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Roles of calsyntenin-3 in synapse development and optimizing conditional gene deletion

University of British Columbia

Synaptic adhesion molecules play central roles in synapse organization and neuronal signal transduction machineries. This dissertation first focuses on calsyntenin-3, a crucial synaptic adhesion molecule identified by our lab, based on in vitro and in vivo biochemistry, immunofluorescence, electron microscopy, and electrophysiological studies. Chapter 2 shows that calsyntenin-3 interacts with α-neurexins, but not β-neurexins, at nanomolar affinity. Being calcium-dependent and regulated by the heparan sulfate modification of α-neurexins, this interaction requires the cadherin and LNS domains of calsyntenin-3, as well as the LNS5-EGFc-LNS6 domains of α-neurexins. Calsyntenin-3 full-length form triggers pre-synapse differentiation, but calsyntenin-3-shed ectodomain suppresses the ability of α-neurexin partners in mediating pre-synapse differentiation. Calsyntenin-3 is present in pyramidal neurons throughout cortex and hippocampus, and is most highly expressed in interneurons. Young adult calsyntenin-3 knockout mice present deficits in both density and transmission for both excitatory and inhibitory synapses. Chapter 3 shows that calsyntenin-3 regulates synaptic transmission in different neuronal types. Conditional calsyntenin-3 knockout in forebrain interneurons does not affect basal inhibitory transmission in CA1 pyramidal neurons, while conditional knockout in excitatory neurons enhances both basal excitatory and inhibitory transmission. Partial calsyntenin-3 knockout in primary hippocampal neuron culture and in juvenile calsyntenin-3 mice exhibit enhanced inhibitory transmission. Juvenile calsyntenin-3 knockout mice, however, show no difference in inhibitory transmission compared to wild-type littermates. Thus, calsyntenin-3 contributes in a developmentally regulated and at least partially non-cell-autonomous manner to synaptic transmission. Chapter 4 discusses our discovery of unexpected germline recombination in distinct mouse Cre driver lines and its profound implications. The Cre-loxP system (and resulting Cre mouse lines) has been widely used for cell-type specific gene manipulations and helps decipher gene functions, especially in neuroscience research. We demonstrated that unexpected germline recombination occurs in many Cre lines based on the studies of two Cre lines within our lab and collected data for fifty-seven Cre lines worldwide. Consequently, this may sway the interpretation of past experimental results as well as moving forward. This work not only provides guidelines for breeding strategies and precautions aimed at future studies, but most importantly for the first time, comprehensively raises awareness of this phenomenon among the neuroscience community.

Text

2020-07-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/71330

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/