bims-proarb 2021-07-11 papers

bims-proarb

Biomed News

on Proteostasis in aging and regenerative biology

Issue of 2021–07–11
28 papers selected by
Rich Giadone, Harvard University

Hormetic endoplasmic reticulum stress in hematopoietic stem cells.
Proteostasis Dysfunction in Aged Mammalian Cells. The Stressful Role of Inflammation.
Two human metabolites rescue a C. elegans model of Alzheimer's disease via a cytosolic unfolded protein response.
Protein S-nitrosylation and oxidation contribute to protein misfolding in neurodegeneration.
The Mitochondrial Hsp90 TRAP1 and Alzheimer's Disease.
IRE1-mTOR-PERK Axis Coordinates Autophagy and ER Stress-Apoptosis Induced by P2X7-Mediated Ca2+ Influx in Osteoarthritis.
Large organellar changes occur during mild heat shock in yeast.
Analysis of proteostasis during aging with western blot of detergent-soluble and insoluble protein fractions.
ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors.
Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence.
AgDD System: A Chemical Controllable Protein Aggregates in Cells.
Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β- selection.
UPRmt activation protects against MPP+-induced toxicity in a cell culture model of Parkinson's disease.
Analytical Challenges Assessing Protein Aggregation and Fragmentation Under Physiologic Conditions.
Diversity of transcriptomic microglial phenotypes in aging and Alzheimer's disease.
Single-nucleus chromatin accessibility and transcriptomic characterization of Alzheimer's disease.
α-synuclein pathogenesis in hiPSC models of Parkinson's disease.
Optogenetic approaches for understanding homeostatic and degenerative processes in Drosophila.
Vascular Endothelial Senescence: Pathobiological Insights, Emerging Long Noncoding RNA Targets, Challenges and Therapeutic Opportunities.
zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.
Vesicular trafficking in cellular responses to stress.
A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation.
Autophagy in vascular dementia and natural products with autophagy regulating activity.
Methyltransferase-like protein 7A (METTL7A) promotes cell survival and osteogenic differentiation under metabolic stress.
Comprehensive analysis of O-glycosylation of amyloid precursor protein (APP) using targeted and multi-fragmentation MS strategy.
Adenine Base Editing Reduces Misfolded Protein Accumulation and Toxicity in Alpha-1 Antitrypsin Deficient Patient iPSC-Hepatocytes.
K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.
Role of heat shock proteins in aging and chronic inflammatory diseases.

Curr Opin Hematol. 2021 Jul 06.

Hormetic endoplasmic reticulum stress in hematopoietic stem cells.

Larry L Luchsinger.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) possess the ability to regenerate over a lifetime in the face of extreme cellular proliferation and environmental stress. Yet, mechanisms that control the regenerative properties of HSCs remain elusive. ER stress has emerged as an important signaling event that supports HSC self-renewal and multipotency. The purpose of this review is to summarize the pathways implicating ER stress as cytoprotective in HSCs.
RECENT FINDINGS: Recent studies have shown multiple signaling cascades of the unfolded protein response (UPR) are persistently activated in healthy HSCs, suggesting that low-dose ER stress is a feature HSCs. Stress adaptation is a feature ascribed to cytoprotection and longevity of cells as well as organisms, in what is known as hormesis. However, assembling this information into useful knowledge to improve the therapeutic application of HSCs remains challenging and the upstream activators and downstream transcriptional programs induced by ER stress that are required in HSCs remain to be discovered.
SUMMARY: The maintenance of HSCs requires a dose-dependent simulation of ER stress responses that involves persistent, low-dose UPR. Unraveling the complexity of this signaling node may elucidate mechanisms related to regeneration of HSCs that can be harnessed to expand HSCs for cellular therapeutics ex vivo and transplantation in vivo.

DOI: https://doi.org/10.1097/MOH.0000000000000668
Front Mol Biosci. 2021 ;8 658742

Proteostasis Dysfunction in Aged Mammalian Cells. The Stressful Role of Inflammation.

Diego Ruano.

  Aging is a biological and multifactorial process characterized by a progressive and irreversible deterioration of the physiological functions leading to a progressive increase in morbidity. In the next decades, the world population is expected to reach ten billion, and globally, elderly people over 80 are projected to triple in 2050. Consequently, it is also expected an increase in the incidence of age-related pathologies such as cancer, diabetes, or neurodegenerative disorders. Disturbance of cellular protein homeostasis (proteostasis) is a hallmark of normal aging that increases cell vulnerability and might be involved in the etiology of several age-related diseases. This review will focus on the molecular alterations occurring during normal aging in the most relevant protein quality control systems such as molecular chaperones, the UPS, and the ALS. Also, alterations in their functional cooperation will be analyzed. Finally, the role of inflammation, as a synergistic negative factor of the protein quality control systems during normal aging, will also be addressed. A better comprehension of the age-dependent modifications affecting the cellular proteostasis, as well as the knowledge of the mechanisms underlying these alterations, might be very helpful to identify relevant risk factors that could be responsible for or contribute to cell deterioration, a fundamental question still pending in biomedicine.

