Functional Studies of SNAP-25 using a knock-out and rescue approach

by Ignacio Delgado Martínez
Doctoral thesis
Date of Examination:2006-10-18
Date of issue:2006-11-01
Advisor:Dr. Jakob Sørensen
Referee:Dr. Jakob Sørensen
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Detlev Schild
Persistent Address: http://dx.doi.org/10.53846/goediss-3299

 

 

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Abstract

English

The neuronal SNARE complex, consisting of SNAP-25, synaptobrevin and syntaxin, is required for presynaptic exocytosis of neurotransmitter-filled vesicles during synaptic transmission. However the SNARE complex undertakes neuronal developmental roles as well. SNAP-25 has been involved in vesicular fusion during axonal outgrowth, synaptic formation and trafficking of glutamate receptors. Its expression is developmentally regulated by alternative splicing from SNAP-25a to SNAP-25b. In GABAergic neurons, SNAP-25 seems to be replaced by SNAP-23, a ubiquitousSNAP-25 homologue, after synaptogenesis. Deletion of SNAP-25 compromises neuronal survival in culture, impeding detailed functional studies. Here, I overcame this difficulty by reintroducing SNAP-25a, SNAP-25b or SNAP-23 using the long-term expression lentiviral system in culture neurons from Snap25 null mice and I was able to dissect the main functions of SNAP-25. I found that that SNAP-25 deficient cultured neurons presented impaired arborization and severe reduction in viability as well as complete arrest of evoked release and reduction in the amplitude and frequency of the spontaneous events. Expression of the SNAP-25 homologues restored neuronal survival, arborization and the properties of spontaneous release. In addition, it rescued evoked release, in both glutamatergic and GABAergic neurons, although SNAP-23 was found to support exclusively asynchronous release. SNAP-25b was superior to SNAP-25a in vesicle priming, which would produce larger releasable pools after synaptic maturation. My results revealed SNAP-25 as key component for neuronal survival and outgrowth, regulation of the synchronous and asynchronous release and spontaneous activity and demonstrate a hierarchical ability of the SNAP-25 homologues to support neuronal function.
Keywords: Lentivirus; autaptic culture; neurite outgrowth; spontaneous release; synchronous release
 
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