Examination of multiple SynGAP isoforms in mammalian central neurons
McMahon, Aoife Christina
The ability of neurons to dynamically regulate their response to changing inputs is essential for the correct development and function of a nervous system capable of learning and memory. The post synaptic compartment of excitatory synapses contains a dense proteinaceous complex of molecules that link excitatory glutamatergic neurotransmission to downstream signalling pathways that ultimately result in modification of the synapse. One of the most abundant of such postsynaptic signalling molecules, synaptic GTPase activation protein, SynGAP, represents a key signalling link between the activation of the NMDA sensitive glutamate receptor to outcomes such as the structural rearrangement of synaptic sites and altered synaptic content of AMPA type glutamate receptors, molecular processes that underly learning and memory. The primary finding of this thesis is that different isoforms of SynGAP, which varies at it N terminus through alternative transcription start sites and at its C terminus through alternative splicing, can differentially affect the function of the synapse. Using primary murine neuronal cultures we show that despite being crucial for the survival of the mouse the absence of SynGAP does not effect mean dendritic spine morphology and density or miniature excitiatory post synaptic currents under a range of experimental conditions (days in vitro 10 – 14, with and without serum, high and low cell plating density). In order to examine the effects of different SynGAP isoforms we cloned two full length transcripts (SynGAP A-alpha-2 and SynGAP Ealpha- 1) which were used to construct a range of isoforms. Whole cell patch clamping of SynGAP transfected neurons revealed that the post synaptic expression of SynGAPs which terminate as an alpha-1 isoform can lead to the elimination of mEPSCs, while isoforms that terminate as an alpha-2 isoform can lead to synaptic strengthening. The magnitude of the effect in both cases is determined by the identity of the N terminus of the protein; SynGAP A-alpha-1 has the largest synaptic weakening effect and SynGAP B-and C alpha-2 strenghten the synapse. The changes in miniature electrophysiological properties are not mirrored by changes in dendritic spine morphology, whole cell AMPA/NMDA currents, or synaptic responsiveness to stimulation suggesting an undefined novel mechanism of action. SynGAPs A, B and C appear to be under the control of different promoters which are differentially regulated by development and synaptic activity, thus the differential function of SynGAP N and C terminal combinations could play a part in the activity dependent regulation of synaptic strength.