isoforms in dendritic spine function: electrophysiological and morphological characterisation

Chanchanok Chaichim School of Medical Sciences Faculty of Medicine & Health UNSW Sydney A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy February 2021

Thesis Title and Abstract

Thesis Title

Tropomyosin isoforms in dendritic spine function: electrophysiological and morphological characterisation

Thesis Abstract

The neuronal is crucial for the formation and function of synapses. The structure and dynamic reorganisation of actin filaments are modulated by a variety of actin-associated . The tropomyosin family of proteins regulates the access of other proteins to the actin filament. There are over 40 isoforms of tropomyosin produced from four in mammalian tissue. Our study aimed to examine the roles of two isoforms, Tpm3.1 and Tpm4.2, that are enriched in the postsynaptic compartment.

We first assayed the role of Tpm3.1 in cultured neurons. We compared synaptic function and structure in neurons from transgenic mice overexpressing Tpm3.1 with WT controls. No differences were seen in mean amplitude or frequency of miniature excitatory postsynaptic currents (mEPSCs), or dendritic spine morphology, suggesting that increasing Tpm3.1 has little impact on basal function. Pharmacological inhibition of Tpm3.1/2 did not alter the distribution of spine morphology types or mean length and width.

Next, we examined the role of Tpm4.2. We used adeno-associated viruses to overexpress the isoform Tpm4.2 in cultured neurons. Spine morphology and mEPSC amplitude and frequency were similar between groups. Deletion of Tpm4.2 altered dendritic spine morphology and significantly reduced mEPSC amplitude and frequency, showing that Tpm4.2 is necessary for normal synaptic function.

Finally, we made extracellular field recordings from ex vivo hippocampal slices prepared from transgenic mice overexpressing Tpm3.1 and Tpm4.2 KO mice. We measured basal synaptic function and induced long term potentiation to test synaptic plasticity. Tpm3.1- overexpressing slices had no changes in basal function or plasticity. Unlike in cultured neurons, all measures were unaltered in Tpm4.2 KO brain slices.

Overall, this study demonstrates that it is possible to alter the expression of Tpm3.1 and Tpm4.2 without serious adverse effects to neuronal function post-maturation, suggesting that individual isoforms in isolation have limited influence on the synapse.

Declarations

ORIGINALITY STATEMENT

I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.

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