot, but not next to each other

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References

    1. Vassar R, Kuhn PH, Haass C, Kennedy ME, Rajendran L, Wong PC, Lichtenthaler SF. Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects. J Neurochem. 2014;130:4–28. doi: 10.1111/jnc.12715. - DOI - PMC - PubMed
    1. Hussain I, Powell D, Howlett DR, Tew DG, Meek TD, Chapman C, Gloger IS, Murphy KE, Southan CD, Ryan DM, et al. Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci. 1999;14:419–27. doi: 10.1006/mcne.1999.0811. - DOI - PubMed
    1. Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, et al. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature. 1999;402:537–40. doi: 10.1038/990114. - DOI - PubMed
    1. Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999;286:735–41. doi: 10.1126/science.286.5440.735. - DOI - PubMed
    1. Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM, Brashier JR, Stratman NC, Mathews WR, Buhl AE, et al. Membrane-anchored aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature. 1999;402:533–7. doi: 10.1038/990107. - DOI - PubMed

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