Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons

In the paper

“because BACE1 also negatively regulates surface levels and/or function of two other substrates, contactin-2 and CHL1 [12, 15, 53].”

What the sources say

• Disputed

  The Discussion (page unavailable) says BACE1 cleavage of CHL1 is necessary for Sema3A-induced growth cone collapse, and that the resulting CHL1 fragment transmits the signal. That is contrary to the claim that BACE1 negatively regulates CHL1 function. Our copy does not address contactin-2 or establish a reduction in surface levels. (LLM, conf=0.84)

  Checked by a reasoning model — may be wrong.

  cites [12] Barao S, Gartner A, Leyva-Diaz E, Demyanenko G, Munck S, Vanhoutvin T, Zhou L, Schachner M, Lopez-Bendito G, Maness PF, De Strooper B. Antagonistic effects of BACE1 and APH1B-gamma-secretase control axonal guidance by regulating growth cone collapse. Cell Rep. 2015;12:1367–76. sourceExperimental — accuracy not yet measured
• Partly supported

  The section “BACE1 inhibition increases cell surface levels of contactin-2” directly states that BACE1 negatively regulates contactin-2 at the neuronal plasma membrane. “Validation of BACE1 substrates in primary neurons” reports reduced CHL1 shedding and increased full-length protein in cell lysates after BACE1 inhibition, but does not establish reduced CHL1 surface levels or function. (LLM, conf=0.86)

  cites [15] Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68. sourceExperimental — accuracy not yet measured
• Supported

  The Results and discussion section (Figure 5) reports that BACE1 overexpression decreased surface contactin-2 in mouse primary neurons, while BACE1 inhibition increased it. This supports the contactin-2 part attributed to this paper; the same section identifies Hitt et al. as the source of the CHL1 finding, which the citing sentence supports with multiple citations. (LLM, conf=0.91)

  cites [53] Gautam V, D’Avanzo C, Hebisch M, Kovacs DM, Kim DY. BACE1 activity regulates cell surface contactin-2 levels. Mol Neurodegener. 2014;9:4. source

In the paper

“BACE1 cleaves the amyloid precursor protein (APP) and thus catalyzes the first step in generation of the amyloid β peptide (Aβ) [2–5]”

What the sources say

• ?Not checked — no full text of the sourcecites [2] 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. source
• ?Not checked — no full text of the sourcecites [3] 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. source
• Supported

  The introductory passage indexed under Abstract (page not provided) states that β-secretase cleaves APP to initiate Aβ formation, before further cleavage by γ-secretase. The purified-BACE section shows that BACE directly cleaves APP-derived substrates at the β-secretase site. (LLM, conf=0.94)

  cites [4] 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. source
• ?Not checked — no full text of the sourcecites [5] 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. source

In the paper

“The three members of the seizure protein 6 (SEZ6) family, namely SEZ6, SEZ6-like (SEZ6L) and SEZ6-like 2 (SEZ6L2) have been identified as candidate BACE1 substrates in different studies [15, 17], but have not yet been validated in detail.”

What the sources say

• Partly supported

  Table I and the Discussion identify SEZ6, SEZ6L1 and SEZ6L2 as BACE1 substrate candidates, with SEZ6L2 showing less dependence on BACE1 for shedding. However, the sections on validation in primary neurons and BACE1-deficient mouse brains report further validation of SEZ6. The claim that the family members had not yet been validated in detail therefore does not hold for SEZ6 in this source. (LLM, conf=0.87)

  cites [15] Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68. sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion (page unavailable) reports that the SEZ6 family had been shown to be cleaved by BACE1 in primary neurons. But this paper does not identify all three named proteins as candidate BACE1 substrates: it investigates SEZ6L and SEZ6L2 and validates them as BACE2-specific substrates in pancreatic islet β-cells. It also does not establish the claim that the BACE1 candidates had not been validated in detail. (LLM, conf=0.86)

  cites [17] Stutzer I, Selevsek N, Esterhazy D, Schmidt A, Aebersold R, Stoffel M. Systematic proteomic analysis identifies beta-site amyloid precursor protein cleaving enzyme 2 and 1 (BACE2 and BACE1) substrates in pancreatic beta-cells. J Biol Chem. 2013;288:10536–47. sourceExperimental — accuracy not yet measured

In the paper

“The SEZ6 family controls synaptic connectivity and motor coordination in mice [18, 19], but little is known about the functions of these proteins at the molecular level.”

What the sources say

• Partly supported

  The Abstract reports fewer excitatory synapses and impaired motor coordination in Sez-6-null mice, supporting both effects. This paper studies Sez-6 and its isoforms, however, rather than establishing that the SEZ6 family as a whole controls these outcomes. (LLM, conf=0.86)

  cites [18] Gunnersen JM, Kim MH, Fuller SJ, De Silva M, Britto JM, Hammond VE, Davies PJ, Petrou S, Faber ES, Sah P, Tan SS. Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons. Neuron. 2007;56:621–39. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [19] Miyazaki T, Hashimoto K, Uda A, Sakagami H, Nakamura Y, Saito SY, Nishi M, Kume H, Tohgo A, Kaneko I, et al. Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family. FEBS Lett. 2006;580:4057–64. source

In the paper

“The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease.”