Keywords:  aging; autophagy; cell stress and aging; inflammation; proteasome; proteostasis

DOI:  https://doi.org/10.3389/fmolb.2021.658742
Commun Biol. 2021 Jul 07. 4(1): 843

Two human metabolites rescue a C. elegans model of Alzheimer's disease via a cytosolic unfolded protein response.

Priyanka Joshi, Michele Perni, Ryan Limbocker, Benedetta Mannini, Sam Casford, Sean Chia, Johnny Habchi, Johnathan Labbadia, Christopher M Dobson, Michele Vendruscolo.

Age-related changes in cellular metabolism can affect brain homeostasis, creating conditions that are permissive to the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Although the roles of metabolites have been extensively studied with regard to cellular signaling pathways, their effects on protein aggregation remain relatively unexplored. By computationally analysing the Human Metabolome Database, we identified two endogenous metabolites, carnosine and kynurenic acid, that inhibit the aggregation of the amyloid beta peptide (Aβ) and rescue a C. elegans model of Alzheimer's disease. We found that these metabolites act by triggering a cytosolic unfolded protein response through the transcription factor HSF-1 and downstream chaperones HSP40/J-proteins DNJ-12 and DNJ-19. These results help rationalise previous observations regarding the possible anti-ageing benefits of these metabolites by providing a mechanism for their action. Taken together, our findings provide a link between metabolite homeostasis and protein homeostasis, which could inspire preventative interventions against neurodegenerative disorders.

DOI: https://doi.org/10.1038/s42003-021-02218-7
Free Radic Biol Med. 2021 Jul 02. pii: S0891-5849(21)00401-9. [Epub ahead of print]

Protein S-nitrosylation and oxidation contribute to protein misfolding in neurodegeneration.

Tomohiro Nakamura, Chang-Ki Oh, Xu Zhang, Stuart A Lipton.

  Neurodegenerative disorders like Alzheimer's disease and Parkinson's disease are characterized by progressive degeneration of synapses and neurons. Accumulation of misfolded/aggregated proteins represents a pathological hallmark of most neurodegenerative diseases, potentially contributing to synapse loss and neuronal damage. Emerging evidence suggests that misfolded proteins accumulate in the diseased brain at least in part as a consequence of excessively generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). Mechanistically, not only disease-linked genetic mutations but also known risk factors for neurodegenerative diseases, such as aging and exposure to environmental toxins, can accelerate production of ROS/RNS, which contribute to protein misfolding - in many cases mimicking the effect of rare genetic mutations known to be linked to the disease. This review will focus on the role of RNS-dependent post-translational modifications, such as S-nitrosylation and tyrosine nitration, in protein misfolding and aggregation. Specifically, we will discuss molecular mechanisms whereby RNS disrupt the activity of the cellular protein quality control machinery, including molecular chaperones, autophagy/lysosomal pathways, and the ubiquitin-proteasome system (UPS). Because chronic accumulation of misfolded proteins can trigger mitochondrial dysfunction, synaptic damage, and neuronal demise, further characterization of RNS-mediated protein misfolding may establish these molecular events as therapeutic targets for intervention in neurodegenerative diseases.

Keywords:  Autophagy; Molecular chaperones; Protein S-Nitrosylation; Protein misfolding; Tyrosine nitration; Ubiquitin-proteasome system

DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.07.002
Front Mol Biosci. 2021 ;8 697913

The Mitochondrial Hsp90 TRAP1 and Alzheimer's Disease.

Françoise A Dekker, Stefan G D Rüdiger.

  Alzheimer's Disease (AD) is the most common form of dementia, characterised by intra- and extracellular protein aggregation. In AD, the cellular protein quality control (PQC) system is derailed and fails to prevent the formation of these aggregates. Especially the mitochondrial paralogue of the conserved Hsp90 chaperone class, tumour necrosis factor receptor-associated protein 1 (TRAP1), is strongly downregulated in AD, more than other major PQC factors. Here, we review molecular mechanism and cellular function of TRAP1 and subsequently discuss possible links to AD. TRAP1 is an interesting paradigm for the Hsp90 family, as it chaperones proteins with vital cellular function, despite not being regulated by any of the co-chaperones that drive its cytosolic paralogues. TRAP1 encloses late folding intermediates in a non-active state. Thereby, it is involved in the assembly of the electron transport chain, and it favours the switch from oxidative phosphorylation to glycolysis. Another key function is that it ensures mitochondrial integrity by regulating the mitochondrial pore opening through Cyclophilin D. While it is still unclear whether TRAP1 itself is a driver or a passenger in AD, it might be a guide to identify key factors initiating neurodegeneration.

Keywords:  mitochondria; molecular chaperones; neurodegeneration; protein aggregation; protein folding; protein quality control; proteostasis

DOI:  https://doi.org/10.3389/fmolb.2021.697913
Front Cell Dev Biol. 2021 ;9 695041

IRE1-mTOR-PERK Axis Coordinates Autophagy and ER Stress-Apoptosis Induced by P2X7-Mediated Ca2+ Influx in Osteoarthritis.

Zihao Li, Ziyu Huang, He Zhang, Jinghan Lu, Yingliang Wei, Yue Yang, Lunhao Bai.

  Moderate-intensity exercise can help delay the development of osteoarthritis (OA). Previous studies have shown that the purinergic receptor P2X ligand gated ion channel 7 (P2X7) is involved in OA development and progression. To investigate the effect of exercise on P2X7 activation and downstream signaling in OA, we used the anterior cruciate ligament transection (ACLT)-induced OA rat model and primary chondrocyte culture system. Our in vivo experiments confirmed that treadmill exercise increased P2X7 expression and that this effect was more pronounced at the later time points. Furthermore, P2X7 activation induced endoplasmic reticulum (ER) stress and increased the expression levels of ER stress markers, such as 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6). At the early time points, IRE1 and PERK were activated, and mTOR was inhibited. At the later time points, mTOR was activated, mediating PERK to promote ER stress-apoptosis, whereas IRE1 and autophagy were inhibited. To confirm our observations in vitro, we treated primary chondrocytes with the P2X7 agonist benzoylbenzoyl-ATP (Bz-ATP). Our results confirmed that P2X7-mediated Ca2+ influx activated IRE1-mediated autophagic flux and induced PERK-mediated ER stress-apoptosis. To further investigate the role of P2X7 in OA, we injected mTOR antagonist rapamycin or P2X7 antagonist A740003 into the knee joints of ACLT rats. Our results demonstrated that mTOR inhibition induced autophagy, decreased apoptosis, and reduced cartilage loss. However, injection of mTOR agonist MHY1485 or Bz-ATP had the opposite effect. In summary, our results indicated that during the early stages of moderate-intensity exercise, P2X7 was activated and autophagic flux was increased, delaying OA development. At the later stages, P2X7 became over-activated, and the number of apoptotic cells increased, promoting OA development. We propose that the IRE1-mTOR-PERK signaling axis was involved in the regulation of autophagy inhibition and the induction of apoptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA, suggesting that the intensity and duration of exercise play a critical role. We also demonstrated that on a molecular level, P2X7 and its downstream pathways could be potential therapeutic targets for OA.

Keywords:  P2X7 receptor; autophagy; endoplasmic reticulum stress; inositol-requiring enzyme-1; mammalian target of rapamycin; osteoarthritis; protein kinase R-like endoplasmic reticulum kinase; treadmill exercise

DOI:  https://doi.org/10.3389/fcell.2021.695041
J Cell Sci. 2021 Jul 07. pii: jcs.258325. [Epub ahead of print]

Large organellar changes occur during mild heat shock in yeast.

Katharina S Keuenhof, Lisa Larsson Berglund, Sandra Malmgren Hill, Kara L Schneider, Per O Widlund, Thomas Nyström, Johanna L Höög.

  When the temperature is increased, the heat shock response is activated to protect the cellular environment. The transcriptomics and proteomics of this process are intensively studied, while information about how the cell responds structurally to heat stress is mostly lacking. Here, Saccharomyces cerevisiae were subjected to a mild continuous heat shock (38°C) and intermittently cryo-immobilized for electron microscopy. Through measuring changes in all distinguishable organelle numbers, sizes, and morphologies in over 2100 electron micrographs a major restructuring of the cell's internal architecture during the progressive heat shock was revealed. The cell grew larger but most organelles within it expanded even more, shrinking the volume of the cytoplasm. Organelles responded to heat shock at different times, both in terms of size and number, and adaptations of certain organelles' morphology (such as the vacuole), were observed. Multivesicular bodies grew to almost 170% in size, indicating a previously unknown involvement in the heat shock response. A previously undescribed electron translucent structure accumulated close to the plasma membrane. This all-encompassing approach provides a detailed chronological progression of organelle adaptation throughout the cellular heat-stress response.

Keywords:  Budding yeast; Electron microscopy; Heat shock; Organelles; Ultrastructure

DOI:  https://doi.org/10.1242/jcs.258325
STAR Protoc. 2021 Sep 17. 2(3): 100628

Analysis of proteostasis during aging with western blot of detergent-soluble and insoluble protein fractions.

Mamta Rai, Michelle Curley, Zane Coleman, Anjana Nityanandam, Jianqin Jiao, Flavia A Graca, Liam C Hunt, Fabio Demontis.

  Defects in protein quality control are the underlying cause of age-related diseases. The western blot analysis of detergent-soluble and insoluble protein fractions has proven useful in identifying interventions that regulate proteostasis. Here, we describe the protocol for such analyses in Drosophila tissues, mouse skeletal muscle, human organoids, and HEK293 cells. We describe key adaptations of this protocol and provide key information that will help modify this protocol for future studies in other tissues and disease models. For complete details on the use and execution of this protocol, please refer to Rai et al. (2021) and Hunt el al. (2021).

Keywords:  Cell Biology; Developmental biology; Model Organisms; Molecular Biology; Organoids; Protein Biochemistry

DOI:  https://doi.org/10.1016/j.xpro.2021.100628
Elife. 2021 Jul 05. pii: e57376. [Epub ahead of print]10

ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors.

Brian D Alford, Eduardo Tassoni-Tsuchida, Danish Khan, Jeremy J Work, Gregory Valiant, Onn Brandman.

  Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.

Keywords:  CRISPR; HSF1; S. cerevisiae; cell biology; chaperones; genetic screens; genetics; genomics; heat shock response; protein quality control

DOI:  https://doi.org/10.7554/eLife.57376
Dev Cell. 2021 Jun 28. pii: S1534-5807(21)00516-5. [Epub ahead of print]

Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence.

Jery Joy, Lara Barrio, Celia Santos-Tapia, Daniela Romão, Nikolaos Nikiforos Giakoumakis, Marta Clemente-Ruiz, Marco Milán.

  Aneuploidy, an unbalanced number of chromosomes, is highly deleterious at the cellular level and leads to senescence, a stress-induced response characterized by permanent cell-cycle arrest and a well-defined associated secretory phenotype. Here, we use a Drosophila epithelial model to delineate the pathway that leads to the induction of senescence as a consequence of the acquisition of an aneuploid karyotype. Whereas aneuploidy induces, as a result of gene dosage imbalance, proteotoxic stress and activation of the major protein quality control mechanisms, near-saturation functioning of autophagy leads to compromised mitophagy, accumulation of dysfunctional mitochondria, and the production of radical oxygen species (ROS). We uncovered a role of c-Jun N-terminal kinase (JNK) in driving senescence as a consequence of dysfunctional mitochondria and ROS. We show that activation of the major protein quality control mechanisms and mitophagy dampens the deleterious effects of aneuploidy, and we identify a role of senescence in proteostasis and compensatory proliferation for tissue repair.

Keywords:  Drosophila; aneuploidy; autophagy; chromosomal instability; mitochondrial dysfunction; mitophagy; proteotoxic stress; senescence; tissue repair

DOI:  https://doi.org/10.1016/j.devcel.2021.06.009
Methods Mol Biol. 2021 ;2312 277-285

AgDD System: A Chemical Controllable Protein Aggregates in Cells.

Yusuke Miyazaki.

  There are increasing evidence and growing interest in the relationship between protein aggregates/phase separation and various human diseases, especially neurodegenerative diseases. However, we do not entirely comprehend how aggregates generate or the clearance network of chaperones, proteasomes, ubiquitin ligases, and other factors interact with aggregates. Here, we describe chemically controllable systems compose with a genetically engineered cell and a small drug that enables us to rapidly induce protein aggregates' formation by withdrawing the small molecule. This trigger does not activate global stress responses induced by stimuli, such as proteasome inhibitors or heat shock. This method can produce aggregates in a specific compartment and diverse experimental systems, including live animals.

Keywords:  Chaperones; Chemical biology; Destabilizing domain; Phase separation; Proteasome; Protein aggregates

DOI:  https://doi.org/10.1007/978-1-0716-1441-9_16
Elife. 2021 Jul 09. pii: e69975. [Epub ahead of print]10

Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β- selection.

Xia Liu, Jingjing Yu, Longyong Xu, Katharine Umphred-Wilson, Fanglue Peng, Yao Ding, Brendan M Barton, Xiangdong Lv, Michael Y Zhao, Shengyi Sun, Yuning Hong, Ling Qi, Stanley Adoro, Xi Chen.

  Signals from the pre-T cell receptor and Notch coordinately instruct b-selection of CD4-CD8- double negative (DN) thymocytes to generate ab T cells in the thymus. However, how these signals ensure a high-fidelity proteome and safeguard the clonal diversification of the pre-selection TCR repertoire given the considerable translational activity imposed by b-selection is largely unknown. Here, we identify the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery as a critical proteostasis checkpoint during b-selection. Expression of the SEL1L-HRD1 complex, the most conserved branch of ERAD, is directly regulated by the transcriptional activity of the Notch intracellular domain. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired mouse ab T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. Accordingly, genetically inactivating PERK rescued T cell development from Sel1l-deficient thymocytes. In contrast, IRE1a/XBP1 pathway was induced as a compensatory adaptation to alleviate Sel1l-deficiency induced ER stress. Dual loss of Sel1l and Xbp1 markedly exacerbated the thymic defect. Our study reveals a critical developmental signal controlled proteostasis mechanism that enforces T cell development to ensure a healthy adaptive immunity.

Keywords:  developmental biology; immunology; inflammation; mouse

DOI:  https://doi.org/10.7554/eLife.69975
Biochem Biophys Res Commun. 2021 Jun 30. pii: S0006-291X(21)01001-9. [Epub ahead of print]569 17-22

UPRmt activation protects against MPP+-induced toxicity in a cell culture model of Parkinson's disease.

Di Hu, Zunren Liu, Xin Qi.

  The pathogenesis of Parkinson's disease (PD) remains elusive, but mitochondrial dysfunction is believed to be one crucial step in its pathogenesis. The mitochondrial unfolded protein response (UPRmt) is an important mitochondrial quality control strategy that maintains mitochondrial function in response to disturbances of mitochondrial protein homeostasis. Activation of the UPRmt and the beneficial effect of rescuing mitochondrial proteostasis have been reported in several genetic models of PD. However, the pathogenic relevance of the UPRmt in idiopathic PD is unknown. The present study examined the link between the UPRmt and mitochondrial dysfunction in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells. Treatment with MPP + induced activation of the UPRmt, reflected by an increase in the expression of UPRmt-related chaperones, proteases, and transcription mediators. UPRmt activation that was induced by overexpressing mutant ornithine transcarbamylase significantly reduced the production of mitochondrial reactive oxygen species (ROS) and improved cell survival in SH-SY5Y cells following MPP+ treatment. Moreover, the overexpression of activating transcription factor 5 (mammalian UPRmt transcription factor) conferred protection against MPP+-induced ROS production and against cell death in SH-SY5Y cells. Overall, our results demonstrate the beneficial effect of UPRmt activation in MPP + -treated cells, shedding new light on the mechanism of mitochondrial dysfunction in the pathogenesis of PD.

Keywords:  ATF5; MPP(+); Parkinson's disease; UPR(mt)

DOI:  https://doi.org/10.1016/j.bbrc.2021.06.079
J Pharm Sci. 2021 Apr 29. pii: S0022-3549(21)00236-7. [Epub ahead of print]

Analytical Challenges Assessing Protein Aggregation and Fragmentation Under Physiologic Conditions.

Joachim Schuster, Hanns-Christian Mahler, Susanne Joerg, Joerg Huwyler, Roman Mathaes.

  Therapeutic proteins are administered by injection or infusion. After administration, the physiologic environment in the desired body compartment - fluid or tissue - can impact protein stability and lead to changes in the safety and/or efficacy profile. For example, protein aggregation and fragmentation are critical quality attributes of the drug product and can occur after administration to patients. In this context, the in vivo stability of therapeutic proteins has gained increasing attention. However, in vivo protein aggregation and fragmentation are difficult to assess and have been rarely investigated. This mini-review summarizes analytical approaches to assess the stability of therapeutic proteins using simulated physiologic conditions. Furthermore, we discuss factors potentially causing in vivo protein aggregation, precipitation, and fragmentation in complex biological fluids. Different analytical approaches are evaluated with respect to their applicability and possible shortcomings when it comes to these degradation events in biological fluids. Tracking protein stability in biological fluids typically requires purifying or labeling the protein of interest to circumvent matrix interference of biological fluids. Improved analytical methods are strongly needed to gain knowledge on in vivo protein aggregation and fragmentation. In vitro models can support the selection of lead candidates and accelerate the pre-clinical development of therapeutic proteins.

Keywords:  Analytical biochemistry; In vitro model(s); Monoclonal antibody(s); Protein aggregation; Stability

DOI:  https://doi.org/10.1016/j.xphs.2021.04.014
Alzheimers Dement. 2021 Jul 05.

Diversity of transcriptomic microglial phenotypes in aging and Alzheimer's disease.

Delphine Boche, Marcia N Gordon.

  The morphological plasticity of microglia has fascinated neuroscientists for 100 years. Attempts to classify functional phenotypes are hampered by similarities between endogenous brain microglia and peripheral myeloid cells that can enter the brain under pathological conditions. Recent advances in single-cell -omic methodologies have led to an explosion of data regarding gene expression in microglia. Herein, we review the diversity of microglial phenotypes in healthy brains, aging, and Alzheimer's disease (AD); identify knowledge gaps in the body of evidence; and suggest areas in which new knowledge would be useful. Data from human samples and mouse models are compared and contrasted. Understanding the molecular complexity of the microglial response repertoire will suggest new avenues for therapeutic treatments in AD.

Keywords:  gene expression; immunity; microarray; microglia; neurodegeneration; sequencing

DOI:  https://doi.org/10.1002/alz.12389
Nat Genet. 2021 Jul 08.

Single-nucleus chromatin accessibility and transcriptomic characterization of Alzheimer's disease.

Samuel Morabito, Emily Miyoshi, Neethu Michael, Saba Shahin, Alessandra Cadete Martini, Elizabeth Head, Justine Silva, Kelsey Leavy, Mari Perez-Rosendahl, Vivek Swarup.

The gene-regulatory landscape of the brain is highly dynamic in health and disease, coordinating a menagerie of biological processes across distinct cell types. Here, we present a multi-omic single-nucleus study of 191,890 nuclei in late-stage Alzheimer's disease (AD), accessible through our web portal, profiling chromatin accessibility and gene expression in the same biological samples and uncovering vast cellular heterogeneity. We identified cell-type-specific, disease-associated candidate cis-regulatory elements and their candidate target genes, including an oligodendrocyte-associated regulatory module containing links to APOE and CLU. We describe cis-regulatory relationships in specific cell types at a subset of AD risk loci defined by genome-wide association studies, demonstrating the utility of this multi-omic single-nucleus approach. Trajectory analysis of glial populations identified disease-relevant transcription factors, such as SREBF1, and their regulatory targets. Finally, we introduce single-nucleus consensus weighted gene coexpression analysis, a coexpression network analysis strategy robust to sparse single-cell data, and perform a systems-level analysis of the AD transcriptome.

DOI: https://doi.org/10.1038/s41588-021-00894-z
Neuronal Signal. 2021 Jun;5(2): NS20210021

α-synuclein pathogenesis in hiPSC models of Parkinson's disease.

Jara M Baena-Montes, Sahar Avazzadeh, Leo R Quinlan.

  α-synuclein is an increasingly prominent player in the pathology of a variety of neurodegenerative conditions. Parkinson's disease (PD) is a neurodegenerative disorder that affects mainly the dopaminergic (DA) neurons in the substantia nigra of the brain. Typical of PD pathology is the finding of protein aggregations termed 'Lewy bodies' in the brain regions affected. α-synuclein is implicated in many disease states including dementia with Lewy bodies (DLB) and Alzheimer's disease. However, PD is the most common synucleinopathy and continues to be a significant focus of PD research in terms of the α-synuclein Lewy body pathology. Mutations in several genes are associated with PD development including SNCA, which encodes α-synuclein. A variety of model systems have been employed to study α-synuclein physiology and pathophysiology in an attempt to relate more closely to PD pathology. These models include cellular and animal system exploring transgenic technologies, viral vector expression and knockdown approaches, and models to study the potential prion protein-like effects of α-synuclein. The current review focuses on human induced pluripotent stem cell (iPSC) models with a specific focus on mutations or multiplications of the SNCA gene. iPSCs are a rapidly evolving technology with huge promise in the study of normal physiology and disease modeling in vitro. The ability to maintain a patient's genetic background and replicate similar cell phenotypes make iPSCs a powerful tool in the study of neurological diseases. This review focuses on the current knowledge about α-synuclein physiological function as well as its role in PD pathogenesis based on human iPSC models.

Keywords:  Parkinson's disease; aggregation; iPSC; α-synuclein

DOI:  https://doi.org/10.1042/NS20210021
Cell Mol Life Sci. 2021 Jul 07.

Optogenetic approaches for understanding homeostatic and degenerative processes in Drosophila.

Wen Kin Lim, Prameet Kaur, Huanyan Huang, Richard Shim Jo, Anupriya Ramamoorthy, Li Fang Ng, Jahnavi Suresh, Fahrisa Islam Maisha, Ajay S Mathuru, Nicholas S Tolwinski.

  Many organs and tissues have an intrinsic ability to regenerate from a dedicated, tissue-specific stem cell pool. As organisms age, the process of self-regulation or homeostasis begins to slow down with fewer stem cells available for tissue repair. Tissues become more fragile and organs less efficient. This slowdown of homeostatic processes leads to the development of cellular and neurodegenerative diseases. In this review, we highlight the recent use and future potential of optogenetic approaches to study homeostasis. Optogenetics uses photosensitive molecules and genetic engineering to modulate cellular activity in vivo, allowing precise experiments with spatiotemporal control. We look at applications of this technology for understanding the mechanisms governing homeostasis and degeneration as applied to widely used model organisms, such as Drosophila melanogaster, where other common tools are less effective or unavailable.

Keywords:  Aging; Cell signalling; Homeostasis; Protein folding; Tissue regeneration

DOI:  https://doi.org/10.1007/s00018-021-03836-4
Front Physiol. 2021 ;12 693067

Vascular Endothelial Senescence: Pathobiological Insights, Emerging Long Noncoding RNA Targets, Challenges and Therapeutic Opportunities.

Xinghui Sun, Mark W Feinberg.

  Cellular senescence is a stable form of cell cycle arrest in response to various stressors. While it serves as an endogenous pro-resolving mechanism, detrimental effects ensue when it is dysregulated. In this review, we introduce recent advances for cellular senescence and inflammaging, the underlying mechanisms for the reduction of nicotinamide adenine dinucleotide in tissues during aging, new knowledge learned from p16 reporter mice, and the development of machine learning algorithms in cellular senescence. We focus on pathobiological insights underlying cellular senescence of the vascular endothelium, a critical interface between blood and all tissues. Common causes and hallmarks of endothelial senescence are highlighted as well as recent advances in endothelial senescence. The regulation of cellular senescence involves multiple mechanistic layers involving chromatin, DNA, RNA, and protein levels. New targets are discussed including the roles of long noncoding RNAs in regulating endothelial cellular senescence. Emerging small molecules are highlighted that have anti-aging or anti-senescence effects in age-related diseases and impact homeostatic control of the vascular endothelium. Lastly, challenges and future directions are discussed including heterogeneity of endothelial cells and endothelial senescence, senescent markers and detection of senescent endothelial cells, evolutionary differences for immune surveillance in mice and humans, and long noncoding RNAs as therapeutic targets in attenuating cellular senescence. Accumulating studies indicate that cellular senescence is reversible. A better understanding of endothelial cellular senescence through lifestyle and pharmacological interventions holds promise to foster a new frontier in the management of cardiovascular disease risk.

Keywords:  DNA damage; SASP; anti-senescent therapies; cellular senescence; long noncoding RNAs; vascular endothelium

DOI:  https://doi.org/10.3389/fphys.2021.693067
J Cell Biol. 2021 Sep 06. pii: e202103186. [Epub ahead of print]220(9):

zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.

Ashley M Bourke, Samantha L Schwartz, Aaron B Bowen, Mason S Kleinjan, Christina S Winborn, Dean J Kareemo, Amos Gutnick, Thomas L Schwarz, Matthew J Kennedy.

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.

DOI: https://doi.org/10.1083/jcb.202103186
Methods Cell Biol. 2021 ;pii: S0091-679X(21)00069-8. [Epub ahead of print]164 xvii-xxi

Vesicular trafficking in cellular responses to stress.

Oliver Kepp, Lorenzo Galluzzi.



Keywords:  Autophagic flux; Endoplasmic reticulum; Golgi apparatus; Immunogenic cell death; Protein trafficking; Xenobiotic detoxification

DOI:  https://doi.org/10.1016/S0091-679X(21)00069-8
Cell. 2021 Jun 30. pii: S0092-8674(21)00710-8. [Epub ahead of print]

A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation.

Yanniv Dorone, Steven Boeynaems, Eduardo Flores, Benjamin Jin, Shannon Hateley, Flavia Bossi, Elena Lazarus, Janice G Pennington, Emiel Michiels, Mathias De Decker, Katlijn Vints, Pieter Baatsen, George W Bassel, Marisa S Otegui, Alex S Holehouse, Moises Exposito-Alonso, Shahar Sukenik, Aaron D Gitler, Seung Y Rhee.

  Many organisms evolved strategies to survive desiccation. Plant seeds protect dehydrated embryos from various stressors and can lay dormant for millennia. Hydration is the key trigger to initiate germination, but the mechanism by which seeds sense water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We find intragenic, intraspecific, and interspecific natural variation in FLOE1 expression and phase separation and show that intragenic variation is associated with adaptive germination strategies in natural populations. This combination of molecular, organismal, and ecological studies uncovers FLOE1 as a tunable environmental sensor with direct implications for the design of drought-resistant crops, in the face of climate change.

Keywords:  adaptation; bet hedging; biomolecular condensate; intrinsically disordered proteins; phase separation; prion-like; salt stress; seed germination; water sensing; water stress

DOI:  https://doi.org/10.1016/j.cell.2021.06.009
Pharmacol Res. 2021 Jul 05. pii: S1043-6618(21)00340-6. [Epub ahead of print] 105756

Autophagy in vascular dementia and natural products with autophagy regulating activity.

Wenzhe Wang, Ou Qiao, Haixia Ji, Xinyu Zhang, Xiaoying Han, Yi Zhang, Juan Wang, Xia Li, Wenyuan Gao.

  Chronic Cerebral Hypoperfusion(CCH)-induced vascular dementia(VD) is a common neurodegenerative disease which seriously affects the patient's quality of life. Therefore, it is critical to find an effective treatment of VD. Autophagy is a natural regulated mechanism that can remove dysfunctional proteins and organelles, however, over-activation or under-activationcan of autophagy can induce the apoptosis of cells. Although autophagy plays a role in the central nervous system is unquestionable, the effects of autophagy in the ischemic brain are still controversial. Some autophagy regulators have been tested, suggesting that both activation and inhibition of autophagy can improve the cognitive function. This article reviews the role of autophagy in CCH-induced VD to discuss whether autophagy has the potential to become a target for drug development and provides several potential compounds for treating vascular dementia.

Keywords:  autophagy; chronic cerebral hypoperfusion; natural products; vascular dementia

DOI:  https://doi.org/10.1016/j.phrs.2021.105756
Cell Death Discov. 2021 Jun 30. 7(1): 154

Methyltransferase-like protein 7A (METTL7A) promotes cell survival and osteogenic differentiation under metabolic stress.

Eugene Lee, Ju-Young Kim, Tae-Kyung Kim, Seo-Young Park, Gun-Il Im.

While bone has an inherent capacity to heal itself, it is very difficult to reconstitute large bone defects. Regenerative medicine, including stem cell implantation, has been studied as a novel solution to treat these conditions. However, when the local vascularity is impaired, even the transplanted cells undergo rapid necrosis before differentiating into osteoblasts and regenerating bone. Thus, to increase the effectiveness of stem cell transplantation, it is quintessential to improve the viability of the implanted stem cells. In this study, given that the regulation of glucose may hold the key to stem cell survival and osteogenic differentiation, we investigated the molecules that can replace the effect of glucose under ischemic microenvironment of stem cell transplantation in large bone defects. By analyzing differentially expressed genes under glucose-supplemented and glucose-free conditions, we explored markers such as methyltransferase-like protein 7A (METTL7A) that are potentially related to cell survival and osteogenic differentiation. Overexpression of METTL7A gene enhanced the osteogenic differentiation and viability of human bone marrow stem cells (hBMSCs) in glucose-free conditions. When the in vivo effectiveness of METTL7A-transfected cells in bone regeneration was explored in a rat model of critical-size segmental long-bone defect, METTL7A-transfected hBMSCs showed significantly better regenerative potential than the control vector-transfected hBMSCs. DNA methylation profiles showed a large difference in methylation status of genes related to osteogenesis and cell survival between hBMSCs cultured in glucose-supplemented condition and those cultured in glucose-free condition. Interestingly, METTL7A overexpression altered the methylation status of related genes to favor osteogenic differentiation and cell survival. In conclusion, it is suggested that a novel factor METTL7A enhances osteogenic differentiation and viability of hBMSCs by regulating the methylation status of genes related to osteogenesis or survival.

DOI: https://doi.org/10.1038/s41420-021-00555-4
Biochim Biophys Acta Gen Subj. 2021 Jul 03. pii: S0304-4165(21)00113-6. [Epub ahead of print] 129954

Comprehensive analysis of O-glycosylation of amyloid precursor protein (APP) using targeted and multi-fragmentation MS strategy.

Jingjing Shi, Xin Ku, Xia Zou, Jingli Hou, Wei Yan, Yan Zhang.

   BACKGROUND: The aberrant proteolytic processing of amyloid precursor protein (APP) into amyloid β peptide (Aβ) in brain is a critical step in the pathogenesis of Alzheimer's disease (AD). As an O-glycosylated protein, O-glycosylation of APP is considered to be related to Aβ generation. Therefore, comprehensive analysis of APP O-glycosylation is important for understanding its functions.
METHODS: We developed a Targeted MS approach with Multi-Fragmentation techniques (TMMF strategy), and successfully characterized O-glycosylation profiling of APP695 expressed in HEK-293 T cells. We calculated relative abundance of glycopeptides with various O-glycosites and O-glycans, and further investigated the alteration of APP O-glycosylation upon TNF-α treatment.
RESULTS: A total of 14 O-glycosites were identified on three glycopeptides of APP, and at least four O-glycans including GalNAc (Tn antigen), core 1, and mono-/di-sialylated core 1 glycans were determinant at the residues of Thr576 and Thr577. We found a dense cluster of truncated O-glycans on the region nearby beginning of E2 domain and high abundance of sialylated O-glycans on the region close to β-cleavage site. Moreover, we also observed that TNF-α could upregulate the expression of APP and the truncated O-glycans on APP in HEK-293 T cell.
CONCLUSION: Our study established an intact O-glycopeptide MS analysis strategy for APP O-glycopeptide identification with enhanced fragmentation efficiency and detection sensitivity. These results provide a comprehensive O-glycosylation map of APP expressed in HEK-293 T cell.
GENERAL SIGNIFICANCE: The accurate O-glycosites and O-glycan structures on APP may lead to a better understanding of the roles O-glycosylation plays in the processing and functions of APP.

Keywords:  APP; Alzheimer's disease; Aβ; Intact O-glycopeptides; Multiple fragmentation MS analysis; O-glycosylation

DOI:  https://doi.org/10.1016/j.bbagen.2021.129954
Mol Ther. 2021 Jul 01. pii: S1525-0016(21)00351-8. [Epub ahead of print]

Adenine Base Editing Reduces Misfolded Protein Accumulation and Toxicity in Alpha-1 Antitrypsin Deficient Patient iPSC-Hepatocytes.

Rhiannon B Werder, Joseph E Kaserman, Michael S Packer, Jonathan Lindstrom-Vautrin, Carlos Villacorta-Martin, Lauren E Young, Yvonne Aratyn-Schaus, Francine Gregoire, Andrew A Wilson.

Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient-induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole genome sequencing. These results reveal the feasibility and utility of base-editing to correct the Z mutation in AATD patient cells.

DOI: https://doi.org/10.1016/j.ymthe.2021.06.021
Nat Chem Biol. 2021 Jul 08.

K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.

Yuanyuan Yu, Qingyun Zheng, Satchal K Erramilli, Man Pan, Seongjin Park, Yuan Xie, Jingxian Li, Jingyi Fei, Anthony A Kossiakoff, Lei Liu, Minglei Zhao.

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

DOI: https://doi.org/10.1038/s41589-021-00823-5
Geroscience. 2021 Jul 09.

Role of heat shock proteins in aging and chronic inflammatory diseases.

Christian R Gomez.

  Advanced age is associated with a decline in response to stress. This contributes to the establishment of chronic inflammation, one of the hallmarks of aging and age-related disease. Heat shock proteins (HSP) are determinants of life span, and their progressive malfunction leads to age-related pathology. To discuss the function of HSP on age-related chronic inflammation and illness. An updated review of literature and discussion of relevant work on the topic of HSP in normal aging and chronic inflammatory pathology was performed. HSP contribute to inflamm-aging. They also play a key role in age-associated pathology linked to chronic inflammation such as autoimmune disorders, neurological disease, cardiovascular disorder, and cancer. HSP may be targeted for control of their effects related to age and chronic inflammation. Research on HSP functions in age-linked chronic inflammatory disorders provides an opportunity to improve health span and delay age-related chronic disorders.

Keywords:  Age; Chronic; Disease; HSP; Inflammation; Therapy

DOI:  https://doi.org/10.1007/s11357-021-00394-2