bims-proarb 2021-08-01 papers

bims-proarb

Biomed News

on Proteostasis in aging and regenerative biology

Issue of 2021–08–01
forty papers selected by
Rich Giadone, Harvard University

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress.
Activation of the Protein Kinase R-Like Endoplasmic Reticulum Kinase (PERK) Pathway of the Unfolded Protein Response after Experimental Traumatic Brain Injury and Treatment with a PERK Inhibitor.
Recent Advances in the Roles of HSFs and HSPs in Heat Stress Response in Woody Plants.
All cells are created equal in the sight of autophagy: selective autophagy maintains homeostasis in senescent cells.
Maintaining proteostasis under mechanical stress.
ER stress-induced modulation of neural activity and seizure susceptibility is impaired in a fragile X syndrome mouse model.
Towards a better understanding of the neuro-developmental role of autophagy in sickness and in health.
Hypoxia and the integrated stress response promote pulmonary hypertension and preeclampsia: Implications in drug development.
Naringenin attenuates endoplasmic reticulum stress, reduces apoptosis, and improves functional recovery in experimental traumatic brain injury.
ER-Phagy: A New Regulator of ER Homeostasis.
Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.
MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells.
Flavan-3-ol Microbial Metabolites Modulate Proteolysis in Neuronal Cells Reducing Amyloid-beta (1-42) Levels.
Rewiring of the ubiquitinated proteome determines ageing in C. elegans.
Integrated stress response control of granulosa cell translation and proliferation during normal ovarian follicle development.
Rutin alleviates cardiomyocyte injury induced by high glucose through inhibiting apoptosis and endoplasmic reticulum stress.
Inhibition of the unfolded protein response reduces arrhythmic risk after myocardial infarction.
Assessment of EGFP-Q74 degradation for the measurement of autophagic flux.
miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging.
Immunoblot-based assays for assessing autophagy in the turquoise killifish Nothobranchius furzeri.
Protonation-Deprotonation Switch Controls the Amyloid-like Misfolding of Nucleic-Acid-Binding Domains of TDP-43.
MyD88-mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms.
Regulation of fractone heparan sulfate composition in young and aged subventricular zone neurogenic niches.
Protein farnesylation is upregulated in Alzheimer's human brains and neuron-specific suppression of farnesyltransferase mitigates pathogenic processes in Alzheimer's model mice.
A 4D single-cell protein atlas of transcription factors delineates spatiotemporal patterning during embryogenesis.
α-Mangostin Induces Apoptosis in Human Osteosarcoma Cells Through ROS-Mediated Endoplasmic Reticulum Stress via the WNT Pathway.
eIF2-dependent translation initiation: Memory consolidation and disruption in Alzheimer's disease.
Effects of dopamine transporter changes in the ventral tegmental area of the midbrain on cognitive function in aged rats.
Development of a specific live-cell assay for native autophagic flux.
Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice.
A Dictyostelium model for BPAN disease reveals a functional relationship between the WDR45/WIPI4 homolog Wdr45l and Vmp1 in the regulation of autophagy-associated PtdIns3P and ER stress.
Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span.
Endoplasmic reticulum stress inhibits 3D Matrigel-induced vasculogenic mimicry of breast cancer cells via TGF-β1/Smad2/3 and β-catenin signaling.
Age-Related Transcriptional Deregulation of Genes Coding Synaptic Proteins in Alzheimer's Disease Murine Model: Potential Neuroprotective Effect of Fingolimod.
A β-Wrapin Targeting the N-Terminus of α-Synuclein Monomers Reduces Fibril-Induced Aggregation in Neurons.
Insane in the membrane: developments in protein folding, protein transport, and signaling by GPCRs.
Biochanin A Alleviates Cerebral Ischemia/Reperfusion Injury by Suppressing Endoplasmic Reticulum Stress-Induced Apoptosis and p38MAPK Signaling Pathway In Vivo and In Vitro.
Stress-primed secretory autophagy promotes extracellular BDNF maturation by enhancing MMP9 secretion.
Multimodal assessment of autophagy in mammalian cells with a novel, LC3-based tandem reporter.
A glutaminyl cyclase-catalyzed α-synuclein modification identified in human synucleinopathies.

Aging Cell. 2021 Jul 30. e13446

The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress.

Ernesto Llamas, Salvador Torres-Montilla, Hyun Ju Lee, María Victoria Barja, Elena Schlimgen, Nick Dunken, Prerana Wagle, Wolfgang Werr, Alga Zuccaro, Manuel Rodríguez-Concepción, David Vilchez.

  The biological purpose of plant stem cells is to maintain themselves while providing new pools of differentiated cells that form organs and rejuvenate or replace damaged tissues. Protein homeostasis or proteostasis is required for cell function and viability. However, the link between proteostasis and plant stem cell identity remains unknown. In contrast to their differentiated counterparts, we find that root stem cells can prevent the accumulation of aggregated proteins even under proteotoxic stress conditions such as heat stress or proteasome inhibition. Notably, root stem cells exhibit enhanced expression of distinct chaperones that maintain proteome integrity. Particularly, intrinsic high levels of the T-complex protein-1 ring complex/chaperonin containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an important requirement for plants to persist under extreme environmental conditions and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to design and breed plants tolerant to environmental challenges caused by the climate change.

Keywords:  chaperones; heat stress; plant stem cells; protein aggregation; protein misfolding; proteostasis

DOI:  https://doi.org/10.1111/acel.13446
Neurotrauma Rep. 2021 ;2(1): 330-342

Activation of the Protein Kinase R-Like Endoplasmic Reticulum Kinase (PERK) Pathway of the Unfolded Protein Response after Experimental Traumatic Brain Injury and Treatment with a PERK Inhibitor.

Rhys D Brady, Stefanie Bird, Mujun Sun, Glenn R Yamakawa, Brendan P Major, Richelle Mychasiuk, Terence J O'Brien, Stuart J McDonald, Sandy R Shultz.

  Neurodegeneration after traumatic brain injury (TBI) is increasingly recognized as a key factor contributing to poor chronic outcomes. Activation (i.e., phosphorylation) of the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway has been implicated in neurodegenerative conditions with pathological similarities to TBI and may be a potential target to improve TBI outcomes. Here, we aimed to determine whether a moderate TBI would induce activation of the PERK pathway and whether treatment with the PERK inhibitor, GSK2606414, would improve TBI recovery. Male mice were administered a lateral fluid percussion injury (FPI) or sham injury and were euthanized at either 2 h, 24 h, or 1 week post-injury (n = 5 per injury group and time point) to assess changes in the PERK pathway. In the injured cortex, there was increased phosphorylated-PERK at 2 h post-FPI and increased phosphorylation of eukaryotic translation initiation factor α at 24 h post-FPI. We next examined the effect of acute treatment with GSK2606414 on pathological and behavioral outcomes at 4 weeks post-injury. Thus, there were a total of four groups: sham + VEH (n = 9); sham + GSK4606414 (n = 10); FPI + VEH (n = 9); and FPI + GSK2606414 (n = 9). GSK2606414 (50 mg/kg) or vehicle treatment was delivered by oral gavage beginning at 30 min post-injury, followed by two further treatments at 12-h increments. There were no significant effects of GSK2606414 on any of the outcomes assessed, which could be attributable to several reasons. For example, activation of PERK may not be a significant contributor to the neurological consequences 4 weeks post-FPI in mice. Further research is required to elucidate the role of the PERK pathway in TBI and whether interventions that target this pathway are beneficial.

Keywords:  GSK2606414; eukaryotic translation initiation factor α; fluid percussion injury; misfolded proteins; neurodegeneration; protein kinase RNA-like ER kinase

DOI:  https://doi.org/10.1089/neur.2021.0001
Front Plant Sci. 2021 ;12 704905

Recent Advances in the Roles of HSFs and HSPs in Heat Stress Response in Woody Plants.

Fengxia Tian, Xiao-Li Hu, Tao Yao, Xiaohan Yang, Jin-Gui Chen, Meng-Zhu Lu, Jin Zhang.