• Also stated in: § Background · plain

  “The β-secretase BACE1 (β-site APP cleaving enzyme) is a key drug target in Alzheimer’s disease (AD) [1].”

What the sources say

• Supported

  The Outlook section says BACE1 has taken center stage in Alzheimer’s disease drug-development programs and that around a dozen companies have BACE inhibitors in clinical trials. This supports calling BACE1 a major drug target. (LLM, conf=0.94)

  cites [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. source

In the paper

“More than 40 substrate candidates for BACE1 were identified in recent proteomic studies in murine neurons or cerebrospinal fluid (CSF), but only a few of them have been validated to date with functional or in vitro assays, including L1, CHL1, ENPP5 and PTPRN2 [12, 14–16].”

What the sources say

• Partly supported

  The Introduction (page unavailable) reports a long list of potential BACE1 substrates, and the paper investigates the functional consequences of BACE1 processing of CHL1. It does not give the figure of more than 40, specify proteomic studies of cerebrospinal fluid, or address validation of ENPP5 and PTPRN2. (LLM, conf=0.85)

  cites [12] Barao S, Gartner A, Leyva-Diaz E, Demyanenko G, Munck S, Vanhoutvin T, Zhou L, Schachner M, Lopez-Bendito G, Maness PF, De Strooper B. Antagonistic effects of BACE1 and APH1B-gamma-secretase control axonal guidance by regulating growth cone collapse. Cell Rep. 2015;12:1367–76. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [14] Dislich B, Wohlrab F, Bachhuber T, Müller SA, Kuhn P-H, Hogl S, Meyer-Luehmann M, Lichtenthaler SF. Label-free quantitative proteomics of mouse cerebrospinal fluid detects β-Site APP Cleaving Enzyme (BACE1) protease substrates in vivo. Mol Cell Proteomics. 2015;14:2550–63. source
• Partly supported

  The ‘Identification of neuronal BACE1 sheddome’ section reports 34 candidate membrane-protein substrates in mouse neurons, and the validation sections confirm L1 and CHL1 as physiological substrates. This paper alone does not establish a total of more than 40 substrate candidates across neuronal and CSF studies, or validate ENPP5 and PTPRN2. (LLM, conf=0.85)

  cites [15] Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [16] Zhou L, Barao S, Laga M, Bockstael K, Borgers M, Gijsen H, Annaert W, Moechars D, Mercken M, Gevaert K, De Strooper B. The neural cell adhesion molecules L1 and CHL1 are cleaved by BACE1 protease in vivo. J Biol Chem. 2012;287:25927–40. source

In the paper

“BACE1 is highly expressed in the nervous system and contributes to additional physiological processes besides its role in AD, e.g. through neuregulin-1 cleavage in myelination and CHL1 cleavage in axon targeting [7–12].”

What the sources say

• ?Not checked — no full text of the sourcecites [7] Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, DeStrooper B, Saftig P, Birchmeier C, Haass C. Control of peripheral nerve myelination by the beta-secretase BACE1. Science. 2006;314:664–6. source
• ?Not checked — no full text of the sourcecites [8] Hu X, Hicks CW, He W, Wong P, Macklin WB, Trapp BD, Yan R. Bace1 modulates myelination in the central and peripheral nervous system. Nat Neurosci. 2006;9:1520–5. source
• Partly supported

  The Introduction says BACE1 processes neuregulin-1 type III, a regulator of peripheral nervous system myelination, and the Discussion reports that a BACE1-liberated neuregulin domain promotes myelination in zebrafish. The passages do not support the sentence’s separate claims that BACE1 is highly expressed in the nervous system or that CHL1 cleavage contributes to axon targeting. (LLM, conf=0.86)

  cites [9] Fleck D, van Bebber F, Colombo A, Galante C, Schwenk BM, Rabe L, Hampel H, Novak B, Kremmer E, Tahirovic S, et al. Dual cleavage of neuregulin 1 type III by BACE1 and ADAM17 liberates its EGF-like domain and allows paracrine signaling. J Neurosci. 2013;33:7856–69. sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction reports that Bace1-dependent shedding of neuregulin-1 is implicated in peripheral nervous system myelination, and the Results say Bace1 is broadly expressed in sensory neurons. The copy does not establish high expression throughout the nervous system. Its References list a study of CHL1 cleavage by Bace1, but no passage links that cleavage to axon targeting. (LLM, conf=0.88)

  cites [10] Cheret C, Willem M, Fricker FR, Wende H, Wulf-Goldenberg A, Tahirovic S, Nave KA, Saftig P, Haass C, Garratt AN, et al. Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles. Embo J. 2013;32:2015–28. sourceExperimental — accuracy not yet measured
• Supported

