Aspartate mutations in presenilin and γ-secretase inhibitors both impair Notch1 proteolysis and nuclear translocation with relative preservation of Notch1 signaling

Oksana Berezovska, Christine Jack, Pamela J McLean, Jon C. Aster, Carol Hicks, Weiming Xia, Michael S. Wolfe, W. Taylor Kimberly, Gerry Weinmaster, Dennis J. Selkoe, Bradley T. Hyman

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in β-amyloid protein (Aβ) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells: Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP γ-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP γ-secretase inhibitors block Notch1 nuclear translocation with an IC50 similar to that reported for APP γ-secretase, Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or γ-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP γ- secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting γ-secretase processing of APP to reduce Aβ production.

Original languageEnglish (US)
Pages (from-to)583-593
Number of pages11
JournalJournal of Neurochemistry
Volume75
Issue number2
DOIs
StatePublished - 2000
Externally publishedYes

Fingerprint

Presenilins
Proteolysis
Amyloid Precursor Protein Secretases
Presenilin-1
Aspartic Acid
Mutation
Amyloid beta-Protein Precursor
Serum Amyloid A Protein
Assays
Notch1 Receptor
Processing
Cells
Ligands
Cricetulus
Transcriptional Activation
Inhibitory Concentration 50
Ovary
Alzheimer Disease
Transcription Factors
Chemical activation

Keywords

  • Delta
  • Notch
  • Nuclear translocation
  • Presenilin

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Aspartate mutations in presenilin and γ-secretase inhibitors both impair Notch1 proteolysis and nuclear translocation with relative preservation of Notch1 signaling. / Berezovska, Oksana; Jack, Christine; McLean, Pamela J; Aster, Jon C.; Hicks, Carol; Xia, Weiming; Wolfe, Michael S.; Kimberly, W. Taylor; Weinmaster, Gerry; Selkoe, Dennis J.; Hyman, Bradley T.

In: Journal of Neurochemistry, Vol. 75, No. 2, 2000, p. 583-593.

Research output: Contribution to journalArticle

Berezovska, Oksana ; Jack, Christine ; McLean, Pamela J ; Aster, Jon C. ; Hicks, Carol ; Xia, Weiming ; Wolfe, Michael S. ; Kimberly, W. Taylor ; Weinmaster, Gerry ; Selkoe, Dennis J. ; Hyman, Bradley T. / Aspartate mutations in presenilin and γ-secretase inhibitors both impair Notch1 proteolysis and nuclear translocation with relative preservation of Notch1 signaling. In: Journal of Neurochemistry. 2000 ; Vol. 75, No. 2. pp. 583-593.
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T1 - Aspartate mutations in presenilin and γ-secretase inhibitors both impair Notch1 proteolysis and nuclear translocation with relative preservation of Notch1 signaling

AU - Berezovska, Oksana

AU - Jack, Christine

AU - McLean, Pamela J

AU - Aster, Jon C.

AU - Hicks, Carol

AU - Xia, Weiming

AU - Wolfe, Michael S.

AU - Kimberly, W. Taylor

AU - Weinmaster, Gerry

AU - Selkoe, Dennis J.

AU - Hyman, Bradley T.

PY - 2000

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N2 - It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in β-amyloid protein (Aβ) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells: Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP γ-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP γ-secretase inhibitors block Notch1 nuclear translocation with an IC50 similar to that reported for APP γ-secretase, Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or γ-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP γ- secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting γ-secretase processing of APP to reduce Aβ production.

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