Molecular Neuroprotection Induced by Zinc-Dependent Expression of Hepatitis C–Derived Protein NS5A Targeting Kv2.1 Potassium Channels

Abstract

We present the design of an innovative molecular neuroprotective strategy and provide proof-of-concept for its implementation, relying on the injury-mediated activation of an ectopic gene construct. As oxidative injury leads to the intracellular liberation of zinc, we hypothesize that tapping onto the zinc-activated metal regulatory element () transcription factor 1 system to drive expression of the Kv2.1-targeted hepatitis C protein NS5A (hepatitis C nonstructural protein 5A) will provide neuroprotection by preventing cell death-enabling cellular potassium loss in rat cortical neurons in vitro. Indeed, using biochemical and morphologic assays, we demonstrate rapid expression of -driven products in neurons. Further, we report that-driven NS5A expression, induced by a slowly evolving excitotoxic stimulus, functionally blocks injurious, enhanced Kv2.1 potassium whole-cell currents and improves neuronal viability. We suggest this form of "on-demand" neuroprotection could provide the basis for a tenable therapeutic strategy to prevent neuronal cell death in neurodegeneration.

Location, location, location: the organization and roles of potassium channels in mammalian motoneurons

“…It is well established that that soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE)-mediated Kv2.1 membrane insertion mediates neuronal apoptosis (Pal et al 2003;Redman et al 2007;Yao et al 2009;Justice et al 2017Justice et al , 2018. Kv2.1-mediated apoptosis is a distinct, and likely much slower, signalling cascade than the rapid calcineurin-dependent homeostatic mechanism that regulates repetitive firing described in this review.…”

“…2009; Justice et al . 2017, 2018). Kv2.1‐mediated apoptosis is a distinct, and likely much slower, signalling cascade than the rapid calcineurin‐dependent homeostatic mechanism that regulates repetitive firing described in this review.…”

“…to oxidative damage at ER-PM junctions in cortical cells (Deutsch et al 2012;Justice et al 2017Justice et al , 2018Schulein et al 2020). It must also be remembered that many Kv2.1 channels are distant from large clusters or ER-PM junctions/SSCs, and even if only a minority of that population is in the conducting mode, uncontrolled cellular calcium levels and consequent dephosphorylation of these channels could depress cell excitability as well (Deardorff et al 2014;Romer et al 2019).…”

Defining the Kv2.1–syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant

“…We next examined whether these molecules could provide neuroprotection against overnight applications of threo-βbenzyloxyaspartate (TBOA; 75 μM), a nonselective glutamate reuptake inhibitor (34). TBOA induces relatively slow (overnight) NMDA receptor-mediated excitotoxicity, characterized by Kv2.1dependent cell death in our cultures (24,35). We found that pretreatment for 1 h and coincubation with 10…”

“…We previously showed that plasmid-mediated overexpression of C1a or use of the TAT-C1aB peptide prevents this Kv2.1-mediated current surge (23,24). To determine whether cpd5 achieves a similar inhibition of current enhancement, we obtained whole-cell patch clamp recordings of rat cortical neurons in vitro after coincubation with TBOA (50 μM for 2 h, followed by a 2-h wash period), a treatment previously shown to cause the canonical Kv2.1 current increase (35). In strong agreement with our neuroprotection assays, we found that cpd5 (10 μM) preloading (for 1 h) and cotreatment with TBOA effectively suppressed the postinjury enhancement of delayed rectifier K + currents in neurons to levels comparable to those in uninjured neurons (Fig.…”