Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic, stress-independent activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic, stress-independent activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that stress-independent activation of these ISR kinases reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic, stress-independent activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Pharmacologic activation of a compensatory integrated stress response kinase promotes mitochondrial remodeling in PERK-deficient cells.

The integrated stress response (ISR) comprises the eIF2α kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2α kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells. We show that the HRI activator BtdCPU and GCN2 activator halofuginone promote ISR signaling and rescue ER stress sensitivity in PERK-deficient cells. However, BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and activation of the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and adaptive mitochondrial respiration, mimicking regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK signaling and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly selective ISR activators.

DELE1 oligomerization promotes integrated stress response activation.

Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

PERK signaling promotes mitochondrial elongation by remodeling membrane phosphatidic acid.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are linked in the onset and pathogenesis of numerous diseases. This has led to considerable interest in defining the mechanisms responsible for regulating mitochondria during ER stress. The PERK signaling arm of the unfolded protein response (UPR) has emerged as a prominent ER stress-responsive signaling pathway that regulates diverse aspects of mitochondrial biology. Here, we show that PERK activity promotes adaptive remodeling of mitochondrial membrane phosphatidic acid (PA) to induce protective mitochondrial elongation during acute ER stress. We find that PERK activity is required for ER stress-dependent increases in both cellular PA and YME1L-dependent degradation of the intramitochondrial PA transporter PRELID1. These two processes lead to the accumulation of PA on the outer mitochondrial membrane where it can induce mitochondrial elongation by inhibiting mitochondrial fission. Our results establish a new role for PERK in the adaptive remodeling of mitochondrial phospholipids and demonstrate that PERK-dependent PA regulation adapts organellar shape in response to ER stress.

Pharmacologic Activation of a Compensatory Integrated Stress Response Kinase Promotes Mitochondrial Remodeling in PERK-deficient Cells.

The integrated stress response (ISR) comprises the eIF2α kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2α kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells. We show that the HRI activator BtdCPU and GCN2 activator halofuginone promote ISR signaling and rescue ER stress sensitivity in PERK-deficient cells. However, BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and activation of the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and adaptive mitochondrial respiration, mimicking regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK signaling and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly-selective ISR activators.

Metabolically Activated Proteostasis Regulators Protect against Glutamate Toxicity by Activating NRF2.

The extracellular accumulation of glutamate is a pathologic hallmark of numerous neurodegenerative diseases including ischemic stroke and Alzheimer's disease. At high extracellular concentrations, glutamate causes neuronal damage by promoting oxidative stress, which can lead to cellular death. This has led to significant interest in developing pharmacologic approaches to mitigate the oxidative toxicity caused by high levels of glutamate. Here, we show that the small molecule proteostasis regulator AA147 protects against glutamate-induced cell death in a neuronal-derived cell culture model. While originally developed as an activator of the activating transcription factor 6 (ATF6) arm of the unfolded protein response, this AA147-dependent protection against glutamate toxicity is primarily mediated through activation of the NRF2-regulated oxidative stress response. We demonstrate that AA147 activates NRF2 selectively in neuronal-derived cells through a mechanism involving metabolic activation to a reactive electrophile and covalent modification of KEAP1─a mechanism analogous to that involved in the AA147-dependent activation of ATF6. These results define the potential for AA147 to protect against glutamate-induced oxidative toxicity and highlight the potential for metabolically activated proteostasis regulators like AA147 to activate both protective ATF6 and NRF2 stress-responsive signaling pathways to mitigate oxidative damage associated with diverse neurologic diseases.

Comparative effectiveness of second-line ipilimumab vs. nivolumab in combination with ipilimumab in patients with advanced melanoma who received frontline anti-PD-1 antibodies.

INTRODUCTION: Anti-PD-1 antibodies are commonly used as frontline therapy for patients with metastatic melanoma. Although these medications can cause long term responses, a significant number of patients will not respond or will lose response. Optimal second-line therapy after losing response to anti-PD-1 antibodies is not well established. Therefore, we retrospectively compared the overall survival of patients who lost response to anti-PD1 antibodies between patients treated with single agent ipilimumab or ipilimumab and nivolumab. METHODS: A de-identified U.S. nationwide electronic health record-derived database was reviewed for patients with advanced melanoma treated with single agent anti-PD1 antibodies in the frontline setting and who subsequently received second-line ipilimumab or combination ipilimumab and nivolumab. Overall survival from initiation of second-line therapy was compared using Kaplan Meier curves and log-rank analysis. Other known prognostic markers for melanoma were analyzed for correlation with survival in a similar fashion. Disease characteristics between the two groups were compared using chi-square analysis. RESULTS: A total of 842 patients with advanced melanoma who received frontline anti-PD-1 antibodies were included for analysis. Of these, 57 received either ipilimumab (n = 22) or ipilimumab in combination with nivolumab (n = 35) in the second-line setting. Median survival from second-line therapy initiation for those treated with ipilimumab alone was 6 months and was 5.6 months for those treated with combination ipilimumab and anti-PD-1 antibodies, p = 0.81. CONCLUSIONS: In this small, retrospective analysis, for patients who lost response to frontline anti-PD-1 therapy, patients treated with ipilimumab had similar survival to those who received ipilimumab in combination with anti-PD-1 antibodies.

Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation.

Tauopathies are a class of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy, which are associated with the pathological aggregation of tau protein into neurofibrillary tangles (NFT). Studies have characterized tau as a "prion-like" protein given its ability to form distinct, stable amyloid conformations capable of transcellular and multigenerational propagation in clonal fashion. It has been proposed that progression of tauopathy could be due to the prion-like propagation of tau, suggesting the possibility that end-stage pathologies, like NFT formation, may require an instigating event such as tau seeding. To investigate this, we applied a novel human induced pluripotent stem cell (hiPSC) system we have developed to serve as a human neuronal model. We introduced the tau repeat domain (tau-RD) with P301L and V337M (tau-RD-LM) mutations into hiPSC-derived neurons and observed expression of tau-RD at levels similar to total tau in postmortem AD brains. Tau aggregation occurred without the addition of recombinant tau fibrils. The conditioned media from tau-RD cultures contained tau-RD seeds, which were capable of inducing aggregate formation in homotypic mode in non-transduced recipient neuronal cultures. The resultant NFTs were thioflavin-positive, silver stain-positive, and assumed fibrillary appearance on transmission electron microscopy (TEM) with immunogold, which revealed paired helical filament 1 (PHF1)-positive NFTs, representing possible recruitment of endogenous tau in the aggregates. Functionally, expression of tau-RD caused neurotoxicity that manifested as axon retraction, synaptic density reduction, and enlargement of lysosomes. The results of our hiPSC study were reinforced by the observation that Tau-RD-LM is excreted in exosomes, which mediated the transfer of human tau to wild-type mouse neurons in vivo. Our hiPSC human neuronal system provides a model for further studies of tau aggregation and pathology as well as a means to study transcellular propagation and related neurodegenerative mechanisms.