bims-cemest 2025-08-24 papers

FASEB J. 2025 Aug 31. 39(16): e70940

Transcriptomic Profiling of Small Molecule Atf6 Modulators in the Retina.

Monica Sophia Diaz-Aguilar, Angela Galdamez, Leon Chea, Masako Le, Soyoung Park, Hyejung Min, Eun-Jin Lee, Jonathan H Lin.

The Unfolded Protein Response (UPR) maintains endoplasmic reticulum (ER) homeostasis and is essential for retinal health. Activating Transcription Factor 6 (ATF6) controls a key UPR branch and upregulates genes that mitigate ER stress. Small molecule modulators of ATF6 have been characterized in cell culture models that increase or decrease the amount of the cleaved, transcriptional activator domain of ATF6 generated from the full-length precursor. However, the effects of these small molecule ATF6 modulators remain unclear in vivo, in part because of the lack of antibodies that robustly detect the cleaved, activated form of ATF6 in model organisms like mice. Here, we used targeted RNA sequencing (RNA-seq) to assess the transcriptional response to intraocular delivery of Ceapin-A7 (an ATF6 inhibitor) and AA147 (an ATF6 activator) in the mouse retina. Using this strategy, we demonstrate that Ceapin-A7 significantly suppressed ATF6 transcriptional targets, whereas AA147 induced ATF6-regulated genes in retinal tissue of the eye. We also show that neither small molecule ATF6 modulator caused retinal cell death, compromised vision, or triggered ER stress by histology, functional testing, and transcriptional analysis. These results identify a transcriptional strategy to sensitively detect and quantify Ceapin-A7 and AA147 modulation of ATF6 in vivo. These findings also identify nontoxic conditions for further in vivo evaluation of small molecule ATF6 modulators in mouse vision loss disease models linked to ER stress.

Keywords: RNA‐seq; activating transcription factor 6; endoplasmic reticulum; retina; transcriptome; unfolded protein response

DOI: https://doi.org/10.1096/fj.202501459R

bioRxiv. 2025 Aug 13. pii: 2025.08.11.669714. [Epub ahead of print]

Proteostasis and Unfolded Protein Response Dynamics in Human Neuron and Mouse Glia Co-culture Reveal Cell-Specific Aging Responses.

Lea A Barny, Sarah K Garcia, Aiden J Houcek, Burak Uzay, Minsoo Kim, Ege T Kavalali, Lars Plate.

Proteostasis, or protein homeostasis, is a tightly regulated network of cellular pathways essential for maintaining proper protein folding, trafficking, and degradation. Neurons are particularly vulnerable to proteostasis collapse due to their post-mitotic and long-lived nature and thus represent a unique cell type to understand the dynamics of proteostasis throughout development, maturation, and aging. Here, we utilized a dual-species co-culture model of human excitatory neurons and mouse glia to investigate cell type- specific, age-related changes in the proteostasis network using data-independent acquisition (DIA) LC-MS/MS proteomics. We quantified branch-specific unfolded protein response (UPR) activation by monitoring curated effector proteins downstream of the ATF6, IRE1/XBP1s, and PERK pathways, enabling a comprehensive, unbiased evaluation of UPR dynamics during neuronal aging. Species-specific analysis revealed that aging neurons largely preserved proteostasis, although they showed some signs of collapse, primarily in ER-to-Golgi transport mechanisms. However, these changes were accompanied by upregulation of proteostasis-related machinery and activation of the ATF6 branch, as well as maintenance of the XBP1s and PERK branches of the UPR with age. In contrast, glia exhibited broad downregulation of proteostasis factors and UPR components, independent of neuronal presence. Furthermore, we quantified stimulus-specific modulation of select UPR branches in aged neurons exposed to pharmacologic ER stressors. These findings highlight distinct, cell-type-specific stress adaptations during aging and provide a valuable proteomic resource for dissecting proteostasis and UPR regulation in the aging brain.
Significance: Understanding how the unfolded protein response (UPR) and proteostasis network change with age is often studied in model organisms, where pathways are assessed across mixed cell types. Such systems can obscure cell-type-specific regulation. Here, we evaluate age-associated remodeling of the UPR and proteostasis network in a dual-species co-culture of human neurons and mouse glia using DIA proteomics. This approach enables species-specific proteomic profiling without physical separation, supported by a customizable data analysis pipeline. We show that neurons and glia exhibit divergent age-related responses, with neurons maintaining adaptive proteostasis and glia showing broader declines. The analytical framework presented here supports future studies to uncover additional cell-type-specific aging phenotypes or to probe the effects of pharmacologic or physical manipulation of biological systems.

