Lysosomal degradation pathways coordinate the clearance of superfluous and damaged cellular components. Compromised lysosomal degradation is a hallmark of many degenerative diseases, including lysosomal storage diseases (LSDs), which are caused by loss-of-function mutations within both alleles of a lysosomal hydrolase, leading to lysosomal substrate accumulation. Gaucher\'s disease, characterized by <15% of normal glucocerebrosidase function, is the most common LSD and is a prominent risk factor for developing Parkinson\'s disease. Here, we show that either of two structurally distinct small molecules that modulate PIKfyve activity, identified in a high-throughput cellular lipid droplet clearance screen, can improve glucocerebrosidase function in Gaucher patient-derived fibroblasts through an MiT/TFE transcription factor that promotes lysosomal gene translation. An integrated stress response (ISR) antagonist used in combination with a PIKfyve modulator further improves cellular glucocerebrosidase activity, likely because ISR signaling appears to also be slightly activated by treatment by either small molecule at the higher doses employed. This strategy of combining a PIKfyve modulator with an ISR inhibitor improves mutant lysosomal hydrolase function in cellular models of additional LSD.

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Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.

Cannabidiol (CBD) is a non-psychoactive component of cannabis with potential applications in biomedicine, food, and cosmetics due to its analgesic, anti-inflammatory, and anticonvulsant properties. However, increasing reports of adverse CBD exposure events underscore the necessity of evaluating its toxicity. In this study, we investigated the developmental toxicity of CBD in zebrafish during the embryonic (0-4 dpf, days post fertilization) and early larval stages (5-7 dpf). The median lethal concentration of CBD in embryos/larvae is 793.28 μg/L. CBD exhibited concentration-dependent manner (ranging from 250 to 1500 μg/L) in inducing serious malformed somatotypes, like shorter body length, pericardial cysts, vitelline cysts, spinal curvature, and smaller eyes. However, no singular deformity predominates. The 5-month-old zebrafish treated with 100 and 200 μg/L of CBD during the embryonic and early larval stages produced fewer offspring with higher natural mortality and malformation rate. Gonadal growth and gamete development were inhibited. Transcriptomic and metabolomic analyses conducted with 400 μg/L CBD on embryos/larvae from 0 to 5 dpf suggested that CBD promoted the formation and transportation of extracellular matrix components on 1 dpf, promoting abnormal cell division and migration, probably resulting in random malformed somatotypes. It inhibited optical vesicle development and photoreceptors formation on 2 and 3 dpf, resulting in damaged sight and smaller eye size. CBD also induced an integrated stress response on 4 and 5 dpf, disrupting redox, protein, and cholesterol homeostasis, contributing to cellular damage, physiological dysfunction, embryonic death, and inhibited reproductive system and ability in adult zebrafish. At the tested concentrations, CBD exhibited developmental toxicity, lethal toxicity, and reproductive inhibition in zebrafish. These findings demonstrate that CBD threatens the model aquatic animal, highlighting the need for additional toxicological evaluations of CBD before its inclusion in dietary supplements, edible food, and other products.

Mitochondrial stress, resulting from dysfunction and proteostasis disturbances, triggers the mitochondrial unfolded protein response (UPRMT), which activates gene encoding chaperones and proteases to restore mitochondrial function. Although ATFS-1 mediates mitochondrial stress UPRMT induction in C. elegans, the mechanisms relaying mitochondrial stress signals to the nucleus in mammals remain poorly defined. Here, we explored the role of protein kinase R (PKR), an eIF2α kinase activated by double-stranded RNAs (dsRNAs), in mitochondrial stress signaling. We found that UPRMT does not occur in cells lacking PKR, indicating its crucial role in this process. Mechanistically, we observed that dsRNAs accumulate within mitochondria under stress conditions, along with unprocessed mitochondrial transcripts. Furthermore, we demonstrated that accumulated mitochondrial dsRNAs in mouse embryonic fibroblasts (MEFs) deficient in the Bax/Bak channels are not released into the cytosol and do not induce the UPRMT upon mitochondrial stress, suggesting a potential role of the Bax/Bak channels in mediating the mitochondrial stress response. These discoveries enhance our understanding of how cells maintain mitochondrial integrity, respond to mitochondrial dysfunction, and communicate stress signals to the nucleus through retrograde signaling. This knowledge provides valuable insights into prospective therapeutic targets for diseases associated with mitochondrial stress.

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons.

