Novel Anti-Stroke Agents Targeting Toxic Protein Aggregation

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This proposal is to study the role of toxic Nascent Polypeptide Chain (NPC) misfolding and aggregation in stroke brain injury. Stroke is a devastating neurological disease, affecting millions of Veterans. Therapies to reestablish perfusion are the current gold standard for treating acute stroke. However, because of the potential hemorrhagic side effect, only about 5% of stroke patients receive reperfusion therapy. Translation of messenger RNA (mRNA) into protein is also known as NPC synthesis. Our laboratory originally discovered that massive NPC aggregation occurs in post-ischemic neurons destined for death. We have published 30 papers on this subject. These studies have shown that NPC synthesis is inhibited in two distinct phases. The first phase of inhibition is transient, abating entirely after 0.5-1 h of reperfusion in all post- ischemic neurons; thus, not correlating with future neuronal death after brain ischemia. This first phase of inhibition is followed by a recovery of >70% of NPC synthesis at 4 h of reperfusion among all post-ischemic neurons. The second phase of NPC synthesis inhibition takes place progressively from 4 h of reperfusion onward and is only in neurons destined to die. Our studies have shown that this second phase of inhibition is due to the post-ischemic loss of co-translational chaperone protection of NPCs. Without this protection, NPCs expose their sticky hydrophobic segments during synthesis and are irreversibly aggregated on ribosomes from 4 h of reperfusion onward after brain ischemia. These studies have led to the novel discovery that targeting massive NPC aggregation by reducing the load of “unprotected” NPCs on ribosomes with eukaryotic initiation factor (eIF) inhibitors can protect brain from stroke injury. By using high throughput technologies, several “direct” eIF4E (interaction) inhibitors have been identified, e.g., a dual inhibitor against eIF4E-to-eIF4G and eIF4E-to-4E-BP1 interactions (4E1Rcat) and ribavirin. These discoveries provide an outstanding opportunity to understand the role of NPC aggregation in stroke brain injury and to develop a novel category of anti-stroke agents. Our laboratory has studied several eIF inhibitors. Among them, 4EGI-1 offers the best anti-stroke efficacy in mouse stroke models. Therefore, we used 4EGI-1 in double-blind, randomized controlled animal studies. These studies clearly demonstrated that mice treated with i.p. injection of 4EGI-1 after 30 min of reperfusion had no stroke mortality compared to the 45% mortality in the vehicle group. 4EGI-1 treatment significantly reduced the infarct volume, improved physical recovery and behavioral performance, and decreased neurological deficits after stroke. The goals of the proposed research are to investigate further the role of NPC aggregation in stroke brain injury and to identify the best targets and inhibitors against stroke brain injury (Aim 1); and to investigate the details of the underlying brain protection mechanisms (Aims 2 and 3). We will test the following hypotheses: (1) Inhibition of eIF4E not only reduces the load of post-ischemic “unprotected” NPCs on ribosomes, but also switches translation from a cap-dependent to a cap-independent state. This switch shuts down cap-dependent NPC translation while prioritizing cap-independent translation of molecular chaperones; thus, protecting post- ischemic “unprotected” NPCs from toxic aggregation during reperfusion. (2) 4EGI-1 treatment leads to a robust upregulation of ATF4 and CHOP transcription factors to induce essential autophagic genes; thus, facilitating the removal of toxic NPC aggregates after brain ischemia. (3) Inhibition of eIF4E mitigates post-stroke inflammation. Although inhibition of other NPC synthesis initiation factors/regulators may also reduce the load of “unprotected” NPCs on ribosomes, these other superior benefits (e.g., upregulation of co-translational molecular chaperones and autophagic proteins) offered by eIF4E inhibition described above give eIF4E an edge as the therapeutic target to reduce toxic NPC aggregation after brain ischemia. The eIF4E inhibitors show an excellent potential to become a new class of anti-stroke drugs for Veterans.