Novel N-Phenyl–Substituted Thiazolidinediones Protect Neural Cells against Glutamate- and tBid-Induced Toxicity

Abstract

Mitochondrial demise is a key feature of progressive neuronal death contributing to acute and chronic neurological disorders. Recent studies identified a pivotal role for the BH3-only protein B-cell lymphoma-2 interacting domain death antagonist (Bid) for such mitochondrial damage and delayed neuronal death after oxygen-glucose deprivation, glutamate-induced excitotoxicity, or oxidative stress in vitro and after cerebral ischemia in vivo. Therefore, we developed new N-phenyl-substituted thiazolidine-2,4-dione derivatives as potent inhibitors of Bid-dependent neurotoxicity. The new compounds 6, 7, and 16 were identified as highly protective by extensive screening in a model of glutamate toxicity in immortalized mouse hippocampal neurons (HT-22 cells). These compounds significantly prevent truncated Bid-induced toxicity in the neuronal cell line, providing strong evidence that inhibition of Bid was the underlying mechanism of the observed protective effects. Furthermore, Bid-dependent hallmarks of mitochondrial dysfunction, such as loss of mitochondrial membrane potential, ATP depletion, as well as impairments in mitochondrial respiration, are significantly prevented by compounds 6, 7, and 16. Therefore, the present study identifies a class of N-phenyl thiazolidinediones as novel Bid-inhibiting neuroprotective agents that provide promising therapeutic perspectives for neurodegenerative diseases, in which Bidmediated mitochondrial damage and associated intrinsic death pathways contribute to the underlying progressive loss of neurons.

BID links ferroptosis to mitochondrial cell death pathways

“…3c). In line with our previous studies [19], detailed analysis of cell viability showed that the protective effect of BI-6c9 against glutamate and erastin is dose-dependent (Supp. 3a and 3b).…”

“…Erastin induced morphological and functional damage of mitochondria such as enhanced fragmentation, increased formation of mitochondrial ROS, loss of MMP and ATP depletion in a very similar pattern as the hallmarks of oxytosis triggered by glutamate in the neuronal HT-22 cells [12], [13], [16], [19], [25]. Further, using CRISPR/Cas9 technology for genetic BID deletion and the pharmacological BID-inhibitor BI-6c9 we identified a key role for BID in mediating the hallmarks of erastin toxicity.…”

“…Hence, we investigated the subcellular distribution of BID in response to erastin and glutamate-induced stress, respectively. To this end, HT-22 WT cells were co-transfected with a red-labeled BID and a mitochondrial-targeted GFP vector as described before [16], [19]. Confocal microscopy pictures reveal a translocation of BID to the mitochondria upon erastin and glutamate exposure indicated by the merging fluorescence shown in yellow and a Pearson's coefficient of r=0.78+0.18 and r=0.67+0.06 respectively.…”

“…Following previously established protocols [19], [25], we transfected HT-22 cells with a tBID-encoding plasmid and analyzed cell viability 24 h after the transfection. Annexin V/PI staining revealed that BI-6c9 significantly reduced tBID-induced cytotoxicity similar to earlier findings [19], while ferrostatin-1 failed to protect HT-22 cells from tBID toxicity (Fig. 6b, c).…”

“…In paradigms of oxytosis in neuronal cells, mitochondrial impairments are mediated through mitochondrial transactivation of the pro-apoptotic protein BID  [13], [16], [25]. Upon translocation to the mitochondria, BID mediates loss of mitochondrial integrity and function and detrimental translocation of mitochondrial AIF to the nucleus [12], [13], [19], [21], [24], [25], [26], [5].…”