Hleihil Mohammad, Bhat Musadiq A, Grampp Thomas, Benke Dietmar
Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Neuroscience Center Zurich, University and ETH Zurich, Zurich, Switzerland.
Front Pharmacol. 2025 Apr 28;16:1576884. doi: 10.3389/fphar.2025.1576884. eCollection 2025.
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 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 receptor function by downregulating the receptors via pathologically altered trafficking events. Previously, we developed interfering peptides to inhibit the interaction of GABA receptors with key interacting proteins, leading to the pathological downregulation of the receptors. These interfering peptides restored GABA receptor expression and function, resulting in reduced excitability and death of neurons in and models of cerebral ischemia. However, the interfering peptides were not effective because of their limited proteolytic stability after systemic application.
METHODS/RESULTS: 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 receptors expression and prevented neuronal death following excitotoxic stress in cultured neurons. 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.
These findings confirm the potential of these peptides as neuroprotective agents and emphasize the importance of proteolytic stability of peptide drugs for their successful application.
脑缺血引发一系列有害事件，主要由于神经元过度兴奋导致脑损伤。目前，临床上可应用的阻止神经元进行性死亡的神经保护治疗方法仍未找到。γ-氨基丁酸（GABA）受体对神经元抑制至关重要，是神经保护的一个有前景的靶点，因为它可抑制否则会导致兴奋性毒性死亡的神经元过度兴奋。然而，缺血状态通过病理改变的转运事件下调GABA受体，从而损害其功能。此前，我们开发了干扰肽来抑制GABA受体与关键相互作用蛋白的相互作用，导致受体的病理性下调。这些干扰肽恢复了GABA受体的表达和功能，减少了大脑缺血模型中神经元的兴奋性和死亡。然而，由于全身应用后其蛋白水解稳定性有限，干扰肽并不有效。
方法/结果：在此，我们旨在通过用D-氨基酸取代天然L-氨基酸，使三种干扰肽对蛋白水解降解具有抗性。此外，我们优化了源自狂犬病病毒糖蛋白（RVG）的血脑屏障穿梭（BBBpS）序列，该序列介导这些干扰肽的神经元特异性摄取和血脑屏障穿越。通过优化这些肽，我们开发出了稳定的、神经元特异性的干扰肽，其成功恢复了GABA受体的表达，并在培养的神经元中防止了兴奋性毒性应激后的神经元死亡。在大脑中动脉闭塞（MCAO）脑缺血小鼠模型中的测试表明，优化后的肽具有神经保护活性，梗死面积显著减小。
这些发现证实了这些肽作为神经保护剂的潜力，并强调了肽药物蛋白水解稳定性对其成功应用的重要性。
