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Design and optimization of small peptides that regulate the balance of synaptic excitation and inhibition

University of British Columbia

The balance of synaptic excitation and inhibition plays a very important role in maintaining the function of central nervous system (CNS) and the imbalance is involved in neurologic diseases such as autism and epilepsy. PSD95 and gephyrin have been studied as scaffolding proteins, having critical functional and structural roles in excitatory and inhibitory synapses, respectively. In the thesis, I have attempted to develop small systemically applicable peptides that can reversibly knock down PSD95 or gephyrin in vitro and in vivo, using the novel peptide-mediated technology recently developed in our lab, as tools for modulating the balance of synaptic excitation and inhibition. The efficacy of the peptides knocking down respective targeted proteins was tested by immunoblots after the cultured neurons were treated with the peptides for the desired time at various concentrations. I found that peptides that target either PSD95 or gephyrin showed toxicity to the neurons in a dose and time dependent manner utilizing the LDH assay. The toxicity may also contribute to the reduction of protein levels. Using one of the peptides, TAT-NR2B9C-CMA that targets PSD95 as example, I systemically investigated the causes of the toxicity and tested several strategies to reduce the toxicity while keeping the efficacy of the protein knockdown. I found that while multiple treatments at low dose could not successfully separate the cell death and knockdown effect, treatment at high doses with shortened durations appeared partially effective in reducing the toxicity and maintaining knockdown efficacy. However, this protocol may not be applicable in vivo. I next modified the intrinsic properties of peptides by shortening CMA targeting motifs and/or adding a linker between the binding sequences and CMA targeting motif. I found that while both strategies could decrease the toxicity with varied degree, peptides with short CMA targeting motif kept the knockdown efficacy. Taken together, my study demonstrated the effective strategies to reduce the toxicity of the peptides one can consider in the process of developing novel protein knockdown peptides as novel research tools and therapeutic reagents.

Text

2017-05-03

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/61467

Neuroscience

University of British Columbia

UBCV

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