Ischemic stroke-induced neuronal cell death leads to the permanent impairment of brain function. The Fas-mediating extrinsic apoptosis pathway and the cytochrome c-mediating intrinsic apoptosis pathway are two major molecular mechanisms contributing to neuronal injury in ischemic stroke. In this study, we employed a Fas-blocking peptide (FBP) coupled with a positively charged nona-arginine peptide (9R) to form a complex with negatively charged siRNA targeting Bax (FBP9R/siBax). This complex is specifically designed to deliver siRNA to Fas-expressing ischemic brain cells. This complex enables the targeted inhibition of Fas-mediating extrinsic apoptosis pathways and cytochrome c-mediating intrinsic apoptosis pathways. Specifically, the FBP targets the Fas/Fas ligand signaling, while siBax targets Bax involved in mitochondria disruption in the intrinsic pathway. The FBP9R carrier system enables the delivery of functional siRNA to hypoxic cells expressing the Fas receptor on their surface—a finding validated through qPCR and confocal microscopy analyses. Through intranasal (IN) administration of FBP9R/siCy5 to middle cerebral artery occlusion (MCAO) ischemic rat models, brain imaging revealed the complex specifically localized to the Fas-expressing infarcted region but did not localize in the non-infarcted region of the brain. A single IN administration of FBP9R/siBax demonstrated a significant reduction in neuronal cell death by effectively inhibiting Fas signaling and preventing the release of cytochrome c. The targeted delivery of FBP9R/siBax represents a promising alternative strategy for the treatment of brain ischemia.

Apoptosis plays a crucial role in neuronal injury, with substantial evidence implicating Fas-mediated cell death as a key factor in ischemic strokes. To address this, inhibition of Fas-signaling has emerged as a promising strategy in preventing neuronal cell death and alleviating brain ischemia. However, the challenge of overcoming the blood–brain barrier (BBB) hampers the effective delivery of therapeutic drugs to the central nervous system (CNS). In this study, we employed a 30 amino acid-long leptin peptide to facilitate BBB penetration. By conjugating the leptin peptide with a Fas-blocking peptide (FBP) using polyethylene glycol (PEG), we achieved specific accumulation in the Fas-expressing infarction region of the brain following systemic administration. Notably, administration in leptin receptor-deficient db/db mice demonstrated that leptin facilitated the delivery of FBP peptide. We found that the systemic administration of leptin-PEG-FBP effectively inhibited Fas-mediated apoptosis in the ischemic region, resulting in a significant reduction of neuronal cell death, decreased infarct volumes, and accelerated recovery. Importantly, neither leptin nor PEG-FBP influenced apoptotic signaling in brain ischemia. Here, we demonstrate that the systemic delivery of leptin-PEG-FBP presents a promising and viable strategy for treating cerebral ischemic stroke. Our approach not only highlights the therapeutic potential but also emphasizes the importance of overcoming BBB challenges to advance treatments for neurological disorders.

The anti-apoptotic B-cell lymphoma 2 (BCL2) family member myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers and has been linked to chemoresistance. Unlike other BCL2 family members, MCL1 displays both well-defined mitochondrial anti-apoptotic activities and emerging nuclear functions. Prior reports suggest that MCL1 enters the nucleus during chemotherapy treatment and confers chemoresistance by influencing cell cycle progression and DNA repair. These nuclear roles of MCL1, however, remain poorly characterized. Using a newly validated monoclonal antibody across several cell lines and treatments, we find no evidence that MCL1 enhances chemoresistance or preferentially accumulates in the nucleus after drug exposure. Indeed, bulk RNA sequencing data suggest that MCL1 loss dampens, rather than potentiates, transcriptional responses to DNA-damaging chemotherapy. Proximity biotinylation experiments identified novel nuclear MCL1 interactors but did not recover previously reported DNA repair or cell cycle partners. Thus, while MCL1 does reach the nucleus and interact with nuclear proteins, our data do not support a role for MCL1 in chemoresistance. Further work is needed to clarify the functional significance of nuclear MCL1.

The anti-apoptotic BCL2 family member MCL1 is overexpressed in many cancers and has been linked to chemoresistance. Unlike other BCL2 family members, MCL1 displays both well-defined mitochondrial anti-apoptotic activities and also emerging nuclear functions. Prior reports suggest that MCL1 enters the nucleus during chemotherapy and promotes chemoresistance by influencing cell cycle progression and DNA repair. These nuclear roles of MCL1, however, remain poorly characterized. Using a newly validated monoclonal antibody across several cell lines and treatments, we find no evidence that MCL1 enhances chemoresistance or preferentially accumulates in the nucleus after drug exposure. Proximity biotinylation identified novel nuclear MCL1 interactors but did not recover previously reported DNA repair or cell cycle partners. Thus, while MCL1 does reach the nucleus and interact with nuclear proteins, our data do not support a role for MCL1 in chemoresistance. Further work is needed to clarify the functional significance of nuclear MCL1. SIGNFIGANCE: Previous studies have implicated MCL1 in promoting chemoresistance via interactions with DNA repair machinery in the nucleus. Using a validated, monoclonal anti-MCL1 antibody, we were unable to replicate these data. We report that MCL1 neither confers chemoresistance, translocates to the nucleus during chemotherapy treatment, nor interacts with DNA repair proteins in live cancer cells.