DOI: https://doi.org/10.1101/2025.08.11.669714

Cell Stress Chaperones. 2025 Aug 15. pii: S1355-8145(25)00054-9. [Epub ahead of print]30(5): 100109

Multifaceted roles of mammalian heat shock factor 1 in the central nervous system.

Nicholas B Rozema, Rocio Gomez-Pastor.

Heat shock factor 1 (HSF1) is a stress-protective transcription factor most associated with transcriptional regulation of genes involved thermal stress response and protein folding. The canonical activation cycle of HSF1, in which HSF1 recognizes a simple promoter binding site known as a heat shock element (HSE) to promote the transcription of molecular chaperones, has been well documented. However, it is now evident that mammalian HSF1 exhibits unexpected complexity and participates in the response to a vast array of cellular stress types. The versatility of HSF1 can be attributed to distinct local protein concentrations, posttranslational modifications (PTMs), and binding partners found in different anatomical regions of the mammalian system. Advances in our knowledge of HSF1 under different types of stress have illuminated its vast array of gene targets, ranging from protein folding to mitochondrial homeostasis to cytoskeletal stability and beyond. In this review, we explore current knowledge of mammalian HSF1 and its gene targets within the central nervous system. While HSF1 has been extensively studied in the context of neurodegeneration, our understanding of its diverse roles in this setting remains limited. We also highlight emerging evidence supporting a physiological role for HSF1 in the healthy brain, an area that has received relatively little attention. Advancing a more comprehensive understanding of HSF1 function in the mammalian brain may aid in the development of novel therapeutics aimed at alleviating symptoms across a range of neurological disorders.

Keywords: Brain; Cellular stress; HSF1; Neurodegeneration; Synaptic stability

DOI: https://doi.org/10.1016/j.cstres.2025.100109

Nat Commun. 2025 Aug 18. 16(1): 7690

Investigational eIF2B activator DNL343 modulates the integrated stress response in preclinical models of TDP-43 pathology and individuals with ALS in a randomized clinical trial.

Neuronal TDP-43 aggregates are a hallmark ALS pathology. The integrated stress response (ISR) occurs downstream of TDP-43 pathology and may promote neurodegeneration. Here we demonstrate that a CNS penetrant small molecule eIF2B activator inhibits the ISR in cellular models of ALS and the brain of an inducible mouse model of TDP-43 pathology, where it transiently slowed progression of locomotor deficits and neurodegeneration. ISR activation was observed in ALS patient spinal cord and CSF. The investigational drug DNL343 was advanced into Phase 1 and Phase 1b randomized, double-blind, placebo-controlled trials in healthy and ALS participants, respectively (NCT04268784/NCT05006352); the primary objective in both studies was to investigate the safety and tolerability DNL343. DNL343 demonstrated a half-life supporting once-daily dosing and showed extensive CSF distribution. DNL343 was generally well tolerated and reduced ISR biomarkers in peripheral blood mononuclear cells and CSF of ALS participants. Therefore, DNL343 is a useful investigational drug to explore the effects of ISR inhibition in ALS models and individuals with neurological diseases.

