Calcineurin and cytoskeleton in low-frequency depression

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Phasic synapses exhibit transmitter release that depresses under sustained stimulation.
• Low-frequency depression (LFD) is a form of synaptic depression observed at crayfish neuromuscular junctions.
• The role of presynaptic protein phosphatases, specifically calcineurin, in LFD remains largely uncharacterized.

Purpose of the Study:

• To investigate the role of presynaptic calcineurin in low-frequency depression (LFD) at the crayfish neuromuscular junction.
• To identify phosphoproteins involved in LFD using a phosphoproteomic approach.
• To elucidate the molecular mechanisms underlying presynaptic depression.

Main Methods:

• Intracellular injection of calcineurin autoinhibitory peptide into presynaptic neurons.
• Phosphoproteomic screening of motor axons and nerve terminals using PAGE and phospho-specific/total protein ratio stains.
• Mass spectrometry analysis of protein bands.
• Immunoreactivity assays for tubulin and phosphoactin.
• Pharmacological manipulation using actin depolymerizing drugs (cytochalasin, latrunculin) and a microtubule stabilizer (taxol).

Main Results:

• Inhibition of presynaptic calcineurin prevented LFD and induced facilitation of transmitter release.
• LFD induction led to decreased phosphorylation of actin and tubulin.
• Blocking calcineurin increased actin and tubulin phosphorylation.
• Reduced tubulin and phosphoactin immunoreactivity was observed in presynaptic terminals after LFD.
• Cytochalasin, latrunculin, and taxol inhibited LFD.

Conclusions:

• Presynaptic calcineurin activity is necessary for low-frequency depression (LFD).
• Dephosphorylation of presynaptic actin and tubulin, leading to cytoskeletal alterations, regulates LFD.
• This study reveals a novel presynaptic depression mechanism distinct from postsynaptic mechanisms in mammalian synapses.