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Pharmacol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Cerebral ischemia triggers a cascade of detrimental events, leading to brain damage mainly due to the over-excitation of neurons. Currently, clinically applicable neuroprotective treatments to stop progressive neuronal death remain elusive. The GABA<jats:sub>B<\/jats:sub> receptor, crucial for neuronal inhibition, is a promising target for neuroprotection because it inhibits neuronal over-excitation which otherwise leads to excitotoxic death. However, ischemic conditions impair GABA<jats:sub>B<\/jats:sub> receptor function by downregulating the receptors via pathologically altered trafficking events. Previously, we developed interfering peptides to inhibit the interaction of GABA<jats:sub>B<\/jats:sub> receptors with key interacting proteins, leading to the pathological downregulation of the receptors. These interfering peptides restored GABA<jats:sub>B<\/jats:sub> receptor expression and function, resulting in reduced excitability and death of neurons in <jats:italic>in-vitro<\/jats:italic> and <jats:italic>ex-vivo<\/jats:italic> models of cerebral ischemia. However, the interfering peptides were not effective <jats:italic>in-vivo<\/jats:italic> because of their limited proteolytic stability after systemic application.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods\/results<\/jats:title><jats:p>Here, we aimed to render three interfering peptides resistant to proteolytic degradation by replacing natural L-amino acids by D-amino acids. Additionally, we optimized a blood brain barrier shuttle (BBBpS) sequence derived from the Rabies virus glycoprotein (RVG) that mediates neuron-specific uptake and blood-brain barrier crossing of these interfering peptides. By optimizing the peptides, we developed stable, neuron-specific interfering peptides that successfully restored GABA<jats:sub>B<\/jats:sub> receptors expression and prevented neuronal death following excitotoxic stress in cultured neurons. <jats:italic>In vivo<\/jats:italic> testing in the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia demonstrated the neuroprotective activity of the optimized peptides by a significantly reduced infarct size.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>These findings confirm the potential of these peptides as neuroprotective agents and emphasize the importance of proteolytic stability of peptide drugs for their successful <jats:italic>in-vivo<\/jats:italic> application.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fphar.2025.1576884","type":"journal-article","created":{"date-parts":[[2025,4,28]],"date-time":"2025-04-28T05:31:50Z","timestamp":1745818310000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Optimization of neuron-specific interfering peptides targeting GABAB receptor downregulation for proteolytic stability for conferring neuroprotection in a mouse model of cerebral ischemia"],"prefix":"10.3389","volume":"16","author":[{"given":"Mohammad","family":"Hleihil","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Musadiq A.","family":"Bhat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Grampp","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dietmar","family":"Benke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,28]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"111","DOI":"10.1016\/j.coph.2017.07.014","article-title":"Excitatory and inhibitory amino acid neurotransmitters in stroke: from neurotoxicity to ischemic tolerance","volume":"35","author":"Amantea","year":"2017","journal-title":"Curr. 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