LongDistance Retrograde Effects of Botulinum Neurotoxin A

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Long-Distance Retrograde Effects of Botulinum
Neurotoxin A

• Flavia Antonucci, Chiara Rossi, Laura
  Gianfranceschi, Ornella Rossetto, and Matteo
  Caleo
• J Neuro, April 2, 2008, 28 (14)3689-3696

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Introduction
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Introduction

• BoNT/A-G, cleave SNARE, prevent vesicle fusion
• BoNT/A and E cleave SNAP-25
• BoNT/A treats
• hyperfunction of cholinergic terminals
• remove wrinkles
• pain management

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Introduction

• BoNT/A enters neurons by binding to synaptic
  vesicle protein SV2
• Internalized by
• vesicle endocytosis,
• translocated to cytosol
• Thought to remain
• at injection area
• Retrograde axonal
• transport?
• Trancytosis to afferent?

Dong et al. 2006
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Results
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Results

• Grew antibodies against A and E cleave SNAP-25
• Inj. dorsal Hipp., found dose-response curve,
  comparing BoNT/A concentration to amount of
  cleaved SNAP-25 (3 days after injection)

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Results

• Inject dorsal hippocampus, analyze over a time
  course for levels of cleaved SNAP-25
• BoNT/E maximal 1-10 days after
• BoNT/A high through day 120
• confirms

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Results

• Analyzed contralateral SNAP-25 cleavage
• BoNT/E cleavage remained on ipsilateral side
• BoNT/A treated animals had contralateral cleaved
  SNAP-25 after 3 days

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Results

• Immonohistochemistry confirms spread
• Schaffer collateral connects CA3 and CA1
• Redcleaved SNAP-25, Greenneuronal
• Not passive spread
• Layer II-III of entorhinal cortex, layers that
  project to hippocampus
• Retrograde propogation?

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Results

• SNAP-25 cleavage has functional repercussions
• Recorded spontaneous spike activity of CA1
  pyramidal cells
• Reduced contralateral activity 3 d after A inj.
• E blocks only ips.
• Be skeptical

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Results

• Superior Colliculus receives 1-way projections
  from cont. retina and ips. primary visual cortex
• Inject BoNT/A into SC, measure cleavage
• Cleavage after 1 day only in SC, in retina and
  cortex after 3 days
• Colchicine MT depolymerizing agent blocks
  spread of cleavage MT-based axonal tranport?

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Results

• Immunostaining showed cleavage in layer V of
  visual cortex (cells that project to SC)
• Retrograde transport, not passive spread
• In retina, cleavage in ON/OFF sublamina of inner
  plexiform layer (project to SC)

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Results

• Analyzed IPL to see type of synapse 3 d after
  inj.
• Cleavage colocalized with choline
  acetyltransferase
• Also colocalized with SV2C
• Cleavage appears in cholinergic, SV2C synapses
  of retina

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Results

• Could it be that just the cleaved SNAP-25 is
  transported, not the toxin?
• Inj. into SC, found expected cleavage 3 d after
• Cut optic nerve, nothing more transported from SC
• Inj BoNT/E into eye
• BoNT/E cleaves off terminal where antibody binds

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Results

• BoNT/E cleaves off terminal where antibody binds
• BoNT/E loses its effectiveness after about 3
  weeks
• Expect initial disappearance of A-cleaved SNAP-25
• If only A-cleaved SNAP-25 had been transported
  from SC to the eye, there would be no increase of
  A-cleaved SNAP-25 after BoNT/E loses its effect
• If BoNT/A had been transported from SC, there
  WOULD be a recovery of A-cleaved SNAP-25

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Results

• Initially a lot of A-cleaved SNAP-25
• With BoNT/E injection, large decrease in amount
  of A-cleaved SNAP-25
• After 25 d, recovery of A-cleaved SNAP-25
• It was BoNT/A that was retrogradely transported
  from SC to retina

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Results

• Natural target of BoNT/A is neuromuscular
  junction so examined transport in motoneurons
• Injected into whisker muscles (no proprioceptive
  or sensory innervation)
• After 3 days, cleavage in facial nucleus in pons

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Conclusions
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Conclusions

• BoNT/A cleaves SNAP-25 at injection site and to
  far regions that project to the infusion site
• No passive spread, only retrograde axonal
  transport and transcytosis
• Functional changes?
• Active BoNT/A is transported, rather than its
  cleaved substrate
• Rather than left in cytosol, some loaded into
  vesicles, released at synapse, and taken up by
  afferent neurons

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Further exploration

• Functional effects?
• Beyond second-order neuron transport?
• Is BoNT/A like tentanus, in that it trancytosis
  is limited to specific synaptic inputs?
  Chonlinergic?
• What is the mechanism for transcytosis
• Implications of BoNT/A used medically?
• Dystonic patients treated with BoNT/A have show
  changes in cortical excitability
• BoNT/A administered in the periphery may directly
  affect central circuits via retrograde transport
  and transcytosis