  A continuous increase in ambient temperature caused by global warming has been considered a worldwide threat. As sessile organisms, plants have evolved sophisticated heat shock response (HSR) to respond to elevated temperatures and other abiotic stresses, thereby minimizing damage and ensuring the protection of cellular homeostasis. In particular, for perennial trees, HSR is crucial for their long life cycle and development. HSR is a cell stress response that increases the number of chaperones including heat shock proteins (HSPs) to counter the negative effects on proteins caused by heat and other stresses. There are a large number of HSPs in plants, and their expression is directly regulated by a series of heat shock transcription factors (HSFs). Therefore, understanding the detailed molecular mechanisms of woody plants in response to extreme temperature is critical for exploring how woody species will be affected by climate changes. In this review article, we summarize the latest findings of the role of HSFs and HSPs in the HSR of woody species and discuss their regulatory networks and cross talk in HSR. In addition, strategies and programs for future research studies on the functions of HSFs and HSPs in the HSR of woody species are also proposed.

Keywords:  heat shock protein; heat shock transcription factor; heat stress; molecular response; signaling network; woody plants

DOI:  https://doi.org/10.3389/fpls.2021.704905
Autophagy. 2021 Jul 27. 1-2

All cells are created equal in the sight of autophagy: selective autophagy maintains homeostasis in senescent cells.

Jaejin Kim, Yeonghyeon Lee, Taerang Jeon, Mi-Sung Kim, Chanhee Kang.

  Macroautophagy/autophagy is a sophisticated quality control program that limits cellular damage and maintains homeostasis, being an essential part of several lifespan-promoting interventions. However, autophagy is also necessary for full establishment of cellular senescence, a causal factor for many age-related diseases and aging. What lies ahead of us to unravel such a paradoxical role of autophagy in senescence is to identify specific targets degraded by autophagy during senescence and determine their importance in the senescence regulatory network. Recently, we developed the "Selective autophagy substrates Identification Platform (SIP)" to advance these goals, providing a rich set of autophagy substrate proteins involved in senescence. Our study demonstrated that selective autophagy coordinates the stress support networks in senescent cells by degrading multiple regulatory components, echoing its homeostatic roles in normal cells. Targeting this type of selective autophagy might provide a unique opportunity to develop non-senescence addiction-based therapeutic strategies for senotherapy by disturbing the homeostatic state of senescent cells.

Keywords:  Autophagy interactome; cellular senescence; inflammation; oxidative stress; proteostasis; regulated protein stability; selective autophagy; stress support networks

DOI:  https://doi.org/10.1080/15548627.2021.1953848
EMBO Rep. 2021 Jul 26. e52507

Maintaining proteostasis under mechanical stress.

Jörg Höhfeld, Thomas Benzing, Wilhelm Bloch, Dieter O Fürst, Sebastian Gehlert, Michael Hesse, Bernd Hoffmann, Thorsten Hoppe, Pitter F Huesgen, Maja Köhn, Waldemar Kolanus, Rudolf Merkel, Carien M Niessen, Wojciech Pokrzywa, Markus M Rinschen, Dagmar Wachten, Bettina Warscheid.

  Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.

Keywords:  autophagy; chaperones; mechanobiology; proteostasis; signal transduction

DOI:  https://doi.org/10.15252/embr.202152507
Neurobiol Dis. 2021 Jul 23. pii: S0969-9961(21)00199-6. [Epub ahead of print]158 105450

ER stress-induced modulation of neural activity and seizure susceptibility is impaired in a fragile X syndrome mouse model.

Dai-Chi Liu, Kwan Young Lee, Simon Lizarazo, Jessie K Cook, Nien-Pei Tsai.

  Imbalanced neuronal excitability homeostasis is commonly observed in patients with fragile X syndrome (FXS) and the animal model of FXS, the Fmr1 KO. While alterations of neuronal intrinsic excitability and synaptic activity at the steady state in FXS have been suggested to contribute to such a deficit and ultimately the increased susceptibility to seizures in FXS, it remains largely unclear whether and how the homeostatic response of neuronal excitability following extrinsic challenges is disrupted in FXS. Our previous work has shown that the acute response following induction of endoplasmic reticulum (ER) stress can reduce neural activity and seizure susceptibility. Because many signaling pathways associated with ER stress response are mediated by Fmr1, we asked whether acute ER stress-induced reduction of neural activity and seizure susceptibility are altered in FXS. Our results first revealed that acute ER stress can trigger a protein synthesis-dependent prevention of neural network synchronization in vitro and a reduction of susceptibility to kainic acid-induced seizures in vivo in wild-type but not in Fmr1 KO mice. Mechanistically, we found that acute ER stress-induced activation of murine double minute-2 (Mdm2), ubiquitination of p53, and the subsequent transient protein synthesis are all impaired in Fmr1 KO neurons. Employing a p53 inhibitor, Pifithrin-α, to mimic p53 inactivation, we were able to blunt the increase in neural network synchronization and reduce the seizure susceptibility in Fmr1 KO mice following ER stress induction. In summary, our data revealed a novel cellular defect in Fmr1 KO mice and suggest that an impaired response to common extrinsic challenges may contribute to imbalanced neuronal excitability homeostasis in FXS.

Keywords:  ER stress; FMRP; FXS; Hyperexcitability; Seizures

DOI:  https://doi.org/10.1016/j.nbd.2021.105450
Cell Stress. 2021 Jul;5(7): 99-118

Towards a better understanding of the neuro-developmental role of autophagy in sickness and in health.

Juan Zapata-Muñoz, Beatriz Villarejo-Zori, Pablo Largo-Barrientos, Patricia Boya.

  Autophagy is a critical cellular process by which biomolecules and cellular organelles are degraded in an orderly manner inside lysosomes. This process is particularly important in neurons: these post-mitotic cells cannot divide or be easily replaced and are therefore especially sensitive to the accumulation of toxic proteins and damaged organelles. Dysregulation of neuronal autophagy is well documented in a range of neurodegenerative diseases. However, growing evidence indicates that autophagy also critically contributes to neurodevelopmental cellular processes, including neurogenesis, maintenance of neural stem cell homeostasis, differentiation, metabolic reprogramming, and synaptic remodelling. These findings implicate autophagy in neurodevelopmental disorders. In this review we discuss the current understanding of the role of autophagy in neurodevelopment and neurodevelopmental disorders, as well as currently available tools and techniques that can be used to further investigate this association.

Keywords:  autism spectrum disorder; developmental disorders; neurodevelopment; neurogenesis; neuronal autophagy

DOI:  https://doi.org/10.15698/cst2021.07.253
Drug Discov Today. 2021 Jul 22. pii: S1359-6446(21)00319-6. [Epub ahead of print]

Hypoxia and the integrated stress response promote pulmonary hypertension and preeclampsia: Implications in drug development.

Xiang-Qun Hu, Lubo Zhang.

  Chronic hypoxia is a common cause of pulmonary hypertension, preeclampsia, and intrauterine growth restriction (IUGR). The molecular mechanisms underlying these diseases are not completely understood. Chronic hypoxia may induce the generation of reactive oxygen species (ROS) in mitochondria, promote endoplasmic reticulum (ER) stress, and result in the integrated stress response (ISR) in the pulmonary artery and uteroplacental tissues. Numerous studies have implicated hypoxia-inducible factors (HIFs), oxidative stress, and ER stress/unfolded protein response (UPR) in the development of pulmonary hypertension, preeclampsia and IUGR. This review highlights the roles of HIFs, mitochondria-derived ROS and UPR, as well as their interplay, in the pathogenesis of pulmonary hypertension and preeclampsia, and their implications in drug development.

Keywords:  Endoplasmic reticulum; Hypoxia; Integrated stress response; Mitochondria; Preeclampsia; Pulmonary hypertension; Reactive oxygen species; Unfolded protein response; Vascular remodeling

DOI:  https://doi.org/10.1016/j.drudis.2021.07.011
Brain Res. 2021 Jul 26. pii: S0006-8993(21)00448-0. [Epub ahead of print] 147591

Naringenin attenuates endoplasmic reticulum stress, reduces apoptosis, and improves functional recovery in experimental traumatic brain injury.

