Examination of multiple SynGAP isoforms in mammalian central neurons
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Date
27/06/2011Author
McMahon, Aoife Christina
Metadata
Abstract
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.