Contribution of Zinc-Dependent Delayed Calcium Influx via TRPC5 in Oxidative Neuronal Death and its Prevention by Novel TRPC Antagonist

Abstract

Oxidative stress is a key mediator of neuronal death in acute brain injuries, such as epilepsy, trauma, and stroke. Although it is accompanied by diverse cellular changes, increases in levels of intracellular zinc ion (Zn) and calcium ion (Ca) may play a critical causative role in oxidative neuronal death. However, the mechanistic link between Zn and Ca dyshomeostasis in neurons during oxidative stress is not well-understood. Here, we show that the exposure of cortical neurons to HO led to a zinc-triggered calcium influx, which resulted in neuronal death. The cyclin-dependent kinase inhibitor, NU6027, inhibited HO-induced Ca increases and subsequent cell death in cortical neurons, without affecting the early increase in Zn. Therefore, we attempted to identify the zinc-regulated Ca pathway that was inhibited by NU6027. The expression profile in cortical neurons identified transient receptor potential cation channel 5 (TRPC5) as a candidate that is known to involve in the generation of epileptiform burst firing and epileptic neuronal death (Phelan KD et al. 2012a; Phelan KD et al. 2013b). NU6027 inhibited basal and zinc-augmented TRPC5 currents in TRPC5-overexpressing HEK293 cells. Consistently, cortical neurons from TRPC5 knockout mice were highly resistant to HO-induced death. Moreover, NU6027 is neuroprotective in kainate-treated epileptic rats. Our results demonstrate that TRPC5 is a novel therapeutic target against oxidative neuronal injury in prolonged seizures and that NU6027 is a potent inhibitor of TRPC5.

Cryo-EM structures of human TRPC5 reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site

“…Intracellular Zn 2+ -dependent activation of TRPC5 channels was recently reported to contribute to oxidative neuronal death, 38 but the molecular mechanism of Zn 2+ regulation of TRPC5 was not identified. We have identified a putative intracellular zinc binding site of TRPC5, which is conserved in all TRPC channels.…”

“…This observation may indicate that zinc is already bound in TRPC5:Pico145, or that the role of zinc in TRPC5 modulation is subtle, which is consistent with the delayed Zn 2+ -mediated [Ca 2+ ]i increase observed in calcium imaging experiments, and the small TRPC5 currents evoked by intracellular application of ZnCl2. 38 Part of the putative zinc binding site has also been implicated in TRPC5 glutathionylation: mutation of C176, C178 or C181 prevented TRPC5 opening in response to glutathionylation. 40 A separate report proposed that palmitoylation of C181 is required for correct trafficking of TRPC5 to the plasma membrane in striatal neurons.…”

“…In addition, Park et al recently reported that intracellular application of Zn 2+ (micromolar concentrations) can open neuronal TRPC5 channels through an unknown mechanism. 38 Therefore, we solved an additional structure of TRPC5 (1 mg·ml -1 ; ca. 7 µM of TRPC5 monomer) in the presence of ZnCl2 (final concentration 20 µM) (Supplementary Figure 3C).…”

Transient Receptor Potential Melastatin 2 (TRPM2) Inhibition by Antioxidant, N-Acetyl-l-Cysteine, Reduces Global Cerebral Ischemia-Induced Neuronal Death

“…In a previous study, it was demonstrated that zinc-treated cultured neurons produce ROS from their mitochondria [ 18 ] and trigger microglial activation [ 26 ]. In addition, previous studies by our group have observed that excessive zinc accumulation within brain hippocampal regions can lead to neurodegeneration after cerebral insult, such as ischemia [ 10 , 27 ], head trauma [ 25 ], hypoglycemia [ 28 ], and cultured neurons [ 29 ].…”

Contribution of TRPC Channels in Neuronal Excitotoxicity Associated With Neurodegenerative Disease and Ischemic Stroke

“…Indeed, TRPC1/C4 and TRPC5 channels have been implicated in seizure-induced neuronal death in mouse lateral septum and hippocampus, respectively, in the pilocarpine-induced model of epilepsy ( Phelan et al, 2012 ). Inhibiting TRPC5 also protected neurons in pyriform cortex, amygdala, and hippocampus from death in kainate-treated rats with prolonged seizures ( Park et al, 2019 ) and in hippocampal CA3 neurons in a tramatic brain injury model ( Park et al, 2020 ). In these models, TRPC5 was thought to be activated by oxidation which first triggered a rise in cytosolic Zn 2+ levels and then opening of TRPC5 channels to mediate Ca 2+ influx, leading to neuronal cell death ( Park et al, 2019 ).…”