Chulei Deng, Renxin Yi, Maoxing Fei, Tao Li, Yanling Han, Handong Wang.

  Traumatic brain injury (TBI) is a significant cause of disability and death worldwide. Accumulating evidence suggests that endoplasmic reticulum (ER) stress would be an important component in the pathogenesis of TBI. Although the neuroprotective effects of naringenin, a natural flavonoid isolated from citrus plants, have been confirmed in several neurological diseases, its mechanism of action in TBI needs further investigation. In ICR mice, we found that TBI induced elevated expression of ER stress marker proteins, including 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) in the perilesional cortex, which peaked at 7 days and 3 days after TBI, respectively. The induction of ER stress-related proteins partly coincided with ER architectural changes at 3 days post-TBI, indicating ER stress activation in our TBI model. Our results also revealed that continuous naringenin administration ameliorated neurological dysfunction, cerebral edema, plasmalemma permeability, and neuron cell loss at day 3 after TBI. Further, Naringenin suppressed TBI-induced activation of the ER stress pathway (p-eIF2α, ATF4, and CHOP), oxidative stress and apoptosis on day 3 after TBI. In summary, our data suggest that naringenin could ameliorate TBI-induced secondary brain injury by pleiotropic effects, including ER stress attenuation.

Keywords:  endoplasmic reticulum stress; naringenin; oxidative stress; traumatic brain injury

DOI:  https://doi.org/10.1016/j.brainres.2021.147591
Front Cell Dev Biol. 2021 ;9 684526

ER-Phagy: A New Regulator of ER Homeostasis.

Ming Yang, Shilu Luo, Xi Wang, Chenrui Li, Jinfei Yang, Xuejing Zhu, Li Xiao, Lin Sun.

  The endoplasmic reticulum (ER) is one of the most important cellular organelles and is essential for cell homeostasis. Upon external stimulation, ER stress induces the unfolded protein response (UPR) and ER-associated degradation (ERAD) to maintain ER homeostasis. However, persistent ER stress can lead to cell damage. ER-phagy is a selective form of autophagy that ensures the timely removal of damaged ER, thereby protecting cells from damage caused by excessive ER stress. As ER-phagy is a newly identified form of autophagy, many receptor-mediated ER-phagy pathways have been discovered in recent years. In this review, we summarize our understanding of the maintenance of ER homeostasis and describe the receptors identified to date. Finally, the relationships between ER-phagy and diseases are also discussed.

Keywords:  ER-phagy; ERAD; autophagy; endoplasmic reticulum (ER); unfolded protein response (UPR)

DOI:  https://doi.org/10.3389/fcell.2021.684526
PLoS One. 2021 ;16(7): e0255355

Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.

Rintaro Kuroda, Kaoru Tominaga, Katsumi Kasashima, Kenji Kuroiwa, Eiji Sakashita, Hiroko Hayakawa, Tom Kouki, Nobuhiko Ohno, Kensuke Kawai, Hitoshi Endo.

Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.

DOI: https://doi.org/10.1371/journal.pone.0255355
Front Cell Dev Biol. 2021 ;9 671461

MicroRNA-494 Regulates Endoplasmic Reticulum Stress in Endothelial Cells.

Namita Chatterjee, Eugenia Fraile-Bethencourt, Adrian Baris, Cristina Espinosa-Diez, Sudarshan Anand.

  Defects in stress responses are important contributors in many chronic conditions including cancer, cardiovascular disease, diabetes, and obesity-driven pathologies like non-alcoholic steatohepatitis (NASH). Specifically, endoplasmic reticulum (ER) stress is linked with these pathologies and control of ER stress can ameliorate tissue damage. MicroRNAs have a critical role in regulating diverse stress responses including ER stress. Here, we show that miR-494 plays a functional role during ER stress. Pharmacological ER stress inducers (tunicamycin (TCN) and thapsigargin) and hyperglycemia robustly increase the expression of miR-494 in vitro. ATF6 impacts the primary miR-494 levels whereas all three ER stress pathways are necessary for the increase in mature miR-494. Surprisingly, miR-494 pretreatment dampens the induction and magnitude of ER stress in response to TCN in endothelial cells and increases cell viability. Conversely, inhibition of miR-494 increases ER stress de novo and amplifies the effects of ER stress inducers. Using Mass Spectrometry (TMT-MS) we identified 23 proteins that are downregulated by both TCN and miR-494 in cultured human umbilical vein endothelial cells. Among these, we found 6 transcripts which harbor a putative miR-494 binding site. We validated the anti-apoptotic gene BIRC5 (survivin) and GINS4 as targets of miR-494 during ER stress. In summary, our data indicates that ER stress driven miR-494 may act in a feedback inhibitory loop to dampen downstream ER stress signaling.

Keywords:  ER stress; UPR – unfolded protein response; cell stress adaptation; endothelial cells; microRNA

DOI:  https://doi.org/10.3389/fcell.2021.671461
Mol Nutr Food Res. 2021 Jul 28. e2100380

Flavan-3-ol Microbial Metabolites Modulate Proteolysis in Neuronal Cells Reducing Amyloid-beta (1-42) Levels.

Valentina Cecarini, Massimiliano Cuccioloni, Yadong Zheng, Laura Bonfili, Chunmei Gong, Mauro Angeletti, Pedro Mena, Daniele Del Rio, Anna Maria Eleuteri.

   SCOPE: Alzheimer's disease (AD) is a progressive neurodegeneration characterized by extensive protein aggregation and deposition in the brain, associated with defective proteasomal and autophagic-lysosomal proteolytic pathways. Since current drugs can only reduce specific symptoms, the identification of novel treatments is a major concern in AD research. Among natural compounds, (poly)phenols and their derivatives/metabolites are emerging as candidates in AD prevention due to their multiple beneficial effects. Here, we investigated the ability of a selection of phenyl-γ-valerolactones, gut microbiota-derived metabolites of flavan-3-ols, to modulate the functionality of cellular proteolytic pathways.
METHODS AND RESULTS: Neuronal SH-SY5Y cells transfected with either the wild-type or the 717 valine-to-glycine amyloid precursor protein mutated gene were used as an AD model and treated with 5-(4'-hydroxyphenyl)-γ-valerolactone, 5-(3',4'-dihydroxyphenyl)-γ-valerolactone and 5-(3'-hydroxyphenyl)-γ-valerolactone-4'-sulfate. Combining in vitro and in silico studies, we observed that the phenyl-γ-valerolactones of interest modulated cellular proteolysis via proteasome inhibition and consequent autophagy upregulation and inhibited cathepsin B activity, eventually reducing the amount of intra- and extracellular amyloid-beta (1-42) peptides.
CONCLUSION: Our findings establish, for the first time, that these metabolites exert a neuroprotective activity by regulating intracellular proteolysis and confirm the role of autophagy and cathepsin B as possible targets of AD preventive/therapeutic strategies. This article is protected by copyright. All rights reserved.

Keywords:  Alzheimer's disease; amyloid; autophagy; polyphenol metabolites; proteasome

DOI:  https://doi.org/10.1002/mnfr.202100380
Nature. 2021 Jul 28.

Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Seda Koyuncu, Rute Loureiro, Hyun Ju Lee, Prerana Wagle, Marcus Krueger, David Vilchez.

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

DOI: https://doi.org/10.1038/s41586-021-03781-z
Mol Hum Reprod. 2021 Jul 26. pii: gaab050. [Epub ahead of print]

Integrated stress response control of granulosa cell translation and proliferation during normal ovarian follicle development.

Evelyn Llerena Cari, Synneva Hagen-Lillevik, Asma Giornazi, Miriam Post, Anton A Komar, Leslie Appiah, Benjamin Bitler, Alex J Polotsky, Nanette Santoro, Jeffrey Kieft, Kent Lai, Joshua Johnson.