  The Introduction reports robust BACE1 expression in hippocampal mossy-fiber terminals and describes reduced neuregulin-1 cleavage as a cause of hypomyelination. The Abstract and Introduction report BACE1-dependent CHL1 processing in the hippocampus and olfactory bulb and link its loss to axon-guidance defects. Together, these passages support the cited examples of nervous-system functions beyond BACE1’s role in Alzheimer disease. (LLM, conf=0.86)

  cites [11] Hitt B, Riordan SM, Kukreja L, Eimer WA, Rajapaksha TW, Vassar R. beta-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)-deficient mice exhibit a close homolog of L1 (CHL1) loss-of-function phenotype involving axon guidance defects. J Biol Chem. 2012;287:38408–25. source
• Partly supported

  The Introduction (page unavailable) identifies neuregulin-1 type III as a BACE1 substrate implicated in myelination, and the Discussion reports that BACE1 cleavage of CHL1 is necessary for growth-cone collapse during axonal guidance. The copy does not establish that BACE1 is highly expressed in the nervous system. The Introduction also says the precise link between neuregulin-1 processing and its function is unknown, so the claim's cleavage-to-myelination mechanism is more definite than the source supports. (LLM, conf=0.87)

  cites [12] Barao S, Gartner A, Leyva-Diaz E, Demyanenko G, Munck S, Vanhoutvin T, Zhou L, Schachner M, Lopez-Bendito G, Maness PF, De Strooper B. Antagonistic effects of BACE1 and APH1B-gamma-secretase control axonal guidance by regulating growth cone collapse. Cell Rep. 2015;12:1367–76. sourceExperimental — accuracy not yet measured

In the paper

“However, the proteomic data for SEZ6L have not been validated by other methods and another proteomic study using pancreatic cells and tissue failed to confirm SEZ6L as a BACE1 substrate. Instead, that study demonstrated that SEZ6L is cleaved by the BACE1-homolog BACE2 in pancreas [17].”

What the sources say

• Supported

  In “Validation and Characterization of SEZ6L and SEZ6L2 as Specific BACE2 Substrates in Pancreatic β-Cells” (page not provided), SEZ6L shedding persisted in BACE1-deficient islets but was nearly undetectable in BACE2-deficient islets. The same section concludes that BACE2 is sufficient and required for SEZ6L cleavage in pancreatic β-cells, supporting the cited sentence’s pancreas-specific contrast. (LLM, conf=0.93)

  cites [17] Stutzer I, Selevsek N, Esterhazy D, Schmidt A, Aebersold R, Stoffel M. Systematic proteomic analysis identifies beta-site amyloid precursor protein cleaving enzyme 2 and 1 (BACE2 and BACE1) substrates in pancreatic beta-cells. J Biol Chem. 2013;288:10536–47. source

In the paper

“which is very efficiently cleaved by BACE1 [4].”

What the sources say

• Supported

  The section “Purified BACE cleaves APP substrates and exhibits the properties of β-secretase” reports that purified BACE cleaves a Swedish-mutant APP peptide with much higher specific activity than a wild-type APP peptide. The cell experiments also report increased β-secretase cleavage of Swedish-mutant APP when BACE is overexpressed. The supplied passages have no page numbers. (LLM, conf=0.93)

  cites [4] 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. source

In the paper

“The previously identified cleavage site in SEZ6L [15] is not identical, but similar to SEZ6, as it also has a hydrophobic amino acid in the P1 and a negatively charged amino acid in the P1’ position (Fig. 5a).”

What the sources say

• Partly supported

  The Identification of neuronal BACE1 sheddome section reports a SEZ6L1 semi-tryptic peptide consistent with direct BACE1 cleavage. The Discussion proposes a leucine–aspartate cleavage site for SEZ6, but our copy does not give the amino acids flanking the SEZ6L1 site or establish the claimed similarity between the two sites. (LLM, conf=0.82)

  cites [15] Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68. sourceExperimental — accuracy not yet measured

In the paper

“Recent proteomic studies have identified more than 40 membrane proteins as potential BACE1 substrates [14–16].”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Dislich B, Wohlrab F, Bachhuber T, Müller SA, Kuhn P-H, Hogl S, Meyer-Luehmann M, Lichtenthaler SF. Label-free quantitative proteomics of mouse cerebrospinal fluid detects β-Site APP Cleaving Enzyme (BACE1) protease substrates in vivo. Mol Cell Proteomics. 2015;14:2550–63. source
• Partly supported

  The 'Identification of neuronal BACE1 sheddome' section reports 34 membrane proteins among 40 proteins whose shedding or secretion was reduced. It supports the identification of potential BACE1 substrates, but does not establish the citing sentence’s total of more than 40 membrane proteins; six of its 40 proteins were soluble. (LLM, conf=0.87)

  cites [15] Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [16] Zhou L, Barao S, Laga M, Bockstael K, Borgers M, Gijsen H, Annaert W, Moechars D, Mercken M, Gevaert K, De Strooper B. The neural cell adhesion molecules L1 and CHL1 are cleaved by BACE1 protease in vivo. J Biol Chem. 2012;287:25927–40. source

In the paper

“BACE1 is a major drug target in AD, but has additional substrates and thus contributes to various biological processes [1, 13], which may limit its therapeutic potential.”

What the sources say

• Supported

  The Outlook identifies BACE1 as a focus of AD drug development and warns that inhibiting its other functions could cause side effects. The section on therapeutic potential says that newly identified substrates and biological functions have raised safety concerns about chronic BACE inhibition. This supports the claim’s qualified statement that those functions may limit its therapeutic potential. (LLM, conf=0.94)

  cites [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. source
• ?Not checked — no full text of the sourcecites [13] Barao S, Moechars D, Lichtenthaler SF, De Strooper B. BACE1 physiological functions may limit its use as therapeutic target for alzheimer’s disease. Trends Neurosci. 2016;39:158–69. source

In the paper

“Notably, both BACE1- and SEZ6-deficient mice have deficits in hippocampal learning paradigms [18, 54–56] and in motor coordination [18, 57].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [18] Gunnersen JM, Kim MH, Fuller SJ, De Silva M, Britto JM, Hammond VE, Davies PJ, Petrou S, Faber ES, Sah P, Tan SS. Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons. Neuron. 2007;56:621–39. source
• ?Not checked — no full text of the sourcecites [54] Ohno M, Sametsky EA, Younkin LH, Oakley H, Younkin SG, Citron M, Vassar R, Disterhoft JF. BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer’s disease. Neuron. 2004;41:27–33. source
• ?Not checked — no full text of the sourcecites [55] Ohno M, Chang L, Tseng W, Oakley H, Citron M, Klein WL, Vassar R, Disterhoft JF. Temporal memory deficits in Alzheimer’s mouse models: rescue by genetic deletion of BACE1. Eur J Neurosci. 2006;23:251–60. source
• Partly supported

  The section “BACE1 is critical for cognitive and emotional functions” reports deficits in hippocampus-dependent tasks in BACE1-deficient mice. The section on APP and PS1 transgenes reports slower swimming, but does not establish a deficit in motor coordination. This paper also does not study SEZ6-deficient mice; the sentence cites other sources for that part. (LLM, conf=0.85)

  cites [56] Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, et al. BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J Neurosci. 2005;25:11693–709. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [57] Kobayashi D, Zeller M, Cole T, Buttini M, McConlogue L, Sinha S, Freedman S, Morris RG, Chen KS. BACE1 gene deletion: impact on behavioral function in a model of Alzheimer’s disease. Neurobiol Aging. 2008;29:861–73. source

In the paper

“Several other previously identified BACE1 substrates, such as CHL1, L1, contactin-2, APP and its homolog APLP2, are not exclusively cleaved by BACE1, but also by other proteases, including ADAM10 [15, 38].”

What the sources say

• Supported

  The Discussion says that, apart from a few nearly exclusive BACE1 substrates, the others are cleaved to varying degrees by additional proteases. The paper reports partial BACE1-dependent shedding of CHL1, L1 and contactin-2, identifies ADAM10 cleavage of APLP2 and L1, and describes APP cleavage by distinct proteases. Page numbers are not available in our copy. (LLM, conf=0.91)

  cites [15] Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68. source
• Partly supported

  The Results section (“Cross-talk between ADAM10 and BACE1”; page unavailable) reports cleavage of CHL1, L1, contactin-2 (CNTN2), and APP by both ADAM10 and BACE1. It does not establish the same point for APLP2; the APLP2 mention found in our copy is in a reference about synaptic function, not proteolytic cleavage. (LLM, conf=0.88)

  cites [38] Kuhn PH, Colombo AV, Schusser B, Dreymueller D, Wetzel S, Schepers U, Herber J, Ludwig A, Kremmer E, Montag D, et al. Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. Elife. 2016; 5, doi: 10.7554/eLife.12748 . sourceExperimental — accuracy not yet measured

In the paper

“Hephaestin is known to be expressed in the brain [45].”

What the sources say

• ?Could not verify

  The Results passages on brain-region and adult mouse brain proteomes describe protein expression generally but do not identify hephaestin. Searches of our copy for “Hephaestin” and “HEPH” found no passage establishing its expression in the brain. (LLM, conf=0.72)

  cites [45] Sharma K, Schmitt S, Bergner CG, Tyanova S, Kannaiyan N, Manrique-Hoyos N, Kongi K, Cantuti L, Hanisch U-K, Philips M-A, et al. Cell type- and brain region-resolved mouse brain proteome. Nat Neurosci. 2015;18:1819–31. sourceExperimental — accuracy not yet measured

In the paper

“However, the function of SEZ6 and SEZ6L may not only be exerted by the full-length proteins, but also by sSEZ6 and sSEZ6L or even by the C-terminal fragments resulting from BACE1 cleavage, as recently found for the BACE1 substrate CHL1 [12].”

What the sources say

• Supported

  The citation supports the sentence’s CHL1 example, not a demonstrated function of SEZ6 or SEZ6L fragments. The Discussion reports that the BACE1-generated, membrane-bound CHL1 C-terminal fragment was necessary and sufficient to restore Sema3A-induced growth cone collapse. The proposed analogous roles for SEZ6 and SEZ6L fragments remain the citing authors’ speculation. (LLM, conf=0.92)

  cites [12] Barao S, Gartner A, Leyva-Diaz E, Demyanenko G, Munck S, Vanhoutvin T, Zhou L, Schachner M, Lopez-Bendito G, Maness PF, De Strooper B. Antagonistic effects of BACE1 and APH1B-gamma-secretase control axonal guidance by regulating growth cone collapse. Cell Rep. 2015;12:1367–76. source

In the paper

“For example, the APP homolog APLP2 is cleaved to about 60 % by BACE1 and to 40 % by ADAM10 in neurons, but the percentages may strongly vary for each substrate [38].”

What the sources say

• Partly supported

  The Results section (‘Cross-talk between ADAM10 and BACE1’; page unavailable) reports that the contribution of the two proteases differs among neuronal substrates, supporting the claim's general qualification. But APLP2 is not among the substrates listed there, and no passage found gives the claimed 60% BACE1/40% ADAM10 split for APLP2. (LLM, conf=0.86)

  cites [38] Kuhn PH, Colombo AV, Schusser B, Dreymueller D, Wetzel S, Schepers U, Herber J, Ludwig A, Kremmer E, Montag D, et al. Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. Elife. 2016; 5, doi: 10.7554/eLife.12748 . sourceExperimental — accuracy not yet measured

In the paper

“Precedents for such a tissue-specific proteolytic cleavage are the BACE1 substrates CHL1 and L1, which are mostly cleaved by BACE1 in the nervous system, but by ADAM proteases in non-neuronal cells [15, 37].”

What the sources say

• Supported

  The discussion passage in the provided copy (labelled “Isolation of primary neurons”; page not supplied) explicitly gives CHL1 and L1 as examples of tissue-specific cleavage. It says both are mainly cleaved by BACE1 in neurons, while CHL1 is mainly cleaved by ADAM8 in non-neuronal tissue and L1 by ADAM10 in non-neuronal cells. (LLM, conf=0.95)

  cites [15] Kuhn PH, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, et al. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J. 2012;31:3157–68. source
• ?Not checked — no full text of the sourcecites [37] Reiss K, Cornelsen I, Husmann M, Gimpl G, Bhakdi S. Unsaturated fatty acids drive disintegrin and metalloproteinase (ADAM)-dependent cell adhesion, proliferation, and migration by modulating membrane fluidity. J Biol Chem. 2011;286:26931–42. source

In the paper

“One example is APP. BACE1 inhibition increases the ADAM10 cleavage of APP, such that total APP cleavage is only mildly reduced [36, 47].”

What the sources say

• Supported

  In Results > Embryonic primary cortical neurons (page unavailable), pharmacological BACE1 inhibition increased APPsα while reducing total shed APP to 78% of control. Results > ADAM10 mediates the increase of α-cleavage (page unavailable) identifies ADAM10 as responsible for that increase. The finding applies to primary neurons; the paper reports no corresponding increase in the cell lines studied. (LLM, conf=0.91)

  cites [36] Colombo A, Wang H, Kuhn PH, Page R, Kremmer E, Dempsey PJ, Crawford HC, Lichtenthaler SF. Constitutive alpha- and beta-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines. Neurobiol Dis. 2013;49:137–47. source
• Partly supported

  The Discussion (page unavailable) reports that BACE1 inhibition reduces the APP cleavage products C99 and sAPPβ. It does not report increased ADAM10 cleavage or establish that total APP cleavage is only mildly reduced. (LLM, conf=0.84)

  cites [47] May PC, Dean RA, Lowe SL, Martenyi F, Sheehan SM, Boggs LN, Monk SA, Mathes BM, Mergott DJ, Watson BM, et al. Robust central reduction of amyloid-beta in humans with an orally available, non-peptidic beta-secretase inhibitor. J Neurosci. 2011;31:16507–16. sourceExperimental — accuracy not yet measured

In the paper

“Moreover, both mouse lines appear to have reduced levels of anxiety and/or cognitive deficits [18, 56], reduced glutamatergic synapse function and reduced dendritic spine densities [18, 58].”

