Stroke, commonly known as stroke, is one of the leading causes of disability and death in the world, and the main causes of stroke are the interruption of cerebral blood perfusion caused by diseases such as atherosclerosis, atrial fibrillation, hypertension, and diabetes. Stroke can be divided into ischemic stroke and hemorrhagic stroke, with the former being the most common, accounting for about 80% of strokes. During ischemic stroke, a large release of glutamate activates NMDARs (N-methyl-D-aspartate receptors), inducing calcium ions to flow into neurons through these ion channels, overloading neurons and disrupting their calcium homology stability. Although scientists have studied ischemic injury extensively, there are no effective drugs yet, so there is an urgent need to identify the mechanism of stroke and find potential new targets for effective**ischemic stroke.
On March 14, 2023, Shankai Yin, Haibo Shi from Shanghai Jiao Tong University, and Wang Lu-Yang from the University of Toronto published the results of a study entitled "Bilirubin Gates the TRPM2 Channel as a Direct Agonist to Exacerbate Ischemic Brain Damage" on Neuron**. It is proposed that bilirubin, as an endogenous agonist, can directly block the opening of TRPM2 channels, thereby aggravating the brain injury of ischemic stroke. Through animal experiments, the research team found that molecular intervention and TRPM2 channel blocking in mouse models can play a good neuroprotective role.
Main results (results):
1. The relationship between cerebral infarction area and bilirubin level in stroke patients and mouse models
The role of bilirubin in stroke is controversial, with patients with high serum bilirubin levels presenting with more severe neurological symptoms, whereas magnetic resonance imaging can accurately determine the location and magnitude of ischemia. An analysis of the clinical records of different stroke patients found that the serum total bilirubin (TB) levels and direct bilirubin (DB) levels in stroke patients with hepatitis B were 2 to 3 times higher than those in the normal group, and the stroke significantly increased the concentration of TB and DB, suggesting that elevated bilirubin levels may be the result of stroke and are positively correlated with infarct volume.
Previous studies have shown that under ischemic conditions, elevated intracellular Ca2+ concentrations lead to excessive ROS production, which activates downstream signaling pathways, which in turn leads to cell death. The role of the TRPM2 channel in ischemia reperfusion was investigated by using an animal model of TMCAO and TRPM2 genotype mice. The experimental results showed that injection of bilirubin increased the infarct volume of TRPm2++ mice with TMCAO, while the infarct volume of TRPm2- mice did not change. This suggests that ischemic injury may induce endogenous bilirubin release, in which the TRPM2 channel plays a role. The investigators also observed that serum and cerebrospinal fluid bilirubin levels were significantly correlated with infarct volume, especially in TRPm2++ mice. Overall, the study concluded that ischemic reperfusion during stroke leads to increased bilirubin concentrations, and that the TRPM2 channel may play a modest role in this process.
2. Bilirubin activates the TRPM2 channel through a novel gating mechanism
The research team discussed the activation mechanism of TRPM2 channels by bilirubin and its structural derivatives. Previous studies have noted that the endogenous agonist ADPR controls its opening through the N-terminal MHR1 2 and the C-terminal NUDT9-H domain binding to two sites of the TRPM2 channel. However, due to the high degree of membrane permeability of bilirubin, the investigators speculate that bilirubin may activate the TRPM2 channel through different mechanisms.
By introducing point mutations at the ADPR binding site and the Nudt9-H domain, the researchers found that these mutants still respond to bilirubin, albeit with reduced activation. In contrast, ADPR failed to excite the mutated TRPM2 channel. Further experiments confirmed that truncation of the N-terminal and C-terminal ADPR binding domains prevented the activation of TRPM2 by ADPR, but did not affect the activation of bilirubin. Bilirubin and its derivatives are observed to activate TRPM2 channels for a relatively long time, while smaller molecules such as XAME activate channels at a faster rate. Through molecular docking simulations, the researchers found that bilirubin may interact with specific binding pockets of TRPM2 channels. This pocket is located near the calcium-binding site of the TRPM2 channel and forms hydrogen bonds and salt bridges with amino acid residues such as D866, K928, and D1069.
Experimentally confirmed that the replacement of these residues with non-polar alanine did not affect the activation of ADPR and calcium ions, but completely prevented the activation of bilirubin. Finally, through molecular dynamics simulations, the researchers compared the state transitions of bilirubin and Xame in the TRPM2 channel complex. The results showed that XAME had a higher affinity than bilirubin, which may explain the difference in their activation speed and effect. Overall, this study reveals a unique mechanism by which bilirubin activates TRPM2 channels, unlike classical intracellular ligands.
Figure Specific recognition of bilirubin-binding HTRPM2.
3. The bilirubin-binding cavity in the TRPM2 channel is an ideal drug site for antagonizing neurotoxicity
TRPM2 is a target for stroke** and uses blockers of different structures, such as clotrimazole, talm2nx, and a23, which are thought to have neuroprotective effects in preclinical studies in animal models. The research team found that the newly published TRPM2 blocker A23 forms a highly stable binding to the lumen, providing a structural explanation for its in vitro nanomolecular semi-maximal inhibitory concentration and effectiveness against ischemic injury in vivo. A23 has a high affinity for bilirubin binding cavity, which can effectively antagonize the effect of bilirubin on neurotoxicity.
Subsequently, the team studied the effect of TMCAO combined with bilirubin injection on the increase in cerebral infarction volume and serum and cerebrospinal fluid bilirubin levels, and identified the possible mechanisms. The experimental results showed that TMCAO combined with bilirubin injection could lead to an increase in the volume of cerebral infarction and an increase in serum and cerebrospinal fluid bilirubin levels, and bilirubin was mainly produced and released in neurons. Experiments have also found that ischemia and hypoxia can directly induce the brain to release endogenous bilirubin, thereby exacerbating neurotoxicity during stroke.
Figure Neurons NEUN, TRPM2 channel, biliverdin reductase BVR, and heme oxygenated 1 (HO-1).MIHC result plot, indicating that during a stroke, bilirubin is mainly produced and released in neurons.
4. Molecular perturbation of bilirubin-binding cavity on TRPM2 eliminates bilirubin-induced excitability upregulation and neurotoxicity
Through various experiments, the research team concluded that bilirubin and its structurally similar derivatives can activate the TRPM2 channel by binding to the cavity near the channel, and A23 blocks the binding of these agonists through competitive antagonism. To verify that a hole on TRPM2 mediates bilirubin neurotoxicity, a study used a knock-in mouse line to replace the key site in TRPM2 with alanine. This site has been shown to be a potent agonist for bilirubin rather than calcium ADPR. D1066A mice were shown in electrophysiological experiments that bilirubin no longer induced neuronal hyperexcitability, and the aggravating effect of bilirubin was completely inhibited in the ischemic brain injury model. This suggests that the binding cavity in the TRPM2 channel plays a key role in the neurotoxicity of bilirubin in stroke.
Conclusion:
This study provides mechanistic insight and principled evidence for the development of new strategies targeting bilirubin-binding cavities in TRPM2 channels to mitigate and prevent stroke- and jaundice-related brain injury in patients.
References
liu hw, gong ln, lai k, yu xf, liu zq, li mx, yin xl, liang m, shi hs, jiang lh, yang w, shi hb, wang ly, yin sk. bilirubin gates the trpm2 channel as a direct agonist to exacerbate ischemic brain damage. neuron. 2023 may 17;111(10):1609-1625.e6. doi: 10.1016/j.neuron.2023.02.022. epub 2023 mar 14. pmid: 36921602; pmcid: pmc10191619.
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