  Mechanisms that directly control mammalian ovarian primordial follicle (PF) growth activation and the selection of individual follicles for survival are largely unknown. Follicle cells produce factors that can act as potent inducers of cellular stress during normal function. Consistent with this, we show here that normal, untreated ovarian cells, including pre-granulosa cells of dormant PFs, express phenotype and protein markers of the activated integrated stress response (ISR), including stress-specific protein translation (phospho-Serine 51 eukaryotic initiation factor 2 α ; P-EIF2 α ), active DNA damage checkpoints, and cell cycle arrest. We further demonstrate that mRNAs upregulated in primary (growing) follicles versus arrested PFs mostly include stress-responsive upstream open reading frames (uORFs). Treatment of a granulosa cell (GC) line with the PF growth trigger tumor necrosis factor alpha results in the upregulation of a 'stress-dependent' translation profile. This includes further elevated P-eIF2 α and a shift of uORF-containing mRNAs to polysomes. Because the active ISR corresponds to slow follicle growth and PF arrest, we propose that repair and abrogation of ISR checkpoints (e.g. checkpoint recovery) drives the GC cell cycle and PF growth activation (PFGA). If cellular stress is elevated beyond a threshold(s) or, if damage occurs that cannot be repaired, cell and follicle death ensue, consistent with physiological atresia. These data suggest an intrinsic quality control mechanism for immature and growing follicles, where PFGA and subsequent follicle growth and survival depend causally upon ISR resolution, including DNA repair and thus the proof of genomic integrity.

Keywords:  aging; eukaryotic initiation factor 2 (EIF2); follicle; integrated stress response; menopause; oocyte; ovary; translational control

DOI:  https://doi.org/10.1093/molehr/gaab050
Exp Ther Med. 2021 Sep;22(3): 944

Rutin alleviates cardiomyocyte injury induced by high glucose through inhibiting apoptosis and endoplasmic reticulum stress.

Jing Wang, Ru Wang, Jiali Li, Zhuhua Yao.

  Diabetic cardiomyopathy is a common complication of diabetes, in which endoplasmic reticulum stress (ERS) serves an important role. Rutin can treat the myocardial dysfunction of diabetic rats. However, to the best of our knowledge, studies on the effects of Rutin on myocardial injury caused by diabetes from the perspective of ERS have not previously been reported. In the present study, the role of rutin in the regulation of ERS in myocardial injury was assessed. Different high glucose concentrations were used to treat H9C2 myoblast cells to establish a myocardial damage model. A cell counting kit-8 assay was used to determine cell viability. A lactate dehydrogenase kit was used to detect cytotoxicity. Apoptosis levels were determined using a TUNEL assay. Western blotting was used to determine the expression levels of apoptosis-related proteins and ERS-related proteins, including heat shock protein A family member 5, inositol-requiring enzyme-1α, X-box binding protein 1, activating transcription factor 6, C/EBP-homologous protein (CHOP), cleaved caspase-12 and caspase-12. The anti-apoptotic and anti-ERS effects of Rutin on H9C2 cardiac cells induced by high glucose were examined after the administration of the ERS activator thapsigargin (TG). The results indicated that rutin could dose-dependently inhibit the level of apoptosis and ERS induced by high glucose in H9C2 cells. After administration of the ERS activator TG, it was demonstrated that TG could reverse the anti-apoptotic and anti-ERS effects of rutin on H9C2 cells stimulated with high glucose. Collectively, the present results suggested that rutin may alleviate cardiomyocyte model cell injury induced by high glucose through the inhibition of apoptosis and ERS.

Keywords:  apoptosis; cardiomyocyte injury; diabetic cardiomyopathy; endoplasmic reticulum stress; rutin

DOI:  https://doi.org/10.3892/etm.2021.10376
J Clin Invest. 2021 Jul 29. pii: 147836. [Epub ahead of print]

Inhibition of the unfolded protein response reduces arrhythmic risk after myocardial infarction.

Man Liu, Hong Liu, Preethy Parthiban, Gyeoung-Jin Kang, Guangbin Shi, Feng Feng, Anyu Zhou, Lianzhi Gu, Courtney Karnopp, Elena G Tolkacheva, Samuel C Dudley.

  Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels. When activated, the protein kinase-like ER kinase (PERK) branch of the UPR reduces protein translation and abundance. We hypothesized that PERK inhibition could prevent ion channel downregulation and reduce arrhythmic risk after myocardial infarct (MI). MI induced by coronary artery ligation resulted in mice exhibited reduced ion channel levels, ventricular tachycardia (VT), and prolonged corrected intervals between the Q and T waves of the ECGs (QTc). Protein levels of major cardiac ion channels were decreased. MI cardiomyocytes showed significantly prolonged action potential duration and decreased maximum upstroke velocity. Cardiac-specific PERK knockout (PERKKO) reduced electrical remodeling in response to MI with shortened QTc intervals, less VT episodes, and higher survival rates (P<0.05 vs. MI). Pharmacological PERK inhibition had similar effects. In conclusion, activated PERK during MI contributed to arrhythmic risk by downregulation of select cardiac ion channels. PERK inhibition prevented these changes and reduced arrhythmic risk. These results suggest that ion channel downregulation during MI is a fundamental arrhythmic mechanism and maintaining ion channel levels is antiarrhythmic.

Keywords:  Arrhythmias; Cardiology; Cell stress; Ion channels

DOI:  https://doi.org/10.1172/JCI147836
Methods Cell Biol. 2021 ;pii: S0091-679X(20)30201-6. [Epub ahead of print]165 31-38

Assessment of EGFP-Q74 degradation for the measurement of autophagic flux.

Yan Wang, Jiao Meng, Qi Wu, Guo Chen, Wei Xie, Zhen Zhang, Guido Kroemer, Oliver Kepp.

  Autophagy plays a major role in physiological and pathological processes. The quantitation of the abundance of autophagy-specific substrates constitutes an efficient strategy for assessing autophagic activity. Here, we provide a detailed protocol for quantifying the decay of a fusion protein composed by enhanced green fluorescent protein (EGFP) and glutamine repeats (Q74) using regular or high-throughput fluorescence microscopy. This method provides a direct measurement of autophagic flux in a Huntington's disease model.

Keywords:  Autophagy; EGFP-Q74; Fluorescence microscopy; Huntington's disease

DOI:  https://doi.org/10.1016/bs.mcb.2020.10.018
Elife. 2021 Jul 27. pii: e66768. [Epub ahead of print]10

miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging.

Isabelle Schiffer, Birgit Gerisch, Kazuto Kawamura, Raymond Laboy, Jennifer Hewitt, Martin Sebastian Denzel, Marcelo A Mori, Siva Vanapalli, Yidong Shen, Orsolya Symmons, Adam Antebi.

  Muscle function relies on the precise architecture of dynamic contractile elements, which must be fine-tuned to maintain motility throughout life. Muscle is also plastic, and remodeled in response to stress, growth, neural and metabolic inputs. The conserved muscle-enriched microRNA, miR-1, regulates distinct aspects of muscle development, but whether it plays a role during aging is unknown. Here we investigated Caenorhabditis elegans miR-1 in muscle function in response to proteostatic stress. mir-1 deletion improved mid-life muscle motility, pharyngeal pumping, and organismal longevity upon polyQ35 proteotoxic challenge. We identified multiple vacuolar ATPase subunits as subject to miR-1 control, and the regulatory subunit vha-13/ATP6V1A as a direct target downregulated via its 3'UTR to mediate miR-1 physiology. miR-1 further regulates nuclear localization of lysosomal biogenesis factor HLH-30/TFEB and lysosomal acidification. Our studies reveal that miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact muscle function and health during aging.

Keywords:  C. elegans; genetics; genomics; lysosomal v-ATPase; miR-1; polyglutamine; proteostasis; vha-13

DOI:  https://doi.org/10.7554/eLife.66768
Methods Cell Biol. 2021 ;pii: S0091-679X(20)30190-4. [Epub ahead of print]165 123-138

Immunoblot-based assays for assessing autophagy in the turquoise killifish Nothobranchius furzeri.

Gerasimos Anagnostopoulos, Léa Montégut, Hui Chen, Vincent Carbonnier, Isabelle Martins, Khady Mangane, Abdelkrim Mannioui, Alex Bois, Elisa Elena Baracco, Maria Chiara Maiuri, Guido Kroemer.

