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Related Experiment Videos

Rapid annexin-V labeling in synaptosomes.

Karen H Gylys1, Jeffrey A Fein, Dorothy J Wiley

  • 1UCLA School of Nursing and Brain Research Institute, Box 956919, Factor Building, Los Angeles, CA 90095, USA. kgylys@sonnet.ucla.edu

Neurochemistry International
|October 22, 2003
PubMed
Summary

Synaptosomes rapidly expose phosphatidylserine (PS) during apoptosis, preceding caspase activation. This cell-free assay models early synapse loss in neurodegenerative diseases.

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Biochemistry

Background:

  • Synaptic terminals are implicated in neurodegenerative disease pathogenesis.
  • Apoptosis and synapse loss occur early in these conditions.
  • A cell-free model is needed to study synaptic apoptosis.

Purpose of the Study:

  • To establish and validate a cell-free assay for studying apoptosis in neuronal terminals.
  • To investigate the temporal relationship between phosphatidylserine (PS) exposure and caspase activation in synaptosomes.

Main Methods:

  • Utilized a rat brain crude synaptosomal preparation (P-2 fraction) as a model system.
  • Employed annexin-V to detect PS exposure, a marker of apoptosis.
  • Used calcein AM as a viability marker to ensure assay of intact terminals.

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  • Quantified fluorescence via flow cytometry and measured caspase-3-like activity fluorometrically.

    • Main Results:

    • Rapid annexin-V labeling indicative of PS externalization occurred within 1 minute of staurosporine incubation.
    • Significant increase in caspase-3-like activity was detected only after 30 minutes.
    • A caspase-3 inhibitor partially blocked PS externalization after 30 minutes but not the initial rapid labeling.

    Conclusions:

    • Demonstrates rapid phosphatidylserine (PS) externalization in synaptosomes, preceding detectable caspase activation.
    • Validates a cell-free synaptosomal preparation as a model for studying early apoptotic events in neuronal terminals.
    • Highlights the utility of this model for neurodegenerative disease research.