What the sources say

• Supported

  The Results report that Sez-6 knockout mice spent more time on the open arms of the elevated plus maze, had reduced dendritic spine density, and showed fewer excitatory synapses. The paper also reports fewer functional excitatory connections and a possible long-term memory deficit. Its anxiety finding is qualified: decreased anxiety was not apparent in the open-field or light/dark tests. This source establishes these findings for the Sez-6 mouse line; the other line is covered by the sentence’s other citations. (LLM, conf=0.88)

  cites [18] Gunnersen JM, Kim MH, Fuller SJ, De Silva M, Britto JM, Hammond VE, Davies PJ, Petrou S, Faber ES, Sah P, Tan SS. Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons. Neuron. 2007;56:621–39. source
• Partly supported

  The sections on cognitive and emotional functions report cognitive deficits and low anxiety in BACE1-null mice. The synaptic-plasticity section reports altered paired-pulse facilitation and de-depression, but normal basal AMPA- and NMDA-mediated transmission; it does not establish a general reduction in glutamatergic synapse function. No passage found in our copy reports reduced dendritic spine density, and this paper alone does not establish findings for both mouse lines. (LLM, conf=0.88)

  cites [56] Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, et al. BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J Neurosci. 2005;25:11693–709. sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [58] Savonenko AV, Melnikova T, Laird FM, Stewart KA, Price DL, Wong PC. Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizophrenia-like phenotypes in BACE1-null mice. Proc Natl Acad Sci U S A. 2008;105:5585–90. source

In the paper

“After initial BACE1 cleavage, the resulting C-terminal, membrane-bound protein fragments of several membrane proteins, including APP and SEZ6L [17], are further processed within their transmembrane domains by γ-secretase, in a process referred to as regulated intramembrane proteolysis [41]”

What the sources say

• Partly supported

  The Discussion identifies APP as a BACE1 substrate, but identifies SEZ6L as a BACE2-specific substrate in pancreatic islets; it also reports BACE1 cleavage of the Seizure 6 family in primary neurons. Thus the paper does not support unqualified BACE1 cleavage of SEZ6L in the studied cells. The later γ-secretase statement is attached to a different citation [41]. (LLM, conf=0.88)

  cites [17] Stutzer I, Selevsek N, Esterhazy D, Schmidt A, Aebersold R, Stoffel M. Systematic proteomic analysis identifies beta-site amyloid precursor protein cleaving enzyme 2 and 1 (BACE2 and BACE1) substrates in pancreatic beta-cells. J Biol Chem. 2013;288:10536–47. sourceExperimental — accuracy not yet measured
• Supported

  The RIP of APP section describes a membrane-bound C-terminal fragment formed after β-secretase cleavage and subsequently cleaved by γ-secretase. The Outlook figures identify sequential cleavage of APP by β- and γ-secretase as regulated intramembrane proteolysis. The separate citation [17] accompanies the mention of SEZ6L, so that identification is not attributed to this source. (LLM, conf=0.92)

  cites [41] Lichtenthaler SF, Haass C, Steiner H. Regulated intramembrane proteolysis--lessons from amyloid precursor protein processing. J Neurochem. 2011;117:779–96. source

In the paper

“Similarly, SEZ6L immunoreactivity (Fig. 1d) appeared strong in the neocortex and hippocampus, and protein localization in these areas was consistent with SEZ6L mRNA expression in the Allen Mouse Brain Atlas [34]. SEZ6L localized to pyramidal neurons throughout the cortex, particularly the apical dendrites (Fig. 1d), and appeared relatively lower in layer IV and VI. All regions of the hippocampus”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [34] Allen Mouse Brain Atlas [ http://mouse.brain-map.org/ ]

In the paper

“Likewise, in P7 BACE1 KO mouse brains sSEZ6L was strongly reduced in the diethylamine soluble DEA brain fraction, while full-length SEZ6L was increased in the membrane fraction (Fig. 2c).”

In the paper

“Taken together, these results demonstrate that SEZ6 and SEZ6L are N-glycosylated proteins and that the newly generated antibodies specifically detect endogenous SEZ6 and SEZ6L.”

• Also stated in: § Validation of new monoclonal antibodies against SEZ6 and SEZ6L · plain

  “Taken together, the newly generated antibodies specifically detect endogenous SEZ6 and SEZ6L by immunohistochemistry as well as Western Blot.”

In the paper

“Interestingly, sSEZ6 was also not reduced upon knock-down of BACE1, but mildly reduced upon knock-down of BACE2.”

In the paper

“This reveals that the 170 kDa band contains complex sugars (referred to as mature SEZ6), whereas the 150 kDa band (referred to as immature SEZ6), contains only high-mannose sugars.”

In the paper

“In fact, treatment of primary neurons with the established BACE1 inhibitor C3 (also known as BACE1 inhibitor IV) [35] strongly reduced sSEZ6L levels compared to the control treatment with a concomitant moderate increase of full-length SEZ6L levels in the cell lysate (Fig. 2b).”