  Autophagy is an evolutionarily conserved biological process required for the turnover of the cytoplasm of eukaryotic cell. Beyond its catabolic nature, autophagy has a plethora of pro-survival functions, thus combatting hypoxia, nutrient shortage, and unfolded protein accumulation. Here, we introduce the naturally short-lived turquoise killifish Nothobranchius furzeri as an emerging model to study autophagic function in vivo, in response to environmental challenges. We show that starvation in killifish is sufficient to increase autophagic flux in the liver, thus enhancing the lipidation of microtubule-associated protein light chain 3 (LC3) and reducing the abundance of the autophagic substrate sequestosome-1 (SQSTM1). We describe an immunoblot-based comprehensive protocol to monitor fluctuations in autophagy in this model organism.

Keywords:  Autophagy; Killifish

DOI:  https://doi.org/10.1016/bs.mcb.2020.10.007
J Phys Chem B. 2021 Jul 28.

Protonation-Deprotonation Switch Controls the Amyloid-like Misfolding of Nucleic-Acid-Binding Domains of TDP-43.

Divya Patni, Santosh Kumar Jha.

Nutrient starvation stress acidifies the cytosol and leads to the formation of large protein assemblies and misfolded aggregates. However, how starvation stress is sensed at the molecular level and leads to protein misfolding is poorly understood. TDP-43 is a vital protein, which, under stress-like conditions, associates with stress granule proteins via its functional nucleic-acid-binding domains (TDP-43tRRM) and misfolds to form aberrant aggregates. Here, we show that the monomeric N form of TDP-43tRRM forms a misfolded amyloid-like protein assembly, β form, in a pH-dependent manner and identified the critical protein side-chain residue whose protonation triggers its misfolding. We systematically mutated the three buried ionizable residues, D105, H166, and H256, to neutral amino acids to block the pH-dependent protonation-deprotonation titration of their side chain and studied their effect on the N-to-β transition. We observed that D105A and H256Q resembled TDP-43tRRM in their pH-dependent misfolding behavior. However, H166Q retains the N-like secondary structure under low-pH conditions and does not show pH-dependent misfolding to the β form. These results indicate that H166 is the critical side-chain residue whose protonation triggers the misfolding of TDP-43tRRM and shed light on how stress-induced misfolding of proteins during neurodegeneration could begin from site-specific triggers.

DOI: https://doi.org/10.1021/acs.jpcb.1c03262
FASEB J. 2021 Aug;35(8): e21821

MyD88-mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms.

Arshiya Parveen, Kyle R Bohnert, Meiricris Tomaz da Silva, Yefei Wen, Raksha Bhat, Anirban Roy, Ashok Kumar.

  Skeletal muscle atrophy is a debilitating complication of many chronic disease states and disuse conditions including denervation. However, molecular and signaling mechanisms of muscle wasting remain less understood. Here, we demonstrate that the levels of several toll-like receptors (TLRs) and their downstream signaling adaptor, myeloid differentiation primary response 88 (MyD88), are induced in skeletal muscle of mice in response to sciatic nerve denervation. Muscle-specific ablation of MyD88 mitigates denervation-induced skeletal muscle atrophy in mice. Targeted ablation of MyD88 suppresses the components of ubiquitin-proteasome system, autophagy, and FOXO transcription factors in skeletal muscle during denervation. We also found that specific inhibition of MyD88 reduces the activation of canonical nuclear factor-kappa (NF-κB) pathway and expression of receptors for inflammatory cytokines in denervated muscle. In contrast, inhibition of MyD88 stimulates the activation of non-canonical NF-κB signaling in denervated skeletal muscle. Ablation of MyD88 also inhibits the denervation-induced increase in phosphorylation of AMPK without having any effect on the phosphorylation of mTOR. Moreover, targeted ablation of MyD88 inhibits the activation of a few components of the unfolded protein response (UPR) pathways, especially X-box protein 1 (XBP1). Importantly, myofiber-specific ablation of XBP1 mitigates denervation-induced skeletal muscle atrophy in mice. Collectively, our experiments suggest that TLR-MyD88 signaling mediates skeletal muscle wasting during denervation potentially through the activation of canonical NF-κB signaling, AMPK and UPR pathways.

Keywords:  ER stress; NF-kappa B; XBP1; denervation; inflammation; skeletal muscle

DOI:  https://doi.org/10.1096/fj.202100777RR
Glycobiology. 2021 Jul 29. pii: cwab081. [Epub ahead of print]

Regulation of fractone heparan sulfate composition in young and aged subventricular zone neurogenic niches.

Aurelien Kerever, Fumina Nagahara, Kazuko Keino-Masu, Masayuki Masu, Toin H van Kuppevelt, Romain R Vivès, Eri Arikawa-Hirasawa.

  Fractones, specialized extracellular matrix structures found in the subventricular zone (SVZ) neurogenic niche, can capture growth factors, such as basic fibroblast growth factor, from the extracellular milieu through a heparin-binding mechanism for neural stem cell presentation, which promotes neurogenesis. During aging, a decline in neurogenesis correlates with a change in the composition of heparan sulfate (HS) within fractones. In this study, we used antibodies that recognize specific short oligosaccharides with varying sulfation to evaluate the HS composition in fractones in young and aged brains. To further understand the conditions that regulate 6-O sulfation levels and its impact on neurogenesis, we used endosulfatase Sulf1 and Sulf2 double knock out (DKO) mice. Fractones in the SVZ of Sulf1/2 DKO mice showed immunoreactivity for the HS epitope, suggesting higher 6-O sulfation. While neurogenesis declined in the aged SVZ of both WT and Sulf1/2 DKO mice, we observed a larger number of neuroblasts in the young and aged SVZ of Sulf1/2 DKO mice. Together, these results show that the removal of 6-O-sulfation in fractones HS by endosulfatases inhibits neurogenesis in the SVZ. Our findings advance the current understanding regarding the extracellular environment that is best suited for neural stem cells to thrive, which is critical for the design of future stem cell therapies.

Keywords:  endosulfatase; extracellular matrix; fractone; heparan sulfate; neurogenesis

DOI:  https://doi.org/10.1093/glycob/cwab081
Acta Neuropathol Commun. 2021 Jul 27. 9(1): 129

Protein farnesylation is upregulated in Alzheimer's human brains and neuron-specific suppression of farnesyltransferase mitigates pathogenic processes in Alzheimer's model mice.

Angela Jeong, Shaowu Cheng, Rui Zhong, David A Bennett, Martin O Bergö, Ling Li.

  The pathogenic mechanisms underlying the development of Alzheimer's disease (AD) remain elusive and to date there are no effective prevention or treatment for AD. Farnesyltransferase (FT) catalyzes a key posttranslational modification process called farnesylation, in which the isoprenoid farnesyl pyrophosphate is attached to target proteins, facilitating their membrane localization and their interactions with downstream effectors. Farnesylated proteins, including the Ras superfamily of small GTPases, are involved in regulating diverse physiological and pathological processes. Emerging evidence suggests that isoprenoids and farnesylated proteins may play an important role in the pathogenesis of AD. However, the dynamics of FT and protein farnesylation in human brains and the specific role of neuronal FT in the pathogenic progression of AD are not known. Here, using postmortem brain tissue from individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), or Alzheimer's dementia, we found that the levels of FT and membrane-associated H-Ras, an exclusively farnesylated protein, and its downstream effector ERK were markedly increased in AD and MCI compared with NCI. To elucidate the specific role of neuronal FT in AD pathogenesis, we generated the transgenic AD model APP/PS1 mice with forebrain neuron-specific FT knockout, followed by a battery of behavioral assessments, biochemical assays, and unbiased transcriptomic analysis. Our results showed that the neuronal FT deletion mitigates memory impairment and amyloid neuropathology in APP/PS1 mice through suppressing amyloid generation and reversing the pathogenic hyperactivation of mTORC1 signaling. These findings suggest that aberrant upregulation of protein farnesylation is an early driving force in the pathogenic cascade of AD and that targeting FT or its downstream signaling pathways presents a viable therapeutic strategy against AD.

Keywords:  Alzheimer’s disease; Cholesterol; Farnesyltransferase; Isoprenoids; Protein prenylation; Small GTPases

DOI:  https://doi.org/10.1186/s40478-021-01231-5
Nat Methods. 2021 Jul 26.

A 4D single-cell protein atlas of transcription factors delineates spatiotemporal patterning during embryogenesis.

Xuehua Ma, Zhiguang Zhao, Long Xiao, Weina Xu, Yahui Kou, Yanping Zhang, Gang Wu, Yangyang Wang, Zhuo Du.

Complex biological processes such as embryogenesis require precise coordination of cell differentiation programs across both space and time. Using protein-fusion fluorescent reporters and four-dimensional live imaging, we present a protein expression atlas of transcription factors (TFs) mapped onto developmental cell lineages during Caenorhabditis elegans embryogenesis, at single-cell resolution. This atlas reveals a spatiotemporal combinatorial code of TF expression, and a cascade of lineage-specific, tissue-specific and time-specific TFs that specify developmental states. The atlas uncovers regulators of embryogenesis, including an unexpected role of a skin specifier in neurogenesis and the critical function of an uncharacterized TF in convergent muscle differentiation. At the systems level, the atlas provides an opportunity to model cell state-fate relationships, revealing a lineage-dependent state diversity within functionally related cells and a winding trajectory of developmental state progression. Collectively, this single-cell protein atlas represents a valuable resource for elucidating metazoan embryogenesis at the molecular and systems levels.

DOI: https://doi.org/10.1038/s41592-021-01216-1
Cell Transplant. 2021 Jan-Dec;30:30 9636897211035080

α-Mangostin Induces Apoptosis in Human Osteosarcoma Cells Through ROS-Mediated Endoplasmic Reticulum Stress via the WNT Pathway.

Shengsen Yang, Fei Zhou, Yi Dong, Fei Ren.

  α-mangostin has been confirmed to promote the apoptosis of MG-63 cells, but its specific pro-apoptosis mechanism in osteosarcoma (OS) remains further investigation. Here, we demonstrated that α-mangostin restrained the viability of OS cells (143B and Saos-2), but had little effect on the growth of normal human osteoblast. α-mangostin increased OS cell apoptosis by activating the caspase-3/8 cascade. Besides, α-mangostin induced endoplasmic reticulum (ER) stress and restrained the Wnt/β-catenin pathway activity. 4PBA (an ER stress inhibitor) or LiCl (an effective Wnt activator) treatment effectively hindered α-mangostin-induced apoptosis and the caspase-3/8 cascade. Furthermore, we also found that α-mangostin induced ER stress by promoting ROS production. And ER stress-mediated apoptosis caused by ROS accumulation depended on the inactivation of Wnt/β-catenin pathway. In addition, α-mangostin significantly hindered the growth of xenograft tumors, induced the expression of ER stress marker proteins and activation of the caspase-3/8 cascade, and restrained the Wnt/β-catenin signaling in vivo. In short, ROS-mediated ER stress was involved in α-mangostin triggered apoptosis, which might depended on Wnt/β-catenin signaling inactivation.

Keywords:  ER stress; ROS; apoptosis; osteosarcoma; α-mangostin

DOI:  https://doi.org/10.1177/09636897211035080
Semin Cell Dev Biol. 2021 Jul 22. pii: S1084-9521(21)00196-8. [Epub ahead of print]

eIF2-dependent translation initiation: Memory consolidation and disruption in Alzheimer's disease.

Mauricio M Oliveira, Eric Klann.

  Memory storage is a conserved survivability feature, present in virtually any complex species. During the last few decades, much effort has been devoted to understanding how memories are formed and which molecular switches define whether a memory should be stored for a short or a long period of time. Among these, de novo protein synthesis is known to be required for the conversion of short- to long-term memory. There are a number translational control pathways involved in synaptic plasticity and memory consolidation, including the phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α), which has emerged as a critical molecular switch for long-term memory consolidation. In this review, we discuss findings pertaining to the requirement of de novo protein synthesis to memory formation, how local dendritic and axonal translation is regulated in neurons, and how these can influence memory consolidation. We also highlight the importance of eIF2α-dependent translation initiation to synaptic plasticity and memory formation. Finally, we contextualize how aberrant phosphorylation of eIF2α contributes to Alzheimer's disease (AD) pathology and how preventing disruption of eIF2-dependent translation may be a therapeutic avenue for preventing and/or restoring memory loss in AD.

Keywords:  Alzheimer’s disease; EIF2α; Integrated stress response; MRNA translation; Memory consolidation; Protein synthesis

DOI:  https://doi.org/10.1016/j.semcdb.2021.07.009
J Chem Neuroanat. 2021 Jul 27. pii: S0891-0618(21)00092-2. [Epub ahead of print] 102009

Effects of dopamine transporter changes in the ventral tegmental area of the midbrain on cognitive function in aged rats.

Ke Xu, Jia Guo, Mingyue Ge, Jiangwen Yin, Han Zhang, Jieting Yin, Yan Li.

  The pathogenesis of Perioperative neurocognitive disorders (PND) is a synergistic effect of many factors. Up to now, the exact mechanism remains unclear. The dopamine pathway in the brain is one of the paths involved in the means of cognitive function. Therefore, the purpose of this study was to investigate the relationship between changes in dopamine transporters in the ventral tegmental area (VTA) of the midbrain and postoperative cognitive dysfunction in elderly rats. In this study, a mental dysfunction model in elderly rats was established after splenectomy under general anesthesia. Eighty male SD rats, aged 18-20 months, with a body mass of 300-500 g. Randomly divided into eight groups: Normal group (Normal, N) and Sham group (sham, S), Model 3 day group(PND, P3), Model 7 day group(PND, P7), Virus 3 days AAV·DAT·RNAi (AAV3), Virus 7 days AAV·DAT·RNAi (AAV7), Virus control for three days AAV·NC(NC3), Virus control for seven days AAV·NC(NC7). The results show that knockdown of dopamine transporter in the VTA region can significantly improve the cognitive dysfunction of elderly rats after surgery. These results suggest that dopamine transporter in the VTA region is involved in cognitive dysfunction in elderly rats. The effect of DAT changes in the VTA region on postoperative cognitive function in elderly rats may be related to the regulation of α-syn and Aβ1-42 protein aggregation in the hippocampus.

Keywords:  Dopamine transporter; Perioperative neurocognitive disorders; The VTA; The hippocampus; α-synuclein; β-Amyloid1-42

DOI:  https://doi.org/10.1016/j.jchemneu.2021.102009
J Biol Chem. 2021 Jul 22. pii: S0021-9258(21)00805-X. [Epub ahead of print] 101003

Development of a specific live-cell assay for native autophagic flux.

Nathaniel Safren, Elizabeth M Tank, Ahmed M Malik, Jason P Chua, Nicholas Santoro, Sami J Barmada.

  Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity, or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.

Keywords:  ALS; FTD; autophagy; high-content; high-throughput; neurodegeneration; photoconvertible; repurposing; screen; stem cell

DOI:  https://doi.org/10.1016/j.jbc.2021.101003
Proc Natl Acad Sci U S A. 2021 Aug 03. pii: e2103425118. [Epub ahead of print]118(31):

Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice.

Kelly E Glajch, Tim E Moors, Yi Chen, Pascal A Bechade, Alice Y Nam, Molly M Rajsombath, Thomas D McCaffery, Ulf Dettmer, Andreas Weihofen, Warren D Hirst, Dennis J Selkoe, Silke Nuber.

  Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson's disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient-derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD-causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)-human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

Keywords:  GBA; cathepsin; glucosylcerebrosidase; tetramer; α-synuclein

DOI:  https://doi.org/10.1073/pnas.2103425118
Autophagy. 2021 Jul 27. 1-17

A Dictyostelium model for BPAN disease reveals a functional relationship between the WDR45/WIPI4 homolog Wdr45l and Vmp1 in the regulation of autophagy-associated PtdIns3P and ER stress.

Alba Tornero-Écija, Luis-Carlos Tábara, Miranda Bueno-Arribas, Laura Antón-Esteban, Cristina Navarro-Gómez, Irene Sánchez, Olivier Vincent, Ricardo Escalante.

  PROPPINs are conserved PtdIns3P-binding proteins required for autophagosome biogenesis that fold into a characteristic group of seven-bladed beta-propellers. Mutations in WDR45/WIPI4, a human member of this family, lead to BPAN, a rare form of neurodegeneration. We have generated mutants for the two PROPPIN proteins present in the model system Dictyostelium discoideum (Atg18 and Wdr45l) and characterized their function. Lack of Wdr45l greatly impairs autophagy, while Atg18 only causes subtle defects in the maturation of autolysosomes. The strong phenotype of the Wdr45l mutant is strikingly similar to that observed in Dictyostelium cells lacking Vmp1, an ER protein required for omegasome formation. Common phenotypes include impaired growth in axenic medium, lack of aggregation, and local enrichment of PtdIns3P as determined by the use of lipid reporters. In addition, Vmp1 and Wdr45l mutants show a chronically active response to ER stress. For both mutants, this altered PtdIns3P localization can be prevented by the additional mutation of the upstream regulator Atg1, which also leads to recovery of axenic growth and reduction of ER stress. We propose that, in addition to an autophagy defect, local autophagy-associated PtdIns3P accumulation might contribute to the pathogenesis of BPAN by disrupting ER homeostasis. The introduction of BPAN-associated mutations in Dictyostelium Wdr45l reveals the impact of pathogenic residues on the function and localization of the protein.

Keywords:  Atg18; autophagosome; membrane contact site; omegasome; proppin proteins

DOI:  https://doi.org/10.1080/15548627.2021.1953262
Science. 2021 07 30. pii: eabc8479. [Epub ahead of print]373(6554):

Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span.

M Grunewald, S Kumar, H Sharife, E Volinsky, A Gileles-Hillel, T Licht, A Permyakova, L Hinden, S Azar, Y Friedmann, P Kupetz, R Tzuberi, A Anisimov, K Alitalo, M Horwitz, S Leebhoff, O Z Khoma, R Hlushchuk, V Djonov, R Abramovitch, J Tam, E Keshet.

Aging is an established risk factor for vascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. Here, we show in aged mice that vascular endothelial growth factor (VEGF) signaling insufficiency, which is caused by increased production of decoy receptors, may drive physiological aging across multiple organ systems. Increasing VEGF signaling prevented age-associated capillary loss, improved organ perfusion and function, and extended life span. Healthier aging was evidenced by favorable metabolism and body composition and amelioration of aging-associated pathologies including hepatic steatosis, sarcopenia, osteoporosis, "inflammaging" (age-related multiorgan chronic inflammation), and increased tumor burden. These results indicate that VEGF signaling insufficiency affects organ aging in mice and suggest that modulating this pathway may result in increased mammalian life span and improved overall health.

DOI: https://doi.org/10.1126/science.abc8479
FEBS Open Bio. 2021 Jul 28.

Endoplasmic reticulum stress inhibits 3D Matrigel-induced vasculogenic mimicry of breast cancer cells via TGF-β1/Smad2/3 and β-catenin signaling.

Huifen Liu, Hao Wang, Dan Chen, Cuirong Gu, Jianming Huang, Kun Mi.

  Endoplasmic reticulum (ER) stress is a cellular stress condition involving disturbance in the folding capacity of the ER caused by endogenous and exogenous factors. ER stress signaling pathways affect tumor malignant growth, angiogenesis and progression, and promote the anti-tumor effects of certain drugs. However, the impact of ER stress on the vasculogenic mimicry (VM) phenotype of cancer cells has not been well addressed. VM is a phenotype which mimics vasculogenesis by forming patterned tubular networks, which are related to stemness and aggressive behaviors of cancer cells. In this study we employed tunicamycin (TM), a UPR-activating agent, to induce ER stress in aggressive triple negative MDA-MB-231 breast cancer cells, which exhibit a VM phenotype in 3D Matrigel cultures. TM-induced ER stress was able to inhibit the VM phenotype. In addition to the tumorspheroid phenotype observed upon inhibiting the VM phenotype, we observed alterations in glycosylation of integrin β1, loss of VE-cadherin and a decrease in stem cell marker Bmi-1. Further study revealed decreased activated TGF-β1, Smad2/3, Phospho-Smad2 and β-catenin. β-catenin knockdown markedly inhibited the VM phenotype and resulted in the loss of VE-cadherin. The data suggest that the activation of ER stress inhibited VM phenotype formation of breast cancer cells via both the TGF-β1/Smad2/3 and β-catenin signaling pathways. The discovery of prospective regulatory mechanisms involved in ER stress and VM in breast cancer could lead to more precisely targeted therapies that inhibit vessel formation and affect tumor progression.

Keywords:  Endoplasmic reticulum stress; TGF-β1; breast cancer; vasculogenic mimicry; β-catenin

DOI:  https://doi.org/10.1002/2211-5463.13259
Front Mol Neurosci. 2021 ;14 660104

Age-Related Transcriptional Deregulation of Genes Coding Synaptic Proteins in Alzheimer's Disease Murine Model: Potential Neuroprotective Effect of Fingolimod.

Henryk Jęśko, Iga Wieczorek, Przemysław Leonard Wencel, Magdalena Gąssowska-Dobrowolska, Walter J Lukiw, Robert Piotr Strosznajder.

  Alzheimer's disease (AD) induces time-dependent changes in sphingolipid metabolism, which may affect transcription regulation and neuronal phenotype. We, therefore, analyzed the influence of age, amyloid β precursor protein (AβPP), and the clinically approved, bioavailable sphingosine-1-phosphate receptor modulator fingolimod (FTY720) on the expression of synaptic proteins. RNA was isolated, reverse-transcribed, and subjected to real-time PCR. Expression of mutant (V717I) AβPP led to few changes at 3 months of age but reduced multiple mRNA coding for synaptic proteins in a 12-month-old mouse brain. Complexin 1 (Cplx1), SNAP25 (Snap25), syntaxin 1A (Stx1a), neurexin 1 (Nrxn1), neurofilament light (Nefl), and synaptotagmin 1 (Syt1) in the hippocampus, and VAMP1 (Vamp1) and neurexin 1 (Nrxn1) in the cortex were all significantly reduced in 12-month-old mice. Post mortem AD samples from the human hippocampus and cortex displayed lower expression of VAMP, synapsin, neurofilament light (NF-L) and synaptophysin. The potentially neuroprotective FTY720 reversed most AβPP-induced changes in gene expression (Cplx1, Stx1a, Snap25, and Nrxn1) in the 12-month-old hippocampus, which is thought to be most sensitive to early neurotoxic insults, but it only restored Vamp1 in the cortex and had no influence in 3-month-old brains. Further study may reveal the potential usefulness of FTY720 in the modulation of deregulated neuronal phenotype in AD brains.

Keywords:  Alzheimer's disease; FTY720/fingolimod; aging; amyloid β; neurodegeneration; sphingolipids; sphingosine-1-phosphate; synaptic proteins

DOI:  https://doi.org/10.3389/fnmol.2021.660104
Front Neurosci. 2021 ;15 696440

A β-Wrapin Targeting the N-Terminus of α-Synuclein Monomers Reduces Fibril-Induced Aggregation in Neurons.

Éva M Szegő, Fabian Boß, Daniel Komnig, Charlott Gärtner, Lennart Höfs, Hamed Shaykhalishahi, Michael M Wördehoff, Theodora Saridaki, Jörg B Schulz, Wolfgang Hoyer, Björn H Falkenburger.

  Reducing α-synuclein pathology constitutes a plausible strategy against Parkinson's disease. As we recently demonstrated, the β-wrapin protein AS69 binds an N-terminal region in monomeric α-synuclein, interferes with fibril nucleation, and reduces α-synuclein aggregation in vitro and in a fruit fly model of α-synuclein toxicity. The aim of this study was to investigate whether AS69 also reduces α-synuclein pathology in mammalian neurons. To induce α-synuclein pathology, primary mouse neurons were exposed to pre-formed fibrils (PFF) of human α-synuclein. PFF were also injected into the striatum of A30P-α-synuclein transgenic mice. The extent of α-synuclein pathology was determined by phospho-α-synuclein staining and by Triton X-100 solubility. The degeneration of neuronal somata, dendrites, and axon terminals was determined by immunohistochemistry. AS69 and PFF were taken up by primary neurons. AS69 did not alter PFF uptake, but AS69 did reduce PFF-induced α-synuclein pathology. PFF injection into mouse striatum led to α-synuclein pathology and dystrophic neurites. Co-injection of AS69 abrogated PFF-induced pathology. AS69 also reduced the PFF-induced degeneration of dopaminergic axon terminals in the striatum and the degeneration of dopaminergic dendrites in the substantia nigra pars reticulata. AS69 reduced the activation of astroglia but not microglia in response to PFF injection. Collectively, AS69 reduced PFF-induced α-synuclein pathology and the associated neurodegeneration in primary neurons and in mouse brain. Our data therefore suggest that small proteins binding the N-terminus of α-synuclein monomers are promising strategies to modify disease progression in Parkinson's disease.

Keywords:  molecular chaperones; nanobodies; pre-formed fibrils; protein aggregation; α-synuclein

DOI:  https://doi.org/10.3389/fnins.2021.696440
Curr Opin Struct Biol. 2021 Jul 23. pii: S0959-440X(21)00098-1. [Epub ahead of print]

Insane in the membrane: developments in protein folding, protein transport, and signaling by GPCRs.

Megan J Maher, Oliver P Ernst.

DOI: https://doi.org/10.1016/j.sbi.2021.06.014
Front Endocrinol (Lausanne). 2021 ;12 646720

Biochanin A Alleviates Cerebral Ischemia/Reperfusion Injury by Suppressing Endoplasmic Reticulum Stress-Induced Apoptosis and p38MAPK Signaling Pathway In Vivo and In Vitro.

Min-Min Guo, Sheng-Biao Qu, Hui-Ling Lu, Wen-Bo Wang, Mu-Liang He, Jian-Lin Su, Jian Chen, Yong Wang.

  We have previously shown that biochanin A exhibits neuroprotective properties in the context of cerebral ischemia/reperfusion (I/R) injury. The mechanistic basis for such properties, however, remains poorly understood. This study was therefore designed to explore the manner whereby biochanin A controls endoplasmic reticulum (ER) stress, apoptosis, and inflammation within fetal rat primary cortical neurons in response to oxygen-glucose deprivation/reoxygenation (OGD/R) injury, and in a rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) injury. For the OGD/R in vitro model system, cells were evaluated after a 2 h OGD following a 24 h reoxygenation period, whereas in vivo neurological deficits were evaluated following 2 h of ischemia and 24 h of reperfusion. The expression of proteins associated with apoptosis, ER stress (ERS), and p38 MAPK phosphorylation was evaluated in these samples. Rats treated with biochanin A exhibited reduced neurological deficits relative to control rats following MCAO/R injury. Additionally, GRP78 and CHOP levels rose following I/R modeling both in vitro and in vivo, whereas biochanin A treatment was associated with reductions in CHOP levels but further increases in GRP78 levels. In addition, OGD/R or MCAO/R were associated with markedly enhanced p38 MAPK phosphorylation that was alleviated by biochanin A treatment. Similarly, OGD/R or MCAO/R injury resulted in increases in caspase-3, caspase-12, and Bax levels as well as decreases in Bcl-2 levels, whereas biochanin A treatment was sufficient to reverse these phenotypes. Together, these findings thus demonstrate that biochanin A can alleviate cerebral I/R-induced damage at least in part via suppressing apoptosis, ER stress, and p38 MAPK signaling, thereby serving as a potent neuroprotective agent.

Keywords:  Biochanin A; apoptosis; cerebral ischemia/reperfusion; endoplasmic reticulum stress; oxygen-glucose deprivation/reoxygenation; p38MAPK

DOI:  https://doi.org/10.3389/fendo.2021.646720
Nat Commun. 2021 Jul 30. 12(1): 4643

Stress-primed secretory autophagy promotes extracellular BDNF maturation by enhancing MMP9 secretion.

Silvia Martinelli, Elmira A Anderzhanova, Thomas Bajaj, Svenja Wiechmann, Frederik Dethloff, Katja Weckmann, Daniel E Heinz, Tim Ebert, Jakob Hartmann, Thomas M Geiger, Michael Döngi, Kathrin Hafner, Max L Pöhlmann, Lee Jollans, Alexandra Philipsen, Susanne V Schmidt, Ulrike Schmidt, Giuseppina Maccarrone, Valentin Stein, Felix Hausch, Christoph W Turck, Mathias V Schmidt, Anne-Kathrin Gellner, Bernhard Kuster, Nils C Gassen.

The stress response is an essential mechanism for maintaining homeostasis, and its disruption is implicated in several psychiatric disorders. On the cellular level, stress activates, among other mechanisms, autophagy that regulates homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway in which autophagosomes fuse with the plasma membrane rather than with lysosomes. Here, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy via the stress-responsive co-chaperone FK506-binding protein 51. We identify the matrix metalloproteinase 9 (MMP9) as one of the proteins secreted in response to stress. Using cellular assays and in vivo microdialysis, we further find that stress-enhanced MMP9 secretion increases the cleavage of pro-brain-derived neurotrophic factor (proBDNF) to its mature form (mBDNF). BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a cellular stress adaptation mechanism that bears the potential of opening avenues for the understanding of the pathophysiology of stress-related disorders.

DOI: https://doi.org/10.1038/s41467-021-24810-5
Methods Cell Biol. 2021 ;pii: S0091-679X(21)00017-0. [Epub ahead of print]165 39-57

Multimodal assessment of autophagy in mammalian cells with a novel, LC3-based tandem reporter.

Dan F Lazar, Amani A Gillette, Samantha R Lewis, Braeden L Butler.

  Autophagy is an important intracellular pathway for the degradation of superfluous or harmful subcellular materials, thereby playing a critical role in the maintenance of cell health under normal and stress-related conditions. Researchers interrogating autophagic activity in mammalian cell lines often leverage complementary assay technologies to confirm observations. The Autophagy LC3 HiBiT Reporter assay system utilizes a tandem reporter module, HiBiT-HaloTag, fused to a key marker of autophagic activity, LC3B protein, to enable multiple, cell-based assay modalities. This novel autophagy reporter expressed in a single cell line supports (a) a bioluminescent, homogeneous, plate-reader assay for rapid and quantitative assessment of changes in the level of the LC3-based reporter, (b) a fluorescence-based imaging approach to monitor reporter subcellular distribution in live cells, and (c) an antibody-free, protein blotting method to detect the relative amounts of the LC3-I and LC-II forms of the reporter associated with modulation of autophagic flux. Here we detail protocols for all three assay modalities applied to a U2OS human osteosarcoma cell line stably expressing the novel autophagy reporter, enabling the identification of modulators of autophagic activity and subsequent confirmation of mechanism of action.

Keywords:  Autophagic flux; Autophagy; Autophagy LC3 HiBiT reporter; HaloTag; HiBiT; LC3B

DOI:  https://doi.org/10.1016/bs.mcb.2021.02.004
Acta Neuropathol. 2021 Jul 26.

A glutaminyl cyclase-catalyzed α-synuclein modification identified in human synucleinopathies.

Maike Hartlage-Rübsamen, Alexandra Bluhm, Sandra Moceri, Lisa Machner, Janett Köppen, Mathias Schenk, Isabel Hilbrich, Max Holzer, Martin Weidenfeller, Franziska Richter, Roland Coras, Geidy E Serrano, Thomas G Beach, Stephan Schilling, Stephan von Hörsten, Wei Xiang, Anja Schulze, Steffen Roßner.

  Parkinson's disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79-α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may-in analogy to pGlu-Aβ peptides in Alzheimer's disease-act as a seed for pathogenic protein aggregation.

Keywords:  Animal models; Dementia with Lewy bodies; Glutaminyl cyclase; Parkinson’s disease; Post-translational modification; Substantia nigra; α-Synuclein

DOI:  https://doi.org/10.1007/s00401-021-02349-5