DOI: https://doi.org/10.1038/s41467-025-63031-y

bioRxiv. 2025 Aug 12. pii: 2025.08.10.669490. [Epub ahead of print]

The Hsp40 co-chaperone DNAJC7 modifies polyglutamine but not polyglycine aggregation.

Biswarathan Ramani, Kean Ehsani, Martin Kampmann.

Polyglutamine (polyQ) diseases, including Huntington's disease and several spinocerebellar ataxias, are caused by abnormally expanded CAG nucleotide repeats, which encode aggregation-prone polyQ tracts. Substantial prior evidence supports a pathogenic role for polyQ protein misfolding and aggregation, with molecular chaperones showing promise in suppressing disease phenotypes in cellular and animal models. In this study, we developed a FRET-based reporter system that models polyQ aggregation in human cells and used it to perform a high-throughput CRISPR interference screen targeting all known molecular chaperones. This screen identified as a strong suppressor of polyQ aggregation the Hsp40 co-chaperone DNAJC7, which has previously been shown to modify aggregation of other disease proteins (tau and TDP-43) and has mutations causative for amyotrophic lateral sclerosis. We validated this phenotype and further established a physical interaction between DNAJC7 and polyQ-expanded protein. In contrast, DNAJC7 did not modify aggregation of polyglycine (polyG) in a FRET-based model of neuronal intranuclear inclusion disease. In addition to establishing new inducible, scalable cellular models for polyQ and polyG aggregation, this work expands the role of DNAJC7 in regulating folding of disease-associated proteins.

DOI: https://doi.org/10.1101/2025.08.10.669490

Biochem J. 2025 Aug 18. pii: BCJ20253237. [Epub ahead of print]482(16):

High tide or low tide: the transport and metabolism of mitochondrial nucleotides.

Thomas MacVicar.

Mitochondria are multifaceted organelles that support numerous cellular metabolic pathways, including the biosynthesis of nucleotides required for cell growth and proliferation. Owing to an ancient endosymbiotic origin, mitochondria contain multiple copies of their own genome and therefore demand sufficient (deoxy)nucleotides in the mitochondrial matrix for DNA replication and transcription into RNA. Disturbed mitochondrial deoxynucleotide homeostasis can lead to a decline in mitochondrial DNA abundance and integrity, causing mitochondrial diseases with diverse and severe symptoms. Mitochondrial nucleotides are not only required for nucleic acid synthesis but also for bioenergetics and mitochondrial enzymatic activity. This review first explores how mitochondria supply energy and anabolic precursors for nucleotide synthesis and how the mitochondrial network influences the spatial control of cellular nucleotide metabolism. Then follows an in-depth discussion of the mechanisms that supply mitochondria with sufficient and balanced nucleotides and why these mechanisms are relevant to human mitochondrial disease. Lastly, the review highlights the emergence of regulated mitochondrial nucleotide supply in physiological processes including innate immunity and discusses the implications of dysregulated mitochondrial and cytosolic nucleotide homeostasis in pathophysiology.

Keywords: metabolism; mitochondria; mitochondrial disease; nucleotide salvage; nucleotide transport; nucleotides

DOI: https://doi.org/10.1042/10.1042/BCJ20253237

bioRxiv. 2025 Aug 11. pii: 2025.08.07.669190. [Epub ahead of print]

Dehydration promotes intracellular lipid synthesis and accumulation.

Lipids can be considered a water reservoir used to offset dehydration stress as their oxidation by the mitochondria generates water. However, whether dehydration directly regulates lipid synthesis is unknown, which is the focus of this study. We found that dehydration stress decreases cellular oxygen consumption, increases intracellular lipid synthesis, and favors glutamine oxidation as a carbon precursor for lipid synthesis via remodeling mitochondrial metabolism. These findings provide a mechanism whereby cellular dehydration leads to intracellular lipid accumulation, functionally linking water availability to lipid storage.

DOI: https://doi.org/10.1101/2025.08.07.669190