What the sources say

• ?Not checked — no full text of the sourcecites [35] Stachel SJ, Coburn CA, Steele TG, Jones KG, Loutzenhiser EF, Gregro AR, Rajapakse HA, Lai MT, Crouthamel MC, Xu M, et al. Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1). J Med Chem. 2004;47:6447–50. source

In the paper

“The non-cleaved, full-length peptide eluted from the nLC column at ~ 22 min (Fig. 5b).”

In the paper

“The same reduction was observed in CSF from BACE1 KO, but not for BACE2 KO mice.”

In the paper

“Importantly, we show that neither SEZ6 nor SEZ6L are substrates for BACE1 in the pancreatic cell line, which is in contrast to brain”

In the paper

“Upon addition of BACE1, the full-length peptide levels were decreased in the chromatogram and two additional peptides with elution times of ~16 and ~21 min were detected (Fig. 5b).”

In the paper

“Among these proteins, SEZ6 (DKO/WT = 13 %, p = 5.99E-06) and SEZ6L (DKO/WT = 20 %, p = 5.10E-05) showed the strongest reduction as well as the highest statistical significance (Fig. 7a).”

In the paper

“Subcellular locations of proteins quantified in all replicates of SDC or urea supported digestions were similar (Additional file 1: Figures S2 and S3).”

In the paper

“Importantly, the new workflow allowed the quantification of SEZ6 and SEZ6L, which were not quantified in the previous study [14].”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Dislich B, Wohlrab F, Bachhuber T, Müller SA, Kuhn P-H, Hogl S, Meyer-Luehmann M, Lichtenthaler SF. Label-free quantitative proteomics of mouse cerebrospinal fluid detects β-Site APP Cleaving Enzyme (BACE1) protease substrates in vivo. Mol Cell Proteomics. 2015;14:2550–63. source

In the paper

“The deglycosylation experiment (Fig. 1b) had revealed that mature SEZ6 and SEZ6L carry complex N-linked sugars and are resistant to EndoH treatment.”

In the paper

“Additionally, the average number of identified and quantified proteins was 10.3 and 7.5 % higher for the SDC supported digestion protocol, respectively.”

In the paper

“We confirm this finding and also extend it to SEZ6.”

In the paper

“Peptides were almost exclusively mapped to extracellular domains of transmembrane proteins (Additional file 3: Tables S1-S6).”

In the paper

“However, cleavage of sFlag-V5-SEZ6 and sSEZ6L was less impacted than Aβ42 upon inhibition with BACE1-selective inhibitors (B and C) and followed the enzymatic inhibition curves of BACE2 rather than BACE1 (Fig. 3b).”

In the paper

“A concentration of 0.1 % SDC was sufficient to improve the digestion efficiency.”

What the sources say

• ?Not checked — no full text of the sourcecites [43] Lin Y, Lin H, Liu Z, Wang K, Yan Y. Improvement of a sample preparation method assisted by sodium deoxycholate for mass-spectrometry-based shotgun membrane proteomics†. J Sep Sci. 2014;37:3321–9. source
• ?Not checked — no full text of the sourcecites [44] Lin Y, Wang K, Liu Z, Lin H, Yu L. Enhanced SDC-assisted digestion coupled with lipid chromatography-tandem mass spectrometry for shotgun analysis of membrane proteome. J Chromatogr B. 2015;1002:144–51. source

In the paper

“Addition of C3 inhibited the production of both peptides, demonstrating that they are BACE1 cleavage products of the full-length peptide.”

In the paper

“Cleavage of Aβ42 and sFlag-V5-SEZ6 and sSEZ6L were similar after addition of nonselective BACE inhibitor A and was consistent with equipotent inhibition of BACE1 and BACE2 in enzymatic assays.”

In the paper

“We found opposite expression patterns of BACE1 and BACE2 in MIN6 cells and in neurons, which correlated with the tissue-specific cleavage of SEZ6 and SEZ6L.”

In the paper

“In contrast to our previous proteomic study of CSF from BACE1 deficient mice [14], we were able to quantify SEZ6 and SEZ6L with the optimized protocol (Additional file 2: Supplementary Data: proteins BACE DKO vs WT CSF).”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Dislich B, Wohlrab F, Bachhuber T, Müller SA, Kuhn P-H, Hogl S, Meyer-Luehmann M, Lichtenthaler SF. Label-free quantitative proteomics of mouse cerebrospinal fluid detects β-Site APP Cleaving Enzyme (BACE1) protease substrates in vivo. Mol Cell Proteomics. 2015;14:2550–63. source

In the paper

“However, digestion efficiency was strongly increased which was detected by the 58.1 % lower number of average missed cleavages per peptide (Table 1).”

In the paper

“The full-length SEZ6 in the cell lysate and the shed ectodomain (sSEZ6) in the supernatant were detected by immunoblots in the transfected cells, but not in control transfected cells (Fig. 6a).”

