High content analysis of gamma-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer's disease

Biochimica Et Biophysica Acta
Cristina FloreanPaola Pizzo

Abstract

gamma-Secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP), Notch and other cellular substrates and is considered a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). We describe here an efficient, new, simple, sensitive and rapid assay to quantify gamma-secretase activity in living cells by flow cytometry using two membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for gamma-secretase. The principle of the assay is based on the fact that the soluble intracellular domain of GFP-tagged APP (AICD-GFP) is released from the membrane into the cytosol following gamma-secretase cleavage. Using this feature, enzymatic activity of gamma-secretase could be deduced from the extent of the membrane retention of the probe observed after plasma membrane permeabilization and washout of the cleaved fraction. By applying two well-known gamma-secretase inhibitors (DAPT and L-685,458), we validated our assay showing that the positional GFP-based probes for gamma-secretase activity behave properly when expressed in different cell lines, providing the basis for the further development of a high-throughput and high content screening for AD targeted drug disc...Continue Reading

References

Dec 18, 2001·The Journal of Biological Chemistry·Helena KarlströmJohan Lundkvist
Jan 5, 2002·The Journal of Biological Chemistry·Bridget E BerechidJeffrey S Nye
Jan 8, 2003·Biochemistry·W Taylor KimberlyMichael S Wolfe
May 6, 2004·Journal of Molecular Neuroscience : MN·Robert Vassar
Jan 13, 2006·Journal of Neurochemistry·Mostafa BentahirBart De Strooper
Feb 7, 2007·Trends in Genetics : TIG·Annette L Parks, Daniel Curtis
Jul 31, 2007·Neurologic Clinics·Nilüfer Ertekin-Taner
Aug 28, 2007·Molecular Neurodegeneration·Can ZhangAleister J Saunders

❮ Previous
Next ❯

Citations

Nov 11, 2008·Proceedings of the National Academy of Sciences of the United States of America·Giovanna PontarinVera Bianchi
May 5, 2010·Proceedings of the National Academy of Sciences of the United States of America·Valentina LissandronTullio Pozzan
Jul 15, 2009·The Journal of Biological Chemistry·Simone EggertEdward H Koo
Apr 9, 2011·Progress in Neurobiology·Joao P Lopes, Paula Agostinho
Apr 23, 2009·Journal of Cellular and Molecular Medicine·Lucia BrunelloCristina Fasolato
Jun 15, 2010·Neurobiology of Aging·Emilia BialopiotrowiczUrszula Wojda
Apr 9, 2014·Journal of Biomolecular Screening·Shantanu SinghAuguste Genovesio
May 21, 2010·Epigenetics : Official Journal of the DNA Methylation Society·Wei-Gang TongGuillermo Garcia-Manero
Jan 30, 2020·International Journal of Molecular Sciences·Luisa GallaElisa Greotti
Apr 12, 2021·The Journal of Biological Chemistry·Ricardo CaponeAnne K Kenworthy

❮ Previous
Next ❯

Related Concepts

Related Feeds

Alzheimer's Disease: Amyloid Beta

Alzheimer's disease is a neurodegenerative disease associated with the accumulation of amyloid plaques in the brain; these plaques are comprised of amyloid beta deposits. Here is the latest research in this field.

Alzheimer's Disease: APP

Amyloid precursor protein (APP) proteolysis is critical for the development of Alzheimer's disease, a neurodegenerative disease associated with accumulation of amyloid plaques in the brain. Here is the latest research on APP and Alzheimer's disease.

Apoptotic Caspases

Apoptotic caspases belong to the protease enzyme family and are known to play an essential role in inflammation and programmed cell death. Here is the latest research.

Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis