Rapalink-1 Increased Infarct Size in Early Cerebral Ischemia–Reperfusion With Increased Blood–Brain Barrier Disruption

Abstract

It has been reported that the mechanistic target of rapamycin (mTOR) pathway is involved in cerebral ischemia–reperfusion injury. One of the important pathological changes during reperfusion after cerebral ischemia is disruption of blood–brain barrier (BBB). Rapamycin, a first-generation mTOR inhibitor, produces divergent effects on neuronal survival and alteration in BBB disruption. In this study, we investigated how Rapalink-1, a third-generation mTOR inhibitor, would affect neuronal survival and BBB disruption in the very early stage of cerebral ischemia–reperfusion that is within the time window of thrombolysis therapy. The middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Of note, 2 mg/kg of Rapalink-1 or vehicle was administered intraperitoneally 10 min after MCAO. After 1 h of MCAO and 2 h of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (104 Da) and the volume of 3H-dextran (70,000 Da) distribution were determined to assess the degree of BBB disruption. At the same time points, phosphorylated S6 (Ser240/244) and Akt (Ser473) as well as matrix metalloproteinase-2 (MMP2) protein level were determined by Western blot along with the infarct size using tetrazolium stain. Rapalink-1 increased the Ki in the ischemic-reperfused cortex (IR-C, +23%, p < 0.05) without a significant change in the volume of dextran distribution. Rapalink-1 increased the percentage of cortical infarct out of the total cortical area (+41%, p < 0.005). Rapalink-1 significantly decreased phosphorylated S6 and Akt to half the level of the control rats in the IR-C, which suggests that both of the mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2) were inhibited. The MMP2 level was increased suggesting that BBB disruption could be aggravated by Rapalink-1. Taken together, our data suggest that inhibiting both mTORC1 and mTORC2 by Rapalink-1 could worsen the neuronal damage in the early stage of cerebral ischemia–reperfusion and that the aggravation of BBB disruption could be one of the contributing factors.

Therapeutic strategies of glioblastoma (GBM): The current advances in the molecular targets and bioactive small molecule compounds

“…Studies also showed that 44 can effectively impair the motility and clonogenicity of GBM stem cells and reduce the expression of stem cell molecules 94 . However, 44 has an effect on the aggravation of BBB disruption, which may lead to serious side effect 95 . The chemical structure of 44 and the mTOR co-crystallized with 44 are presented in Fig.…”

Dysregulation of mTOR Signaling after Brain Ischemia

“…Several studies have revealed a positive neuroprotective effect of mTORCs upregulation after cerebral ischemia [ 105 , 106 ]. In the tMCAo model, mTORC1 and mTORC2 activity suppression using Rapalink-1, a third-generation mTOR inhibitor, has been reported to exacerbate neuronal damage induced by ischemia in the short-term, worsen BBB stability, and increase the area of damage [ 107 ]. These observations suggest that the upregulation of mTORC activity after cerebral ischemia has beneficial effects on the CNS.…”

Commentary: Rapalink-1 Increased Infarct Size in Early Cerebral Ischemia–Reperfusion With Increased Blood–Brain Barrier Disruption

“…Approval for this new indication could therefore take less time at a substantially reduced cost (Ashburn and Thor, 2004 ). Chi et al provide the first experimental stroke study of the mTOR inhibitor, Rapalink-1 (Chi et al, 2021 ). Rapalink-1 combines rapamycin with the mTOR kinase inhibitor, MLN0128 (molecular weight of Rapalink-1= 1,784 g/mol vs. rapamycin = 914 g/mol), with the goal of leveraging the high affinity of rapamycin for mTORC1 (via FK506 Binding Protein 12) to selectively deliver MLN0128 to the ATP-site of the mTORC1 complex (Rodrik-Outmezguine et al, 2016 ).…”

“…Given the increased potency of Rapalink-1, it would be expected that doses of Rapalink-1 needed for brain cytoprotection in stroke would be much lower than rapamycin (optimal dose for rapamycin <2 mg/kg). Chi et al used 2 mg/kg of Rapalink-1, which was associated with mTORC1 and mTORC2 inhibition and increased infarct volume (Chi et al, 2021 ). These findings further highlight the importance of mTORC2 signaling for cell survival during ischemia and suggest that although the dose of Rapalink-1 used is not too dissimilar to cytoprotective doses of rapamycin, the increased potency of Rapalink-1 may have resulted in mTORC2 inhibition at a lower dose.…”

“…We read with great interest the article by Chi et al investigating the effect of inhibiting mammalian target of rapamycin (mTOR) with a third generation mTOR inhibitor, Rapalink-1, in an experimental model of stroke (Chi et al, 2021 ). Rapalink-1 increased stroke size which was associated with reduced activity of both mTORC1 and mTORC2.…”