In the paper

“However, the number of quantified membrane proteins was 8.9 % higher for the samples digested in the presence of SDC (135 vs. 124).”

In the paper

“Additionally, the average number of identified and quantified proteins was 10.3 and 7.5 % higher for the SDC supported digestion protocol, respectively.”

In the paper

“However, sSEZ6 and sSEZ6L were not consistently detected and could not be quantified in murine CSF, potentially because their levels were below the detection limit.”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Dislich B, Wohlrab F, Bachhuber T, Müller SA, Kuhn P-H, Hogl S, Meyer-Luehmann M, Lichtenthaler SF. Label-free quantitative proteomics of mouse cerebrospinal fluid detects β-Site APP Cleaving Enzyme (BACE1) protease substrates in vivo. Mol Cell Proteomics. 2015;14:2550–63. source

In the paper

“The number of identified unique peptides was 6.6 % lower for the SDC supported protocol (Table 1).”

In the paper

“Here, we improved the digestion efficiency of murine CSF in comparison to our previous protocol by using 0.1 % SDC in 50 mM ammonium bicarbonate as digestion buffer.”

In the paper

“This shows that both SEZ6 and SEZ6L are not substrates for BACE1 in the pancreatic cell line.”

In the paper

“Moreover, several phenotypic changes were described in BACE1-/- mice, such as epileptic seizures, schizophrenic symptoms, increased mortality and altered insulin metabolism, but most of the BACE1 substrates contributing to these phenotypes still need to be determined [13].”

What the sources say

• ?Not checked — no full text of the sourcecites [13] Barao S, Moechars D, Lichtenthaler SF, De Strooper B. BACE1 physiological functions may limit its use as therapeutic target for alzheimer’s disease. Trends Neurosci. 2016;39:158–69. source

In the paper

“Label-free quantification (LFQ) of proteins required at least two ratio counts of unique peptides.”

In the paper

“The correct sequence was verified by MS/MS-based fragmentation (Fig. 5c).”

In the paper

“False discovery rate for both peptides and proteins was adjusted to less than 1 % using a target and decoy approach (concatenated forward/reverse database).”

In the paper

“A previous proteomic study demonstrated that the soluble ectodomains of other BACE1 substrates, such as APLP1, PLXDC2 and CHL1, were reduced in the CSF of BACE1-deficient mice [14].”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Dislich B, Wohlrab F, Bachhuber T, Müller SA, Kuhn P-H, Hogl S, Meyer-Luehmann M, Lichtenthaler SF. Label-free quantitative proteomics of mouse cerebrospinal fluid detects β-Site APP Cleaving Enzyme (BACE1) protease substrates in vivo. Mol Cell Proteomics. 2015;14:2550–63. source

In the paper

“The improved method may be of wide relevance for studying murine CSF in the context of different neurological and neurodegenerative diseases.”

In the paper

“All curves are based on biological replicates with at least two technical replicates.”

In the paper

“To ensure the specificity of the immunoblot signals, brains from wild type (WT) as well as from SEZ6-/- (SEZ6 KO) or SEZ6L2-/- (SEZ6L2 KO) mice were used.”

In the paper

“This demonstrates that sSEZ6 is generated specifically by BACE1, but not by BACE2 in murine CSF.”

In the paper

“Only unique peptides were used for quantification.”

In the paper

“A p-value less than 5 % was set as significance threshold.”

In the paper

“but BACE1 preferentially cleaves C-terminally to leucine or other hydrophobic amino acids [40].”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [40] Gruninger-Leitch F, Schlatter D, Kung E, Nelbock P, Dobeli H. Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases. J Biol Chem. 2002;277:4687–93. source

In the paper

“Primary or secondary antibodies were omitted on sections in each experiment to confirm staining specificity.”

In the paper

“Using different techniques, our study validates SEZ6 and SEZ6L as BACE1 substrates in vitro and in vivo and demonstrates that, in contrast to other BACE1 substrates, SEZ6 and SEZ6L are nearly exclusively cleaved by BACE1 and not by other proteases in the brain.”

In the paper

“For example, in APP the ADAM10 and BACE1 cleavage sites are 16 amino acids apart from each other and yield APP ectodomains with diverging functions [48, 49].”

What the sources say

• ?Not checked — no full text of the sourcecites [48] Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA, Herms J, Buchholz C, Eckman CB, Korte M, et al. The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci. 2007;27:7817–26. source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [49] Li H, Wang B, Wang Z, Guo Q, Tabuchi K, Hammer RE, Sudhof TC, Zheng H. Soluble amyloid precursor protein (APP) regulates transthyretin and Klotho gene expression without rescuing the essential function of APP. Proc Natl Acad Sci U S A. 2010;107:17362–7. source

In the paper

“We demonstrate that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and that, in contrast to most other BACE1 substrates, these two proteins are nearly exclusively cleaved by BACE1.”