For bipolar disorder (BD), the inconsistency of treatment guidelines and the long phases of pharmacological adjustment remain major challenges. BD is known to be comorbid with many medical and psychiatric conditions and they may share inflammatory and stress-related aetiologies, which could give rise to this association. The integrated stress response (ISR) responds to various stress conditions that lead to alterations in cellular homeostasis. However, as a causative mechanism underlying cognitive deficits and neurodegeneration in a broad range of brain disorders, the impact of ISR on BD is understudied. Mendelian randomization has been widely used to repurpose licensed drugs and discover novel therapeutic targets. Thus, we aimed to identify novel therapeutic targets for BD and analyze their pathophysiological mechanisms, using the summary data-based Mendelian Randomization (SMR) and Bayesian colocalization (COLOC) methods to integrate the summary-level data of the GWAS on BD and the expression quantitative trait locus (eQTL) study in blood. We utilized the GWAS data including 41,917 BD cases and 371,549 controls from the Psychiatric Genomics Consortium and the eQTL data from 31,684 participants of predominantly European ancestry from the eQTLGen consortium. The SMR analysis identified the EIF2B5 gene that was associated with BD due to no linkage but pleiotropy or causality. The COLOC analysis strongly suggested that EIF2B5 and the trait of BD were affected by shared causal variants, and thus were colocalized. Utilizing data in EpiGraphDB we find other putative causal BD genes (EIF2AK4 and GSK3B) to prioritize potential alternative drug targets.

Endoplasmic reticulum (ER) stress in oligodendrocyte (OL) linage cells contributes to several CNS pathologies including traumatic spinal cord injury (SCI) and multiple sclerosis. Therefore, primary rat OL precursor cell (OPC) transcriptomes were analyzed using RNASeq after treatments with two ER stress-inducing drugs, thapsigargin (TG) or tunicamycin (TM). Gene ontology term (GO) enrichment showed that both drugs upregulated mRNAs associated with the general stress response. The GOs related to ER stress were only enriched for TM-upregulated mRNAs, suggesting greater ER stress selectivity of TM. Both TG and TM downregulated cell cycle/cell proliferation-associated transcripts, indicating the anti-proliferative effects of ER stress. Interestingly, many OL lineage-enriched mRNAs were downregulated, including those for transcription factors that drive OL identity such as Olig2. Moreover, ER stress-associated decreases of OL-specific gene expression were found in mature OLs from mouse models of white matter pathologies including contusive SCI, toxin-induced demyelination, and Alzheimer\'s disease-like neurodegeneration. Taken together, the disrupted transcriptomic fingerprint of OL lineage cells may facilitate myelin degeneration and/or dysfunction when pathological ER stress persists in OL lineage cells.
The ER stress response compromises the transcriptomic identity of the OL lineage. Therefore, persistent, pathological ER stress may have a negative impact on structural and/or functional integrity of the white matter.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-βH1 human β cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

ONC201 (originally discovered as TRAIL-Inducing Compound #10 or TIC10) and analogue ONC206 have been found to induce an integrated stress response with suggested primary targets and mechanisms involving targeting mitochondrial protein ClpP and antagonism of dopamine receptors D2/3 (DRD2/3). We hypothesized that dopamine, the agonist of DRD2, may counteract ONC201 or ONC206 for DRD2/3 and impair the anti-cancer effect of ONC201 or ONC206, thus protect the tumor cells from the cytotoxic effect of ONC201 or ONC206. We therefore pre-treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, and diffuse midline glioma (DMG) with dopamine, followed by treatment of ONC201, ONC206 or ONC212. We observed that 48 hours of pre-treatment with dopamine impaired the cell viability suppression effect of ONC201, ONC206 and ONC212 in pancreatic cancer cells and colorectal cancer cells. We pre-treated multiple cancer cell lines with dopamine for one week followed by ONC201, ONC206, or ONC212 treatment and performed colony assays. Pre-treatment with dopamine impaired the anti-cancer effect of ONC201 or ONC206 in pancreatic cancer and colorectal cancer. Impairment of ONC212 effect by pre-treatment with dopamine was also seen in colony assay for colorectal cancer, but not in pancreatic cancer cells by colony assay. No protection from killing by imipridones was observed with DRD2 agonist sumanirole in tumor cells, or with brain tumor cell lines pretreated with dopamine. Immunoblotting was conducted to investigate whether dopamine pre-treatment impacts signaling pathways reported to be affected by ONC201. The dopamine pre-treatment did not impact changes in ATF4, CHOP, DR5 and ClpX which were reported to be affected by ONC201. The mechanism of impairment of ONC201/206/212 effect caused by dopamine pre-treatment appears to involve upregulation of anti-apoptotic p-Bad, XIAP, FLIP and pAkt. Our results shed light on mechanisms of cancer cell protection by dopamine after imipridone treatment, heterogeneity among different tumor cell types, and suggest that effects of dopamine adaptation on tumor cells may impact on cell survival pathways in ways that may or may not depend on expression of dopamine receptors.