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Peptide-mediated neuroprotection against excitotoxicity and cerebral ischemia by regulation of the JNK-activation cascade

University of British Columbia

Ischemic stroke is one of the leading causes of death and disability worldwide. One of the major pathological processes that causes brain damage after an ischemic insult is excitotoxicity, which initiates inflammation and oxidative stress and eventually leads to apoptotic and necrotic neuronal death. C-Jun-NH₂-Terminal kinases (JNKs) are activated after an ischemic event and contribute to excitotoxicity, inflammation, oxidative stress, apoptosis and necrosis. Targeting JNKs is a promising strategy to mediate the deathly excitotoxic cascade and reduce infarct volume after a stroke. The aim of this Master’s thesis was to employ the selective native protein eradication (SNIPER) method to degrade multiple players in the JNK activation pathway as a novel neuroprotective approach. SNIPER peptides consist of a cell-penetrating domain, a protein-binding domain (PBD) derived from a natural binding partner of the protein of interest, and a motif that is recognized by chaperone-mediated autophagy. With these three segments SNIPER peptides can enter cells, bind to the protein of interest and target it to the lysosome for degradation. We designed three peptides with a PBD based on the key JNK-binding site (T1A) of Arrestin-3, which has been shown to bind JNK3 as well as its upstream activators Apoptosis signal-regulated kinase 1 (ASK1) and mitogen-activated protein kinase kinase 7 (MKK7). We hypothesized that these peptides can mediate the JNK activation cascade and act as potent neuroprotectants. We found that a peptide with a protein-binding domain consisting of residues 12 to 25 of T1A, named T1A-3, decreases levels of active JNK and protects cultures of cortical neurons against excitotoxic insult. Furthermore, T1A-3 dramatically reduces infarct volume in an endothelin-1 model of stroke in rats. The peptide did not reduce protein levels of JNK in vitro or in the brains of rats in vivo, indicating that T1A-3 likely exerts its protective function by regulating kinases upstream of JNK phosphorylation. Thus, we developed a novel and potent peptide-based tool to inhibit JNK activation and effectively protect against excitotoxicity and ischemic stroke. This tool has great potential to be a more effective and clinically feasible treatment for stroke than previously developed therapies.

Text

2020-05-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/69758

Neuroscience

University of British Columbia

UBCV

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